Diss. ETH No. 14234
Cloning
and functional
analysis
of bacterial genes
involved in alkane oxidation
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
for the
degree
of
Doctor of Natural Sciences
Presented
by
THEODORUS HENRIKUS MARIA SMITS
M.Sc.
Bioprocess technology
Born June
22, 1973
Citizen of The Netherlands
Accepted
of the recommendation of
Prof. Dr. B.
Prof. Y.
Dr. N.
Dr. J.B.
Witholt, examiner
Barrai, co-examiner
Mantei, co-examiner
van
Beilen, co-examiner
Zürich 2001
ZÜRICH
ii
Ill
Acknowledgements
"De laatste
it is hard to
Looking back,
I
wegen het zwaarst" is
loodjes
simply
At the first
place
they
to Zürich to
Yves
Barrai, who kindly agreed
perform
Eppenberger
The person I
expression
at this
owe a
as
that
fits at this
place.
Biotechnology
where
really
I would like to thank all
position,
people
were.
I have to thank Prof. Bernard Witholt to
come
Dr. H.M.
Dutch
turn your back to the Institute of
spent four very fine years. Therefore,
that made these years what
a
this work.
on
My
give
me
opportunity
to
thanks also go to Dr. Ned Mantei and Prof.
co-examinator for the
being
the
exam.
I also thank Prof.
head of the examination committee.
lot to is Dr. Jan
nearly daily "group-meetings"
in
Beilen. He
van
our
taught
at the corridor
lab,
me
or
research at the best. Our
in his office
brought
me
many
aspects of the real lab-life: experimental setup and planning, self-criticism, logic thinking,
and
doing
His
patience
taught
me
those control
the valuable lesson that theories
more
only
our
to be
lab: Urban
practical
our
are
ever
think of but that should be done.
preliminary
good,
ideas and conclusions
but first you have to prove them.
new
help
with
sequence and
greatly acknowledged.
They brought
"Lehrlinge"
work. A
for her
sequencing.
an even
As this thesis
larger
amount of control
Thanks also to the students who
Fritsche, Jasminka Kovac, Alessandro Franchini, Markus
and Daniel Lüscher.
thanks goes to
of the
Röthlisberger
about 65 kilobases of
reactions, her help has
Seeger
would
I will take for the rest of my life.
I have to thank Martina
worked in
no-one
with my often very enthousiastic and
This and much
contains
experiments
big
Stefanie
"thank
you"
the
right atmosphere
Balada, who helped
to all
me
to the lab. A very
with
a
special
substantial amount
present and former IBT members, for their
support, discussions, encouragement, lunches, basketball games and other fun activities.
IV
Special
thanks to Helena Zuber for the technical and administrative aspects and her
encouraging
role in
organizing
Mosenbacher and Monica
solving
Hartwig
for
cleaning
the
glass
ware
has, against all public opinion, still
acknowledge
my
family
a
private
holidays
were
and
are
have had
nothing
to do:
our
endless discussions about your
still very welcome.
weekends. I also want to thank Ruth's
especially
when I needed
This work
was
National
early
Foundation, project
at age 26.
family,
My
dear
who became
position
numerous
I would
of letters and
Ruth, without you I would
work,
a
our common
second
family
walks, the
for me,
one.
supported by
This work is dedicated to
life. At this
in Holland. Their support, care,
common
too
and to Peter Koller for
the technical failures.
A Ph.D. student
like to
lots of social events for the lab. Thanks also to Helen
the Swiss
nr.
Priority Program
in
Biotechnology
of the Swiss
5002-037023.
Martijn
S.
van
der
Gaag (1973-2000),
who
passed
away much
V
Table of contents
Page
Summary
vi
Zusammenfassung
vii
Abbreviations
viii
Chapter
1
Introduction
Chapter
2
Molecular
1
screening
Gram-negative
Chapter 3
New
and
for alkane
hydroxylase
Gram-positive
alkane-responsive expression
genes in
bacteria
21
vectors for
Escherichia coli
and Pseudomonas
Chapter 4
Cloning
and functional
Gram-negative
Chapter
5
43
Functional
and
analysis
analysis
of alkane
hydroxylases
from
55
Gram-positive organisms
of rubredoxin reductases involved in
alkane oxidation
Chapter
6
Functional characterization of genes involved in alkane oxidation
by
Chapter 7
Pseudomonas
Cloning
8
Factors
9
by
influencing
hydroxylase
Chapter
83
aeruginosa
and characterization of genes involved in
alkane oxidation
Chapter
75
Summary
long-chain
Pseudomonas fluoréscens CHAO
the substrate range and
of Pseudomonas
and conclusions
activity
putida (oleovorans)
93
of the alkane
GPol
117
129
References
141
Curriculum vitae
171
VI
Summary
bacteria
Many
Pseudomonas
an
linear n-alkanes
can use
putida (oleovorans) GPol,
to clone
new
alkane
hydroxylases
highly degenerate oligonucleotides
genes
homologous
to the GPol alkane
medium
(C6-C11)
peptides
with 37.1-100 % sequence
AlkB.
Using
several
or
the PCR
Gram-negative
expressing
all
proteins
and
GPol alkB
substrate
give
on
and
probes,
All
alkanes.
catalysis.
were
newly
alkanes
to the
analysis.
amplify
growth.
study
For this purpose,
internal
PCR
In
catalyzed by
fragments
bacteria able to grow
yielded
of
on
fragments encoding
corresponding fragment
of the GPol
homologs
from
strains. Four recombinant host strains
on
alkanes except
developed
an
alkane
for the functional
cloned alkane
Sequence comparisons
as
for
The aim of this
have cloned the alkB
we
growth
vectors
first indications
binding
identity
necessary for
hydroxylase homologs.
of the hosts to grow
as
to
hydroxylase. Many
Gram-positive
alkane-responsive expression
alkane
developed
long-chain (C12-C16)
fragments
hydroxylase (AlkB).
for structure-function
were
source
the first step of alkane oxidation is
non-heme iron alkane
integral-membrane
was
sole carbon and energy
as
hydroxylases
and in vivo
to which amino acid residues
hydroxylase
analysis
are
of the
enable at least
mutagenesis
important
and
one
of the
for
Vil
Zusammenfassung
Viele Bakterien wachsen auf n-Alkanen. Der erste Schritt des Alkanmetabolismus wird in
Pseudomonas
Eisen
putida (oleovorans)
GPol
von
einer
Alkan-Hydroxylase (AlkB) katalysiert.
Hydroxylasen
analysieren.
zu
klonieren
Das Ziel dieser Studie
welche der GPol
sind. Aus einer Vielzahl
Gram-negativen
mittellangen (C6-C11)
PCR-Fragmenten,
Bestätigung,
von
oder
deren
korrespondierenden
aus
Teil der GPol
dass die
Gram-positiven
Alkanen
Alkan-
zu
37.1-100 % Identität
und zwei
Colony
Bakterien die auf
zu
dem
erhalten werden. Mittels
Blotten wurden
Gram-positiven
homologe Alkan-Hydroxylasen
ähnlich
wachsen, konnten solche
Alkan-Hydroxylase haben,
Gram-negativen
homologe
Bakterien kloniert. Zur
auch wirklich Alkanen
oxidieren,
entwickelt, welche mit Ausnahme der Alkan-Hydroxylase alle
für das Wachstum auf Alkanen
auf die für
und
als Sonden für das Southern und
fünf
Alkan-Hydroxylasen
Sequenzanalysen
neue
Alkan-Hydroxylase Gensequenz
langkettigen (C12-C16)
Proteinsequenzen
wurden vier Wirt-Stämme
klonierten
es,
beziehungsweise Struktur-Funktionsbeziehungen
Genfragmenten amplifiziert,
alkB Gene
war
nicht-Häm-
wurden, mittels hoch degenerierter Oligonucleotiden, interne
Dazu
PCR-Fragmenten
membrangebundenen
und in vivo
Substratbindung
notwendig
des GPol alkBs
Katalyse wichtige
Die Funktion der
exprimieren.
wurde in den vier Wirt-Stämme
Mutagenese
und
Proteine
neu
erfolgreich getestet.
ergaben
Aminosäuren.
zudem erste Hinweise
Vlll
Abbreviations
Ap
ampicillin
bp
base
C5
pentane
C6
hexane
C8
octane
CIO
decane
Cll
undecane
C12
dodecane
C14
tetradecane
C16
hexadecane
C18
octadecane
C20
eicosane
C24
tetracosane
C28
octacosane
C30
triacontane
C32
dotriacontane
Cm
chloramphenicol
(k)Da
(kilo)dalton
DCPK
dicyclopropylketone
DMMZ
Department
DOPh
dioctylphthalate
EMBL
European
dNTP
deoxynucleotide triphosphate
FAD
flavin adenine mononucleotide
G+C
guanine-plus-cytosine
Gm
gentamycin
HPLC-MS
high-performance liquid chromatography-mass spectroscopy
IPTG
isopropyl-ß-D-thiogalactopyranoside
pair(s)
of Medical
Molecular
Microbiology
Zürich
Biology Laboratory
IX
kb
kilobasepairs
Km
kanamycin
LB
Luria-Bertani broth
MALDI-TOF
matrix-assisted laser
desorption/ionisation, time-of-flight mass
spectrometry
Chemotaxis
MCP
methyl-accepting
protein
MT
metal traces
NAD(H)
nicotinamide adenine dinucleotide
(reduced form)
NCBI
National Centre for
Information
OMP
outer membrane
ORF
open-reading-frame
PCR
polymerase
SDS-PAA
sodium
dodecyl sulfate-polyacrylamide
SDS-PAGE
sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
Tc
tetracycline
v/v
volume per volume
w/v
weigth
Biotechnology
protein
chain reaction
per volume
X
Chapter
1
INTRODUCTION
Theo H. M. Smits
2
1. INTRODUCTION
1.1
of
Origin
hydrocarbons
Hydrocarbons
atoms.
chemistry
chain
or
geochemical
n-alkanes
alkanes, terpenes and aromatic compounds
processes, while others
as
constitutent of the
present in plant
wax.
plant
wax
has
a
different
The
gas methane is
greenhouse
organic
occurs
methane has doubled
A
large
amount of
by large
annum, but
hydrocarbons
tanker
comes
from
and storage tanks and
natural sources,
The
consists of
resins and
this feature
can
communities
large quantities by
over
a
the last 200 years, the
the
major
length n-alkanes,
are as
well found
be used
marker
as a
(54).
microbial
constituent of
major
(212). The
being
decomposition
conditions, e.g. in landfills, marshlands and
ends up in nature
petroleum
enters the
sea.
by
oceans.
It
natural-gas deposits.
atmospheric
human activities.
The
major part
accidents, which constitute 'only'
municipal
discharge
principally
composition
even-chain alkanes
complete plant
in
odd-chain
very-long-
concentration of
(166).
35 million metric tons of
released
of
in many coal formations and is
due to human activities
Largely
though
composition,
produced
material under anoxic
mainly very-long
are
between 25 and 37 carbon atoms,
As each
varies from trace amounts to
waxes
Plant n-alkanes
species composition
also
produced
produced by synthetic organic
sterols and flavonoids
triterpenes,
esters,
to determine the
of
are
are
natural constituents of waxes, which also contain
fatty acids, alcohols, ketones,
amount of n-alkanes
(54).
like
hydrogen
processes.
produce
varying
of carbon and
ubiquitous organic compounds consisting
Many hydrocarbons
by biological
Plants
are
in the environment
of
and industrial wastes and
dirty
seepages,
of mineral oil is
ballast and
only
bilge
dependent
on
million metric tons per
runoffs, leaks in pipelines
waters. Oil
the oil well
around
of this oil is not
input
accounts for 0.5 million tons
paraffins, cycloalkanes (naphthenenes),
asphaltenes) (101).
one
Annually,
to the
sea
from
annually (218).
(206). Mineral oil generally
aromatics and other
compounds (e.g.
3
1.2
Biodegradation
of
hydrocarbons
Many petroleum hydrocarbons
components
are
are more
degraded
are
less
decomposed
readily degraded
more
readily
readily
than
aromatic
polycyclic
Its recalcitrance is
due to the low
rates
are
compounds
directly proportional
determined
by
and
structure and molecular
than aromatic
chain
for
Saturated
and branched chains
compounds,
reflects the
biodegradability. Particularly
petroleum
to the molecular
availability
hydrocarbons.
weight.
compounds (37).
fraction of
hydrocarbon (PAH)
solubility
on
than unsaturated
straight
The residence time of individual oil
the
the oil
readily degraded by microorganisms. However,
degraded differentially depending
are
Aliphatic paraffins
compounds
are
weight
of the
biological uptake (9).
the balance of the substrate
easily degraded.
compound, mainly
Microbial
degradation
and substrate transfer to the
cells. The substrate transfer in turn is limited
by
soil matrix. As
low, halflife times of individual compounds
degradation
vary between less than
1.2.1
Microbiology
The presence of
one
of oil
after
an
oil
soil
(225).
spill,
in
pristine
fungi
and
occurs
soil
by
studies
frequently brings
can
increase
populations
mixed cultures
isolates with
are
usually Gram-positives
are
found less
frequently.
or
The
In nature,
an
in situ
relatively
low level
magnitude
biodégradation
of
and in the presence of other
are rare.
can
One
example
is
overgrow bacteria in
hydrocarbon-degrading species belonging
yield
about
three orders of
slowly (218).
mixed
on
by
alkane-degrading yeasts
have been isolated in the past
polluted
soil
and then decreases
which showed that
Numerous
(105).
in the environment
organic compounds. However,
study,
of the substrate from the
spills
hydrocarbons predominantly
recent
desorption
hydrocarbon-utilizing microorganisms.
hydrocarbon degraders
directly
are
year and infinite
hydrocarbons
selective enrichment for
of
rates of PAH
the
uptake
is not
to the
a
sandy
bacteria, yeasts,
(37). Enrichments of microorganisms from pristine
varying
substrate
pseudomonads,
specificities (217).
These isolates
while members of other bacterial
species
4
1.2.1.1 Genetic
approaches
to
Several authors have tried to find
presence of genes
fragments
gene
the
xylE
occurs
gene,
in
degradation potential
assess
a
relation between
coding
for aromatic and
probes
for Southern
as
the aromatic
encoding
large populations
of
biodégradation capacity
aliphatic degradation pathways using
colony blotting (98, 243, 247).
or
ring-cleaving
oil-degrading
is the alkane
probe
section
1.3.4.1), which
enrichment
highly
on
was
crude oil
1.2.2 Anaerobic
bacteria isolated from contaminated
gene alkB of P.
putida (oleovorans)
GPol
(see
organisms
after
(243). The results indicated that hexadecane mineralization is
occurred when strains
long
of
degradation
been assumed that
degradation
of
hydrocarbons
aromatic and
able to
degrade
ß- and 3-subclasses of
proteobacteria (reviewed
The initial attack of the anaerobic
succinate)
to the
degradation
The anaerobic
degradation
bacteria
proteobacteria.
understood.
(247, 273).
not take
in
have been
in the absence of
and
belong
to the
(108)).
a
compounds
does not involve
cellular metabolite
the initial enzyme
This mechanism is
sequenced.
under
microorganisms
sulphate reducing
by coupling
encoding
place
activity
general
(for
toluene
for toluene
for all
degraders (108).
between hexane and
degrading
Genes
or
of aromatic
degradation
have been cloned and
anaerobic toluene
denitrifying
but is achieved
hydrocarbon.
decane
aliphatic hydrocarbons
organisms
are
can
the last ten years several
oxygen. Most of these
oxygen-dependent step,
on
hydrocarbons
isolated that
are
able to grow
were
over
as
aquifers
at much lower levels. One
found in up to two third of the culturable
anaerobic conditions. However,
an
found that
was
correlated to the presence of alkB. However, other authors found that
hybridization only
It has
hydroxylase
It
known
catechol-2,3-dioxygenase,
enzyme
(98, 243). Other gene probes also give specific signals, but
such
and the
of n-alkanes has been
eicosane, although only
were
obtained
a
reported
for alkanes in the range
few pure cultures of anaerobic alkane
(2, 71, 242), all of which belong
to the
3-subclass of
The biochemical basis of anaerobic alkane metabolism is still
Dehydrogenation
described for P.
of the alkane to
aeruginosa
196Aa
(205)
a
1-alkene and
most
probably
hydratation
was an
to
artefact
poorly
an
1-alcohol
(11). Based
on
5
the
fatty
acid pattern of strain
from even-chain
alkanes,
Hxd3, which shows formation of odd-chain fatty acids
carboxylation
a
(2). This would then correspond
pathway
in
which
can
coupling
enrichment
an
unknown component to the alkane
culture, that fumarate
dodecylsuccinnic
acid
Degradation
Several
of
shown for
was
pathways
was
coupled
known for the aerobic
The TOL
plasmid
xylene
encodes the
monooxygenase
XylMA,
a
of benzoic
is oxidized to form
mt-2 is
A
a
dioxygenases.
The upper
pathway (38).
todABClC2)(94),
The lower
pathway
and the
genes
for the
ß-ketoadipate pathway,
pathway
to
give
for the
which in the
degradation
case
of P.
of
putida
(182).
compounds
a
is
large
initially
gene
oxidized to
family
of
a
dihydrodiol by
multicomponent
Rieske
non-
(39, 93), of which toluene dioxygenase (encoded by
benzene
place
level.
acid, the end product of the upper pathway (38). Here,
dioxygenase (encoded by bedABClC2)(252)
(encoded by nahAaAbAcAd)(231)
takes
These
encodes the
meta-pathway, responsible
can
systems. The dihydrodiol is subsequently converted
opening
genetic
pathway
benzyl alcohol dehydrogenase XylB
These enzymes form
heme iron oxygenases
di oxygenase
or
located
number of aromatic
compounds.
catechol, which is subsequently opened
ortho-pathway
chromosomally
large
fatty acid,
molecule, forming
of aromatic
intermediates that enter the central metabolism. A second
benzoate is the
a
bacterial
the biochemical and the
on
encode the enzymes of the
subsequent degradation
ring
in
via the ß-oxidation
degraded
to the dodecane
degradation
benzaldehyde dehydrogenase XylC (102, 251).
the
resulting
which oxidize toluene and its dérivâtes. These
xy/-genes
divided in the upper and lower
xylXYZLTEGFJQKIH
is
or
strain AK-
hydrocarbons
have been well characterized both
are
acids
biosynthesis
reducing
sulfate-reducing, dodecane-grown
a
pathways
genes
fatty
proposed
(146).
of aromatic
are
reaction has been
reversed mechanism of the alkane
then be found back in the cellular
(241). Recently, it
1.2.3
a
carbonylation
A different mechanism found in the sulfate
plants (212).
01 involves the
to
or
similar to that of the
xylene
be considered
to
lower
a
as
and
naphthalene
prototype enzyme
catechol, after which
pathway.
a
ring
6
1.2.4
Degradation
of
alicyclic hydrocarbons
Although cyclic
alkanes
isolated that
degrade
grow
on
can
cyclic
are a
these components
the past years
system, yielding
a
cyclic
cyclic
oxidized
be further metabolized
key steps
in
alkane
induced
strains
by
a
The
can
breaks
a
dehydrogenation step yields
or
by
an
yielding
esterase,
cycloalkane
oxidation
oxidize
ring
are
the
is done
cyclic compounds
enzymes
not
are
monooxygenase is
an
oxidation of the
the
keton, which is
The
resulting
ring-
lactone is
which
co-hydroxy fatty acid,
ring-oxidation
by
can
a
an
fungi (48, 254, 279). Recently,
several
able to grow
Baeyer-Villiger
monooxygenase. Several alkane-
on
because
cyclic compounds,
present.
oxygen-dependent
one
atom of oxygen.
monooxygenase which
Baeyer-Villiger
species belonging
Baeyer-Villiger
on
and the
when the alkane oxidation system is
monooxygenases have been isolated from several
organisms
an
these strains do not grow
carbon-carbon bond and inserts
cloned from
utilized via
are
(48, 206).
metabolizing
Baeyer-Villiger
involves
Baeyer-Villiger monooxygenäse.
(259, 264), although
downstream
to enrich strains able to
degradation usually
reaction. The initial oxidation of the
oxidizing
(217). The failure
(101). However, several pure cultures have been isolated
alcohol. A
hydrolysed spontaneously
The two
few bacteria have been
(206, 227)(van Beilen, unpublished results).
The initial reaction of
subsequently
petroleum,
alkanes led to the conclusion that in nature these substrates
cooxidation and commensalism
over
of
major component
to the bacteria and
were
monooxygenase genes
cycloalkanes (36, 47)(J.
B.
van
Beilen,
unpublished results).
1.3 Aerobic
The
degradation
degradation
attention from
of linear
and bioconversion of
microbiologists
microorganisms
aliphatics
to
very common, and
use
a
aliphatic hydrocarbons
and biochemists for many decades. The
aliphatic hydrocarbons (alkanes)
large
number of
species beloning
as
to
been described to utilize alkanes. Some excellent reviews
published
in the past
(31, 37).
has received
a
great deal of
capacity
carbon and energy
of
source
is
bacteria, yeasts and fungi have
on
this field have been
7
1.3.1
The
for alkane oxidation
Pathways
of n-alkanes
degradation pathway
alcohol. In most cases, this takes
generally
place
at the terminal carbon
oxidation of alkanes has also been described
perform
and soluble non-heme iron alkane
hydroxylases
can
bacteria, cytochrome P450s perform
some
while for
monooxygenation (section 1.3.2),
an
atom, but subterminal
Several enzyme systems
(section 1.3.5).
this initial attack. In yeasts, but also in
the initial
starts with oxidation of the alkane to
some
bacteria
integral-membrane
have been described
(sections
1.3.3 and
1.3.4).
The terminal
fatty
alcohol, the product of the first monooxygenase reaction, is converted
acid via
(CoA) by
an
aldehyde.
fatty
acyl-CoA synthetase,
an
the ß-oxidation
cycle
dioxide and energy
phospholipids,
chain-length
while
The
is
acid is
and enters the ß-oxidation
acetyl-CoA,
which
can
they
alkanes will
even-chain-length
reflect the
yield
more
alkanes
chain-length
odd-chain
yield
a
higher
The ratio between saturated and unsaturated
shifts
1.3.2
more
towards saturated
Cytochrome
Cytochrome
P450
fatty
cycle.
enter the Krebs
(23). Alternatively, the fatty acids
where
activated with
subsequently
on
fatty
are
to
yield
a
A
product
of
carbon
directly incorporated
into the
which the strain is grown: odd-
acid moieties in the
amount of even-chain
fatty
Co-enzyme
The end
cycle
to
acid moieties in the
acids when n-alkanes
are
used
as
phospholipids,
fatty
acid moieties.
phospholipids
carbon
source
(63).
systems
P450 enzymes
are
ubiquitous
in nature
(180). While broad substrate
spectrum cytochrome P450s in mammals oxidize many xenobiotics and steroids,
cytochrome
P450s in
microorganisms
Bacterial P450s often
reaction of the
occur
growth
can
in catabolic
substrate
Alkane-oxidizing cytochrome
have
a more narrow
pathways, catalysing
substrate
the initial
specifity (130).
hydroxylation
(180).
P450s
are
nearly exclusively
found in yeasts, but
some
reports describe the presence of alkane-oxidizing cytochrome P450s in bacteria. In the
yeasts Candida rugosa and Yarrowia lipolytica, several orthologs have been found, but
only
few of those
maltosa
are
cytochrome
proven to be involved in alkane oxidation
P450
(122, 196). The Candida
52A3, which is involved in the initial oxidation of n-alkanes
to
8
alcohols,
also
can
catalyze
more
than
one
step, yielding also aldehydes, fatty acids,
a-co-
diols, ro-hydroxy-fatty acids and a-co-dicarboxylic acids (221). Bacterial cytochrome
P450s involved in alkane oxidation
19067
identified in Rhodococcus rhodochrous ATCC
are
(44) and in Acinetobacter calcoaceticus
EB104
(179).
1.3.3 Non-heme iron oxygenases
The
large
group of non-heme iron oxygenases
can
be divided in two
chain non-heme iron monooxygenase systems and the
subgroups:
integral-membrane
the short-
non-heme iron
oxygenases.
1.3.3.1 Short-chain non-heme iron monooxygenase
Several short-chain
hydrocarbon
detail. Several enzyme systems
systems
oxidation systems have been characterized in great
can
be
distinguised:
soluble and
particulate
methane
monooxygenases and alkene monooxygenases.
Methane is oxidized to methanol
monooxygenase
soluble MMOs
extensively
components:
a
MMO is
an
a
and
a
and
soluble
or a
particulate
site of the enzyme
hydroxylase
The a-subunit contains
(181). The diiron
an
to date possess
a
enzyme
a
those from
OB3b
(reviewed
complex consisting
serves an
"effector"
consists of three subunits
non-heme
in
of three
or
arranged
oxygen interact to form methanol at the active
including
conserved
glutamate
or
(EX^-(D/E)EXRH-XJ00-EX^-(D/E)EXRH)(33).
by
a
aspartate and
This enzyme
system is expressed only when the copper-to-biomass ratio is low. Soluble MMO has
extremely
broad substrate
substrates such
as
specificity
in
bis-/^-hydroxo-bridged
centre in soluble MMO is coordinated
conserved amino acid sequence motif
histidine residues
are
Methylosinus trichosporium
the so-called Protein B, which
binuclear iron centre where methane
methane
widespread (181).
iron-containing
reductase. The
a2ß2Y2 configuration.
less
are
non-heme
hydroxylase,
regulatory role,
a
characterized soluble MMO enzymes
Methylococcus capsulatus (Bath)
(160)). Soluble
either
(MMO). While virtually all methanotrophs studied
particulate MMO,
The two most
by
and
can
oxidize
a
wide range of
alkanes, alkenes and aromatic compounds.
non-growth
an
9
Particular MMO is
mainly expressed
copper-to-biomass
ratio is
subunits, and contains
binding compounds
MMO has
relatively
a
likely
narrow
carbons but not aromatic
Xanthobacter sp.
a
approximately
15 copper atoms per mol.
substrate range,
oxidizing
alkanes and alkenes of up to 5
compounds (181).
(238):
a
chain-length
monomeric
coupling protein
are
alkanes
reductase,
required
a
as
B-276 consists of
reductase component
epoxidize
a
variety
a
ferredoxin,
for full
two-component epoxygenase,
alkenes, but
not
can
Both epoxygenases mentioned above have
that of methane monooxygenases
and histidine. Based
on
the
hydroxylate
can
a
an
a2ß2Y2
structured
(formerly
Nocardia
coupling protein
B-276
alkanes
can
and
a
enantioselectively
(89).
binuclear non-heme iron center similar to
a
(90, 291) with iron coordination via glutamate/aspartate
homology
of the monoxygenase component of these enzyme
systems and the coordination of the non-heme iron center,
heme iron oxygenases
epoxides,
activity.
(178). Whole cells of R. rhodochrous
of
propene to
(267). The enzyme system consists
The alkene monooxygenase system of Rhodococcus rhodochrous
corallina)
Copper-
to be involved in the stabilization of the enzyme. Particular
but is not able to oxidize similar
epoxygenase and
under conditions where the
is able to oxidize short-chain alkenes such
Py2
of four components
methanotrophs
The membrane-bound enzyme system consists of three
high.
two iron and
are
in
a
large family
be defined which also includes the benzene-
of soluble
(bmo),
non-
toluene-4-
(tmo), toluene/benzene- (tbu) and phenol (phi) monooxygenases and soluble acyl-ACP
desaturases
(232, 292).
1.3.3.2 Non-heme iron
integral
membrane oxygenases
All members of the non-heme iron
hydrophobic
substrates
using
one
integral
membrane oxygenases oxidize
atom oxygen
originating
from molecular oxygen. The
other oxygen atom is reduced to water
using
includes
similar enzyme families
a
large
number of
monooxygenases
251), fatty
fatty
acid
structurally
relatively
two electrons from
NAD(P)H. This group
(232): alkane
(143, 207, 213), xylene monooxygenase and related enzymes (66, 124,
modifying
enzymes such
acid monooxygenases and
as
double- and
triple-bond forming desaturases,
epoxidases (35, 156, 233),
steroid oxygenases
(19, 162),
10
ß-carotene
hydroxylases
and ketolases
(120, 128, 176, 177) and aldehyde decarbonylases
(1).
The electrons necessary for the
NADPH. These
reductase
are
b5 in the
case
rubredoxin reductase in the
case
ferredoxin reductase fusion
protein
Three, four
or
six membrane
acid sequences of
with
varying
probably
xylene
for
proteins belonging
also contain
family
from NADH
(233), rubredoxin and
hydroxylases (91, 147)
xylene
monooxygenases
elements
can
and
or
either ferredoxin and ferredoxin
acid desaturases
fatty
or a
ferredoxin-
(66, 124, 251).
be identified in all
primary
amino
to the membrane-bound non-heme iron oxygenases,
eight highly
involved in iron coordination.
are
(233, 261). All
conserved histidine residues which
They
grouped
are
are
in three sequence motifs
essential for the function of these enzymes
Wubbolts, personal communication). For the Pseudomonas putida mt-2
W.
XylM,
monooxygenase
directed
of alkane
spanning
(HX3.4H, HX2.3HH, HX2.3HH)
(233)(M.
of
by
come
numbers of amino acids between each transmembrane helix
members of this
most
reaction
transferred to the monooxygenase
cytochrome
or
monooxygenation
mutagenesis (M.
is also conserved in all
W.
a
ninth essential histidine residue
was
identified
by
site-
Wubbolts, personal communication). This histidine residue
that
proteins
can
be classified
as
integral-membrane
non-heme
iron monooxygenases.
It is not known which residues
are
involved in the substrate
bound non-heme iron enzymes. For the soluble
five amino acids
formed
was
sufficient to
(40). Replacement of
seven
converted
a
strict desaturase to
activity
a
unmodified
to
The Pseudomonas
the
coupled
bifunctional
acid
position
of the membrane-
desaturases, the mutagenesis of
at which the double bond
one
was
of the membrane-bound enzymes
desaturase-hydroxylase comparable
in
hydroxylase (35).
GPol alkane
hydroxylase (207)
is
a
prototype of
non-heme iron monooxygenases. The enzyme contains six
spanning segments (261).
Mössbauer studies have shown that
dinuclear iron cluster is present in the
heme iron
proteins
a
the
amino acids in
putida (oleovorans)
integral-membrane
membrane
change
fatty
specificity
proteins (231).
It
was
proposed
contain these diiron clusters.
protein
that all
an
exchange-
of the type present in soluble
integral-membrane
non-
non-heme iron
11
1.3.4 Non-heme iron
1.3.4.1 The P.
The most
integral
membrane alkane monooxygenases
putida (oleovorans)
thoroughly
described
GPol alkane
microorganism growing
putida (oleovorans) GPol, previously
organism
hydroxylase system (18).
catalyse:
ranging
the alkane
of branched
terminal olefins
(oleovorans)
methyl ethers;
(228, 260). This
hydroxylation
hydroxylase system
the oxidation of terminal alcohols to the
demethylation
n-alkanes is Pseudomonas
from pentane to dodecane
In addition to the
alicyclic compounds (158, 173, 259),
on
named P. oleovorans GPol
is able to oxidize linear alkanes
the alkane
hydroxylase system
of
by
virtue of
aliphatic
and
has been shown to
corresponding aldehydes;
sulfoxidation of
thioethers, and epoxi dation of
(131, 132, 171, 172). Although the substrate range of the P. putida
GPol
(TF4-1L) alkane hydroxylase system has been investigated in detail,
it is not clear to what extent it is determined
solubility, uptake
and
by
factors such
as
host
strain, substrate
regulation.
AlkL
Œ0-
Chemotactic
response?
,^—
.
f
alle
F
G
alWH
alkJ
alkK
alkL
alkN
alkT
alkS
J'
inducer
Figure
1.1: alkane
degradation by
Pseudomonas putida
AlkS
(oleovorans)
GPol.
J
^—**
AlkN
12
All genes involved in the conversion of alkanes to CoA-activated
by
two gene clusters
clusters
cloned in
were
pGEc47 (69, 260).
fragment
alkane
contains
located
(figure 1.1),
pLAFRl
Mini cell
as
hydroxylase system (the
and
encodes the third component of the alkane
and
start of the
alkBFGHJKL operon
levels in the
transcription during
Interestingly,
S1 nuclease
mapped by
was
the
controls the
OCT-plasmid
a
components of the
AlkG)
16.9 kb
and
fragment
rubredoxin reductase
an
whole
flanking regions
PI alk genes, the latter strain
1.3.4.2 Alkane
which allows
increased
identified
as
well
(42).
GPol is
putida (oleovorans)
45
only
alk genes and the
(74, 143, 202). This suggests that the alk genes originate from
found in the
putida (oleovorans)
was
content of DNA around the
host, and entered the OCT-plasmid
was
considerably
(42,
overlapping AlkS-dependent promoter,
the G+C content of the alk genes of P.
as a
and
of the alkS gene
expression
studies
protection
as-dependent promoter,
exponential phase,
%, which is clearly lower than the G+C
putida
to create
AlkB and the rubredoxin
hydroxylase system (the
stationary phase (43). Recently,
positively
hypothesis
The gene
AlkS, which regulates expression of the alkBFGHJKL operon (68, 202). The
low levels of
low G+C
two
(70, 142, 143, 258). The
143), while the alkS gene itself is under control of
which
encoded
are
revealed that the 18 kb
partial sequencing
hydroxylase
enzymes involved in further metabolic steps
AlkT)
fragments,
alkBFGHJKL, which encodes
alkane
acids
OCT-plasmid (20, 45, 73).
18 kb and 16.9 kb EcoRl
experiments
operon named
an
the
on
fatty
GPol
as a
transposon. Evidence for this
of the P.
containing
a
putida (oleovorans)
alk genes
closely
GPol and P.
related to those of P.
(260).
hydroxylase systems
related to the P.
GPol alk-
putida (oleovorans)
system
Biochemical studies
on
P.
strains isolated in the 60's and 70's describe
aeruginosa
enzyme systems that
are
similar to those from P.
(269)). These strains
are
able to grow
on
chain alkanes. Chemical mutants of P.
grow either
on
medium-chain alkanes
concluded that P.
aeruginosa
putida (oleovorans)
medium-chain
aeruginosa
or on
ATCC 17423 contains
(reviewed
alkanes, but also grow
ATCC 17423
long-chain
GPol
alkanes
more
than
were no
(186).
one
It
on
longer
was
alkane
in
long-
able to
therefore
hydroxylase
13
system. More recently, the medium-chain alkane-degrading P. aeruginosa strains
shown to contain alk systems
(262). The
GPol alkB gene
(nearly)
probe
chromosomal DNA from the
identical to that of P.
did not detect any
strictly long-chain
putida (oleovorans)
specific signal
alkane
were
GPol
in Southern blots with
dégrader P. aeruginosa
PAOl
(262)(chapter 6).
Several other strains able to
degrade long-chain
alkanes
systems similar
putida (oleovorans)
GPol
to that of P.
(reviewed
Acinetobacter calcoaceticus 69-V
(49, 138), where
alkane-inducible rubredoxin and
rubredoxin reductase
hydroxylase system
hydroxylase,
called
of Acinetobacter sp. ADP1
was
particulate
GPol AlkB
on
the
of the strain
regulator AlkR
was
by
by
a
hydroxylase
gene
downstream genes estB and
XcpR,
ADP1,
was
as
(204). This protein
esterase
(91), and form
oxyR (92).
were no
impaired growth
was
longer
It
an
on
an
are
operon
A subunit of the
degradation
secreted. The rubAB and
shown
that,
hydroxylase (214).
not encoded in close
together
The
proximity
with the two
general secretory pathway,
of dodecane of Acinetobacter sp.
hexadecane and abolished
shown to be necessary for
was
alkanes, the substrate range
the substrate range of the alkane
found to be necessary for the
deletion
monooxygenase,
amino acid level to the P.
broad range of
rubredoxin rubA and rubredoxin reductase rubB genes
to the alkane
as
(213), and is regulated by AlkR, the positive regulator of
is inducible
determined
(263)), such
(91, 213). The alkane
(214), which is encoded directly upstream of alkM (213).
although
in
to contain enzyme
found. The alkane
were
cloned
AlkM, has 41 % sequence identity
putida (oleovorans)
alkM
a
a
reported
were
growth
on
general protein secretion,
xcpR
genes
are
dodecane
as
lipase
and
constitutively expressed
(91, 204).
1.3.4.3
Catalytic
model of the alkane
hydroxylase
Three classes of diiron clusters have been
class II
as
recognized:
class I
as
is present in
hemerythrin,
in ribonuclease reductase R2, methane monooxygenase and soluble
desaturases, and class III
as
molecules in the latter class
in membrane-bound non-heme iron
are
most
probably
coordinated
by
proteins (33).
the
eight
The iron
histidine
residues,
14
as
concluded from the isomer shifts of reduced alkane
hydroxylase
in Mössbauer studies
(231).
For the alkane
been
hydroxylase
of P.
putida (oleovorans) GPol,
proposed (12, 85, 86) (figure 1.2). First,
generated (figure 2).
leaving
a
subsequently
C-OH bond. The second electron of the
form back the initial situation.
During
oxygen attacks the substrate from the
Fell!
excesses
ranging
FelV
«Fein
radical mechanism has
high-valence FeIV-oxo
This activated oxygen attacks
carbon radical. This radical
enantiomeric
a
a
a
carbon atom and extracts
attacks the Fem-0
Fem-0 bond shifts
to the
the oxidation of terminal
re
face,
to
from 70 to 100 %
^FelV
intermediate is
give mainly
a
H»,
to form the
bond,
neighbouring iron,
to
olefins, the activated
the R enantiomers with
(85, 86).
Fein
\
FelV
^FelH
Fell!
(JL»
Figure
1.2:
catalytic
model for alkane
hydroxylases.
1.3.5 Subterminal oxidation of alkanes
Most enzymes
oxidizing
n-alkanes
catalyze
the reaction at the
1-position
of the n-alkane
(see above). However, subterminal oxidation of n-alkanes also takes place.
conjecturable
what is the overall
terminal oxidation
importance
(84, 169). P. aeruginosa
in addition to
of tridecane
by
compared
to
(169).
The subterminal oxidation of alkanes
products
of subterminal oxidation
It is
NCIB 9904
products
Burkholderia
yields secondary
produces significant
of terminal oxidation
cepacia
alcohols and ketones
and P.
as
products
amounts of subterminal
(84). The pathway for degradation
aeruginosa
Sol 20 included
a
primary
15
oxidation of the alkane to the
tridecanone
undecyl
(82). This in
acetate
turn is oxidized
acid, which
hydroxylases
compounds
molecules like
(137). Li
et
of interest for
of P.
versatility,
methyl
al.
(158)
groups
pregrown
Other
on
octane
acid,
a
an
intermediate of
production
Several
problems
substrates
are
requiring
GPol has
can
be
heterocycles
of
production
high regio-
and
of
to
a
fatty
The
the
regio-
products
and
are
complex organic
(89).
broad substrate
a
The
produced.
has been
optically
active
Lonza AG
iV-benzyl-3alkane-oxidizing
be used to
can
specificity
enzymatic
patented by
GPol and other
produce
when the strain is
stereospecificity
strains used in commercial
NRRL B-16531 for the
pharmaceuticals
production
limit the
only poorly
As most alkane
aromatic
of herbicides
(bio)surfactants (see
compounds
putida (oleovorans)
very
erythropolis
NCIMB12566 for the
for the
esterase,
(46, 158).
alkane-degrading
Rhodococcus
other other fine chemicals
many
on
P.
manufacturing
strains, Sphingomonas sp. HXN-200,
alcohols with
alicyclic
acetate
they perform
hydrocarbons (281).
putida (oleovorans)
described the
hydroxypyrrolidine using
strains. One of these
or
because
biocatalysis,
precursors for the
as
e.g.
hydroxylase
oxidation of
monooxygenase to 1-
biocatalysis
pharmaceuticals
Due to this
(259).
are
to 2-
cycle.
oxidation of non-functionalized
stereospecific
The alkane
Baeyer-Villiger
a
(234, 235). Undecanol is further degraded
enters the ß-oxidation
1.4 Alkane oxidation and
valuble
by
dehydrogenated
was
(32, 81, 83), which is the substrate for the undecyl
1-undecanol and acetate
yielding
Alkane
2-alcohol, which subsequently
use
of oxidized
of
products
are
2-phenyl-l-propionic
and Rhodococcus rhodochrous
which
phenoxy propanoic acids,
are
intermediates
(119).
of alkane
hydroxylases
1.5; chapter 8)
hydroxylases
production
ibuprofen,
water soluble. This
section
are
the cofactor NADH for
reactors will be limited
of
as
production
problem
or
the
biocatalysis. First,
could be solved
by
most of the
the addition of
two-liquid phase systems (29, 222, 268).
membrane-bound
catalysis,
for
use
multi-component
of
purified
(222). This would make it necessary
enzyme systems
enzymes in enzyme
to
use
whole-cell systems for
16
the
production
of the
of target
product by
compounds. Here,
a
suitable host is
required
to avoid
degradation
downstream metabolism.
1.5 Additional factors involved in alkane oxidation
As described
above, the oxidation of alkanes takes place
membrane. One of the
uptake
limiting
of n-alkanes. The
factors in
solubility
alkanes. Medium-chain n-alkanes
growth
(245). However,
Generally,
are
biosurfactant-mediated
to have
a
hydrophobic
uptake
supernatant fluids have
high
or
called accommodated
or
used
by
of the
extent that the aqueous
phase
traverse the outer membrane of
a
alkanes.
The interfacial accession
contact to substrate
in
although
some cases
interfacial
but culture fluids have
hydrocarbon uptake,
solubilized
culture supernatant fluids is
chain-length
surface tension. This mechanism is
hydrophobicity,
For biosurfactant-mediated
an
long-chain
production (biosurfactant-enhanced
medium
generally
simply
surface, allows direct
high
a
cytoplasmic
have been considered: interfacial accession and
the rhodococci and Acinetobacter spp.,
biosurfactant
can
hydrocarbon uptake (28).
cell
in the
alkanes is the solubilization and
on
soluble to such
this may not be true for
two modes of
or
of n-alkanes decreases with the
concentration is sufficient that the alkanes
cell
at
the
hydrocarbons.
a
requires
droplets.
widespread
cell
a
Culture
e.g. among
it is combined with
uptake). Here,
cells have
a
low to medium surface tension.
microorganism
has contact with
so-
In this case, the surface tension of
low, and cells remain hydrophilic (28). This mechanism is
Pseudomonas spp., but also
by
other genera.
1.5.1. Biosurfactants
Surfactants
are
amphiphatic
hydrocarbon)
moieties that
with different
degrees
of
molecules with both
partition preferentially
polarity
interfaces. Biosurfactants
are a
and
at the
hydrogen bonding
structurally
synthesized by microorganisms (77).
structures:
hydrophilic
and
hydrophobic (generally
interphase
such
as
between fluid
oil/water
or
air/water
diverse group of surface-active molecules
A biosurfactant may have
one
of the
following
mycolic acid, glycolipids, polysaccharide-lipid complex, lipoprotein
lipopeptide, phospholipid
or
phases
the microbial cell surface itself
or
(129). These molecules
17
reduce surface and interfacial tensions in both aqueous solutions and
mixtures. Biosurfactants have several
lower
toxicity, higher biodegradability,
foaming, high selectivity
(57).
Potential
and food
play
1.5.1.1
The
better environmental
specific activity
at extreme
cover
of biosurfactants
compatibility, stronger
temperatures, pH and salinity
the
genetics, biochemistry, production
(17, 77, 129, 165). Here,
discussed in
are
more
specific
two
aeruginosa
cultures:
health
and
possible
biosurfactants that
biosurfactant
strains
(59, 193, 265).
rhamnolipids
moiety
2) respectively.
have also been
derived from
they
rhamnolipids
enhance the
interaction is
more
major glycolipids
Two
rhamnolipid
short, precursors
of
of
the
bioavailability (290).
with
hydrophobic
LPS from the outer
substrates
are
produced
in
as
rhamnolipids
1 and
ß-hydroxydecanoyl
two modes of action
increase the
hydrocarbons,
are
as, in contrast to
required
more
of
solubility
The second mode is
to become
by
by
hydrophobic
(289). The mechanism of this
membrane, increasing the relative
more
important
for in
solubilization, only low
to alter the cell surface
has been described in detail
withdrawn from the ß-oxidation
units from
one
hydrocarbons. First, they
cell, causing the cell surface
rhamnolipids
are
products having
to
(3). The second mode of action may be
concentrations
biosynthesis
rhamnosyl
easily
of the cell
situ bioremediation of
more
(193). Rhamnolipids have
degradation
by extracting
hydrophobicity
Two
1 and 2
with the bacterial
and to associate
produced by
described, although these may represent degradation products
hydrocarbons, thereby increasing
interacting
are
L-rhamnosyl-L-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate and
1 and Rhl
(Rhl
The
pollutants,
detail.
L-rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate, referred
which
as
Rhamnolipids
Pseudomonas
2
surfactants, such
of biosurfactants include enhanced oil recovery, crude oil
rhamnose-containing glycolipid
liquid
chemical
processing (17).
role in this thesis
a
over
surfactant-aided bioremediation of water-insoluble
Several recent reviews
applications
and
applications
drilling, lubricants,
care
advantages
hydrocarbon
by
RhlG
thymidine-diphospho-L-rhamnose by
(165).
(41, 192, 194, 195).
(41) and coupled
the
In
to two
rhamnosyltransferases
1
18
and 2, encoded
by
rhlAB and rhlC,
regulated by RhlR,
autoinducer
Rhll
synthetase
network also
regulatory
which in turn is activated
LuxR-type regulator (194),
a
is
respectively (165, 192). Rhamnolipid biosynthesis
The RhlR/Rhll system is part of
(195).
the
regulating
expression
a
by
the
complex
very
of virulence-associated traits
(30, 165).
1.5.1.2 "Protein activator of alkane oxidation"
In the
case
of Pseudomonas
oxidation", PA, plays
a
activator of the alkane
protein,
an
120
which has
pig/ml)
hexadecane
1.5.2
A
made
oxygen-uptake
It has
PA
(103). This
role
a
was
exported
a
large
cloned from P.
less
studies
dominating
50
growth phase
on
slower
PG201 and
on
a
hexadecane
role in hexadecane
rhamnolipids.
as
barrier for
consists of
a
uptake
cell wall and
addition to the
of the
one or
lipopolysaccharides (LPS).
shows
a
hydrophobic
two
lipid
cytoplasmic membrane,
peptidoglycan layer (190).
very low
an
substances is the cell
membranes.
The two membranes
are
toward
separated by
lipophilic LPS,
hydrophobic compounds.
molecular
mass
smaller than 600 Da to diffuse
(189). The cytoplasmic membrane shows
molecules, but apolar compounds
Specific transport
hydrocarbons,
diffusion of the
processes could
can
a
passively
low
permeability
easily penetrate
play
a
The
the
a
have, in
phospholipids
thin
the outer membrane
Low
specificity porins
polar
solutes with
in
a
the outer membrane
for
polar
and
charged
lipid bilayer (236).
role in the mineralization of low concentrations
but the transport activities may not
compounds.
across
which
bacteria
outer membrane that consists of
Due to the presence of the
permeability
envelope,
Gram-negative
the outer membrane form water-filled channels which allow small
of
(between
amounts
aeruginosa
only slightly
as
(114). The
in the initial
especially
mutant grew
which proves that it has
in
aeruginosa S7Bl5
Uptake
major
and
liquid.
in
power, is
emulsifying
activator of alkane
"protein
isolated first from P.
was
hydroxylation activity
weak
was
the so-called
protein
(113). The gene encoding
wildtype,
assimilation
role. This
to the culture
knockout mutant
than the
a
aeruginosa,
general opinion
easily
be revealed due to
is thus that
uptake
of
passive
hydrocarbons
is
a
19
process, i.e.
passive
represents
a
dissolve in the cell membrane. The cell membrane of bacteria
they
hydrophobic phase
surrounding
aqueous
which accumulates
nonpolar compounds
phase (236).
Recent studies have shown the presence of genes
encoding
(OMPs) in hydrocarbon catabolic opérons (125, 127, 274).
homologous
fatty
acids
to the E.
coli FadL
involved in toluene utilization
protein
is
upon exposure to toluene
protein (AlkL),
alkL
negative
specific transport
of
are
long-chain
(22). By analysing the expression levels of tbuX,
it
was
that the
speculated
levels, and that the protein levels increase significantly
of the mutant
alk-system
which is
proteins
(127). Although deletion of the tbuX gene resulted in
significantly impaired growth
not been shown. In the
outer membrane
Some of these OMPs
by Ralstonia pickettii PKOl,
at low
always present
which allows
protein,
the outer membrane
across
from the
of P.
homologous
mutants did not show
on
toluene, the
exact role of the
to
putida (oleovorans) GPol,
OmpW
of Vibrio
cholerae,
an
a
protein
has
outer membrane
was
found. However,
phenotypic changes (258).
1.6 Aim and scope of this thesis
The initial aim of the
aspects of growth
Chapter
and
on
alkanes
2 describes the
fragments
of alkane
Gram-negative
Chapter 3,
we
use
Chapter 4,
regions
is
the
n-alkanes
members of the non-heme iron alkane
of
a
catalytic properties
broader range of
organisms
are
of
an
enzyme
known. All
considered.
highly degenerate
gene
homologs
PCR
primers
from
a
to clone internal gene
broad range of
Gram-positive
strains.
cloning
alkane
by
new
describe the construction of
reported.
constructing
are
hydroxylase
heterologous expression
on
to clone
most effective when sequences from
are
In
was
Studies about the structural and
family.
monooxygenase
In
project
of
proteins
in both E. coli and Pseudomonas
of novel alkane
Functional
alkane-responsive expression
hydroxylase homologs
expression
of alkane
hydroxylase negative hosts,
alkane
hydroxylases
from
that could be
Gram-negative
species.
and their
hydroxylases
vectors for
was
flanking
achieved
complemented
and
Gram-positive
by
for
growth
strains.
20
Chapter
5 describes the functional
coli strains
containing
all
analysis
of rubredoxin reductases in recombinant E.
of P.
GPol
alk-genes
putida (oleovorans)
except for the
rubredoxin reductase gene.
The
Chapters
6 and 7
two
Gram-negative
flanking regions
ability
to grow
is
on
give
a more
strains. For P.
detailed
description
aeruginosa
PAOl
of the alkane oxidation genes of
(Chapter
6)
a
shown, and environmental and clinical isolates
alkanes and for the presence of alkane
detailed
are
hydroxylase
analysis
of
tested for their
genes. For P.
7), characterization of the knockout mutants KOB2 and
fluorescens
CHAO
KOB2A1 is
described, while characterization of culture supernatant and overexpression
(Chapter
of PraA and PraB shows that P. fluorescens CHAO
hexadecane. In the
knockout
In
biocontrol
on
Chapter
8,
rhamnolipids
hydroxylase
Appendix
we
to
activity
chapter
are
7,
some
produces only
and
in
on
initial data about the effect of the alkB
shown.
describe additional intrinsic and extrinsic factors
including
the effect of
and the host strain that influence the apparent substrate range of the alkane
of P.
putida (oleovorans)
GPol. We also describe mutations in the AlkB
which allows P. fluorescens KOB2A1 recombinants to grow
Finally,
PraB when grown
Chapter
Gram-negative
9,
we
summarize all
strains collected
knowledge
over
about
the last years.
on
hexadecane.
alk-systems
in
Gram-positive
21
Chapter
2
MOLECULAR SCREENING FOR ALKANE HYDROXYLASE
GENES IN GRAM-NEGATIVE AND GRAM-POSITIVE STRAINS
Theo H.M.
Smits, Martina Röthlisberger, Bernard Witholt and Jan B.
Parts of this work
are
published
van
in Smits et al. 1999. Environ Microbiol 1
Beilen
(4):
307-316
22
SUMMARY
We have
developed highly degenerate oligonucleotides
genes related to the Pseudomonas
alkane
chain n-alkanes
fragments
sequence
alkane
were
and
identity
with
to the
on
Strains that
were
to alkane
those isolated
phylogenetic
PCR
products
on
of the
and found to encode
of the P.
unable to grow
hydroxylase
short-chain
on
on
medium
expected
peptides
n-alkanes did not
yield
shows that all Acinetobacter sequences
tree, like the sequences from
size. The PCR
putida (oleovorans)
not
or
with 37.1-100 %
genes. Several alkane
alkanes, did
(C6-C11)
GPol
yield
PCR
degraders,
PCR
are
fragments
clustered in
Gram-negative
either.
one
strains able to
medium-chain alkanes. In contrast, sequences obtained from the fluorescent
pseudomonads
divergent as
The alkane
were
sequenced
strains able to grow
corresponding fragment
phylogenetic analysis
group of the
as
(C12-C16), yielded
homology
predominantly
grow
GPol and Acinetobacter sp. ADPl
putida (oleovorans)
Gram-positive
cloned and
hydroxylase.
products
The
internal segments of
hydroxylases.
Many Gram-negative
long
amplify
to
or
sequences found in
the
complete
hydroxylase
cloned
using
containing
all P.
single Gram-negative
or
Gram-positive
strain
are
collection.
genes of Acinetobacter calcoaceticus EB104 and P.
the PCR
hydroxylase negative
a
products
mutant of
as
probes.
The two genes allow
Acinetobacter sp. ADPl and
an
an
putida
PI
alkane
E. coli recombinant
putida (oleovorans) alk-genes except alkB, respectively,
to grow
on n-
alkanes, showing that the cloned genes do indeed encode alkane hydroxylases.
INTRODUCTION
Many
bacterial strains
are
able grow
on
medium
or
long chain-length
strains, P. putida (oleovorans) GPol (TF4-1L), which
pentane
to dodecane
can
grow
on
(228), has been studied in detail with respect
enzymology (173, 208, 255, 256)
and the
genetics
n-alkanes. Of these
alkanes
ranging
from
to both the
of n-alkane metabolism
(142, 143,
23
263), mainly because the strain has proven
(85, 136, 210, 259).
of non-heme iron
xylene
to be
an
interesting
and versatile
biocatalyst
addition, the alkane hydroxylase AlkB is the prototype of
In
membrane monooxygenases which includes the P.
integral
(251)
monooxygenase
and the alkane
hydroxylase
class
a
putida
mt-2
of Acinetobacter sp. ADPl
(213).
Genes with
high
sequence
fraction of the microbial
colony
blots
aeruginosa,
identity
to the alkane
population
putida,
P.
aureofaciens
to
distantly
that
they
or
substituted
phenoxy
of
production
other
high
acid
large
by
On the other
as
alkB-gene probe,
an
unrelated
are
biocatalysts
synthesis
of
or
to convert
pharmaceuticals
(89). Examples include Rhodococcus rhodochrous
propane to
herbicides,
stereoselective oxidation
propionic
with
hybridize
into valuable intermediates for the
NCIMB12566, which, when pregrown
the
not
related to alkB. These enzyme systems could be useful
other fine-chemicals
a
demonstrated
(226, 262, 273).
contain alkane oxidation enzyme systems that
inexpensive compounds
as
in
occur
alkB have been cloned from P.
and P. mendocina
hand, DNA of other alkane-degrading strains does
suggesting
(alkB)
gene
in oil-contaminated environments
(243), while genes almost identical
P.
hydroxylase
or
on
can
to
stereoselectively
phenoxy propanoic acids, compounds
R.
erythropolis
reactions, such
(119), which
n-alkanes, is able
are
used for
B-16531, which carries
the oxidation of
as
be used to
NRRL
that
cumene
to
oxidize
out similar
2-phenyl-l-
produce pharmaceuticals (e.g. ibuprofen)
or
value fine-chemicals. None of the enzyme systems involved have been
characterized.
As
only
few enzymes that oxidize medium-
characterized,
a
hopefully
with
degradation
or
superior properties.
soil-ecology
can
In
One method that
can
be used to isolate
hydroxylases
or
convenient methods
distantly
well
with
new
putida (oleovorans)
GPol
are
more
help
us
available to isolate their genes.
comparison
degenerate
between the alkane
(70) and Acinetobacter sp.
to
biocatalysts,
members of this
related genes is PCR with
conserved sequence elements. A
of P.
us
be studied in greater detail when
known,
on
are
addition, the role of alkane hydroxylases in oil
are
based
n-alkanes
function, evolution and structure, and provide
class of enzymes
primers,
long-chain
with related enzyme systems in other bacteria will
comparison
understand their
or
ADPl
(213)
24
allowed
were
us
to
previously
desaturases
(oleovorans)
on
shown to be essential for the function of the
we
n-alkanes used in this
gene
while such genes
n-alkanes. In
study
addition,
we
can
related
large proportion
are
plasmids
not be
amplified
related to the P.
from strains that
hydroxylase
by complementation
used in this
Strain
putida
are
genes of P.
unable to
putida
PI
that these indeed encode
study
Growth
on
alkanes3
C8
Pseudomonas putida
(oleovorans)
GPol
Pseudomonas putida
(oleovorans)
GPol2
Pseudomonas oleovorans ATCC 8062
PpGl
C12
PCR
Plasmid
product15
derived
550
(228)
bpc
-
289
bpc
Pseudomonas
aeruginosa
PAOl
Pseudomonas
aeruginosa
PG201
«800bpcg
550 bpe
550 bpe
550 bpc
550 bpc
Pseudomonas
aeruginosa
KSLA473
not tested
Pseudomonas putida PI
Pseudomonas fluorescens CHAO
Burkholderia
cepacia
ATCC 25416
550
Burkholderia
cepacia
RR 10
550
Comamonas testosteroni DSM 50244
Stenotrophomonas maltophilia DSM
Ralstonia eutropha JM134 (pJP4)
50170
350
bpc
bpc
bpc
-
(141)
-
(155)
-
(216)
-
pPl
pCHAO
pTS4
pTS2
-
p25416
pRRlO
(118)
(97)
(253)
(198)
(288)
(246)
-
(246)
-
-
(62)
-
(126)
-
(213)
Acinetobacter sp. WH405
not tested
Acinetobacter calcoaceticus 69-V
550
p69-V
Acinetobacter calcoaceticus EB102
bpd
«600bpfg
Acinetobacter calcoaceticus EB 104
550
pEB104
Acinetobacter calcoaceticus EB 114
«600bpfg
bpc
-
Acinetobacter calcoaceticus NCIB 8250
550
Acinetobacter calcoaceticus CCM 2355
«600bpfg
Acinetobacter sp. 2796A
550
Alcanivorax borkumensis SK2
550
Xanthobacter flavus strain 5
550
Rhodococcus rhodochrous NCIMB 12566
289
NRRL B-16531
550
erythropolis
study
(250)
-
not tested
Prauserella rugosa NRRL B-2295
(14)
This
-
Acinetobacter sp. ADPl
Acinetobacter calcoaceticus EB6
Reference
C16
-
Pseudomonas putida KT2442
Rhodococcus
of bacteria
hydroxylases.
Table 1: strains and
Pseudomonas putida
a
possess genes that
cloned the alkane
and A. calcoaceticus EB104 and showed
functional alkane
could show that
structurally
alkB, which encodes the catalytic component of the alkane
hydroxylase system,
on
centered around two histidine clusters that
regions, basically
(233). Using this method
able to grow
grow
such
identify
559
bpc
bpc
bpc
bpc
bpc
bpc
bpc
-
(139)
(139)
-
(139)
-
(139)
p8250
h
(139)
-
p2769A
pSK2
pXf5 (484 bp)
-
pl6531
p2295
(76)
(139)
This
study
(284)
This
study
(50)
(119)
(119)
25
Rhodococcus sp. 1BN
R.
erythropolis
550
n.t.
550
23-D
bpc
bpc
(6)
plBN
p23-Dl, p23D-2,
(226;
p23-D3
(226;
p35-0
erythropolis 35-0
Gram-positive 42-0
R. erythropolis 50-V
R. erythropolis 62-0
R.fascians 115-H
R.fascians 154-S
FfXN 100 (CNM group)
HXN 200 (Sphingomonas sp.)
FfXN 300 (Gram-negative)
550
FfXN 400
(P. aeruginosa)
550
bpc
pFfXN400
FfXN 500
(Mycobacterium?)
(Mycobacterium sp.)
550
bpc
bpc
bpc
pFfXN600
pFfXNlOOO
pFfXNUOO
R.
FfXN 600
550
550
550
550
550
bpc
bpc
bpc
bpc
bpc
bpc
(226;
p42-0
p50-V
h
(226;
(226;
p62-0
pll5-H(240bp) (226;
(226;
pl54-S
(211
(211
(211
(211
(211
(211
FfXN 1000
(CNM group)
550
FfXN 1100
(P. mendocina lineage)
550
FfXN 1200
(211
FfXN 1300
(Rhodococcus sp.)
(not identified)
FfXN 1400
(Comamonas?)
(211
FfXN 1500
(Mycobacterium sp.)
(Ochrobactrum?)
(211
(211
FfXN 1900
(Alcaligenes?)
(not identified)
(Rhodococcus sp.)
FfXN 2000
(CNM group)
FfXN 1600
FfXN 1700
FfXN 1800
(211
(211
(211
(211
(211
(211
550
bpc
pFfXN2000
(211
Plasmids:
Genotypea
Reference
pGEc47
pGEc47AB
Tc, alkBFGHJKL!alkST (P. putida (oleovorans) GPo 1) in pLAFR 1
(69)
Tc, alkFGHJKLIalkST (P. putida (oleovorans) GPo 1) in pLAFR 1,
deletion in alkB
(261)
Ap, ColEl, alkBFGH (P. putida (oleovorans) GPol)
Tc, alkB (Mycobacterium tuberculosis Ff37Rv)
pGEc48
pSCYB 11
pGEM7-Zf(+)
Cloning vector, Ap
Ap, 3.0 kb EcoVl-Hind\ll
pEB 1
alkRM
(A. calcoaceticus
in
(209)
Promega
insert in
EB
pGEM7-Zf(+),
This
104)
pEB7
pWH1274
pWH968
pEB 1 AEcoRV-Ecom, 1.7 kb insert, alkM (A. calcoaceticus
Ap, Tc, oriA, ColEl, shuttle vector for Acinetobacter
Ap, oriA, ColEl, alkRM (Acinetobacter sp. ADPl), rubAB
pWFfEB7
Ap, oriA, ColEl,
alkM
Cloning
Ap, ColEl,
pBR322
pPlEH
1.7 kb insert of
(A. calcoaceticus
vector,
EB
pEB7
in
tetracycline,
n.t.: not tested.
combinations used
f
are:
-
This
104)
: no
PCR
study
study
(121)
(214)
This
104)
Ap, Tc, ColEl
alkBFGHJ (P.
b
EB
pWH1274,
study
(25)
putida PI)
in
This
pBR322
Footnotes:a Abbreviations: C8: octane, C12: dodecane, C16: hexadecane,
Tc:
(70)
pBR322
fragment; Fragment
study
bp: basepairs, Ap: Ampicillin,
fragment observed. Primer
size: PCR
cTS2S/DeglRE;dTS2Smod/DeglRE;eTS2Smod/(DeglRE/DeglRE2, 1:1; mix);
TS2Smod2/DeglRE.8 PCR fragment was observed,
(length in parentheses behind plasmid name)
but not cloned.
h
smaller size
fragment
cloned
26
MATERIAL AND METHODS
Strains, growth media and materials. Strains and plasmids used
study
listed in Table 1. E. coli JM101
are
and the
production
n-alkanes
of
plasmid
used
or were
as
DNA for
(151), supplemented
throughout.
growth
medium
octane
were
on
an
and n-hexadecane
open
erlenmeyer with
a
of the Petri dish. All cultures
were
transformed
used at
with carbon
«-octane, n-dodecane
by placing
grown
by electroporation (64).
100/^g/ml.
sources or
or
was
supplied through
«-octane in
aerobically
a
sealed
at 30°C
To select E. coli
done
on
was
or
Boehringer Mannheim
synthesized by Microsynth,
and I
from
=
and used
as
done
as
previously
the
tested
was
and
from the
gel,
and EcoRl,
PCR
products
and isolated
purified again
by
were
purified
electroelution
over a
1% agarose
cloned between the Sacl and EcoRl sites of
according
Isolation Kit if
sequenced
on a
to Birnboim and
sequencing grade
gel,
on
(204).
=
T
was
Li-Cor 4000L sequencer
PCR
gel
in
+
C, R
or
fragments
isolated from
using
required.
using
was
were
were
+
G
obtained
cut out
cut with Sacl
gel by electroelution,
and
Plasmid DNA
was
the Roche
High
Pure Plasmid
Both strands of the inserts
IRD41-
were
A
=
TBE-buffer,
pGEM7-Zf(+) (Promega).
Doly (21),
DNA
1% agarose
(220). The
Growth of
supplier. Oligonucleotides
Y
was
described
dideoxynucleotides
oligonucleotides
over a
n-
n-alkane in the lid
Inosine, basepairing with all four nucleotides. Taq DNA polymerase
Promega.
isolated
ligase
phase (for
piglm\ ampicilline.
n-dodecane
specified by
Switzerland. In the
used
37°C. E. coli strains
with metal solution 44
from
were
transformants, ampicillin
LB with 300
Restriction enzymes, T4-DNA
(Luria Broth) (220)
\i\
Acinetobacter minimal medium
biology.
on
container, for n-dodecane
on
Molecular
cloning
able to grow
the vapor
Acinetobacter sp. ADPl, and mutants thereof,
supplemented
used for
n-hexadecane, Petri dishes with E2
Transformation of Acinetobacter
(199). Selection of transformants
are
antibiotics
Whatman 3MM filter disc with 200
were
were
All other strains
sequencing.
incubated at 30°C with n-alkanes
by placing
(Gibco BRL)
controls in the PCR reactions. LB
negative
and E2 medium
For
constructed in this
(endA, hsdR, supR, thi-1, A(lac-proAB)
and DHIOB
V(traD36,proAB, lacP, lacZM15) (285)
or
(IRD800-) labelled
were
-40 forward
27
and -40
(agggttttcccagtcacgacgtt)
Biotech)
and the Amersham
Chromosomal DNA
according
first step
was
to Desomer et
(58).
If strains failed to
0.2%, and/or cells
identified
fragments
were
restriction
fragments
isolated
(Promega)
case
resulting
in
Southern
a
blotting.
of A. calcoaceticus EB104,
Plasmid
plasmid pEBl.
Xhol and BamHl,
a
a
pEB7
over
shuttle vector for
Navigator
amino acid sequences
BLAST
Cancer
(5).
fragment
on
an
were
were
was
appropriate
identified
gel
enriched
genebank,
sites of
all
preparative
a
pGEM7-Zf(+)
(Gibco BRL).
by colony blotting.
To
made.
and
and
restriction
cut out from
fragment
constructed from
was
religated.
ligated
cloned,
was
pEBl by digestion
The insert of
in Sall-BamHl
pEB7
with
was
cut
digested
Acinetobacter, resulting in pWHEB7. For P. putida
cloned, resulting in pPlEH.
was
were
from DNASTAR
compared
BLAST searches
analyzed
and
compared using
(Madison, Wisconsin, USA).
Nucleotide and
with the EMBL, SwissProt and GenBank databases
were
carried out at ISREC
(Swiss
Foundation for
Research).
PCR. PCR reactions
Elmer
easy-to-clone
3.0 kb EcoRl-Hindlll
The nucleotide and amino acid sequences
LASERGENE
were
using Klenow,
purified
7.5 kb EcoRl-Hindlll
using
the EDTA concentration in the
To construct
were
inserts, deletion and subclones
,
genes,
between the
the desired genes
containing
pWH1274 (121)
PI
strains
Gram-negative
and transformed into E. coli DHIOB
EcoRl and EcoRW, made blunt
out with
lyse,
in the range around the desired size
pBR322 (25)
or
Transformants
In the
by
hydroxylase
by electroelution, ligated
sequence the
and
incubated at 60°C for 30 minutes.
were
To clone intact and functional alkane
gel,
Gram-positive
kit.
increased to 10 mM, the end concentration of SDS in the second step
was
doubled to
Thermosequenase cycle sequencing
isolated from
al.
(gagcggataacaatttcacacagg) primers (MWG-
reverse
GeneAmp
were
PCR
carried out
System
9600.
as
described in Innis et al.
Degenerate oligonucleotides
amino acid sequences conserved between the P.
Acinetobacter sp. ADPl alkane
a
Sacl
were
putida (oleovorans)
hydroxylases (Figure 1).
TS2Smod and TS2Smod2 contain
(123) using
The forward
a
Perkin
designed
based
GPol and the
primers TS2S,
site, and four additional bases
to allow reliable
28
of the
cleavage
site, while the
reverse
site and three additional bases. For PCR
used: 4'
and
primers DeglRE
these
using
primers,
95°C, 25x(45" 95°C, 1' 40°C, 1' 72°C), 5' 72°C,
PCR, 1 ]A (out of 20 \il) of product
was
used in
contain
DeglRE2
oo
the
following
4°C. After
second round of PCR
a
a
an
EcoRl
was
program
first round of
using
the
same
primers.
sequencing
16S rDNA
amplification
were
was
done
16F27 and
according
to Karlson et
(130). The primers used for
al.
16R1525; primers used for sequencing
were
16F355 and
16R1488(107).
Southern and
appropriate
colony
blots. Chromosomal DNAs
restriction enzymes and loaded
were
digested
1% agarose
on a
to
completion
in TAE-buffer
gel
Acetate-EDTA) (167). After washing the gel for
10 minutes in 0.25 N
minutes washes with 0.5 N NaOH, the DNA
blotted to
membrane
min. For
After
(Roche Diagnostics).
blotting,
colonies
colony hybridisation,
was
were
the filters
a
Diagnostics) according
to the manufacturer. Colonies
picked
from the
Nylon
original plates.
using
the Roche DIG-kit
using
standard
Membrane for
and
phosphatase coupled
baked at 120°C for 30
or
colony
to Anti-DIG antibodies
were
blots
colony
blots
-
were
carried out
was
Hybridizations
at 68°C
with about 250
plates
in the
buffer without formamide at 60° C
alkB)
HCl, and three 15
Plaque Hybridisation (Roche
positive
to the manufacturer.
Southern blot with M. tuberculosis H37Rv
Alkaline
were
Detection of Southern and
according
hybridization
Colony
(Tris-
positively charged nylon
transferred from LB
500 transformants onto
with
carried out
were
(non-stringent, only
(stringent,
all other
used with CSPD
as
blots).
the
chemiluminescent substrate.
Probes
were
prepared
as
follows: alkB
pGEc48 (70) using primers
(gcactctttgtgagagaattcaac),
sp. ADPl
using primers
B5/1
cosmid
(gatgtcgagagtgagagtg)
the alkM
alkMfE2
(aataggcctgcagtcacttagactctctt),
fragment
the alkB gene
amplified
from
from chromosomal DNA of Acinetobacter
homolog
MTalkBfw2
(gtagaagcttcccgggcaccggtagg).
was
and B3R/EcoRI
(ccggaattcactatgaatgcacctgta)
pSCYB 11 (209) using primers
MTalkBrv
(P. putida (oleovorans) GPol)
Other
and alkMrPS
of M. tuberculosis H37Rv from
(cggaattcatatgaccacgcaaatcggc)
fragments
were
obtained from the
and
29
and
pl6531 (R.
fragment
with EcoRl
Plasmids pTS2 (P. aeruginosa PG201), pCHAO (P. fluorescens CHAO)
erythropolis
NRRL
and Sacl. All DNA
labeled
by
B-16531) by excising
fragments
the random
Sequences. Sequences
priming
GenBank under the
study
P.
degenerate
following
and AJ293344
putida
PCR
gel,
kit
labeling
comparisons
products
electroeluted and
(Roche Diagnostics).
available in EMBL under
are
AJ002316
determined in this
accession numbers: AJ009579
NRRL
(alkM,
study
deposited
are
in
(P. fluorescens CHAO);
NCIB
8250);
B-16531),
AJ009585
AJ009587
(Pr.
(Acinetobacter
rugosa NRRL
(B. cepacia ATCC 25416). The sequence of the A. calcoaceticus
deposited
under accession number AJ233398. The sequence of
PI alkBFGHJ genes is
deposited
The 16S rDNA sequences determined in this
AJ009588
the DIG
69-V); AJ009584 (A. calcoaceticus
EB104 alkRM genes is
the P.
for
1% agarose
putida (oleovorans) GPol),
2769A); AJ009586 (R. erythropolis
B-2295)
using
PCR
(P. aeruginosa PAOl); AJ009581 (P. aeruginosa PG201); AJ009582 (A.
calcoaceticus
sp.
(alkB,
over a
bp
ADPl), Z95121 (alkB, M. tuberculosis H37Rv).
The sequences of the
AJ009580
purified
method
used in this
accession numbers X65936
Acinetobacter sp.
were
the cloned 550
under accession number AJ233397.
study
are
available under accession numbers
(A. calcoaceticus 69-V); AJ009589 (A. calcoaceticus EB104); AJ009590
(Acinetobacter
sp.
2769A) and AJ009591 (R. erythropolis
NRRL
B-16531).
As the 16S
rDNA sequences of strains Acinetobacter sp. ADPl and A. calcoaceticus NCIB 8250
were
identical to the
previously
determined sequences, these
were
not submitted.
RESULTS
Selection of strains. We tested 55
n-dodecane
(C12) and n-hexadecane (C16). Twenty-six strains
collections, five
were
isolated
by
enrichment with «-octane
isolated from oil-contaminated soil
were
strains, listed in Table 1, for growth
isolated from
a
near a
hexane air filter in
as
were
n-octane
(C8),
obtained from strain
the C-source,
gas station in Bremen
Stuttgart (211).
on
(226), and
Several strains
seven were
seventeen
reported
to be
n-
30
alkane
degraders
were
not able to grow
8062 and A. calcoaceticus strains
belong
of which
species
to
on
n-alkanes in
hands
our
(P. oleovorans
ATTC
EB102, EB114, EB6, and CCM 2355). Other strains
strains
specific
were
found to be able to grow
previously
on n-
alkanes.
Design
of
degenerate
iron alkane
PCR
hydroxylases
primers.
have been
(oleovorans) GPol) (70),
AlkM
Three sequences of
integral
in the sequence databases: AlkB
deposited
(Acinetobacter
sp.
H37Rv. As the
to this
homolog
gene
functionality
its sequence
study,
with it either. The S.
in the
and
on
the chromosome of
In
not considered in the
N246 alkane
maltophilia
nor
of this enzyme
in the
are
was
primer design,
hydroxylase
alkane
an
tuberculosis
not confirmed
prior
but does not conflict
sequence
phylogenetic analysis,
significantly
addition,
Mycobacterium
of the M. tuberculosis H37Rv gene
was
primer development
properties
is present
(P. putida
ADPl) (213) and AlkBpm
(Stenotrophomonas (Pseudomonas) maltophilia N246) (157).
hydroxylase
membrane non-heme
not considered
was
because both the sequence
different from those of other alkane
hydroxylases.
Sequence alignments
of the amino acid sequences of the P.
and the Acinetobacter sp. ADPl alkane
conserved. We chose two
320
(HSDHH)
The forward
develop
to
primer TS2S
lysine codon,
three
regions
the
hydroxylases
glycine codons,
mainly
on
AlkM
replace
the
codons.
The
contains the TCI serine
DeglRE
length
alkM
a
of the
sequences)
and
DeglRE2
expected
is 557
PCR
are
well
and amino acid
Figure
the AlkB sequence, and
GPol
1.
ignores
the AAA
two leucine codons and the serine codons of the
codons. TS2S has
TS2Smod and TS2Smod2 have
DeglRE
shown in
lysine codon,
and additional
TS2Smod2, the TCI serine codons corresponding
glycine
regions
(HEL(G/S)HK)
degenerate oligonucleotides
AlkM sequence. TS2Smod includes the AAA
leucine codons. In
show that several
around amino acid 145
is based
putida (oleovorans)
degree
of
a
degree
of
degeneracy
degeneracy
to
glycine
position
and
153 of
of 64, whereas
of 1064. The
reverse
primer
codons, while DeglRE2 contains the AGY serine
have
a
degree
of
degeneracy
product including
basepairs (bp).
the
of 64 and 32,
primers (based
on
respectively.
the alkB and
31
primers
AlkB
134
alkM
436
alkB
40
A: Forward
AlkM
146
55'
438
146
L..N..T..G..H..E..L..G..H..K..K..E..T.
55'
3'
gtgaataccgcgcatgaattgagccataaagcagatcga
474
158
V..N..T..A..H..E..L..S..H..K..A..D..R.
TS2S
5
.
TS2Smod
5
.
TS2Smod2
5
.
B: Reverse
3'
ctcaatacaggacacgaactcggtcacaagaaggagact
aayagagctcaygarytrggtcayaacf
3
'
aayagagctcaygaritiggicayaar
3
'
aayagagctcaygarititcicayaar
3
'
primers
alkB
925
AlkB
309
alkM
961
AlkM
321
5'
3'
cttcagcggcactcggatcaccacgcgcatccaacacgt
963
321
.L..Q..R..H..S..D..H..H..A..H..P..T..R.
5'
3'
ttacaacgacattcagatcatcacgcttatccgacgcgt
999
333
.L..Q..R..H..S..D..H..H..A..Y..P..T..R.
deglRE
3'
gtracfictrgtrgtrcgcttaaggtg.
deglRE2
3'
gtrtcrctrgtrgtrcgcttaaggtg.
...
5'
...
5'
1: conserved
regions of AlkB (P. putida (oleovorans) GPol) and AlkM (Acinetobacter sp. ADPl)
design the degenerate primers. The amino acids are shown below the second nucleotide of
the codon. Nucleotides coding for restriction sites are shown in italic print. The differences between TS2S,
TS2Smod and TS2Smod2, and DeglRE and DeglRE2 are underlined. Primer TS2S does not include 2
leucine, 3 glycine, 6 serine and 1 lysine codons, whereas primer TS2Smod includes all codons except 6
serine codons. Primer TS2S includes 4 serine codons, and does not include 4 glycine codons. Primer
DeglRE does not include 3 alanine and 2 serine codons, whereas DeglRE2 does not include 3 alanine and
4 serine codons. A: forward primers; B: reverse primers.
Figure
that
were
Using
used to
the PCR program described in the material and methods
sequences from 27 strains that
primer
combination
clearly
different sizes
grow
on
able to grow
were
TS2S/DeglRE (see
were
obtained
as
Table
Southern blots
Another 8 alkB gene
(chapter 4;
J. B.
sequences. These sequences
van
are
1).
n-alkanes, in
most
we
strains,
In several cases, PCR
homologs
we
were
obtained 29
cases
using
products
well. From the eleven strains that
n-alkanes and 12 hexane air filter
expected length.
on
section,
did not obtain PCR
were
of
unable to
products
obtained from 6 strains
of the
using
Beilen, unpublished results)(168, 273) and genome
also included in the
phylogenetic analysis.
32
generated with CLUSTAL and manually optimized.
by roman capitals I, II and III. The hydrophobic
stretches are marked by solid bars. Peptide numbering refers to the position in the cloned PCR fragment
rather than to that of the full-length enzyme.
Figure
2:
of alkane
alignment
The three histidine-boxes
hydroxylase
sequences,
underlined and marked
are
DNA sequence determination and
pGEM7-Zf(+),
and
bp including primers),
contained
fragments
fragment
cloned from
unique
one
encode
the P.
while
an
sequence. BlastX
peptides
that have
genes. Other
were
cloned and
were
fragments
not cloned. In
fragment
was
encoding
a
we
ignored
A 550
was
the
bp
this
cloned and
gene did not
based
on
sequenced,
case,
a
fragment,
of
correspond
was
only
fragment
probably originates
identified among
from
our
For DNA sequence
a
for
that
binding
at
from this
use as a
cepacia
yielded
corresponding region
proteins (see below).
was
not
primer
was
to
hydroxylase
shown)
in addition to the 550
of the forward
of
In
these, ranging from 289
primers (data
appeared
than
to
a
and
bp
region
167 in AlkB. In further work
obtained
as
well.
subsequently
used
as a
template,
as
probe
to clone
strain, but the sequence of the complete alkB
probe
RR 10
of the
more
fragments
RR10 chromosomal DNA
cepacia
fragment
length (557
fragment
PCR
one
to the
position
bp fragment
sequence did not
fragment (168),
in Southern and
yield
(data
the
not
even
colony
though
shown).
the
blots. Primers
expected fragment
The PCR
contaminating Gram-positive strain,
with
fragment
most
which could not be
strain collection.
alignments
primer annealing
identity
one
to that of the cloned PCR
high enough
the PCR
than
more
showed that all of cloned PCR
(EHXXGHH)
The PCR
hydroxylase
same
cloned in
In two cases, the PCR
and in two cases, this
bp fragment
obtained with B.
sequenced.
alkane
bp longer.
were
and found to be unrelated to the alkane
product
350
of the
were
obtained. Three of
were
when the 550
fragment
chromosomal DNA from B.
the
the
fragments
site, respectively, and only part of the
level of sequence
cloned and found to be due to
PCR
homology
alignments (5)
similar histidine motif
complete
EcoRl
9
was
In several cases,
fragments
were
one
or
products
AlkB and Acinetobacter sp. AlkM
putida (oleovorans)
bp,
product
sequenced,
were
high
a
several cases, different size
350
PCR
sequenced.
strain
particular
a
one
internal Sacl
cloned and
was
Most of the PCR
sequenced.
The PCR
comparisons.
sites
were
and
phylogenetic analysis,
removed before
the sequences
comparing
corresponding
the sequences, because
they
to
P.putida (oleovorans)
GPol
GHH ....T..D
L
.
III
.LQRH.DHHA.
RYERCAPAHSWNSDHIVTNIFLYHLQRHSDHHAN
LMSVVLFGALIAIFGPVVIPFLIIQAVYGFSLLETVNYLEHYGLMRQKTASG
97
.
SYVRCSPEDSWNSDHVVSNLFLYQLQRHSDHHAN
AMSVVLFGSLIAAFGWEIAPWLAVQAIAGFTFLETANYLEHYGLLRAKRPDG
97
N.
SFVKARPEDSWNSDHLVSNLFLYQLQRHSDHHAN
SLSAALFGTLITLFGWQILPWLLLQTLAGITFLEAANYLEHYGLLRVRRQDG
97
SWN
RYERAAPEHSWNSDHICTNIFLYHLQRHSDHHAN
LMSVVLFGVLVAVFGLSVLPFLVLQAVFGFCLLETVNYLEHYGLKRRRLDSG
97
NY.EHYG. .R
RYERCTPAHSWNSDRICTNVFLYHLQRHSDHHAN
LMSVVLFGVALAVFGIGIAPYLVIQAFIGFSLLEAINYLEHYGLLRQKTASG
97
Q
RYERCTPQHSWNSNHWTNLFLYQLQRHADHHAN
LMSAVLWSVMIAWLGIGVLPYLLIQAVVGFSLLEIVNYMEHYGMLRQKRGSPERRRYERVDPSHSWNSNNIATNVLLYHLQRHSDHHAN
97
G
RYERCSPRHSWNSNRIVTNIFLFQLQRHSDHHAN
RYERTTPEHSWNSNFLLTNLFLFHLQRHSDHHAY
AISALFLLGFSLAFGWLGAIFFLGQSVMAFTLLEIVNYVEHYGLHRRRLDNG
97
LYNVVLWGVLIAWLGAAVIPFLVIQGIYGFSLLEVVNYVEHYGLLRQKQPNG
RYERTNHTHSWNSNFVFTNLVLFHLQRHSDHHAF
LLSLALLVGFGWAFGWLGMVFFLGQAFVAVTLLEIINYVEHYGLHRRKGEDG
97
AMTVVVWGIAIAIGGVWIPFLVIQAVYGASLLEVVNYVEHYGLGRRRLPDG
RYEHQKPHHSWNSNHIVSNLVLFHLQRHSDHHAH
VITWLYTLLLAFFGPKMLVFLPIQMAFGWWQLTSANYIEHYGLLREKMADG
97
97
QYERTMPEHSWNNNNVVTNLFLYQLQRHSDHHAN
GMSAAFHASMVGIFGKGTIPYLATQAFYGISLFEIINYIEHYGLLRQKKENG
97
RYERCAPVHSWNSDHIVTNLFLYHLQRHSDHHAN
LMSVVLWGGLIAVFGPALIPFVIIQAVFGFSLLEAVNYLEHYGLLRQKSANG
I
180
101
I
170
97
I
I
E..R
NYERTMPEHSWNNNNIVTNLFLYQLQRHSDHHAY
160
150
F
GMSAAFHSSIIAIFGKGTIPYLVTQAFYGISLFEIINYIEHYGLKRQKRADG
I
II
.
140
EH.
I
.
130
F.
97
.
RYEHQKPHHSWNSNHIVSNLVLFHLQRHSDHHAH
I
I
Y.
IITVILYAVLLALFGPKMLVFLPIQMAFGWWQLTSANYIEHYGLLRQKMEDG
120
110
.
97
I
.
N
IHNDVLNAW
AHELGHKKTELERWLAKITLAQTFYGHFYIEHNRGHHVRVATPEDPASSRFGESFWTFLPRSVWGSLKSSWELEKTRMQRLGKSTWS
1
.
Rhodococcus sp
YRNNILNAW
AHELGHKSEKLEKWLAKVALAQSFYGHFYVEHNHGHHVRVATPEDPASSRYKENFWFFLPRSVIGGLRSGWRIESARLRRQGRRTWS
1
L.
R-
PRNNIVQAW
AHELGHKSSRLERRLAKIVLAQSFYGHFYIEHNYGHHVNVATPRDPASARFGESFWLFLPRSVFGGLKSGWRIESARLRRQGSPALS
1
HK
R.
IRNDVLHSW
AHELGHKKDDLERWLSKITLAQSFYGHFYIEHNRGHHVRVATPEDPASSRFGESFWTFLPRSVWGSLRSSWSLEKARLDRLGKKPWT
1
.
NRRL B-16531 AlkB2
NRRL B-16531 AlkB3
erythropolis
erythropolis
R.
IKNDVLNAW
AHELGHKKESVERWLSKIVLAQSAYGHFYLEHNRGHHVRVSTPEDPATSRFGETLYGFWPRSVGGGLKSAWHLEKTRFERLGTTHWS
1
HE.
NRRL B-1653 1 AlkBl
erythropolis
R-
IGNDVLNAW
AHELGHKKESHERWLSKIALAQSFYGHFYIEHNRGHHVRVATPEDPASSRVGESFYRFWPRTVVGSLRSAWRLERKRYARRDRHPFR
1
.
Pr. rugosa NRRL B-2295
erythropolis
B.
WRNEVLHAW
AHELGHKTNRFERWLAKITLAPVAYGHFFVEHNRGHHVRVATAEDPASARYGESFWAFLPRTVVGSVRSAWRLERTRLARLGRSPWT
1
CHAO
1BN
Pi
aeruginosa
PAOl AlkBl
cepacia
RR 10
Consensus
1BN
erythropolis NRRL B-16531
erythropolis NRRL B-16531
erythropolis NRRL B-16531
erythropolis NRRL B-16531
Rhodococcus sp
R.
R.
R.
R.
Pr. rugosa NRRL B-2295
B.
aeruginosa PAOl AlkB2
P. fluorescens CHAO
P.
P.
P.putida
A. calcoaceticus EB 104
M. tuberculosis H37Rv
Acinetobacter sp ADPl
GPol
AlkB4
AlkB3
AlkB2
AlkB 1
NRRL B-16531 AlkB4
RR10
P.putida (oleovorans)
Consensus
cepacia
aeruginosa
P. fluorescens
P-
PAOl AlkB2
WKNGVLSAW
AHELGHKNRGMAKFLAKLALASTFYGHFFVEHNRGHHVRVATPEDPASSRLGESFWSFLPRTVWFSLSSAWHLESQRLEKLGLPTLH
1
PAOl AlkB 1
WRNELIWWY
SHELIHKDPQLEQNAGGLLLAAVCYAGFKVEHVRGHHVHVSTPEDASSSRYGQSLYSFLPHAYKHNFLNAWRLEAERLKRKGLPALH
Pi
aeruginosa
1
P.
FDNEILQPM
WQNELIWWY
GHELGHKKEAFDRWMAKIVLAWGYGHFFIEHNKGHHRDVATPMDPATSRMGENIYKFSTREIPGAFRRAWGLEEQRLSRRGQSVWS
AHELIHKDSALEQAAGGILLAAVCYAGFKVEHVRGHHVHVSTPEDASSARFGQSVYQFLPHAYKYNFLSAWRLEAVRLRKKGLPVFG
1
P.putida
A. calcoaceticus EB104
M. tuberculosis H37Rv
Acinetobacter sp ADPl
1
LDNELLQGW
AHELGHKHDRIDHILSHLALVPTGYNHFRIEHPYGHHKRAATPEDPASSRMGETFYEFWPRTVIGSFKSAIEIEKNRLKRKGKEFWS
L
100
AHEMGHKKDSLERWLSKITLAQTCYGHFYIEHNRGHHVRVSTPEDPASARFGETLWEFLPRSVIGGLRSAVHLEAQRLRRLGVSPWNPMTYLRNDVLNAW
I
90
1
I
80
1
I
70
KDNELLQGW
I
60
AHELSHKADRLDHILSHLALVPTGYNHFRIEHPYGHHKRAATPEDPASSQMGETFYEFWPRTVFGSLKSAIEIETHRLKRKGKKFWS
I
50
1
I
40
FDNEILQPM
I
30
GHELGHKKETFDRWMAKIVLAVVGYGHFFIEHNKGHHRDVATPMDPATSRMGESIYKFSIREIPGAFIRAWGLEEQRLSRRGQSVWS
I
I
1
20
10
34
differ from the
the
original
chromosomal sequence. The nucleotide
peptide alignment (see below),
fragments using
of sequence
the
because in many
Wilbur-Lipman
identity.
The
resulting
method
(278)
cases
was
pairwise alignment
levels of DNA sequence
based
on
of PCR
due to the low level
possible
not
was
alignment
identity
range from 45.0 to
100 %.
In
a
number of cases, three PCR clones from the
showed that
on
average less than
due to the PCR. Possible
errors
analysis significantly, except
length
introduced
for
by
change
were
per PCR
the PCR will not
microorganisms,
point by sequencing
organism
related genes. Since
closely
genes from several selected
artefacts at this
nucleotide
one
same
sequenced.
fragment
had occurred
the
phylogenetic
change
we
This
intend to clone full
did not attempt to resolve PCR
we
additional clones.
R
R
H
erythropolis
erythropolis
erythropolis
NRRL B-16531 AlkBl
23-D AlkB3
35-0
P rugosa NRRL B-2295
HXN1000
R
Rhodococcus sp 1BN
erythropolis 23-D AlkBl
Gram positive 42-0
erythropolis 50-V
IRR
NRRL B-16531 AlkB2
erythropolis
R
M tuberculosis H37Rv
M bovis AF2122/97
HXN600
unknown source
B cepacia ATCC 25416
j
•— B
cepacia RR10
h:
P
fluorescens CHAO
R erythropolis 62-0
è
c^
R
R
HXN2000
R
erythropolis
NRRL B-16531 AlkB3
R fascians
erythropolis 23-D AlkB2
B-16531
AlkB4
NRRL
erythropolis
R fascians 154-S
115-H
—
Acinetobacter sp 2769A
A calcoaceticus EB104
A calcoaceticus NCIB 8250
Acinetobacter sp ADPl
A calcoaceticus 69-V
HXN1100
_J
P
putida (oleovorans)
I HXHXN400
P
putida
GPol
Pi
P aureofaciens RTWH 529
A borkumensis API AlkBl
X flavus strain 5
^
L
1166100
aeruginosa PAOl AlkBl
P aeruginosa PG201
P aeruginosa PAOl AlkB2
LP
^^—^^—
A borkumensis API AlkB2
pneumophila Philadelphia-1
80
60
40
20
Figure 3: phylogenetic tree generated by CLUSTAL from the alignment of peptide sequences obtained
this study and published sequences. The primer encoded sequences are excluded from the alignment.
in
35
For
comparison
a
sequences
were
of the
translated. In all
contiguous open-reading-frame
compared
based
the
a
series of
Southern
The
blotting.
Figure 3,
probe
figure
and
remaining
hydroxylase
by
the
were
to
use as
only
enough
gave similar results
M. tuberculosis H37Rv
gene
acid
phylogenetic
signals
homologs (chapter 6),
we
tree
by
phylogenetic
(data
probes
a
probe
not
detect
experiments.
aeruginosa
putida
alkB gene
aeruginosa
source
strain.
strains
probe
derived
PI and in P.
strains, which
previously
to
shown
(262), in addition
probe
the alkM
can
be
blotting.
probe
to
derived
weak
explained by
identity ranging
in Southern
signal
was
PG201. The
Using
tree
in Southern
while the
n-hexadecane, and
putida (oleovorans)
to detect
in Southern
is, unlike other P. aeruginosa strains, able
detects the
rugosa NRRL B-2295. These two
mycolic
alignment,
probes
PG201 is selective for the P.
derived from P.
not be detected in other strains. A
fragment
2. For the
gene
should detect
they
detected in the other Acinetobacter
alkM sequence,
and with the
alignment produced using
different branches of the
presence of related genes with levels of DNA sequence
to the
a
were
the amino acids encoded
GPol detects related genes in P.
probe
from P. fluorescens CHAO
signals
aeruginosa
gene identical to the P.
the genes detected
cloned,
sequences
(AlkB, AlkM),
excluding
fragments
are on
KSLA 473. The latter strain
a
was
3.
«-octane in addition to n-dodecane and
to contain
resulting peptide
sequences
of all sequences,
chosen such that
putida (oleovorans)
on
bp (559 bp) fragment
strains. Table 2 summarizes the result of these
derived from P.
aeruginosa
grow
are
the nucleotide
sequences from all branches of the
We selected six DNA
and detects both alkane
from P.
fragments,
from M. tuberculosis H37Rv. An
alignment
shown in
in the
blotting
550
obtained. The
(see Table 2). The six sequences
shown in
a
hydroxylase
representative
the manual
on
primers,
blots
hydroxylase
the PCR
(111), and manually optimized, is shown in Figure
CLUSTAL
selected
was
by
when
cases
with the known alkane
alkane
putative
encoded
peptides
the
from 68 to 80 %
Related genes could
derived from the A. calcoaceticus 69-V PCR
shown).
The R.
fragments
probes
containing actinomycetes (J.
in the R.
also gave
B.
erythropolis
van
NRRL B-16531 and
erythropolis
signals
strain and in P.
with several
high G+C,
Beilen, unpublished results).
36
Table 2: summary of Southern blots.
Strain
Growth
P.
putida (oleovorans)
GPol
P.
putida (oleovorans)
GPo 12
Probe2
on
«-alkanes1
GPol3
C5-C12
+++
P. oleovorans ATCC 8062
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
no
-
P.
putida
PI
C8
P.
fluorescens
CHAO
C12, C16
P.
aeruginosa
PAOl
C12-C16
P.
aeruginosa
KSLA 473
+++
C8-C16
C12-C16
C12-C16
Acinetobacter sp. 2769A
C12, C16
ATCC 25416
C10-C16
eutropha
JM134
R.
erythropolis
-
-
+++
-
-
+++
*
-
*
-
*
+
-
*
-
*
-
++
-
-
-
+
-
-
*
*
-
*
-
-
-
-
-
-
-
-
*
*
-
*
-
-
-
-
no
-
-
-
-
-
-
C8,C12
-
-
-
-
-
-
-
-
-
-
+
++
-
-
-
-
++
+
C8,C12,C16
Pr. rugosa NRRL B-2295
-
no
(pJP4)
NRRL B-16531
-
-
-
no
R. rhodochrous NCIMB 12566
+++
-
+++
Acinetobacter sp. ADPl
R.
*
no
KT2442
DSM 50170
.
*
putida
maltophilia
.
-
P.
S.
.
-
no
C. testosteroni DSM 50244
.
-
putida PpGl
cepacia
.
165313
-
P.
B.
CHAO3 PG2013 H37RV3
no
-
A. calcoaceticus 69-V
ADPl3
C12
-
': Abbreviations: C5: pentane,
C8: octane, CIO: decane, C12: dodecane, C16: hexadecane;
strong signal, + signal, *, aspecific signal, no signal;3: probes derived from:
GPol: P. putida (oleovorans) GPol; ADPl: Acinetobacter sp. ADPl; CHAO: P. fluorescens CHAO;
Footnotes:
2:
+++
very strong
PG201 : P.
signal,
aeruginosa
++
-
PG201 ; H37Rv: M. tuberculosis cosmid
pSCYB 11 ;
16531 : R.
erythropolis
NRRL
B-16531.
Cloning
alkane
alkane
and functional
hydroxylase
hydroxylase
hybridisation,
restriction
analysis
gene
gene
homologs.
homolog
cloned from
of the A. calcoaceticus EB104 and P.
an
A 3.0 kb EcoRl -Hindlll
of A. calcoaceticus EB104
enriched
genebank containing
fragments by colony blotting,
open-reading-frames
was
identified
alkM, respectively. To establish the function of alkM,
by
the
Southern
all 2.5-3.5 kb EcoRl-Hindlll
sequenced. Sequence analysis
respectively (213).
PI
fragment, containing
that have 59.6 % and 72.6 % DNA sequence
Acinetobacter sp. ADPl alkR and alkM,
and
and
putida
identity
The ORFs
we
revealed two
to
were
transformed
labelled alkR
an
alkM
37
negative
mutant of
contains
only
containing
Acinetobacter sp. ADPl
alkM (A. calcoaceticus
8.5
same
an
WH405 for
EB104) encodes
calcoaceticus
from
As
EB104).
a
with
a
way,
enriched
growth
alkane
an
positive control, plasmid pWH968,
used
(214). Both
dodecane, proving that AlkM (A.
fragment
of P.
putida
with EcoRl-Hindlll restriction
kb, and sequenced. This fragment has
alkBFGHJ genes of P.
was
hydroxylase.
7.5 kb EcoRl -Hindlll
genebank
on
which
pWHEB7,
the Acinetobacter sp. ADPl alkRM and rubAB opérons,
plasmids complemented
In the
(WH405) (213)
on
PI
was
identified, cloned
fragments ranging
average 80 % DNA sequence
putida (oleovorans) GPol,
and
complemented
an
from 6.5-
identity
to the
E. coli
recombinant, containing all P. putida (oleovorans) GPol aWc-genes except alkB
(GEcl37[pGEc47AB]) (261)
16S rDNA
sequencing.
on
(2769A) has
a
growth
Nocardia
NRRL culture collection. On
could grow
for
globerula
plating
n-alkanes after
octane.
on
two
was
The PCR
fragment
(Nocardia strains:
Therefore,
strain 2769A, and found that it had
haemolyticus
ATCC 17922
Acinetobacter
to
(100 %
(Ace.
8250, Ace.
to
DN-22
nr.
nr.
(Ace.
Ace.
determined
sequence
nr.
a
60 %
G+C) and is
fragment
partial
identity
of which
to
more
closely
obtained from
16S rDNA sequence of
Acinetobacter
Z93436), indicating that
2769A is
an
species.
X81660)),
X81659)
nr.
were
obtained from Acinetobacter strains Acinetobacter sp.
Acinetobacter sp. DSM587
Acinetobacter sp. 10090
30007
highest
(99.9 %,
Partial 16S rDNA sequences
ADPl
we
one
obtained from this strain
related to the Acinetobacter sp. ADPl alkM gene than the PCR
A. calcoaceticus strain 69-V.
obtained from the
types of colonies appeared, only
purification.
G+C-content of 37.1 %
NRRL B-2769
(Ace.
Z93449)),
NCIB 8250
and R.
X89240))
nr.
(=ADP1,
(100 %
erythropolis
EB 104
to
Ace.
nr.
(99.8 %
X89709)),
to A.
69-V
baumannii DSM
Acinetobacter sp. DSM590
NRRL B-16531
to confirm their classification.
(100 %
(100 %
to
(=NCIB
Rhodococcus sp.
38
DISCUSSION
We have
developed degenerate oligonucleotides
related to the P.
homologous
internal segments of genes
amplify
GPol alkB and the Acinetobacter sp. ADPl alkM
putida (oleovorans)
genes, which encode
to
membrane-bound non-heme iron alkane
We found that most
Gram-negative
contain
genes related to alkB and alkM. Strains unable to grow
one or more
those that
including
did not
yield
PCR
were
reported
fragments
Several strains able to grow
fragment
nor a
subsets of the
were
negative
regions
that
were
calcoaceticus EB104
soluble alkane
actinomycetes,
contain
integral-membrane
the
TS2S and
n-alkanes,
in
our
bp
PCR
dimer
an
as was
develop
alkane
expected
or
properties
550
amplifying
hairpin
the
primers
certain gene may be
a
formation.
hydroxylase
are
specific
that interfere with the
not
hydroxylase belonging
proposed
hands,
Alternatively,
gene
homologs
in
well conserved.
so
to
a
different enzyme
for R. rhodochrous ATCC 19067 and A.
in Acinetobacter sp. Ml
as
the data
presented
(164),
or
other
here show that many
n-alkanes, including several high G+C, mycolic acid containing
homologous
but
related enzymes
distantly
to the class
belonging
non-heme iron monooxygenases.
peptide alignment
primers
neither the
be effective in
(44, 179), dioxygenases
on
so
on
n-alkanes
alkM.
may have
hydroxylases. Nevertheless,
strains able to grow
or
yielded
primer
used to
cytochrome
P450
n-alkanes but failed to do
in the PCR may contain alkane
class:
The
on
on
in Southern blots. This may be due to the fact that
because of
pool
the strain may contain
of
n-alkanes
principle
Finally,
a
to grow
degenerate oligonucleotides
selected out of the
which the
on
bacteria able to grow
Gram-positive
related to alkB
positive signal
PCR. Primers that could in
strains that
and
hydroxylases.
shows that certain
DeglRE
allowed
regions
amplification
this paper, the first and the third histidine
boxes,
be well conserved. The second histidine-box
AlkB sequence, is also
highly
NYXEHYG(L/M) starting
motif is conserved in all
at
are
on
well conserved in all sequences. As
of the PCR
which these
proteins
that
primers
(EHXXGHH), starting
conserved in all sequences,
position
fragments
as
at
are
described in
based,
position
must
167 in the
is the motif
269 in AlkB. The histidine residue of the latter
can
be classified
as
integral-membrane
non-heme
39
iron
proteins
cases
it is
of
case
and contain the three histidine motifs defined
preceded by
a
stretch of
AlkB, has been shown
Site-directed
mutagenesis
hydrophobic
complete
for the function of the related alkane
tree based
on
mean
that the
hydroxylases
to the low G+C content of these
are
Pseudomonas
P.
from
high
are as
a
role. The
the G+C content of the P.
may constitute
a
putida
putida (oleovorans)
from
on
identical
or
genes that
strain,
as
(M.G. Wubbolts,
residues
more
a
a
(figure 3).
are
(111),
This could be due
%, while nearly all other
case
of the
clearly
G+C content that is much lower than
low G+C host
gene
(260, 263). The
fragments
to sequences from
related to each other than to
and
OCT-plasmid,
have
a
high
Gram-positive
some
of the sequences
strains.
observed for
some
higher homology
Gram-positive
strains. P.
than sequences found
aeruginosa
strains able
medium-chain n-alkanes and A. borkumensis API contain genes that
closely
essential
horizontal gene transfer
cepacia alkB
higher homology
are
with CLUSTAL
chromosome and the
found in different strains often show
same
to grow
genes
only slightly
Gram-negative
Sequences
in the
are
xylene
sequences within the genus
aWc-genes,
transposon which originates from
sequences of the P. fluorescens CHAO and B.
strains, which
group
which is around 40
PI
in the
well.
one
from these strains have
G+C content, and appear to have
mt-2
sequences from two different genera. In the
GPol and P.
aWc-genes
putida
generated
organisms. However,
divergent as
putida (oleovorans)
plays
G+C
fragments,
In all
(261) (figure 2, solid bars).
corresponding
as
the DNA sequences,
(233).
shown), which,
not
inactivation of the enzyme
shows that all Acinetobacter sequences cluster in
sequences
Shanklin et al.
to traverse the membrane twice
which could
personal communication),
phylogenetic
(data
of this histidine residue in the P.
monooxygenase leads to the
The
residues
by
related to the P.
identical
or
closely
putida (oleovorans)
related to the P.
are
GPol alkB gene, in addition to
aeruginosa
PAOl alkBl and alkB2
(262) (chapter 6).
To evaluate the
metabolism,
fragments
as
we
use
of the cloned PCR
fragments
carried out Southern blots
probes.
We found that
probes
using
in
detecting
genes involved in alkane
several different PCR
derived from
a
particular
fragments
gene
or
only give
gene
40
information about the presence
as
the cloned alkane
hydroxylase
to each other.
homology
isolates able to grow
genes that
are
too
probably
on
n-alkanes,
related genes in
positive
strains
(J.
B.
that
alkB2
only
was
one
one
strain,
on a
gene is
by
probably
probes
as
is the
case
from
a
single
PCR with the alkB
When strains
able to grow
study
are
amplified
For
genes.
Therefore,
we
putida PI,
additional genes from
strains,
purpose
we
hydroxylase
set up several in vivo
homologs
demonstrate that the alkane
can
or
and
n-alkanes.
pseudomonads,
sequenced.
example,
cloned the alkane
and showed
then, it is
in A. borkumensis API
the
homologs.
gene
fragments
and Gram-
fragments
hydroxylase
part of
are
genes of A.
by complementation experiments
hydroxylases.
constructing
We
are
currently
hydroxylases.
For this
in which the alkane
This allowed
from A. borkumensis API
(chapter 4),
and P. fluorescens CHAO
that
knock-out mutants of
functionally expressed (chapter 4).
hydroxylase homologs
AlkB2) (chapter 6)
Even
Gram-positive
complementation systems
be
and Gram-
(significant)
a
long-chain n-alkanes,
from many
M. tuberculosis H37Rv, P. rugosa NRRL B-2295
(AlkBl
is the P.
exception
very useful to clone alkB gene
number of strains and
a
hydroxylase
Gram-negative
to establish that these genes also encode alkane
gene
environmental
degenerate primers. Nevertheless,
the cloned genes did indeed encode functional alkane
these
used. An
strains. However, this does not prove that these gene
calcoaceticus EB104 and P.
cloning
screen
contains alkane
for several
strain is
either medium-
on
related to alkB and alkM could be
hydroxylase
strains,
plasmid.
amplified efficiently.
described in this
related
do not show clear DNA sequence
Beilen, unpublished results; chapter 6), unless
oligonucleotides
alkane
the
closely
is that this method does not show the presence of two
fragments
obtained
are
by
or
cannot be used to
each strain
be detected
approach
van
number of cloned PCR
negative
as
due to the fact that it is located
or more
only
homologs
gene
host
original
GPol alkB gene, which has been found in several
A limitation of the PCR
possible
absence of the
Therefore, the probes
divergent to
putida (oleovorans)
or
P.
aeruginosa
(chapter 7)
us
(AlkBl),
PAOl
do indeed oxidize
to
41
ACKNOWLEDGEMENTS
We wish to thank Dr.
sp. ADPl
prior
to
Wolfgang
publication.
Hillen for
access
to the sequence data of
We also thank Dr. Michael Kertesz
Switzerland),
Suzanne Schorcht
Plaggenmeier
and Prof. Karl
(University
of Bremen,
Engesser (University
of
Moore
(National
Research Centre for
for
(ETH, Zürich,
Germany),
Thorsten
Stuttgart, Germany),
Ratajczak (Friedrich-Alexander University, Erlangen, Germany)
(Karl-Marx University, Leipzig, Germany)
Acinetobacter
providing
us
with
Andreas
and Dr. Otmar
Asperger
strains, and Dr. Edward
Biotechnology, Braunschweig, Germany)
for
support with 16S rDNA sequencing.
This research
was
supported by
National Science Foundation
the Swiss
Priority Program
in
Biotechnology
of the Swiss
42
43
Chapter
3
NEW ALKANE-RESPONSIVE EXPRESSION VECTORS FOR E.
COLI AND PSEUDOMONAS
Theo H. M.
Smits, Markus A. Seeger, Bernard Witholt and Jan B.
Published in Smits et al. 2001. Plasmid 46
(1):
16-24
van
Beilen
44
SUMMARY
We have
the
developed
Escherichia coli and Pseudomonas
alkane-responsive
expression
vectors
Pseudomonas
were
KOB2A1 with
fluorescens
putida (oleovorans)
tested in several E. coli
pKKPalk
but
pCom8, pCom9
were
levels of
clearly
more
lower for
strains, P. putida GPo 12 and P.
in E. coli and
than 10 % of total cell
and
protein
on
promoter PalkB. The
GPol
catechol-2,3-dioxygenase (XylE).
between 100 and 2700 for
vectors based
expression
pCom8
pComlO
were
Induction factors
in Pseudomonas
in E. coli.
ranged
strains,
XylE expression
obtained for E. coli
well
as
as
for
Pseudomonas strains.
INTRODUCTION
Escherichia coli is the most
production
of
frequently
content of
target genes may lead
as
they
have
In
can
high-level
not be used for all
incomplete processing
or
due
proteins
and export of
addition, the different codon usage and G+C
to low
a
it
host for
levels. Pseudomonas strains
expression
high
G+C content and
are
export extracellular proteins
large quantities (79).
Unfortunately, only
are
the nahR
(55),
the
a
few Pseudomonas
regulated expression
and the tac -based vectors
all based
or
bodies,
proteins (100, 116).
suitable alternatives
in
prokaryotic
heterologous proteins. However,
to the formation of inclusion
extracellular
used
the
on
pUCP
specially
total cell
on
available.
the lac-based
and
Higher
additional
are
pMMB
Examples
pVLT
series
series
(8, 15),
copy number vectors like
plasmids containing
a
lad gene
constructed host strains.
putida (oleovorans)
GPol alkB
promoter (PalkB) (143) controls
of the alkBFGHJKL operon, which encodes
degradation (reviewed
expressed
pHAlO, pHA12, pKT240
(53, 229, 277) depend
The Pseudomonas
expression
vectors
pKMY319 (286),
number vector RSF1010.
low-copy
series
vector
expression
in
a
correctly
protein,
E. coli and P.
in
(263)).
In E. coli
extremely high
putida (oleovorans),
involved in alkane
W3110, alkane hydroxylase could be
folded form from PalkB
which is
proteins
for
the PHA
an
(its
own
integral
polymerase
promoter)
to 10-15 % of
membrane
protein (185).
PhaCl could be
In
expressed
45
from the
same
to 15 and 10 % of total cell
promoter
de Roo, B. Witholt and B.
expressed
styrene monooxygenase (StyAB)
active form
The
of
and is most
regulatory proteins,
from PalkB in E. coli in
an
as
haloalkanes, ethylacetate, ethylether and
AlkS is
closely
member of the
a
related to
LuxR-UhpA family
MalT, the regulator of the
regulon (202).
Expression
from PalkB is
wild type strain P.
coli,
expressed
PalkB, AlkS (67, 202), is activated by C7-C12 n-alkanes,
dicyclopropylketone (DCPK) (96, 283).
E.
levels from other promoters, and
high
(202, 203).
positive regulator
maltose
to
also
were
alkenes, and gratuitous inducers such
of
G.
Kessler, unpublished results)(144). Xylene monooxygenase
which could not be
(XylMA),
protein, respectively (Q. Ren,
by
carbon catabolite
putida (oleovorans) GPol, especially
catabolite
no
influenced
negatively
repression
was
repression
in LB-medium
observed with the carbon
sources
in the
(244, 287).
In
glucose,
lactate, glycerol and pyruvate (244).
Several vectors based
which
PalkB
be used for the
can
detection and
integration
strains
on
production
purification (184).
of the
expression
(201, 202).
constructed
were
of VSV-G
previously,
such
tagged proteins
Another PalkB vector system
developed
complementation
hydroxylase systems
are
their counterparts from other enzyme systems
(chapter 4), easy-to-use
and E. coli
putida (oleovorans)
promoter
were
for E. coli
characterized
cytoplasmic
constructed. These
well
as
vectors based
as
for
on
the P.
plasmids
Pseudomonas,
as
are
useful
as
for
coli and Pseudomonas
Because these vectors could not be used for
expression
E. coli vector,
to allow easy
was
cassette in the chromosome of E.
studies in which components of various alkane
as an
replaced by
Pseudomonas
GPol alkB
general expression
the PalkB function in both
vectors
species
is well-
(69, 143, 287) and yields high induction and expression levels of both
and membrane
proteins (184, 202).
MATERIALS AND METHODS
Strains, plasmids and media. Strains and plasmids used in this study
table 1. E2 medium
glucose (0.5
%
w/v)
supplemented
or
citrate
with metal traces
(0.2 % w/v) and
LB
(151)
(220)
and
were
are
listed in
glycerol (0.3
used
%
throughout
w/v),
this
46
study.
the
0.1 % Yeast extract
following
concentrations:
tetracycline (12.5 //g/ml)
(oleovorans)
transformed
(oleovorans)
was
or
added to the E. coli cultures. Antibiotics
gentamicin (10 piglml
by electroporation (64, 117),
Table 1: strains and
by
three
plasmids
Strain:
used at
ampicillin (200/^g/ml), kanamycin (50/^g/ml),
for E.
coli, 100 piglml for P. putida
and P. fluorescens). E. coli and P. fluorescens KOB2A1
GPo 12
were
while
plasmids
parental mating using
used in this
were
RK600
as
(chapter 4)
introduced in P.
the
were
putida
helper plasmid (61).
study.
Reference:
Genotype:
E. coli strains:
strain
Gibco BRL
DHIOB
cloning
GEcl37
TGI fadR, thi
(69)
W3110
prototroph
Lab collection
Pseudomonas strains:
P.
P.
GPol cured of OCT
putida (oleovorans) GPo 12
fluorescens KOB2A1
Plasmid:
plasmid
(141)
CHAO, alkB gene deleted
chapter 4
Characteristics:
pBGHEAN
pBGl 1
Reference:
with EcoRl site and mutated
Ndel site, alkS
(202)
pSPZ2E
pEXlOOT
pEX18Tc
pCK217
pGEc47
pGEc74
pKK223-3
pUCP25
pSPZ2 harbouring xylE
Knockout plasmid, oriT
Knockout plasmid, TcR
Knockout plasmid, ApR, KmR
(201)
alkBFGHJKL, alkSTm pLAFRl
(69)
aZfcST in
(69)
RK600
Helper plasmid, tra+, mob*
pKK223-3 derivative with PalkB, ApR
pKKPalk, xylE in Ndel-Pstl sites
pKKPalk, rep and aacCl from
pUCP25, GmR
pCom5, oriT from pEXlOOT
pCom7, alkS from pBGl 1EAN
pCom8, xylE in Ndel-Hindlll sites
pCom8; TcR instead of GmR
pCom9, xylE in Ndel-Hindlll sites
pCom8; KmR instead of GmR
pComlO, xylE in Ndel-Hindlll sites
pLAFRl
Expression vector, Vtrc, pMB 1
(115)
(145)
ori
(34)
E. coli-Pseudomonas shuttle vector
pKKPalk
pKKPalk-XylE
pCom5
pCom7
pCom8
pCom8-XylE
pCom9
pCom9-XylE
pComlO
pComlO-XylE
DNA methods. Restriction enzymes, T4 DNA
fragment),
(230)
T4 DNA
polymerase
and
ligase,
(277)
(78)
this
this
this
this
this
this
this
this
this
this
DNA
dideoxynucleotides
study
study
study
study
study
study
study
study
study
study
polymerase (Klenow
were
from Roche Molecular
47
Biochemicals
(Rotkreuz, Switzerland). Oligonucleotides
Microsynth (Balgach, Switzerland). PCR-products
gel,
cut with
appropriate
and cloned into
restriction enzymes,
purified
purified again
suitable vector. Plasmid DNA
a
were
synthesized by
were
1% agarose
over a
1% agarose
over a
isolated with the Roche
was
gel,
High
Pure Plasmid Isolation Kit.
PCR reactions
using
was
a
were
carried out with the Roche
Perkin Elmer
used
(4' 95°C,
GeneAmp
25x
(45" 95°C,
Tm), 1' 72° C), 5' 72° C,
Cor 4000L sequencer
PCR
oo
4°C).
1'
All cloned PCR
(gccagctcgtgtttttccagcagacg)
pKKPalk
derived
pKKPalk
was
and
was
LASERGENE
fragments
were
sequenced
as
pKKRev (gagttcggcatggggtcaggtg)
follows:
ATG of
and
polymerase,
ligated
to
alkB), generated
Bglll site)
with Bamrll and
containing
a
with
was
digested
The
remaining part
pUCP25 (277)
Plasmids
orientation of the
pKKPalk
Fspl
was
of the
Sspl,
to
plasmid
digested
contains the Pseudomonas
were
a
and
PCR
with
digested
was
The
ligated.
with
origin
Fspl
resulting
fragment containing
primers
pGEc47 (69)
PalkB
PalkRBS
as
the inserted PalkB
replicate
remove
was
of
with
with Ndel
EcoRl
template,
fragment
sites)
and
and cut
were
insert, and sequenced using primer pKKRev.
derivative able to
and
for
Plasmid
pComlO.
plasmid pKK223-3 (34)
Palk5 ( g g gttttg gagatctccaatcgtg,
right
kit
(ccctcggtcccccagatagcc),
(cgggatcccgggcgcgccaagcatatggaattctcc, Bamrll, Smal, Ascl, Ndel,
Bglll.
Li-
Navigator (DNASTAR, Madison, Wisconsin, USA).
linearized with Bamrll and
(271 bp upstream of the
To construct
on a
Thermosequenase cycle sequencing
pKKPalk, pCom7, pCom8, pCom9
constructed
checked for the
5°C below
plasmids (MWG-Biotech, Ebersberg, Germany). Sequences
and Smal, made blunt with Klenow DNA
plasmid
PCR, the following program
and IRD800-labelled PalkFw2
PalkFw3
Construction of
9600. For
annealing temperature (usually
the Amersham
using
(Amersham, Rainham, UK)
analyzed using
System
kit
Expand High Fidelity Polymerase
in
pseudomonads,
most of the
ampicillin
replication (rep)
plasmid
resistance gene.
purified by gel electrophoresis.
and SacII, and the 2.8 kb
the
Plasmid
fragment,
and the aacCl gene,
which
was
isolated, made blunt with T4 DNA polymerase and ligated in the large fragment of
pKKPalk, resulting
restriction
analysis.
in
pCom5.
The orientation of rep and aacCl
was
determined
by
48
A 566
the
bp fragment containing
and traK
(88))
was
from
amplified
of transfer
origin
(oriT, intergenic region of traJ
with
pEXlOOT (230)
primers
oriTfw3
(cattgatgcatgccaggtaccgctcgagctcatagtccac, Sphl, AspllSl, Xhol,
oriTrv2
cloned in the
sequenced
sites of
Sphl-Bcll
with the
mutagenized
to
in
pCom5, resulting
with Bamrll and
internal Ndel
site,
was
The onT
pCom7.
and PalkFw2. The
primers pKKRev
remove an
site), digested
Bamrll
(ctttgggatcctctctcgcct,
regulatory
amplified
from
and
sites)
Sacl
and
Sphl
was
gene
then
alkS,
plasmid pBGllEAN
(202) with primers alkSfw3 (ggaaatgggtaccgcgagctact, AspllSl site) and alkSrv
Xhol
(cgggatcctcgagtatacttttcactatatc, Bamrll,
the alkS gene and 248
and cloned in
called
part of alkS
A
pCom9
the aacCl gene,
Clal
site)
and
and
was
was
amplified
with
pCom9.
In the
same
Clal
from
as
manner, the
the
pCom8
npt gene
was
To construct
with Clal and
PCR
was
fragment
was
Clal
which resulted in
pKKPalk-XylE,
the
xylE
was
and cloned in the
gene
To construct
gene
was
the
vector,
assays. Strains
pCom8, pCom8-XylE, pCom9-XylE
containing
appropriate
1.0) and induced with 0.05 %
pCom8, except
The
tetracycline
and
restriction
by
to
a
Clal
site).
was
analysis.
digested
PCR
pCK217 (145), amplified
site)
and
pComlO.
cut from
Clal
pCK217rv
The orientation of the
pSPZ2E (201)
with Ndel
plasmid pCom8-XylE, pCom9-XylE
cut from
respective expression
Catechol-2,3-dioxygenase
medium
ligated
analysis.
xylE
and PalkFw2.
resulting plasmid
determined
restriction
the
site).
The
ligated.
by
pComlO-XylE,
was
primers pEX18Tcfw
resistance gene npt of
site),
pKKPalk.
Clal
with
also determined
and Pstl and cloned in
primers pKKrev
pEX18Tcrv (gattggcatcgattcttggagt,
primers pCK217fw (ctggtatcgatgggaagccctgc,
Clal
and Xhol
resulting plasmid
follows: most of
pEX18Tc (115)
kanamycin
AspllSl
primers pCom8fw (cgcttategattggatgcccgaggc,
The orientation of the tet gene
(gttgggcatcgattggtcggtca,
-
with the
fragment, containing
with
enzymes. The
constructed
and
site)
digested
were
fragment containing
0.5
digested
was
same
sequenced
were
amplified
was
fagments
called
and
with the
pComlO
(atacatcgatatatgtatccgc,
with
promoter,
own
pCom8rv (tgattatcgattggtaactgtcagacc,
resistance gene
The PCR
of its
pCom7, digested
pCom8.
Plasmids
bp
This PCR
sites).
pKKPalk-XylE with Ndel
digested
with the
same
and Hindlll
enzymes.
harbouring plasmids pKKPalk-XylE,
pComlO-XylE
antibiotics to the
were
grown in E2 minimal
mid-logarithmic phase (OD450
DCPK. After 2 and 5
hours, 30 ml samples
~
were
49
harvested, washed
phosphate
buffer
once
with 10 mM
The cells
(pH 7.5).
recovered
extract
was
protein
concentration
MgS04
determined
in
bead-mill and the crude cell
a
minutes in
using
cooled
a
Bradford reagent
München, Germany). Catechol-2,3-dioxygenase activity
Nozaki
One unit
(191).
in 0.6 ml 50 mM
resuspended
disrupted
were
by centrifugation (10
was
and
(U) of activity corresponds
was
(Biorad Laboratory,
measured
according
to
catechol converted per
pimol
to 1
The
microfuge).
minute.
SDS-Polyacrylamide gel electrophoresis
stacking gels.
About 10 pig
protein
Coomassie Brilliant Blue R-250
The sequences of
Sequences.
deposited
in
was
was
carried out
using
loaded per lane. Gels
12 %
were
running
and 6 %
stained with
(104).
and
pKKPalk, pCom7, pCom8, pCom9
pComlO
were
GenBank, and received the accession numbers AJ299425, AJ299426,
AJ299427, AJ302086 and AJ302087, respectively.
RESULTS AND DISCUSSION
Construction of
pKKPalk, pCom7, pCom8, pCom9
pKKPalk (figure la)
resistance, and
can
is
a
2.7 kb
be used in E. coli strains
trans, either inserted in the chromosome
such
as
pGEc74 (69).
The
the ribosome
binding
terminator of
pKK223-3,
readthrough
derivative
pKK223-3
site and
an
Ndel site
or on a
site
from the PalkB into
to the deletion of the rop gene
regions
during
overlapping
amounts isolated with standard
procedures (data
pCom7 (figure la)
gentamicin
resistance and
P. fluorescens and P.
is
an
E. coli
replicates
and the
-
regulator
second
compatible plasmid,
includes
replication
based
of the
plasmid.
on
has
Due
a
plasmid
Pseudomonas shuttle vector which confers
in Pseudomonas
putida (197, 277), by
plasmid pCom7
EcoRl site at
shown).
strains, such
virtue of the
as
P.
aeruginosa,
pUCP25 (pRO 1600)(197,
277) origin of replication. The origin of transfer (oriT) of plasmid
enables transfer of
an
plasmid, pKKPalk
pUC series,
not
gene alkS in
with the start codon. The rrnB
construction of the
pBR322
Plasmid
multiple cloning site, prevents
necessary for
copy number between those of
Plasmid
the
(figure lb)
located downstream of the
pComlO.
(34) which confers ampicillin
harbouring
(201)
multiple cloning
and
from E. coli to Pseudomonas
RP4
by
(78, 88)
three
parental
50
tet
Ndel.
tac
BamHl'
lac
pKK223-3
4586 bp
rrnB
lacZa
•Smal
pUCP25
4036 bp
term.
aacCl
bla
Fspl
rep
Delete Ndel-Smal
PalkB PCR
\
fragment
in BamHl site
BcR
Sphl
MCS
Fspl
fragment
Sphl and
oriT PCR
between
BcR sites
Pstl
Pstl
alkS PCR
between
fragment
Asp! 181
and Xhol sites
MCS
BamHl
B
EcoRl
Ndel
Ascl
Smal
j
Sail
Pstl
Hindlll
TTGGAGAATTCCATATGCTTGGCGCGCCCGGGATCCGTCGACCTGCAGCCAAGCTT
RBS
Start
•Sacl
51
1. A: construction of
Figure
ribosomal
binding
site
additional Pstl sites
are
and
pKKPalk, pCom7
(RBS) and
start codon
are
of transfer of
helper plasmids.
To
use
gene, located either
on
(oleovorans) GPol)
or
Two derivatives of
7675
(pComlO;
pCom7
this
growth
are
plasmid,
pCom8
was
factors, xylE
or on a
inserted in
were
plasmid
coli strain
or
glucose-
and
cloned in
was
the
cause
pCom8
assays. To test
and
a
and
used
as a
bp)
use a
strain
kanamycin
or
gentamycin
resistance
xylE
levels and induction
pComlO.
gene showed
no
measurable
Recombinant
showed very low
were
tested
containing
XylE activity.
XylE
result, glycerol
was
were
E.
activities under
measured 2 hours after
used for all further E. coli cultures.
carbon-source for Pseudomonas recombinants
repression
was
pseudomonads
of PalkB
as
it does not
(244).
tested in the E. coli strains DHIOB, GEcl37 and W3110,
P.
putida (oleovorans)
containing pCom8-XylE
showed
circumstances, with values of up
were
to
pCom8 (figure la).
Recombinants
to 4.2
GPol2 and P.
high XylE
or
fluorescens
activities under
U/mg protein.
induction, specific activities of around 10 U/mg protein
protein
(P. putida
number of control strains
glycerol-grown cells, respectively,
E. coli strains
induced
in
same
copy of the alkS
possible
expression
pKKPalk, pCom8, pCom9
without the
pCom8-XylE
and in the
a
OCT
8121
constructed because the
with the
circumstances, while activities of 0.34 and 0.75 U/mg protein for
catabolite
Plasmid
be
tetracycline (pCom9;
resulting plasmids
induction. Because of this
was
as
pCom7, resulting
GEcl37[pGEc74, pKKPalk-XylE]
non-induced
such
always
catechol-2,3-dioxygenase (XylE) activity (table 2).
Citrate
or
rates and final cell densities.
containing
pKKPalk
pLAFRl
host strains must also contain
that confer
bp) resistance,
than those of
As it may not
pGEc74 (69).
Catechol-2,3-dioxygenase activity
for
site of the vectors. The
the tra and mob genes, like RK600
higher
the chromosome
alkS in trans, alkS
containing
strains
multiple cloning
(78).
Frequencies
reduces
B:
present in alkS.
mating using helper plasmids containing
RK2013
pCom8.
underlined and marked below the sequence. Two
KOB2A1.
non-
5 Hours after
10 to 25 % of total cell
found, with induction factors ranging from 2.3
to 16
(table 2, figure 2).
GEcl37[pGEc74, pKKPalk-XylE]
Table 2: overview of results from the
Strain
E. coli
[pCom8-XylE]
induction
experiments.
Time after
XylE
Carbon
induction
52
Specific activities
(U/mg total cell protein)
Induction
factor
(%
Percent
XylEa
protein)
of total cell
n.d.
Induced
-20
>25
Non-induced
-5
Induced
2-5
-10
>25
Non-induced
-5
>25
n.d.
8.3
-5
2-5
-10
2-5
n.d.b
7.6
1
850
1.9
9.1
16
<
-10
0.34
1.1
10.3
2.3
3.1
<1
-20
0.0004
0.25
6.0
15
2-5
2
5
0.63
9.7
35
2-5
>5
-20
2
1.9
1.2
7.0
<0.1
-5
5
4.2
2.8
27.3
<0.1
n.d.
2
0.08
3.6
<0.1
4.3
5
0.08
13.9
1566
2700
<0.1
1500
2
0.51
2.7
111
4.7
5
0.51
4.7
56.7
0.75
2
0.001
2.0
1.1
5
0.003
3.8
0.0005
2
0.018
2
5
0.067
>
10
>5
10
2
>
5
2
(h)
source
Glucose
Glycerol
Glycerol
Glycerol
Glycerol
W3110[pCom8-XylE]
GEcl37[pCom8-XylE]
E. coli DH10B
E. coli
E. coli
Glycerol
Glycerol
[pCom9-XylE]
DH10B[pComlO-XylE]
E. coli DH10B
E. coli
Citrate
not determined
putida (o/eovora«s)GPol2[pCom8-XylE]
b
n.d.:
P.
from SDS-PAGE;
Citrate
estimated
fluorescens KOB2Al[pCom8-XylE]
a
P.
Footnotes:
53
Similar activities but
obtained with
induction factors
pCom9-XylE and pComlO-XylE
calculated with the
U/mg protein),
protein.
slightly higher
specific
activities of
the measured
from 7.0 to 35
in E. coli DHIOB
(table 2).
were
When
highly purified catechol-2,3-dioxygenase (227
activity corresponds
The difference with the
ranging
expression
to
approximately
4-6 % of total cell
level estimated from SDS-PAGE may be due
to the formation of inclusion bodies.
Figure
2:
protein gels
of the
pCom8-XylE
recombinants.
Non-induced
(-) and induced (+) samples, 2 and 5 hours after induction, were loaded.
strain containing pCom8 without insert, after 5 hours induction with DCPK.
The
high background activities,
and
in E. coli strains may be due to the
Previously
constructed
copy number
expression
consequently
high
plasmid pBR322 (202),
low induction factors of
copy number of the
vectors
containing
or were
M: marker. No
PalkB
dependent
on
pCom8-XylE
positive regulator
were
based
on
alkS encoded
xylE:
alkS.
the medium-
on
the
54
chromosome
or on a
pGEc74 (184).
study
In
for
separate low-copy number plasmid, such
These vectors showed
control of
tight
the
pLAFRl-derived
also observed in this
as was
pKKPalk-XylE.
pseudomonads,
induction factors
2700 for P.
where
pUCP25
derivatives have
a
lower copy number
clearly higher (up
to 110 for P.
putida (oleovorans) GPol2).
The absolute
U/mg protein.
were
In non-induced cultures of
XylE], XylE
is not visible
as a
activity data,
while
XylE
a
clear
visible in induced cultures
band
on
band
the SDS-PAA
accounting
(figure 2). Previously
activities for Pseudomonas
were
(229), the
and up to
fluorescens KOB2A1,
expression
gels,
levels reached 4.7
and
GPol2[pCom8-XylE]
KOB2Al[pCom8-
in accordance with the
for up to 10 % of total cell
described Pseudomonas
systems only reached induction values of around 12
In
PalkB,
as
to
protein
expression
40, although the absolute XylE
comparable (8, 175, 286).
conclusion, plasmids pKKPalk, pCom7, pCom8, pCom9 and pComlO constitute
useful alternative
expression system
Pseudomonas. The
medium to
high
as
high
plasmids
large scale,
expensive
such
as
high
as
sites,
probably
as
expression
fully sequenced,
yield expression
are
compatible
IPTG,
or
ethylacetate
to methods that
background
or
are
DCPK
levels
as
can
be
activities and
an
on a
high
or
pComlO
are
pseudomonads.
ACKNOWLEDGEMENTS
We thank Martina
the Swiss
project
nr.
Röthlisberger for sequencing.
Priority Project
5002-037023.
in
Biotechnology
a
with
cumbersome
coli, pKKPalk is well suited, while pCom8, pCom9
in
have
also with RSF1010 based Pseudomonas
PalkB with n-alkanes,
inducers such
a
in E. coli and
are
2700. The vectors
heat shock activation. For low
induction factors in E.
useful for
as
and most
vectors. Induction of
alternative to
heterologous expression
have convenient restriction
25 % with induction levels
expression
for
copy number in E. coli and Pseudomonas, and
pLAFR-derivatives (RK2),
is
This research
project
was
of the Swiss National Science
supported by
Foundation,
55
Chapter
4
CLONING AND FUNCTIONAL ANALYSIS OF ALKANE HYDROXYLASES
FROM GRAM-NEGATIVE AND GRAM-POSITIVE ORGANISMS
Theo H. M.
B.
van
Smits, Stefanie B. Balada, Alessandro G. Franchini, Bernard Witholt and Jan
Beilen
Manuscript
in
preparation
56
SUMMARY
We have cloned
hydroxylase
of the Pseudomonas
homologs
from Pseudomonas
Alcanivorax borkumensis API,
rugosa NRRL B-2295.
Pseudomonas alkane
remaining
alkane
related to the P.
case
hydroxylases
are as
developed
and
of the
express all
alkane
necessary for
hydroxylases
supplied by
growth
enable at least
hydroxylase, suggesting
alkane
strains, based
rubredoxins from other alkane
proteins
as
to the
that in this
place.
on
electron transfer components of the GPol alkane
replaced by
related to each other
hand, the A. borkumensis enzyme is closely
GPol alkane
(putative)
three recombinant host
show that the three
comparisons
distantly
On the other
horizontal gene transfer has taken
analysis
tuberculosis H37Rv and Prauserella
Mycobacterium
putida (oleovorans)
For the functional
GPol alkane
aeruginosa PAOl, Pseudomonas fluorescens CHAO,
Sequence analysis
hydroxylases.
putida (oleovorans)
one
hydroxylase
genes,
the observation that
have
one
of the
hydroxylase system, rubredoxin,
hydroxylase systems.
on
we
alkanes except
an
of the hosts to grow
on
can
be
The three host strains
alkane
hydroxylase.
All
alkanes, using electrons
the rubredoxin and rubredoxin reductase of the host strain.
INTRODUCTION
Bacterial oxidation of n-alkanes is
and is
a
major
recycled
process in
common
phenomenon
in soil and water
even more
the entire
significant
and other
biosphere. Apart
hydroxylases
are
versatile
are
the alkanes
important
biocatalysts
(mainly
because
organisms,
from their
(37),
terms: the estimated amount of alkanes that is
geochemical
produced by plants, algae
they
waxes or
are
a
paraffins)
available to bacteria in
role in the carbon
which carry out
oil-spills
cycle,
alkane
wide range of useful oxidation
(119,281).
Biochemical and
alkane
very
per year amounts to several million tons from natural oil seepage and
(218). Perhaps
reactions
a
genetic
hydroxylases,
studies have thus far focussed
such
as
the Pseudomonas
on a
very limited number of
putida (oleovorans)
GPol alkane
57
which oxidizes C5-C12 n-alkanes to
hydroxylase (207),
host to grow
on
these
is the prototype of
compounds.
It has become
(251), fatty
The P.
acid
putida
integral
membrane
of Acinetobacter sp. ADPl
hydroxylases
Acinetobacter calcoaceticus EB104 and P.
oxygenases and
apparent only recently that this enzyme
very diverse collection of related non-heme iron
a
oxygenases which includes the alkane
monooxygenases
1-alkanols, thereby allowing the
(213),
(239), but also xylene
PI
desaturases, fatty acid monooxygenases, steroid
decarbonylases (232).
putida (oleovorans)
GPol alkane
consists of three
hydroxylase system
components: alkane hydroxylase (AlkB), rubredoxin (AlkG) and rubredoxin reductase
(AlkT).
AlkB is
a
non-heme iron
transfers electrons from AlkT to
(256).
The
are
detected in
a
closely
large
membrane
AlkB, and AlkT is
protein (143, 173, 261).
an
related to the alkane
fraction of the microbial
hydroxylase
population
AlkG
(207)
NADH-dependent flavoprotein
of this enzyme system has been reviewed
genetics
Genes that
integral
gene
by
van
Beilen et al.
(263).
of GPol have been
(alkB)
in oil-contaminated environments
(243) and in several fluorescent pseudomonads (226, 239, 262, 273).
More
related genes
strains able to grow
on
n-alkanes
which
found in many
ranging
amplify
these PCR
from
we
and genome
and
Gram-negative
results),
fragments
Gram-negative
(239)(chapter 2) using highly degenerate primers
of alkane
hydroxylase homologs.
sequencing
Gram-positive
data to clone
alkane
on
degrading
hydroxylases
expression
a
In this
study,
we
used
number of alkane
the observation that rubredoxins
GPol rubredoxin in alkane oxidation
have constructed hosts and
the novel alkane
and
diverse group of bacteria. Based
a
putida (oleovorans)
Gram-positive
from C6 to C30
internal
fragments
hydroxylases
from
were
distantly
strains
(J.
B.
can
van
replace
the P.
Beilen, unpublished
vectors that allow
us
to test whether
oxidize n-alkanes.
MATERIAL AND METHODS
Strains, plasmids and media. Strains used in this study
Bertani
broth) (220),
sources or
antibiotics
E-medium
were
used
are
listed in table 1. LB
(Luria
(272) and E2-medium (151), supplemented with carbon
throughout.
MT trace elements
(151)
were
added to
58
minimal media. All cultures
were
grown
alkanes, Petri dishes with E2 medium
through
octane
the vapor
]A
in the
were
a
at 30°C
sealed
by placing
(C14) and «-hexadecane (C16) by placing
flasks with E2 medium and 1 %
Rhamnolipids (97)
were
added to
a
growth
an
open
(v/v)
on n-
supplied
erlenmeyer with
n-
n-
Whatman 3MM filter disc with
were
«-alkanes
concentration of 0.1 %
a
37°C. For
container, for «-dodecane (C12),
«-alkane in the lid of the Petri dish. Recombinants
Erlenmeyer
or
incubated at 30°C with «-alkanes
of «-octane
case
(C8) and n-decane (CIO) in
tetradecane
200
phase,
aerobically
as
cultured in baffled
carbon
(w/v)
source.
to solubilize
long-chain
«-alkanes.
Table 1: list of strains and
plasmids
Strain
P.
P.
used in this
Relevant
aeruginosa PAOl
putida (oleovorans)
study
Reference
phenotype
alk
(118)
GPol cured of the OCT
GPo 12
plasmid
(141)
CHAO
alk
(250)
KOB2A1
alkB knockout mutant of CHAO
This
Acinetobacter sp. ADPl
alk
(126)
Alcanivorax borkumensis API
alk
(284)
Prauserella rugosa NRRL B-2295
alk
(119)
E. coli JM101
endA, hsdR, supR, thi-1, A(lac-proAB)
E. coli DHIOB
P'(traD36,proAB, lacf, lacZM15)
cloning strain
E. coli SF800
W3110
P.
P.
fluorescens
fluorescens
(285)
Gibco BRL
Laboratory
polA
thi,fadR
E. coli GEcl37
study
(67)
Plasmid
Characteristics
pGEM7-Zf(+)
Cloning
vector,
Ap
Promega
pUC18Sfi
Cloning
vector,
Ap
(110)
pZero2.1
Cloning
vector, Km
pKKPalk
Expression
vector
using PalkB, Ap
pCom5
Expression
vector
using PalkB,
Gm
pCom7
Expression
vector
using PalkB,
Gm
pCom8
pCK217
pEX18Tc
pUCP24
Expression
using PalkB,
pUC18Sfi containing res-npt-res
gene replacement vector
pJMSB8
Ap
pUCP24ParA
pCHAO
pPFl
Reference
vector
Invitrogen
Gm
(240)
(240)
,
,
oriT
(240)
oriT, alkS
(240)
(145)
(115)
E. coli-Pseudomonas shuttle vector
,
(277)
(145)
PaI/04/03'-'-ParA mpJMSA8
EcoRl-Hindlll
in
fragment
pUCP24
fragment
4.5 kb Xhol-BamHl fragment of P. fluorescens
CHAO in pGEM7-Zf(+), alkB
Gm
,
parA
as
CHAO PCR
This
study
(239)
This
study
collection
59
pPFlAS/'/I-S'tal, res-npt-res in alkB
pPFlrnrB Pvul fragment in pEX18Tc
Plasmid containing alkG (API)
5.0 kb Ncol-EcoRV fragment of A. borkumensis
API in pZero2.1, alkS, alkBl
7.5 kb Bglll fragment of A. borkumensis API in
pZero2.1,a/fcGff/
4.6 kb Nhel fragment of A. borkumensis API in
pZero2.1, 'alkS
pPFlrnrB
pEXPFlrnrB2
pAFl
pAPUl
pAPlSl
pAPlTl
Tc
pPrul
3.0 kb Sacl-BamHl
fragment
fragment of P. rugosa NRRL
B-2295 in pGEM7-Zf(+), alkB
alkBFGHJKLIalkST (GPol) in pLAFRl
pGEc47, deletion in alkB
alkB gene (CHAO) with own promoter in pCom5
alkBl gene (PAOl) in pCom7
alkB2 gene (PAOl) in pCom7
alkB gene (GPol) in pCom7
alkBl gene (API) inpCom7
alkM gene (ADPl) in pCom7
alkBl gene (PAOl) in pCom8
alkB2 gene (PAOl) in pCom8
alkB gene (GPol) in pCom8
alkM gene (ADPl) in pCom8
alkB gene (H37Rv) in pCom8
alkB gene (2295) in pCom8
pPF21
pCom7Bl (PAOl)
pCom7B2 (PAOl)
pCom7B (GPol)
pCom7alkBl (API)
pCom7M (ADPl)
pCom8Bl (PAOl)
pCom8B2 (PAOl)
pCom8B (GPol)
pCom8M (ADPl)
pCom8MT (H37Rv)
pCom8B (2295)
E. coli strains and P.
putida (oleovorans) GPol2[pGEc47B]
electroporation according
transformed
according
to
H0jberg (117).
(API) and pCom7M (ADPl)
GPol2[pGEc47AB] by
three
coli CC118[RK600]
the
containing
grown with
as
the
appropriate
was
10
used at 100
manipulations.
fragment),
helper
T4 DNA
strain
antibiotics
tetracycline (12.5 piglml)
DNA
parental matings
and
For P.
12.5
fluorescens
Restriction enzymes, T4 DNA
and
study
This
study
van
Beilen
(69)
(261)
This
This
This
This
This
This
This
This
This
This
This
This
study
study
study
study
study
study
study
study
study
study
study
study
transformed
by
were
the donor and E.
selected
on
pillml; ampicillin (Ap)
were
ligase,
E-
harbouring plasmids
KOB2A1
dideoxynucleotides
as
were
putida (oleovorans)
gentamycin (100 piglml)
polymerase
This
J. B.
with E. coli DHIOB
(tetracycline (Tc)
piglml.
study
putida (oleovorans)
(61). Transconjugants
Vox P.
This
(239)
were
antibiotics. E. coli strains
piglml).
van
pCom7B2 (PAOl), pCom7alkBl
transferred to P.
appropriate
piglml; gentamycin (Gm)
gentamycin
were
Plasmids
Beilen
J. B.
CHAO and KOB2A1
(64). P. fluorescens
to Dower
study
study
(209)
P. rugosa NRRL B-2295 PCR
pGEc47
pGEc47AB
medium
This
alkB, rubA, rubB (M. tuberculosis H37Rv)
pSCYBll
p2295
,
This
were
100
recombinants,
GPol2
recombinants,
used.
DNA
were
polymerase (Klenow
from Roche
Diagnostics
60
and used
(Rotkreuz, Switzerland)
isolated with the Roche
was
to Sambrook et
al.
(220).
High Fidelity Polymerase
kit
on a
Perkin Elmer
used
(4' 95°C,
following
below
PCR program
was
Tm), 1' 72°C), 5' 72°C,
cut with the
respective
4°C).
oo
enzymes,
PCR reactions
possible
4000L sequencer
using
labelled -40 forward
primers
for
PCR artefacts
clones
and
(gccagctcgtgtttttccagcagacg)
or
PCR
1'
were
System
were
isolated
Expand
annealing temperature (5°C
1% agarose
over
gel,
All PCR
reverse
1% agarose
gels,
and cloned into
fragments
were
on a
Li-Cor
kit and IRD800-
(gagcggataacaatttcacacagg)
(tggcgcaagcgtccgattag),
pKKRev (gagttcggcatggggtcaggtg)
PalkFw3
for
pKKPalk-derived
plasmids (MWG-Biotech, Ebersberg, Germany).
Nucleotide and amino acid sequences
Navigator
from DNASTAR
sequences
were
BLAST
Cloning
(5).
compared
were
To clone
fragments
were
fragments
as
fragments
of the desired size from
between the
(Invitrogen, Leek,
Transformants
same
complete
probes.
by
alkane
homologs
hydroxylase
Southern
The
sites of
a
and construction of
using
genes,
easy-to-clone
were
constructed
preparative gel.
expression
restriction
cloned DNA
by isolating
The DNA
restriction
fragments
were
pGEM7-Zf(+) (Promega, Madison, USA)
Netherlands)
containing
LASERGENE
Nucleotide and amino acid
blotting (220) using previously
Enriched gene banks
appropriate
compared using
carried out at NCBI.
gene
plasmids.
probes.
and
with the EMBL, SwissProt and GenBank databases
hydroxylase
identified
analyzed
(Madison, Wisconsin, USA).
BLAST searches
of alkane
were
and transformed into E. coli DH10B
the target genes
Both strands of the inserts
were
were
identified
sequenced.
to
9600. The
both strands of the inserts
and -40
according
the Roche
Thermosequenase cycle sequencing
PalkFwd
or
was
using
purified
pKKPalk (240).
(agggttttcccagtcacgacgtt)
pGEM7-ZF(+)
over a
by sequencing
the Amersham
carried out
(45" 95°C,
products
purified again
or
were
GeneAmp
25x
PCR
pGEM7-Zf(+) (Promega, Madison, USA)
checked for
Pure Plasmid Isolation Kit
High
for Pseudomonas recombinants. Chromosomal DNA
Birnboim-Doly (21)
according
supplier. Oligonucleotides
Switzerland.
synthesized by Microsynth, Balgach,
Plasmid DNA
the
specified by
as
or
ligated
pZero2.1
(Gibco BRL).
by colony blotting using
the
61
The alkBl and alkB2 genes of P.
aeruginosa
chromosomal DNA
combinations
using primer
and
AlkBpaRv2 (ctgcccgaagcttgagctat)
PAOl
amplified by
AlkBpaBfw (ggagaattctcagacaatct)
and alkMrPS
alkM of Acinetobacter sp. ADPl
amplify
were
PCR from
AlkBpaFwd (aactggaattcacgatgtttga)
AlkBpaBrv (gaggcgaatctagaaaaaactg) respectively.
(ccggaattcactatgaatgcacctgta)
(249)
To
and
Primers alkMfE2
(aataggcctgcagtcacttagactctctt)
(213).
and
amplify
used to
were
alkBl of A. borkumensis
API, primers alkBlfw (caaggtgatccatatgtcagagaac) and alkBlrv
(gcggatcctcaaagtgtgaaagc)
2295fwEco
were
were
used. For alkB of P. rugosa NRRL B-2295
(ggagaattcagatgagcgacgacgcac)
and 2295rvBam
used. The M. tuberculosis H37Rv alkB gene
was
primers
(cggcgaggatccggtccagctc)
amplified
from cosmid
pSCYBll
(209) using primers MTalkBfw2 (cggaattcatatgaccacgcaaatcggc) and MTalkBrv3
The P.
(cagaccgggatccggtaggcgg).
from
pGEc47 (69) using primers
(tttgtgaaagctttcaacgcc).
All PCR
putida (oleovorans)
B5-Eco
pCom7
EcoRl and Xbal sites of
pUC18Sfi,
or
alkB gene of P. fluorescens CHAO
including
sites of
133
bp upstream
digested
with
with T4 DNA
(Klenow)
a
res
pCom8 (240);
alkB2
and recloned in
was
cloned
with the
(PAOl)
as a
respective
was
pCom8 using
directly
and B3-Hind
restriction
cloned first in the
EcoRl and Hindlll. The
1.6 kb Munl-Sall
P. fluorescens CHAO alkB knockout mutant. Plasmid
and Stul to
Sfil
remove a
fragment,
the
fragment
2.1 kb res-npt-res cassette, cut from
digestion
of the
Km selection. A
Southern blot
was
in Smal
and PCR to have
(data
not
was
shown).
to
a
blunt-ended
with Hindlll and Ecll36ll.
with Pvul and treatment with
digested pEX18Tc (115).
Km-resistant, Tc-sensitive colony
alkB gene
ligated
introduced into P. fluorescens CHAO
hybridisation
cassette in the
ligated
was
pCK217 (145)
resulting plasmid (pPFlrnrB2)
was
pPFl
0.5 kb internal segment of alkB and blunt-ended
polymerase. Subsequently,
plasmid, pEXPFlrnrB2,
by
digested
amplified
of the alkB gene, and inserted in the EcoRl-Sall restriction
Klenow, the large fragment
using
were
was
pCom5 (240).
Construction of
After
(ggagaattccaaatgcttgag)
fragments
enzymes and inserted in
GPol alkB gene
a
was
The final
by electroporation
obtained, which
was
shown
chromosomal insertion of the res-npt-
This mutant
was
named P. fluorescens
62
KOB2. To
remove
parA gene
was
pJMSB8 (145)
isolated
over
kanamycin
supplied
on
resistance gene with its
The latter
pUCPParA.
with EcoRl and Hindlll. The
gel,
resistant colonies
Km. One
the
and
ligated
were
in
plasmid
transcriptional terminator,
was
fragment containing
pUCP24 digested
with the
same
tested for absence of the Km-cassette
cured of
positive colony, subsequently
fluorescens
KOB2A1.
Sequences.
Unfinished genome searches
constructed
were
pUCPParA,
done at NCBI
study
available at EMBL under the
are
following
sensitivity
designated
to
P.
(http://
Nucleotide sequences
www.ncbi.nlm.nih.gov/Microb_blast/unfinishedgenome.html ).
used in this
was
Gentamycin
enzymes.
was
by digesting
the parA gene
PCR and
by
the
accession numbers:
Acinetobacter sp. ADPl: alkRM: AJ002316; Alcanivorax borkumensis API: AJ295164;
Mycobacterium
tuberculosis H37Rv: alkB
(Rv3252c): Z46863; Prauserella
NRRL B-2295:
AJ009587; Pseudomonas aeruginosa PAOl: alkBl (PA2574):
rugosa
AE004685; alkB2 (PA 1525): AE004581 ; Pseudomonas fluorescens CHAO: AJ009579;
Pseudomonas
putida (oleovorans)
Pseudomonas
putida
PI:
GPol:
alkBFGHJKL, alkN, alkST: AJ245436;
alkBFGHJKL, alkST: AJ233397
RESULTS
Selection of strains. For this
study,
strains from different environments
fields
organisms,
putida
strains GPol and PI, which
the alkane
hydroxylase
260). Alcanivorax borkumensis
almost
exclusively
on
habitats
API is
«-alkanes, but is of interest
CHAO
as a
marine
a
same
and forms
(101). P. fluorescens
number of
In the
are
case
a
and different research
common
soil and water
sequenced
y-Proteobacterium (284),
bacterium
major part
was
alkane-oxidizing
of Acinetobacter sp.
isolates of
genes have been cloned and
«-alkanes. The
geographic locations,
a
(soil, seawater, cow-rumen),
(biodégradation, biocatalysis, pathogenicity).
ADPl and P.
many
have selected
we
originally
was
isolated from
of the biomass in
(213,
which grows
sea
water at
oil-polluted
not studied for the
biocontrol strain which excretes
before
ability
secondary
marine
to grow
on
metabolites
63
toxic to soilborne
is of clinical
importance
(27), but is also
found in the
plant pathogens (99).
in
common
selected because it is
propionic
Cloning
P.
acid
aeruginosa
alkB2)
cloned
one
as
water and
a
alkane
useful
a
are
biocatalyst that
were
Of these genes,
with
a
other alkane
among the
example
was
of the
isolated from
cumene
analysis
(249) encodes
of the
two alkane
These
putative
alkane
Mycobacterium
included in the
hydroxylase
fluorescens
CHAO
Southern and
an
alkane
encodes two
which
were
cloned
amplify
2-phenyl-l-
and
(51)
hydroxylase
genes
were
hydroxylase homologs
not further
were
The
hydroxylase (alkBl
investigated
and Burkholderia
each. These
but not in the functional studies.
two rubredoxin genes and
proteins
a
are
likely
to
regulatory
encoded downstream of
using PCR-fragments
and rubredoxin
a
550
bp
CHAO alkB
blots. Downstream of the alkane
PraB) with homology
aeruginosa
OmpPl proteins
(239)(J.
B.
are
van
previously
homologous
Beilen,
segment containing the alkane hydroxylase of P.
using
and
obtained
internal segments of genes that
hydroxylase
cloned
proteins (PraA
to the
by
that is similar to other
genes
for alkane oxidation" of P.
homologous
rumen, and
cow
flanking regions.
hydroxylase homolog
phylogenetic analysis,
A 7.8 kb DNA
was
colony
to
slow-growing
genes.
GPol alkane
unpublished results).
pseudomonads
the H37Rv alkB gene is located close to other genes that
TetR-signature
putida
strains
Legionella pneumophila Philadelphia-1
degenerate primers
which
identical to the M. tuberculosis H37Rv alkB. The unfinished
hydroxylase
Other alkane
species
while the genome sequence of M. tuberculosis H37Rv
(nearly)
only
a
In contrast, M. tuberculosis is not
converts
genes, and
hydroxylase
be involved in alkane oxidation: it is followed
to the P.
represents
«-alkanes.
on
K96243 also contain
pseudomallei
with
plants.
and notorious
hydroxylase homolog.
genome sequences of
protein
PAOl
described in the Materials and Methods section. Alkane
they
sequences
on
P. rugosa NRRL B-2295
in the genome sequences of other
because
typical
PAOl genome sequence
homologs,
gene
encodes
soil,
(119) if it is grown
of novel alkane
aeruginosa
primary opportunistic pathogen
environment, but is
pathogenic Mycobacteria.
was
the
as
P.
of
PAOl
fragment (239)
hydroxylase,
an
"protein
outer membrane
Haemophilus influenzae.
probe
in
the cloned DNA
to the so-called
(103), and
as a
activator
protein
64
Figure 1: organization of a/fc-genes in different organisms. The scale is in basepairs.
putida (oleovorans) GPol and P. putida PI. alkB: alkane hydroxylase, alkF: rubredoxin 1, alkG:
A: P.
rubredoxin 2, alkH:
aldehyde dehydrogenase, alkf: alcohol dehydrogenase, alkK: acyl-CoA synthetase,
protein, alkS: regulatory protein, alkT: rubredoxin reductase.
B: A. borkumensis API. alkS: putative regulatory protein, alkBl: alkane hydroxylase, alkG: rubredoxin,
alkH: putative aldehyde dehydrogenase, alkf: putative alcohol dehydrogenase.
C: P. fluorescens CHAO. 'estFl: esterase, alkB: alkane hydroxylase, praA, praB: "protein activators of
alkane oxidation", ompPl: outer membrane protein, yaflT: acyl-CoA dehydrogenase.
D: P. aeruginosa PAOl. PA2575: hypothetical protein, alkBl: alkane hydroxylase 1, tlpS: methylaccepting Chemotaxis protein, hupRl: regulatory protein of hydrogen uptake, PA 1526: regulatory protein of
GntR family, alkBl: alkane hydroxylase 2, xdhA: xanthine dehydrogenase homolog, glcEFG: genes
homologous to the glcEFG genes of E. coli; rubAl: rubredoxin 1, rubAl: rubredoxin 2, rubB: rubredoxin
reductase, PA5347, PA5346: hypothetical proteins.
E: P. rugosa NRRL B-2295. alkB: alkane hydroxylase, tetR: putative regulatory protein of TetR family,
reg: putative regulatory protein.
F: M. tuberculosis H37Rv. MTCY20B 11.28c: cationic transporter, alkB: alkane hydroxylase, rubA:
rubredoxin 1 ; rubB: rubredoxin 2, tetR: putative regulatory protein of TetR family.
alkL: outer membrane
In the
case
of A. borkumensis API,
fragment encoding
cloning
and
assembled.
strain is
an
Sequence analysis
the rubredoxin gene alkF
(258),
immediately upstream
Instead,
by
a
of this
(142)(J.
fragments,
van
are
not
sequence of 11.2 kb
encoding
an
organization
was
in this
(figure 1)(260). However,
as
well
outer membrane
of alkBl. It is transcribed in the
opposite direction,
as
the
protein
acylof
and is not
alkT, unlike in P. putida strains GPol and PI.
terminator
(a strong inverted repeat) is located directly
alkS, while further downstream DNA has homology with
a
Vibrio
antiporter.
chromosomal DNA
van
contiguous
Beilen, unpublished results)
The Prauserella rugosa NRRL B-2295 alkB gene
fragment using
probe. Sequence analysis
hydroxylase
a
Beilen, unpublished results). By
present in API. More surprisingly, alkS is located
possible transcriptional
cholerae Na7H+
as a
van
3.5 kb £coRI-.BamHI
a
strains GPol and PI
the rubredoxin reductase gene
downstream of
cloned
showed that the alk gene
region
putida
B.
B.
(J.
gene alkK and the alkL gene,
unknown function
followed
DNA
similar to that of P.
CoA-synthetase
previously
AlkG-like rubredoxin
sequencing adjacent
quite
we
with
a
the 559
of the 3 kb
bp
was
cloned
as a
EcoRl-Sacl alkB
fragment
C-terminal extension that is
3.0 kb Sacl-BamHl
fragment
of
showed that it encodes
highly homologous
p2295 (239)
an
alkane
to rubredoxins
(J.
B.
Beilen, unpublished results). The region immediately downstream of the alkB gene
encodes
a
possible regulatory protein
with
a
TetR
signature
at its N-terminus
F
E
B
-
alkB
500
I
t
1000
I
2000
I
alkG
glcF
alkB
alkS
alkB
alkBl
alkS
alkF
|i
alkBl
alkT
rubAl
xdhA
praB
rubA
| rubB tetR
>
rubB
alkH
PA5347 PA5346
hupRl
ompPl
alkG
*
5500
I
Pi
5000
I
alkJ
4500
_J_
y//////?t
*
4000
I
>^H
rubAl
glcG
glcG
3500
I
alkH
3000
I
praA
2500
I
ÎSSSSÏ8SSSSS
1500
I
MTCY20B11.28c
glcE
PA1526
'estFl
k
I
.
I
alkJ
'
f-
'
)
l
9000
aeruginosa
PAO 1
GPol
M. tuberculosis H37Rv
P. rugosa NRRLB-2295
P.
P. fluorescens CHAO
A. borkumensis API
P.putida (oleovorans)
P. putida VI
l
l
•-
8500
8000
alkL
7500
~y-\
7000
I
yafil'
alkK
6500
I
r
6000
I
0\
66
to the
(pfam00440), quite homologous
TetR-protein
tuberculosis H37Rv alkB-rubAB genes
(26%
encoded downstream of the M.
sequence
full-length
sequence
identity)
(figure 1).
Comparison
alkane
of alkane
hydroxylases (figure 2),
hydroxylases
sequence
was
identity
generated
monooxygenase
sequence
peptide
was
alignment
of the full
length
new
alkane
from 37 % to 99 %. The closest
hydroxylases ranged
that does not oxidize
hydroxylases,
and the P.
of known and
(111) and manually optimized. The peptide
also included in the
to the alkane
pneumophila Philadelphia-1
phylogenetic analysis
a
sequence
with Clustal
hydroxylases
(XylM),
identity
a
among the alkane
relative of the alkane
For
hydroxylases.
«-alkanes, xylene
analysis.
and is most
aeruginosa
It has 20 % to 26 %
closely
peptide
related to the L.
PAOl enzymes.
P. rugosa NRRLB-2295
A
M. tuberculosis H37Rv
B.
pseudomallei
K96243
P. fluorescens CHAO
Acinetobacter sp. ADPl
'— A. calcoaceticus EB104
ï—
putida PI
aureofaciens RWTH529
P. putida (oleovorans) GPol
J
P.
l_P
P.
1
*-
'
A. borkumensis API
pneumophila Philadelphia-1
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L.
P.
ï
P.
167.2_
T
160
Figure
2:
P.
putida
mt-2
XylM
T
T
T
T
T
T
T
140
120
100
80
60
40
20
1
0
phylogenetic tree of published and hypothetical alkane hydroxylases, generated by
alignment of the (putative) alkane hydroxylases. XylM is included as outgroup.
Clustal from
the manual
The
alignment
(oleovorans)
shows that the six membrane
GPol alkane
spanning segments
hydroxylase (figure 3)(261),
identified in the P.
putida
three histidine motifs conserved
67
among the
hydrocarbon
NYXEHYG(L/M)
oxygenases and desaturases
motif identified in the alkane
(233), and the additional
hydroxylases (239)
are
well conserved in
all sequences. Most insertions and deletions of each sequence, relative to the other AlkBsequences,
are
located in
transmembrane helix.
alkane
predicted cytoplasmic
Only
periplasmic
are
located between the conserved histidine motifs. A 40
amino acid insertion in the P. fluorescens CHAO AlkB is
periplasmic
domain between the third and fourth
terminal end of the alkane
domains before the third
in the M. tuberculosis H37Rv and P. rugosa NRRL B-2295
insertions
hydroxylases
or
hydroxylases
2
probably
putative
also varies
located in the
transmembrane helices. The C-
strongly
in
length
and
composition.
3
^AJ^V
COOH
NH^
Figure
3:
topology
model of the P.
putida (oleovorans)
GPol alkane
localization of the histidine motifs. Numbers indicate the membrane
phoA
fusion
Alkane
protein
studies
(273))
are
P.
putida PI,
clustered in the
hydroxylases
from
Interestingly,
alkane
divergent as
quite closely
alkane
hydroxylase
tree
(figure 2),
degrading
strains
aureofaciens
while the
are
(P. putida
putative
RWTH529
alkane
highly divergent.
genes cloned from fluorescent
pseudomonads
are as
collection, while the A. borkumensis API alkane hydroxylase is
related to the P.
analysis
expression
A. borkumensis API and P.
phylogenetic
long-chain
the entire
In vivo functional
functional
(261)).
from strains that oxidize medium-chain alkanes
hydroxylases
(oleovorans) GPol,
hydroxylase. Grey dots indicate the
spanning segments (based on lacZ and
putida
alkane
of alkane
of alkane
hydroxylases.
hydroxylases.
hydroxylases
that the second component of the P.
were
Three different systems for the
developed,
putida (oleovorans)
based
on
GPol alkane
the observation
hydroxylase
68
system, the electron transfer protein rubredoxin (AlkG),
from other alkane
hydroxylase systems (J.
broad-host range cosmid
of the alkane
pGEc47AB,
gene alkB
hydroxylase
rubredoxin and rubredoxin
a/adi?-mutant
chain-length fatty
acids and «-alkanes of the
as
in Pseudomonas
GPol2 is
cells,
activity (<
0.01 U g
the alkane
hydroxylase
or
gene of P.
length,
GPol
used in this
GPol could
strains, based
on
on
growth
study.
medium
if the strain contains
activity)(261).
putida (oleovorans)
not
Alkane
same
less than 0.06% of full
activity
Table 2: functional
were
pGEc47.
Both
OCT-plasmid.
did not show any detectable alkane
of both recombinant
(data
Two hosts
species.
derivative of GPol, cured of the
a
hydroxylase activity
assays
that lacks part
pGEc47 (69)
of E. coli DH-1, which allows it to grow
containing pGEc47AB
'
Beilen, unpublished results). The
reductase, and other proteins involved in the degradation of
E. coli GEcl37 is
host strains
rubredoxins
replaced by
(261), expresses the P. putida (oleovorans)
as
putida (oleovorans)
van
deletion derivative of
alkanes, in E. coli
P.
well
a
B.
be
can
hydroxylase
Plasmids
fully
rates
encoding
restore alkane
«-octane and
on
shown).
analysis
of alkane
hydroxylases.
hydroxylase
System
for functional
E. coli GEcl37
P.
[pGEc47AB]
(oleovorans)
analysis
'
P. fluorescens
putida
KOB2A1
GPol2[pGEc47AB]
P.
putida (oleovorans)
GPol alkB
++
(C8- C12)
++
PI alkB
++
(C8)
n.t.
A. borkumensis API alkBl
++
(C8-C12)
++
P.
putida
Acinetobacter sp. ADPl alkM
P.
aeruginosa
PAOl alkBl
P.
aeruginosa
PAOl alkBl
P.
fluorescens
CHAO alkB
-
Footnote:
-
: no
between brackets
All alkB gene
(oleovorans)
(240), and
a
growth;
were
+ :
poor
homologs
GPol alkane
gratuitous
(C8
-
C12)
n.t.
-
(C12
-
C20)
(C12-C16)
++
(C12
-
C20)
-
+
(C12-C16)
++
(C12
-
C20)
n.t.
++
(C12
-
C14)
n.t.
++
(C12)
:
good growth;
expressed
from
hydroxylase, using
inducer of
(C10-C16)
+
growth; ++
positive.
were
+
n.t.
-
n.t.
tested and
C12)
n.t.
-
P. rugosa NRRL B-2295 alkB
-
-
-
M. tuberculosis H37Rv alkB
(C8
++
n.t.: not tested. The substrates indicated
PalkB, the promoter of the P. putida
the vectors
pKKPalk, pCom7
and
pCom8
PalkB, dicyclopropylketone (DCPK)(96). The medium-
69
chain
length
alkane
hydroxylases
the alkB deletion in E. coli
GPol2[pGEc47AB]
recombinants
for
on
alkane
putida (oleovorans)
these
on
alkanes,
even
fluorescens KOB2A1,
as
PI and A. borkumensis API
C12-C16
and P.
«-alkanes, did
containing
if
added
an
were
similar E. coli
genes cloned from strains able to grow
GPo 12 recombinants
rhamnolipids
complement
putida (oleovorans)
(table 2). However,
«-octane
hydroxylase
long-chain «-alkanes, including
P.
putida
GEcl37[pGEc47AB]
growth
containing
of P.
not grow
the
same
on
C12-C16 «-alkanes.
genes
(table 3). Therefore,
alkB deletion mutant of P. fluorescens CHAO,
only
a
grew
third
was
«-alkanes, unlike the wild-type, but could be complemented for growth
alkanes
the
by
the native
long-chain
constructed
or
hydroxylases,
P.
putida
P. fluorescens KOB2A1
strains: the P.
of P. fluorescens KOB2A1 recombinants
albeit often with
CHAO
(table 2,
growth
table
rates
doubling
Strain
CHAO
GPol2[pGEc47AB]
times of
on
at least
below that of the
one
a
«-
For
much better
all allowed
of the «-alkanes
wild-type
«-octane, but showed weak
wild-type
tested,
strain P. fluorescens
-
-
alkBl
(PAOl)
Doubling
alkBl
on
on
times
CIO and C12.
«-alkanes
(td) (hours)
C14
C16
19
b
20
31
10
33
63
>200
19
28
78
>200
(PAOl)
176
37
44
(PAOl)
21
22
15
(CHAO)
alkBl
growth
and alkB recombinant strains
Additional gene
alkB
KOB2A1
these
P. fluorescens CHAO, M.
hydroxylases
C12a
PAOl
C10-
3). The KOB2A1 recombinant containing the P. putida (oleovorans)
GPol alkB gene did not grow
Table 3:
clearly
on
to be
proved
aeruginosa PAOl,
tuberculosis H37Rv and P. rugosa NRRL B-2295 alkane
growth
on
on
(CHAO) alkane hydroxylase gene, cloned in pCom5 (table 3).
alkane
host than E. coli
poorly
host, P.
described in the Material and Methods section. KOB2A1 is not able to grow
C16
on
alkB
(CHAO)
42
24
13
alkB
(2295)
72
n.g.
n.g.
alkB
(H37Rv)
80
Footnotes:a C12: dodecane; C14: tetradecane; C16: hexadecane.
176
b
-:
not
tested; n.g.:
n.g.
no
growth
70
However, short-chain alkane hydroxylases did
on
Possibly,
«-octane.
not cloned
yet,
are
not allow KOB2A1 recombinants to grow
the CHAO rubredoxin and rubredoxin
reductase, which
not induced under these conditions. The related P.
we
fluorescens
Pf0-1, the genome of which has been sequenced, possesses such genes, but does
encode
an
reductase
alkane
are
hydroxylase homolog.
related to
closely
hydroxylation (J.
B.
van
proteins
have
strain
not
As the Pf0-1 rubredoxin and rubredoxin
that
were
shown to function in alkane
Beilen, unpublished results), this strain may have lost its alkane
hydroxylase.
DISCUSSION
Functional
analysis
of alkane
hydroxylases.
The alkane
GPol and Acinetobacter sp. ADPl have been cloned
(oleovorans)
cloning
methods. In both cases, cosmids that restored
alkanes
were
selected from
development
of
Firstly,
strains
some
overlapping
a
using
were
cosmid
library (69, 213).
found to contain
more
Two main
long-chain
than
one
medium-chain
length
the conversion of alkenes to
possibly
because
strongly
limited
developed
substrate
by
based
hydroxylases
epoxides
mass
on
are
or
poorly
transfer.
enzyme
reasons
hydroxylase
can
be
assayed
alkane
soluble in water, and
and
not
was
use reverse
with
in vitro,
(259),
possible
such
hydroxylases,
activity
genetics
genes.
difficult.
the co-oxidation of NADH
long-chain
on
necessitated the
hydroxylase
genetic approach
Therefore, it
activity,
alkane
putida
traditional
using
alkane
hydroxylases
for membrane-bound
substrates
long-chain
alkane
genes of P.
of chemical mutants
growth
substrate ranges, which made the classical
methods could not be
alkane
a
PCR method to clone additional
Secondly, although
e.g.
hydroxylase
to
is
purify
the
to clone the
genes.
Here,
we
used PCR
products
obtained with
and information from genome
hydroxylases
genes
were
from
a
quite
cloned based
highly degenerate primers
sequencing projects
to clone
a
diverse collection of strains. This
on
sequence
similarity,
not
on
described
earlier,
number of alkane
implies
that all of these
function. Knockout mutants of
71
the alkB gene
homologs
could in
encode functional alkane
for molecular
principle
genetic studies,
exclusively
as
pneumophila
«-alkanes
on
tools
several strains contain
or
methods
recently
not
are
(yet)
pathogens,
multiple (at
least up to
sum
Heterologous expression
only
or
in
a
few
cases
of all
and
available. This is the
sea
(induced)
unequivocally
can
be
hydroxylase
hydroxylases
is
generated,
van
may not
only give
complicated by
hydroxylase systems
putida (oleovorans)
J. B.
activities.
from genome sequence data.
with the P.
addition,
five) alkB homologs (chapter 2;
alkane
of the novel alkane
complementation experiments
case
water, and grows
difficult to cultivate. In
are
all three components of the alkane
could be identified
accessible
easily
while in vivo substrate range studies would
phenotypical changes,
information about the
not
(284). Other strains, like M. tuberculosis and L.
Beilen, unpublished results). Here, knockout mutants, if they
show
are
been isolated from
human
important
are
many strains
hydroxylases. However,
for A. borkumensis API, which has
almost
be used to prove that these genes indeed
the fact that
were
Fortunately,
GPol alkane
cloned,
earlier
hydroxylase
system have shown that rubredoxins from various Gram-positive and Gram-negative
alkane
degraders
oxidation
(J.
B.
can
van
replace
Based
these
on
equivalent proteins
considerations,
hydroxylases.
«-alkanes. Here,
growth
or
electron donors for novel alkane
novel alkane
on
putida (oleovorans)
of
a
we
we
have
E. coli
a
developed
on
and P.
any
growth
from GPol2
on
also able to grow
factors
(porins
or
so
an
quite slowly.
similar
expression
in vitro
assay)
activity
is
of
growth
needed for
proteins)
of the recombinants
on
«-alkanes
putida (oleovorans) GPol2[pGEc47AB]
hydroxylase
added, but do
several hosts for the
hydroxylase activity.
hosts to test medium-chain alkane
are
related to the GPol enzyme.
the absence of
(in
should be able to
«-octane is around 10-20 % of the full GPol alkane
level of alkane
GEcl37[pGEc47AB]
are
sources
have observed that the minimum level of
recombinant
significant
from other
hydroxylases
The best assay
hydroxylase activity (257). Therefore,
indicates
GPol rubredoxin in alkane
Beilen, unpublished results). This implies that the GPol rubredoxin
and rubredoxin reductase
serve as
the P.
alkanes
It is
that
likely
are
genes
ranging
that
not
(table 2).
of
suitable
Recombinants derived
from C12 to C16 if
uptake
are
longer
rhamnolipids
«-alkanes
present in GPol2. For this
requires
reason,
we
72
constructed
non-polar
a
alkB knockout mutant of P. fluorescens CHAO, in which the
alkane solubilization and
allow
quite
complementation
diverse
makes it
type
integral
and
expression
possible
However,
growth
on
alkanes
longer
to compare sequences to
strains
gain insight
of this class of enzymes. Alkane
our
present knowledge
hydroxylases
that
are
but the membrane
divergence,
on
membrane
(261)
histidine boxes
hydroxylases,
are
highly
binding
analyzed
folding
and
are
likely
are
likely
exchanged
are
This
putida (oleovorans)
(231).
study
of these alkane
are
the four medium
show
It is not
be
chain-length
likely
by
likely
appears to follow the
to span the
same
(231).
sequence
cytoplasmic
is true for the four
nitrogen-rich ligand sphere
yet possible
distinguished
residues conserved
and the
essential for enzymes related to the alkane
to
identify
are
hydroxylase systems
alkanes
of this class of
significant
hydroxylases
to be conserved between all alkane
easily
relationship
of the two active-site iron atoms
in this
to contribute to the
between alkane
these residues cannot
distinguish
hydroxylases.
folding topology (261)
aspects of alkane hydroxylase function. Residues that
to
to demonstrate that
us
of the P.
conserved in all sequences. The
(233, 239), which
di-iron center
rubredoxin
3).
in the structure-function
the structure-function
pattern: the six hydrophobic stretches that
be
allow
table
cloned from
well-studied, mainly because of their potential applications in biocatalysis.
are
The alkane
can
hydroxylases
(table 2,
hydroxylases
membrane oxygenases is limited to the
catalytic
alkane
by
complementation systems
involvement of conserved histidines in
same
is left intact. This host does indeed
of the GPol alkB gene indeed encode functional alkane
relationship
GPol
for
organisms, including Gram-positive
In summary, the
homologs
(putative) uptake system
residues involved in other
involved in
binding
hydroxylases,
as
are
of
rubredoxins
from all strains tested. However,
from other conserved residues.
hydroxylases
of the
too
closely
Similarly,
related to each other
chance from residues conserved because of
functional constraints.
The
organization
different alkane
degradation
of genes involved in alkane oxidation varies
degrading
seem
bacteria
to be distributed
(figure 1).
over
In most
strongly
among the
strains, genes involved in alkane
the genome. None of the rubredoxin reductases is
73
located close to
and
pathways,
are
located
require
Interestingly,
the
because
regulation.
downstream of the alkane
encoded
alk-genes
directly upstream
of A. borkumensis
have
encoded
putative
on a
A. borkumensis
significantly
a
alk-genes
well. However, in this
essential enzyme,
as
B.
organization
organisms.
Alkanes
to utilize these
why
are
highly
polluted
with oil
on
unrecognized
While the
compounds
earlier
an
PCR-studies
previous
putida (oleovorans)
homologs
constructed in this
are
in the
alkane-degrading
relic of
aeruginosa
as
functional, and allow
long-chain (C12-C16)
us
us
and
degrading
this
to demonstrate that
«-alkanes. These
pneumophila
as common
soil
or
source.
hydroxylase
are
available
J. B.
shows that
hydroxylases.
nearly
experiments
are
likely
explains
of M.
M. tuberculosis still has
strains possess
study
they
This
water
pristine soil, aquifers recently
lifestyle. Alternatively,
to show that
as
PAOl may reflect the
water. The alkane
hydroxylase,
as
almost
carbon-sources, such
carbon and energy
strains from
sea
an
environment, and microorganisms
indeed encode functional alkane
allowed
sequence. The P.
seawater.
pathogens
show that many alkane
study
of P.
horizontal gene transfer
(239), and later results (chapter 2;
GPol alkane
as
be considered
reservoir in the environment where alkanes
unpublished results)
of such
a
can
by
very few other
and P.
human
(217) and oil-polluted
tuberculosis may be
an
omnipresent
reduced
it is easy to isolate
as
alk-genes
well
as
in the genome sequences of L.
pseudomallei K96243,
organisms
are
(260). These comparisons suggest that the
may have ended up in this strain
this bacterium grows
double nature of these
genes. Those that
very similar to the
pyruvate, and is found mainly in oil-contaminated
Philadelphia-1,
In contrast, most rubredoxin genes
lower G+C content than the rest of the genome, and
catabolic transposon
hydroxylases
also involved in other
are
hydroxylase
strain, the alkane hydroxylase
The presence of alkane
they
of rubredoxin reductase genes.
are
strains GPol and PI, with respect to gene
putida alk-genes
are
are
hydroxylase, perhaps
different type of
a
immediately
located elsewhere
putida
alkane
an
all alkane
a
as
van
C-sources.
Beilen,
homologs
of the P.
random selection
The three host systems
hydroxylase homologs
oxidize medium-chain
also show that
(C5-C11)
long-chain
alkane
or
74
hydroxylases
show little
thereby provide
cloned alkane
us
with
activity
a
with medium-chain
powerful
hydroxylases,
and
length alkanes,
selection method to
identify
change
and vice versa, and
the substrate range of the
residues involved in substrate
binding.
ACKNOWLEDGEMENTS
The authors wish to thank Martina
of A.
for
Röthlisberger
for
sequencing,
P.
Golyshin
for his
gift
borkumensis, H. P. Schweizer for pUCP24 and pEX18Tc, J. M. Sanchez-Romero
pCK217
the Swiss
project
nr.
and
pJMSB8
and S. T. Cole for
Priority Program
5002-037023.
in
pSCYBll.
Biotechnology
This research
was
supported by
of the Swiss National Science
Foundation,
75
Chapter
5
FUNCTIONAL ANALYSIS OF RUBREDOXIN REDUCTASES
INVOLVED IN ALKANE OXIDATION
Theo H. M.
Smits, Stefanie B. Balada, Bernard Witholt and Jan B.
van
Beilen
76
SUMMARY
The rubredoxin reductase AlkT of Pseudomonas
component of the alkane hydroxylase system. Homologs of AlkT
alkane
degrading strains,
but also
show that rubredoxin reductase
occur
in other bacteria. In vivo
homologs
GPol is
putida (oleovorans)
are
an
essential
present in several
complementation
cloned from Pseudomonas
aeruginosa
assays
PAOl
and Acinetobacter sp. ADPl transfer electrons to the rubredoxin component of the GPol
alkane
hydroxylase system. However,
the flavorubredoxin reductase from E. coli K-12
and the ferredoxin reductase subunit of the chlorobenzene
sp.
P51, which
ADPl
closely
are as
reductases,
cannot
dioxygenase
related to the GPol rubredoxin reductase
replace
of Pseudomonas
as
the PAOl and
AlkT in vivo.
INTRODUCTION
The Pseudomonas
putida (oleovorans)
electrons from NADH to rubredoxin
GPol rubredoxin reductase
(68, 256), is
The other two components
hydroxylase system.
protein (AlkB) (143, 173, 261)
and rubredoxin
an
AlkT, which transfers
essential component of the alkane
are a
non-heme iron
(AlkG) (207),
a
integral
small red
membrane
protein
which
transfers electrons from AlkT to AlkB.The enzyme system oxidizes C5-C12 «-alkanes to
1-alkanols, thereby allowing the host
Spinach
ferredoxin and ferredoxin reductase
reductase of P.
(20, 207).
putida (oleovorans)
In in vivo
pGEc47AT,
rubredoxin
activity
octane
GPol
assays with
which contains all
as
an
alk-genes
on
can
these
compounds.
replace
(257).
degrading
strains
oxidation,
even
In
this
activity
in in vitro assays
growth
was
on
pGEc47,
alkanes except the
found in E. coli and P.
sufficient to allow
growth
of the recombinant
addition, rubredoxins from Gram-negative and Gram-positive alkane
can
replace
though they
unpublised results).
proteins
AlkT deletion derivative of
necessary for
was even
the rubredoxin and rubredoxin
electron transfer
reductase, considerable background activity
putida recombinants,
on
to grow
the P.
show
putida (oleovorans)
as
little
as
GPol rubredoxin in alkane
43 % sequence
identity (J.
This suggests that reductases cloned from other
B.
van
Beilen,
organisms might
also
77
be able to
we
replace
investigated
the rubredoxin reductase of P.
alkane
degrading
rubredoxin reductase gene
putida (oleovorans)
Therefore,
GPol.
strains and genome sequences for the presence of
and tested whether
homologs,
they
can
the GPol
replace
rubredoxin reductase.
Rubredoxin reductases involved in alkane oxidation.
Several rubredoxin reductases have
91, 260)(J. B.
putida
the
van
now
been cloned from alkane
are
of alkane
positive regulator
rubredoxin reductase gene is in
located
directly
downstream of the alkS gene
degradation (260).
an
In Acinetobacter sp.
downstream of
immediately
(rubAl and rubA2) genes (J.
B.
van
alkane
an
hydroxylase (alkBl)
encoding
ADPl, the
operon structure with rubredoxin genes
6). The rubredoxin reductase gene rubB of Rhodococcus erythropolis
(91)(chapter
NRRL
B-16531)
is
and two rubredoxin
Beilen, unpublished results).
We also searched relevant genome sequences for rubredoxin reductase
NCBI :
(68,
Beilen, unpublished results). The rubredoxin reductase genes of P.
strains GPol and PI
located
strains
degrading
homologs (at
http: //ww w.ncbi. nlm. nih. gov/MicrobjDlast/unfinishedgenome. html ) .The
rubredoxin reductase of Pseudomonas
of two rubredoxin genes
structure,
are
Two genes
the genome sequence of
homology (36.1
erythropolis
tuberculosis H37Rv
identity, respectively)
NRRL B-16531
These
(figure 1).
hydroxylase
Interestingly,
the genomes of several
its C-terminus
hydroxylase homologs,
oxygen stress
during
This
protein
anaerobic
that
operon
hydroxylases
and Rv 1869c) in
have
significant
are
not encoded in the
is
growth
are
not able to grow
on
proximity
n-alkanes,
do contain rubredoxin reductase
(24) encodes
of flavorubredoxin
(95, 275).
(51)
putative
to the rubredoxin reductase of R.
proteins
organisms
named flavorubredoxin reductase
stop-codon
a
downstream
and the rubredoxins.
The genome sequence of E. coli K-12
with the
directly
encoding hypothetical proteins (Rv0688
of the alkane
homolog,
of either of the two alkane
vicinity
Mycobacterium
and 35.8 %
do not possess alkane
PAOl is encoded
(rubAl and rubA2). These genes, organized in
not located in the
(249)(chapter 4, 6).
aeruginosa
a
(orf377).
homologs.
rubredoxin reductase gene
The start codon of
orf377 overlaps
(orf479),
which has
possibly
involved in the response of E. coli to
or as
or
a
rubredoxin-like domain at
regeneration system
for NADH
during
78
0.05
l—l
ORF377
RubB 1
RubB
_P
(P. syringae pv.
RubB
AlkT
AlkT
DC3000)
(P. putida KT2440)
(A. calcoaceticus S19)
RubB
*—
tomato
(P. fluorescens Pf0-1)
RubB2
r
\
(P. aeruginosa PAOl) (+)
RubB
I
(-)
(P. putida KT2440)
RubB
I
(E. coli K-12)
(Acinetobacter
sp.
ADPl)
(+)
(P. putida PI) (+)
(P. putida (oleovorans) GPol) (+)
-HcaD(£. coli K-12)
RubB
—
t
—
—
—
{
(M. tuberculosis H37Rv)
CumA4
(P. fluorescens IP01)
TcbAd
(Pseudomonas sp. strain P51)
TodA
(P. putida Fl)
BedA
(P. putida ML2)
TerA
CamA
(-)
(Pseudomonas sp.)
(P. putida
ATCC
17453)
FAD-containing reductases, generated with ClustalX from the
alignment
homologous proteins. Not all known peptide
of
ferredoxin
reductase
have
been
included in the phylogenetic tree. The results of the
homologs
sequences
with
coli
E.
complementation assays
GEcl37[pGEc47AT] is indicated by (-) and (+).
Names and descriptions of the proteins (accession numbers):
TerA: terpredoxin reductase (P450-TERP) of Pseudomonas sp. (P33009); CamA: putidaredoxin reductase
(P450-CAM) of P. putida ATTC 17453 (P16640); CumA4: cumene dioxygenase, ferredoxin reductase
subunit of P. fluorescens IPO 1 (BAA07078); TodA: toluene 1,2-dioxygenase, ferredoxin reductase subunit
of P. putida Fl (P13452); BedA: benzene 1,2-dioxygenase, ferredoxin reductase subunit of P. putida ML2
(Q07946); TcbAd: chlorobenzene dioxygenase, ferredoxin reductase subunit of Pseudomonas sp. P51
(U15298); Rv0688: hypothetical protein of M. tuberculosis H37Rv (C70640); Rv 1869c: hypothetical
protein of M. tuberculosis H37Rv (E70667); RubB: rubredoxin reductase of R. erythropolis NRRL B16531 (AJ009586); HcaD: 3-phenylpropionate dioxygenase, ferredoxin reductase subunit of E. coli K-12
(P77650); AlkT: rubredoxin reductase of P. putida PI (CAB69078.1); AlkT: rubredoxin reductase of P.
putida (oleovorans) GPol (CAB54063.1); ORF377: flavorubredoxin reductase of E. coli K-12; RubBl:
putative rubredoxin reductase 1 of P. putida KT2440; RubB2: putative rubredoxin reductase 2 of P. putida
KT2440; RubB: putative rubredoxin reductase of P. syringae pv. tomato DC3000; RubB: putative
rubredoxin reductase of P. fluorescens Pf0-1 ; RubB : rubredoxin reductase of P. aeruginosa PAO 1
Figure
manual
1: unrooted
B-16531)
(M. tuberculosis H37Rv)
—
—
Rv0688
NRRL
Rvl869
—
—
(R. erythropolis
phylogenetic
tree of
of the rubredoxin reductases with
(AAG08734.1); RubB: rubredoxin reductase of Acinetobacter calcoaceticus
rubredoxin reductase of Acinetobacter sp. ADPl
(CAA86926.1).
S19
(CAA75809);
RubB:
79
aerobiosis
via
a
Another E. coli
ferredoxin to the
putative
one
(95).
AlkT-homolog, HcaD,
3-phenylpropionate dioxygenase (60) (figure 1).
homologs
were
a
also found in the genomes of Pseudomonas
proteins
findings
the function of these
indicate that rubredoxin reductase
roles in addition to alkane
hydroxylases
because the
levels need not be very
expression
GPol rubredoxin reductase AlkT
was
Phylogenetic analysis.
proteins (>
proteins
homologs
they
are
A
is unknown.
have diverse
The P.
oxyR,
that
are
identity
(245).
oxidation)
%). Within
higher (typically
In Acinetobacter sp.
(flavo)rubredoxin
30
%).
not
pathways,
or
ADPl, the
transcriptionally
degradation (92).
of the rubredoxin reductases and related
one
of the rubredoxin reductases known to
showed that the rubredoxin reductases
one
another
each of the two groups, the levels of sequence
over
are
cultures, but its
not necessary for alkane
to at least
physiological
putida (oleovorans)
also detected in non-induced
phylogenetic analysis
20 % sequence
role in alkane
rubredoxin
KT2440 do not contain
also involved in other
high (245).
two groups which have low sequence identities with
and 45
putida
of rubredoxin and rubredoxin reductase is constitutive and
linked to two genes, estB and
a
because
rate increased 4-fold after induction
expression
related to the P.
most rubredoxin reductases
degradation. Possibly,
located close to alkane
play
quite closely
pv. tomato DC3000, P. fluorescens Pf0-1 and P.
hydroxylase homologs,
synthesis
are
pv. tomato DC3000
syringae
PAOl rubredoxin and rubredoxin reductase. As the genome sequences of P.
aeruginosa
syringae
putida KT2440,
rubredoxin domain. Rubredoxin and rubredoxin reductase gene
and Pseudomonas fluorescens Pf0-1. These
These
We identified two
rubredoxin reductases in the unfinished genome sequence of P.
of which is fused to
alkane
transfers electrons from NADH
can
(ranging
be divided in
between 18
homology
are
much
One group contains the chromosomal encoded
reductases of
Gram-negative bacteria, including
the PAOl and ADPl
reductases, while the second group includes the rubredoxin reductase of R.
erythropolis
NRRL B-16531
(J.B.
van
Beilen, unpublished results) and the alk-
transposon encoded rubredoxin reductases of P. putida strains GPol and PI (260). The
second group has
highest (up
to 37
%)
but all the
same
low sequence identities to FAD-
80
which transfer electrons to other oxygenases like
containing reductases,
P450s and class II aromatic
Functional
ring dioxygenases (39)(figure 1).
of rubredoxin reductases. Strains and
analysis
in table 1. For the functional
derivative of
analysis
which lacks
pGEc47,
a
of rubredoxin
355
used
plasmids
reductases,
an
hydroxylase activity
significant
of the
at 10% of the
remaining activity
chromosome
can
was
basepair fragment corresponding
introduced in E. coli GEcl37
recombinant E. coli
resulting
activity
measured with E. coli
primer
(rubB) (91)
were
and rubB IE
The alkane
GEcl37[pGEc47AT]
by
recombinants
GEcl37[pGEc47AT]
rubB of P.
supplemented
amplified by
was
quite
The
the E. coli
and cloned in
PCR from chromosomal DNA
(151)
pKKPalk (240).
and
growth
exposed
on
and RubCrev
and rubBrB
plasmid encoding
a
PAOl showed
with MT trace elements
and of
(caatcacagaggaacgcatatgagc)
containing
aeruginosa
(rubB) (249)
(ctacggagaattctaatgcacc)
(aaaaaggatcctcagatgaatg), respectively
or
of P.
(AlkT)
GEcl37[pGEc47],
PAOl
aeruginosa
combinations RubCfwd
(ctgcgcaagcttcgtccgacaa)
sp. ADPl
to the
(257).
Acinetobacter sp. ADPl
the
(257).
be attributed to unknown reductases encoded
The rubredoxin reductase genes of P.
using
described
are
alkT deletion
translation start site and amino acids 1-113 of the rubredoxin reductase
putida (oleovorans) GPol,
cytochrome
E. coli
rubB of Acinetobacter
E2 minimal medium
plates
to «-octane vapor after three
days.
These results show that these two rubredoxin reductases
deletion,
even
GPol AlkT
as
though
these
to other
proteins
are as
FAD-containing
distantly
reductases
(figure 1),
strain GEcl37 with
we
amplified
primers
Ecoli-AlkT-RV-Hin
complement
related to the P.
the alkT
putida (oleovorans)
(figure 1).
As the flavorubredoxin reductase of E. coli K-12 has
reductases
can
high homology
to rubredoxin
the gene from chromosomal DNA of the K-12 derived
Ecoli-AlkT-FW-Eco
(gtcgcatccgaaagcttaggcacag)
(ggcatcgaattcaaaatgagtaacgg)
and cloned it in
and
pKKPalk (240).
However, when expressed from the PalkB promoter, the flavorubredoxin reductase did
not
complement
the AlkT deletion
although
the
protein
can
transfer electrons from
81
NADH to the rubredoxin of
clear what
causes
the
Based
Desulfovibrio gigas (95).
background activity
these
on
results, it is still
in E. coli. Possible candidates for this role
HcaD, the ferredoxin reductase subunit of the 3-phenylpropionate dioxygenase (60)
other
reductases, which have
even
not
are
or
lower sequence identities to the rubredoxin reductases
involved in alkane oxidation.
Table 1: list of strains and
Strain
P.
plasmids
Relevant
name
aeruginosa
putida (oleovorans)
or
pGEc47,
pGEc47,
as
(257)
deletion in alkT
(257)
(276)
(PAOl) in
This
rubB gene
(ADPl) in pKKPalk
This
gene
(K-12) in pKKPalk
proteins
from
can
distantly
replace
related
and ferredoxin reductase
TcbAd has 30.4 %
(276)
or
RubB
in the E. coli strains
from
replace
dioxygenase
which
sequence
identity
to the
(ADP1)(29.0 %) (figure
the tcbAaAbAcAd genes, which
(pTCB144)(276),
(20,
can
dioxygenases,
protein
Because
(TcbAd)
reductase, which is similar
(PA01)(31.7 %)
plasmid containing
systems.
assay. The chlorobenzene
GPol rubredoxin
study
study
study
AlkG and AlkT in vitro
to the benzene and toluene
dioxygenases (39).
between AlkT and RubB
This
of Pseudomonas sp. strain P51
complementation
highly homologous
more
(TcbAc)
dioxygenase system
from the lacZ promoter
(69)
deletion in alkG
rubB gene
orf377
a
(240)
pLAFRl
pUC19
pKKPalk
class IIB
We introduced
ApR
tcbAaAbAcAd genes in
putida (oleovorans)
homology
1).
alkBFGHJKL/alkST (GPol) in
AlkT in the in vivo
classified
to the P.
Reference
tested whether the ferredoxin
enzyme system is
are
Expression
of electron transfer
the chlorobenzene
AlkG
(67)
using PalkB,
vector
ferredoxin and ferredoxin reductase
we
(141)
Gibco BRL
Characteristics
pKKPalk
pGEc47
pGEc47AG
pGEc47AT
pTCB144
pRubB (PAOl)
pKKRubB (ADPl)
pKKEcRubB (K-12)
207),
(126)
plasmid
cloning strain
thi,fadR
name
spinach
(118)
GPol cured of the OCT
GPo 12
£. co//GEcl37
Exchange
Reference
phenotype
alk*
E. coli DHIOB
Plasmid
study
alk*
PAOl
Acinetobacter sp. ADPl
P.
used in this
can
be
expressed
GEcl37[pGEc47AG]
and
82
E. coli recombinants
GEcl37[pGEc47AT].
cultivated
on
containing pTCB144 produced indigo
LB, which indicates that the dioxygenase system is functionally intact.
However, the electron transfer proteins of the chlorobenzene dioxygenase did
the deletion in either alkG
complement
«-octane. The results show that alkane
components
are
not able to interact in
transfer of electrons to the alkane
AlkG 1-type rubredoxins
(J.
B.
van
AlkG/AlkT in vivo
replace
or
in alkT to the extent necessary for
hydroxylase
and chlorobenzene
not
growth
on
dioxygenase
vivo, possibly because structural features prevent
The
hydroxylase.
same
conclusion
was
reached for
Beilen, unpublished results) and for XylA, the
electron transfer component of the P.
cannot
when
putida
xylene
mt-2
monooxygenase, which also
(257).
Conclusions
Rubredoxin reductases
can
transfer systems in in vivo
It thus
seems
so
differ
that these
reductase of the alkane
degradation
tree
(J.
B.
structural
replaced by
experiments, although
the
that both rubredoxins and rubredoxin
hydroxylase system,
geometry
not be
are more
van
significantly
homologs
from
homologs
protein
from other electron
sequences
reductases, that
homologs present
are
part of the alkane
or
Beilen, unpublished results), could also be
mutagenesis experiments
are
studies
necessary to
docking
rubredoxin
The fact that rubredoxins involved in alkane
related to each other than to other rubredoxins in the
phenomenon. Therefore, crystallization
homologous.
in nature in the
cannot interact with either rubredoxin
hydroxylase system.
are
on
an
indication for this
the alkane
investigate
phylogenetic
hydroxylase system
these interactions in
more
and
detail.
ACKNOWLEDGEMENTS
The authors wish to thank Martina
for
plasmid pTCB 144.
Biotechnology
Röthlisberger
This research
was
for
sequencing
supported by
of the Swiss National Science
and Hans-Peter Köhler
the Swiss
Foundation, project
Priority Program
nr.
in
5002-037023.
83
Chapter
6
FUNCTIONAL CHARACTERIZATION OF GENES INVOLVED IN
ALKANE OXIDATION BY PSEUDOMONAS AERUGINOSA
Theo H. M.
Smits, Bernard Witholt and Jan B.
van
Beilen
84
SUMMARY
Many
Pseudomonas
aeruginosa
PAOl indeed do oxidize
(rubAl and rubA2) and
putida (oleovorans)
hydroxylase
genes
GPol
are
strains tested in this
counterparts in
«-alkanes.
long-chain
hydroxylase (alkBl
long-chain alkanes,
rubredoxin reductase
a
medium- and
on
shows that two alkane
Heterologous expression
of P.
strains grow
aeruginosa
and
alkB2) homologs
while two rubredoxin
(rubB) homologs
replace
can
«-octane oxidation. The two
their P.
alkane
long-chain
present in all environmental and clinical isolates of P. aeruginosa
study.
INTRODUCTION
Pseudomonas
among the
aeruginosa
pseudomonads,
is of clinical
but is also
importance
a common
(27). Environmental and pathogenic isolates
and molecular
techniques (80, 112).
property. Enrichments from soil
P.
aeruginosa
carbon
source
strains.
or
Conversely,
is used to
fluorescence and oxidase assays
degradation by
P.
aeruginosa.
make them available for
"protein
Most
the
of P.
ability
water and
soil,
as
to
plants
chemotaxonomic
the carbon
aeruginosa
on
use
relevant
a
source
often
paraffins
as
yield
sole
isolates, in combination with other methods like
not much is known about «-alkane
fact, only compounds which solubilize «-alkanes and
as
rhamnolipids (140, 290)
activator of alkane oxidation" PraA
and the so-called
(103, 114) have been studied in
some
detail.
reports dealing with the biochemistry of alkane degradation by P. aeruginosa
concern
strains able to grow
these P.
aeruginosa
P.
in
indistinguishable by
water with «-alkanes
such
uptake,
primary opportunistic pathogen
organism
(170). Nevertheless,
In
the
In both contexts, alkane oxidation is
clinical
identify
are
as
GPol alkane
not involved in
(oleovorans)
C6-C10 «-alkanes
strains contain alkane
putida (oleovorans)
probably
on
growth
GPol alkane
(269). Recently, it
hydroxylases
hydroxylase (262).
of these strains
hydroxylase only
on
that
are
shown that
was
virtually
identical to the
This enzyme system,
long-chain alkanes,
oxidizes alkanes with
a
as
chain
however, is
the P.
putida
length
from
85
hexane
to dodecane
(C6) up
(C12) (259). P. aeruginosa long-chain alkane hydroxylases
have not been characterized
biochemically
or
genetically.
Growth of Pseudomonas
aeruginosa
and clinical P.
isolates listed in table 1 for the
long-chain
aeruginosa
alkanes. P.
minimal medium
aeruginosa
plates (151)
strains
on
alkanes. We tested the environmental
ability
to grow
medium- and
on
PAOl and most of the clinical isolates grew
with
on
«-dodecane, «-tetradecane and «-hexadecane
carbon and energy source, which is consistent with
previous
data
(170).
E2
as
None of the
clinical isolates grew with «-octane and «-decane vapor. In contrast, the environmental
isolates described earlier
hexane to «-decane
as
that it showed weak
Cloning
well
growth
yielded
based
PG201 PCR
that the P.
aeruginosa
independent
cosmids
Cosmid
degenerate
alkBl but
significant
conserved
fragment
homolog
regions
,
used
as a
contains
alkB
an
which
was
cross-reactivity
identity
The two genes
are
(249).
the
designated
with
aeruginosa
in Southern
PAOl
with the P.
blots, which showed
to the GPol alkane
No additional alkB
components for activity:
a
homologs.
homologs
GPol alkane
with the
pTSlOO
contains
65 % overall DNA sequence
hydroxylase
were
a
was
a
used
as
no
GPol alkB gene in
a
37.4 %
and 67.7 % to each other.
aeruginosa
found
same
identity
(278). AlkBl and AlkB2 show
Genome
(figure
hydroxylase requires
rubredoxin and
Two
to the PG201
putida (oleovorans)
method
Highly
homologs
in the internal gene segment that
also present in the Pseudomonas
putida (oleovorans)
in
PAOl and PG201
genebank (271)
alkBl. Cosmid
only
gene
in the Southern blot. Both genes show
Wilbur-Lipman
identity
a
PAOl.
hydroxylase
homolog, corresponding
designated alkB2,
DNA sequence
amino acid sequence
probe
by screening
significantly higher homology
the
«-
exception
was an
aeruginosa
of alkane
with P.
fragments
was
isolated
were
pairwise alignment by
The P.
on
fragment,
probe, explaining
sequence
«-alkanes. Strain PG201
longer
PAOl genome contains two related alkB gene
pTS200
PCR
second alkB
to
medium-chain «-alkanes from
with octane and decane.
almost identical PCR
(239). The
probe.
the
as on
on
of genes involved in alkane oxidation from P.
degenerate primers
earlier
(13, 163, 266, 269) grew
la and
Project (PAGP)
lb).
two electron transfer
rubredoxin reductase. We
inspected
the
86
PAGP database for the presence of
consisting
gene
of two rubredoxin
Acinetobacter sp. ADPl
65 % sequence
identity
rubredoxin sequences
most
closely
and
rubA2)
one
are
most
possible
rubredoxin reductase
closely
operon
(rubB)
related to RubA of
-
with other rubredoxins involved in alkane oxidation. All extant
are more
distantly
related. The
sequence
identity) (91)
rubredoxin reductase is
putative
(RubB)
of Acinetobacter sp. ADPl
and the rubredoxin reductase
(AlkT)
of P.
putida
(37.1 %) (68).
GPol
Table 1: list of Pseudomonas
aeruginosa
medium with 0.2 % citrate
«-alkane vapor
Labname
and
a
(70-72 % protein sequence identity) (91) and show between 50
related to the rubredoxin reductase
(40.1% protein
(oleovorans)
(rubAl
The two rubredoxins
homologs (figure lc).
and found
corresponding homologs,
or
Official name3
strains and
as
growth
carbon
Isolated from
behavior
on
E2 minimal
source
Growth
Citr.
on
C8
withb
E2 medium
Reference
CIO
C12
C14
C16
PAOl
ATCC 15692
Infected wound
++
+
++
+++
+++
(118)
PG201
DSM 2659
Soil
++
+
+
++
+++
+++
(97)
KSLA 473
KSLA 473
Y-harbor, Amsterdam
++
+++
++
++
+++
+++
(266)
Sol 20
NCIMB 8704
Soil
++
+++
++
++
+++
+++
(13)
196Aa
NCIMB 9571
Soil
++
+++
++
++
+++
+++
(269)
ATCC 17423
ATCC 17423
Soil
++
+++
++
++
+++
+++
(163)
CPAld
DMMZV10 18600
Urine
++
-
-
+
++
++
This
CPA2
DMMZV07 19924-1
Ethmoid tissue, CF
++
-
-
CPA3
DMMZ V07 19924-4
Ethmoid tissue, CF
CPA4
DMMZ V07 19925-2
Ethmoid tissue, CF
++
CPA5
DMMZ V07 19939
Urine
++
++
++
This
CPA6
DMMZ V07 19941
Urine
++
++
++
This
CPA7
DMMZ V07 19965
Pleural fluid
++
CPA8
DMMZ V09 20207
Urine
++
CPA9
DMMZ V09 20227-1
Bronchial secretion
++
CPA10
DMMZ V05 20348-2
Urine
++
CPA11
DMMZ V05 20391-2
Tracheal
CPA12
DMMZ V07 21517
Tracheal
a
b
c
d
DMMZ:
Department
of Medical
CF:
-
This
-
This
aspirate
aspirate
Microbiology Zürich;
-
This
This
This
This
++
++
++
cystic
This
++
++
This
++
This
study
study
study
study
study
study
study
study
study
study
study
study
fibrosis
Citr.: 0.2 % citrate; C8: octane; CfO: decane; Cf2: dodecane; Cf4: tetradecane; C16: hexadecane
Growth
was
Clinical P.
after 90 h. incubation.
aeruginosa
Functional
alkane
analyzed
strains
analysis.
are
Based
hydroxylase systems
identified
on
good growth, ++ average growth,
according to (f f 2) at DMMZ, Zürich
+++
+
weak
growth,
-
growth
no
the observation that the electron transfer components of
can
be
exchanged (J.
B.
van
Beilen, unpublished results),
we
87
have
developed
recombinant hosts that express two of the three alkane
components (chapters 4 and 5; J. B.
us
to test novel alkane
for the
or
ability
to
E. coli
were
expression
(PAOl)
were
fluorescens
or
alkane
transfer electrons from NADH to
hydroxylase.
The alkane
cloned in the EcoRl and Hindlll sites of
vector
KOB2A1,
(chapter 4)
E2 minimal medium
homologs
rubredoxin,
hydroxylase
pCom8,
a
genes
Pseudomonas-
(240). The resulting plasmids pCom8Bl (PAOl) and pCom8B2
introduced in
CHAO
an
Beilen, unpublished results). These strains allow
rubredoxin and rubredoxin reductase gene
hydroxylate «-alkanes,
from rubredoxin reductase to
alkBl and alkB2
on
hydroxylase,
van
hydroxylase
plates
Table 2: other strains and
as
an
alkane
hydroxylase negative
described before
with 0.5 %
mutant of P.
(117). Both recombinants
a-cyclodextrin
plasmids oligonucleotides
were
and 0.05 % «-alkanes
used in this
plated
ranging
study
Strains
E. coli DH10B
strain
E. coli GEcl37
P.
Reference
Phenotype
Name
fluorescens
KOB2Af
cloning
thi,fadR
Gibco BRL
alkB~
chapter 4
(69)
Plasmids
Name
Characteristics
Reference
pGEM7-Zf(+)
pKKPalk
pCom8
pGEc47
pGEc47AG
pGEc47AT
pGEc48
pTS2
pTSlOO
pTS200
pRubAl (PAOl)
pRubA2(PA01)
pRubB (PAOl)
pCom8Bl (PAOl)
pCom8B2 (PAOl)
Cloning vector, Apr
Expression vector using a/fc-promoter, Apr
pCom7 with alkS from pBGf f EAN
alkBFGHJKL/alkST in pLAFR f
pGEc47, deletion in alkG
pGEc47, deletion in alkT
alkBFGH'inpER322
PG201 PCR fragment in pGEM7-Zf(+)
Cosmid harboring alkBl gene
Cosmid harboring alkBl gene
rubAl gene in pKKPalk
rubAl gene in pKKPalk
rubB gene in pKKPalk
alkBl gene in pCom8
alkBl gene in pCom8
Promega
from C12 to C24 under
promoter(96).
«-alkanes
dicyclopropylketone
The recombinant strain
ranging
vapor,
(240)
(240)
(69)
(263)
(263)
(70)
(239)
This
study
study
This study
This study
This study
chapter 4
chapter 4
This
a
gratuitous
inducer of the alkB
KOB2Al[pCom8Bl (PAOl)]
from C16 to C24, while
was
KOB2Al[pCom8B2 (PAOl)]
able to grow
grew
on
C12
on
-
88
C20 «-alkanes.
specificities.
This trend
Thus, the
also obvious in
fast
as
as
PAOl
P. fluorescens CHAO.
To
analyse
reductase
three
whether the P.
(rubB)
proteins
alkane
in the
are
were
same
aeruginosa
able to function
tested for their
hydroxylase system
derivatives of
where
of P.
pGEc47 having
rubredoxin reductase gene
a
on
all substrates tested
PAOl rubredoxin
as
electron transfer
ability
the
replace
to
growth
wild-type
proteins
were
B.
van
cloned
minimal medium with «-octane
coli
GEcl37[pGEc47]
and
GEcl37[pGEc47AT].
constructs and the
negative
controls.
Table 3:
doubling
on
was
recombinants
the gas
phase.
used, while the negative controls
Growth
lag
resulting
on
«-octane
while very
times of P.
was
were
were
was
PAOl and P.
we
of the
used
or
the
Beilen, unpublished
a
into the
pKKPalk,
and
plated
rubA2)
on
or
E2
positive control,
E.
GEcl37[pGEc47AG]
observed after three
slight growth
aeruginosa
oxidation, the
separately
As
The
aeruginosa
(pGEc47AG)
GEcl37[pGEc47AG] (rubAl
supplied through
positive control,
and
The
grew
(table 3).
corresponding components
a/&r(pGEc47AT), respectively (J.
GEcl37[pGEc47AT] (rubB).
plates.
and rubredoxin
in alkane
EcoRl and Ascl, the EcoRl and Pstl and the Ndel and Hindlll sites of
E. coli
P.
(rubAl, rubA2)
deletion in the rubredoxin gene alkG
and transferred to E. coli
on
GPol. For this purpose,
putida (oleovorans)
results; chapter 5). The rubAl, rubA2 and rubB genes
respectively (240)
substrate
KOB2Al[pCom8B2 (PAOl)]
those found with the
as
range
overlapping
showed stronger
KOB2Al[pCom8Bl (PAOl)]
times
were
appear to have
consistently
liquid cultures,
doubling
or
hydroxylases
The AlkB2 recombinants
was
about twice
two alkane
days
with all
observed with the
fluorescens
recombinants
alkanes.
Strain
Additional
gene
time
(td)
C14
and
lag
time
(in parenthesis)
C16
in hours
C18(10%)
C20(10%)
in DOPh
in DOPh
19.1(150)
-b
19.5(240)
35.2(80)
10.6(40)
alkBl
(PAOl)
176.2(40)
36.6(180)
43.7(20)
18.8(100)
19.2(100)
alkBl
(PAOl)
20.6(8)
21.6(8)
15.4(30)
12.9(45)
11.9(20)
PAOl
KOB2A1
Doubling
C12a
-
Footnotes:a C12, dodecane; C14, tetradecane; C16, hexadecane; C18
dioctylphthalate;
C20
(10%)
in DOPh, 10% eicosane in
(10%)
dioctylphthalate;
b
-,
in DOPh, 10% octadecane in
not tested.
89
Gene
organization
and
flanking
the initial alkane oxidation step
over
the chromosome of P.
possible exception, tlpS,
a
are
directly
indirectly
or
(alkBl, alkB2, rubAlA2B,praA, rhlABRI)
aeruginosa
genes indicates that these have
one
genes. Genes that
no
coding
dispersed
are
(249). Analysis of regions flanking these
PAOl
obvious relation to alkane
gene
involved in
for
a
Chemotaxis
methyl-accepting
protein
(MCP) (282) (figure la). The intergenic region between alkBl and tlpS is only
basepairs,
and
no
clear inverted repeats, which could
terminator, could be found between the
I
lkb
I
B
PA2576
PA2577
PA2575
rC
Y
PA1527
c
a
111
r«o-dependent
downstream of the MCP, two
I
PA2578\
PA2579
towards
Directly
two ORFs.
I^J0C^><^ZH
a
point
with
degradation (figure 1),
glcC
alkBl
H
PA1526
)lZZ>
-^=H
glcD
glcE
%
>J
"
tlpS
Jr
I
alkBl
xdhA
1)—
H
hupRl
hupTl
)Œ^J~
j£
xdhB
PA1522
)fo^LZ^fo>^
glcF glcG I
rubAl rubB
rubAl
I
h
PA5347 PA5346
PA5348
1: analysis of the open reading frames surrounding the P. aeruginosa PAOf genes involved in
degradation. The data were obtained from the P. aeruginosa genome sequence
(www.pseudomonas.com) (249).
A: Alkane hydroxylase 1 (alkBl ; PA2574; from 29f f876 to 29f0728) and flanking region. Other genes:
tlpS: methyl-accepting Chemotaxis protein; hupRl: two-component regulatory system involved in the
regulation of the fNiFe] hydrogenase activity; hupTl: sensor protein involved in repression of hydrogenase
synthesis. PA2577: putative transcriptional regulator of the AsnC family; PA2579: homologous to human
tryptophan-2,3-dioxygenase; PA2578, PA2576, PA2575: hypothetical proteins.
B: Alkane hydroxylase 2 (alkBl; PA1525, from f 660546 to f 6594f3) and flanking region. Other genes:
PAf 527: homologous to yeast chromosome separation protein SMC; PA 1526: putative transcriptional
regulator of the GntR family; xdhA: homologous to the N-terminal domain of eukaryotic xanthine
dehydrogenases (XDH); xdhB: homologous to internal fragments of eukaryotic XDHs and total XDH of
Rhodobacter capsulatus; PAf522: homologous to the N-terminal domain of R. capsulatus XDH.
Figure
alkane
C: The rubAlAlB gene cluster
60f 8996 to 60f 8829; rubB:
(rubAl: PA5351; from 60f9347 to 60f9f80; rubAl: PA5350; from
PA5349; from 60f 8777 to 60f 7623) and flanking region. Other genes:
homologous to the glcRDEFG genes of E. coli involved in glycolate oxidation; PA5348:
protein HU from P. aeruginosa; PA5347, PA5346: hypothetical proteins.
glcCDEFG:
genes
homologous
to histone-like
90
clear inverted repeats
opposite
as
P.
orientation.
aeruginosa
hexadecane
(J.
TlpS
B.
van
in the
gene, which is transcribed in the
hupRl
could be involved in Chemotaxis towards
PAOl and other P.
Genes involved in the
not be
located at the end of the
are
aeruginosa
strains show strong Chemotaxis towards
Beilen, unpublished results).
of the P.
regulation
aeruginosa
PAOl
identified, although the alkB2 gene is preceded by
opposite
alkBl, alkB2
PAOl alkBl IalkBl and P.
aeruginosa
carried out Southern blots
using
GPol alkBFGH'
procedures, digested
membranes
probe.
bands
octane
(data
detected
were
not
shown),
in all P.
fragments
alkanes
Most clinical P.
because
in accordance with
they
are
PAOl, which is
not
a
ability
(figure 2).
It is
genes that
are
possible
directly
mutations in the
are
to grow
on
and
isolated
to
on «-
(262). PAOl, PG201, and
The alkBl
probe.
putida
P.
according
probe hybridized
the strains that did not grow
able to grow
long-chain «-alkanes,
on
as
on
these substrates
well,
were
on
isolated
a
not grow
with
on
in
well
as
on
longer lag-time, perhaps
(170). All other strains, except
on
«-alkanes, and thus
these substrates. Even
though
were
some
of the
alkanes, they do contain both alkane hydroxylase genes
that these strains have mutations in the alkB genes
or
a
positively charged nylon
(4). However, the clinical strains did
to grow well
clinical strains did not grow
probe
we
With the GPol alkBFGH'-
results
the environmental strains and had
adapted
was
aeruginosa,
study
1).
isolates
clinical isolate
for their
band with this
a
table
(figure 2,
as
previous
aeruginosa strains, including
aeruginosa
alkanes
preselected
Chromosomal DNA
strains. To
in the four environmental strains able to grow
only
accordance with literature data
long-chain
PG201 alkBl
with BamHl, and blotted onto
the clinical strains did not show
long-chain
aeruginosa
(Roche Diagnostics, Rotkreuz, Switzerland).
probe,
two
the P.
putida
aeruginosa
the presence of alkB genes in environmental and clinical isolates of P.
standard
gene, transcribed
putative regulatory
a
GPol alkB in environmental and clinical P.
(oleovorans)
rubAlA2B could
or
direction.
Southern blot detection of P.
(oleovorans)
long-chain «-alkanes,
indirectly
or
in other
involved in the initial alkane oxidation. For
rhamnolipid biosynthesis pathway
can
lead to reduced
or
example,
abolished
91
growth
%
on
«-alkanes
(140, 192).
citrate, and may have
an
One strain
(CPA3) did
not grow
on
E2 medium with 0.2
auxotrophy.
©
^h
r*
Q.Q.Q.Q.Q.Q.Q.Q.Q.Q-
Q.
Q.
UÜUUUÜUUUÜ
uu
""'it
*
^ff£m"**fê
"-I
êêé
,;
M
"
~-
Figure 2: Southern blot of chromosomal DNA of environmental and clinical isolates of P. aeruginosa
digested with BamHl. As a probe, the 550 bp internal segment of alkBl from P. aeruginosa PG20f was
used. The marker (M) is digoxigenin-labeled lambda DNA digested with Hindlll; marker sizes are
indicated in kb. For strains PG20I, f 96Aa and ATCC f7423, weak bands of around 0.7, 0.8 and 0.9 kb,
respectively,
were
CPA6 and CPA9
of P.
as
visible
well
on
aeruginosa
physiological
role than
(not shown). The signal for the large fragment (±23 kb) of strains
the film, but could not be enhanced for the
remarks. Based
Concluding
ability
observed
was
on
the results of this
strains to grow
on
only degradation
long-chain
of oil
alkanes. The genome sequences of the clinical
pseudomallei
common
soil
K96243 and
or
water
on
aeruginosa
medium
are
are
chain-length
often P.
pollutants
or
be concluded that the
have
might
a
larger
naturally occurring
microorganisms
«-alkanes is
aeruginosa
such
as
clearly
a
«-
Burkholderia
which
are
also
hydroxylase homologs (chapter
not found in the genome sequences of anaerobic
isolates: strains isolated from
carbon source,
might
alkanes
do also contain alkane
parasitic clinically important microorganisms,
to grow
it
Legionella Pneumophilia Philadelphia-1,
organisms,
4), while AlkB homologs
study,
figure.
or
strictly
Clostridium. In contrast, the
ability
property of environmental P.
gasoline spills
with hexane
strains that contain
or
octane
alk-genes (nearly)
as
sole
identical to
92
the P.
putida (oleovorans)
degradation
GPol
alk-syslem (243, 262).
The relevance of alkane
in clinical and natural environments has therefore to be studied in
more
detail.
ACKNOWLEDGEMENTS
We thank Dr. G. Funke of the
providing
us
with the clinical isolates of P.
Microbiology,
Martina
Department
ETH Zürich for
Röthlisberger
for
providing
sequencing
of Medical
aeruginosa,
us
with the
Zürich
Microbiology,
(DMMZ) for
Dr. M. Kertesz of the Institute of
genebank
of P.
aeruginosa PAOl,
and Alessandro G. Franchini and Stefanie B.
Balada for technical assistance.
This research
project
was
supported by
the Swiss National Science
the Swiss
Foundation, project
Priority Program
nr.
5002-037023.
in
Biotechnology
of
93
Chapter
7
CLONING AND CHARACTERIZATION OF GENES INVOLVED IN
LONG-CHAIN ALKANE OXIDATION BY PSEUDOMONAS
FLUORESCENS CHAO
Theo H. M.
Smits, Raquel Sanjuân, Bernard Witholt and Jan B.
van
Beilen
94
SUMMARY
Pseudomonas fluorescens CHAO is
chromosomal DNA
hydroxylase
(alkB),
gene
«-alkane
fragment containing
was
it contains two
outer membrane
an
cloned and
"protein
protein
gene
CHAO alkB knockout mutant
a
degrading
of the
homolog
analysed.
biocontrol strain. A 7.8 kb
GPol alkane
P.putida
In addition to the alkane
activator of alkane oxidation" genes
(ompPl),
was
and two
incomplete
found to have lost the
alkanes, but could be complemented for growth
on
(praA
gene
and praB),
an
genes. A P. fluorescens
ability
to grow
these «-alkanes
on
C12-C16
«-
by plasmids
the P. fluorescens CHAO alkB gene.
containing
A 16 kDa
protein
identified
as
in the supernatant of
PraB. PraA
hexadecane-grown
P. fluorescens cells
on
gels
SDS-PAA
In the presence of Triton
was
not detectable. When inoculated in the presence of
was
supernatant containing PraB, P. fluorescens CHAO cultures showed
However,
hydroxylase
PraB
was
detectable
only
X-100, the alkB knockout
in the
a
shorter
spent
lag-phase.
stationary phase.
mutant showed weak
growth
on
hexadecane, while the addition of dioctylphthalate allowed the wild-type P. fluorescens
as
well
as
the alkB knockout strain to grow
This indicates that CHAO possesses
a
on
very-long-chain
second alkane
«-alkanes
(C18-C28).
hydroxylase.
INTRODUCTION
While the
biochemistry
studied in strains such
and
as
about membrane-bound
genetics
Pseudomonas
long-chain
Acinetobacter sp. ADPl alkane
alkane
hydroxylation by
because it inhibits
grows
on
PCR with
of
putida (oleovorans)
alkane
hydroxylases,
hydroxylase (213).
ranging
fungal pathogens
In this
in the
based
putida (oleovorans)
on
alkane
study,
a
degradation
is well
(263), little is known
GPol
with the
exception
we
focus
of the
on
long-chain
strain which is of interest
plant rhizosphere (99, 250).
from dodecane to hexadecane
degenerate primers
genes of P.
length
Pseudomonas fluorescens CHAO,
growth
«-alkanes
of medium-chain
CHAO
(239).
the sequences of the alkane
GPol and Acinetobacter sp.
hydroxylase (alkB)
ADPl, yielded PCR
95
fragments
to grow
be
of alkB
on
and
Such alkanes
microorganisms
all factors necessary for
practically
must
were
reported
containing
lipopeptides
to have surfactant
16 %
fluorescens
long-chain
use
species, including
P. fluorescens, two
(215),
elucidated. Pseudomonas
alkanes
P. fluorescens,
growth
or
properties
as
protein
well
on
uptake problems,
long-chain
alkanes
it is accessible for
long-chain
on
(77, 193), but also
excrete the so-called
source
(viscosin
(183, 188).
lipids
and
(28). Several
small extracellular
protein
case
was
of
viscosinamide)
A surfactant called
AP-6,
isolated from the P.
produce rhamnolipids
"protein
genetic
the latter mechanism. In the
and 40 %
strains
was
alkanes.
but the molecular structure of this surfactant
aeruginosa
an
hydrophobicity. Therefore,
their carbon
as
use
of these genes could not
biosurfactant-mediated
with antibiotic function
34 %
carbohydrate,
isolate PG-1
as
insoluble in water due to their
direct interfacial contact
use
solubilization to be able to
Pseudomonas
strain able to grow
strains able
Gram-negative
due to alkane
We chose CHAO for this purpose,
produces
are
a
and
expression
putida, presumably
knockout mutant of
required (chapter 4).
Gram-positive
As functional
(239).
in E. coli and P.
hydroxylase
engineering
from several
alkanes
long-chain
accomplished
alkane
homologs
was
not
to solubilize alkanes
activator of alkane oxidation" PraA,
which stimulates hexadecane oxidation in
a
respiratory
experiments (103, 114).
In this
study,
CHAO alkB gene
gene alkB and
hydroxylase
of the P.
a
aeruginosa
knockouts and
hydroxylase,
flanking DNA,
PG201 PraA
complementation
while the
overexpression
fragment (239)
of the
used to clone the CHAO alkane
which encode two
(103), and
an
and
PraB)
protein (OmpPl).
Gene
homologs (PraA
outer membrane
show that the CHAO alkB encodes
alkane-solubilizing
proteins
was
a
functional alkane
effect of both PraA and PraB
was
shown after
in E. coli.
MATERIAL AND METHODS
Strains, plasmids and media. Strains and plasmids used in this study
1. LB
(Luria
with carbon
Bertani
broth) (220),
sources or
antibiotics
E-medium
were
used
are
listed in table
(272) and E2-medium (151), supplemented
throughout this study.
MT trace elements
96
Table 1: strains and
Strain
plasmids
used in this
study
Reference:
Properties:
:
P.
CHAO
alk*
(250)
P.
KOB2
alkB-, KmR
This
KOB2A1
alkB"
KOT1
alkB"
praAB" ompPl"
KOT2
alkB"
praAB" ompPl"
fluorescens
fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
study
Chapter 4
This study
This study
E. coli DHIOB
cloning
E. coli GM48
thr, leu, thi, lacY, galK, galT,
strain
Gibco
E.coli JM101::alkS-Km
JM101, alkS integrated
ara,
J. Davison
tonA, tsx, dam, dem, supE44
Plasmids:
Relevant genotype:
pGEM7Zf(+)
pZeROf
pKKPalk
pCom5
pEXI8Tc
pCK217
pPFl
Cloning
Cloning vector, ZeocinR
Expression vector, ApR
Expression vector, GmR
vector,
on
chromosome, KmR
(200)
Reference:
ApR
Promega
fnvitrogen
(240)
(240)
TcR
(115)
res-npt-res cassette in
(145)
Knockout vector,
pUC18Sfi
4.5 kb Xhol-BamPH fragment of P. fluorescens
CHAO in pGEM7Zf(+), 'estFl-alkB-praAB-ompPl
alkB gene in pGEM7, deletion clone of pPFf
ompPl fragment in pGEM7, deletion clone of pPFf
4 kb Pstl fragment of P. fluorescens CHAO in
pZeROl, ompPl-yafH'
7.8 kb fragment of P. fluorescens CHAO in pGEM7Zf(+),
'estFl -alkB-praAB-ompP 1 -yafH'
1.8 kb Munl-Sall fragment in pCom5, alkB
2.8 kb Munl-Pstl fragment in pCom5, alkB-praAB
4.0 kb Munl-Xhol fragment in pCom5, alkB-praAB-ompPl
res-npt-res cassette in Sfil-Stul sites of pPFf
pEXf8TC with Pvul fragment of pPFrnrB
Sail deletion clone of pPF20
Aspll 8-EcoRV deletion clone of pPF3f
npt-gene. of pCK217 as Xbal fragment in pPF32
praA in pKKPalk
praB in pKKPalk
praAB in pKKPalk
This
'
pPF7
pPF8
pPFHO
pPF20
pPF21
pPF22
pPF23
pPFrnrB
pEXPFrnrB2
pPF3f
pPF32
pPF32npt2
pPFf05
pPFf06
pPFf07
(151)
were
37°C. For
added to all minimal media. All cultures
growth
with «-alkanes
on
grown
«-alkanes, Petri dishes with E2 medium
supplied through
erlenmeyer with
were
«-octane
the vapour
(C8) in
a
sealed
phase (for
\A
study
This
study
study
study
study
study
study
study
study
study
study
study
study
This
This
This
This
This
This
This
This
This
This
aerobically
at 30°C
or
incubated at 30°C
by placing
an
open
container, for «-decane (C10), «-dodecane
(C12), «-tetradecane (C14) and «-hexadecane (C16) by placing
disc with 200
This
This
were
«-octane
This
study
study
study
This
«-alkane in the lid of the Petri dish.
a
Whatman 3MM filter
97
P. fluorescens CHAO and recombinants derived from this strain
medium.
«-alkanes
Liquid
alkanes
Very-long-chain
cases,
(w/v)
to
and added to
rhamnolipids (97)
improve
E. coli strains
strains
the
were
uptake
of
P. fluorescens CHAO
a
liquid
concentration of 1 %
(v/v).
dissolved to 10%
concentration of 0.2 %
long-chain
added to
were
grown with
appropriate
pillml; ampicillin (Ap)
transformed
was
a
(v/v)
(v/v)
E2
in
to the medium. In
concentration of 0.1 %
alkanes.
by electroporation according
were
12.5
piglmï), tetracycline (Tc)
a
were
Triton X-100
or
transformed
harbouring plasmids
piglmï).
added to the medium to
(C18 and longer)
dioctylphthalate (DOPh)
some
were
grown in
were
to Dower
antibiotics
(64). E. coli
(kanamycin (Km)
piglml; gentamycin (Gm)
100
according
to
H0jberg (117).
50
10
To select
transformants, kanamycin (50 piglmï), gentamycin (100 piglmï) and tetracycline (100
piglmï)
DNA
were
added to the medium.
Restriction enzymes, T4 DNA
manipulations.
fragment),
T4 DNA
polymerase
and
Diagnostics (Rotkreuz, Switzerland)
Oligonucleotides
were
PCR-products
were
purified again
over a
dideoxynucleotides
and used
as
purified
over
1% agarose
1% agarose
gel,
Plasmid DNA
according
isolated
Pure Plasmid Isolation Kit to obtain
were
sequenced
on a
cycle sequencing
(agggttttcccagtcacgacgtt)
pGEM7-Zf(+)
clones
or
and -40
PalkFwd
(gagttcggcatggggtcaggtg)
for
(IRD800-) labelled
reverse
pKKPalk
from DNASTAR
sequences
were
(5).
compared
BLAST searches
were
according
Doly (21),
enzymes,
or
to Desomer
were
High
the Amersham
Thermosequenase
-40 forward
and
for
pKKRev
plasmids (MWG-Biotech).
analyzed
and
(Madison, Wisconsin, USA).
compared using
LASERGENE
Nucleotide and amino acid
with the EMBL, SwissProt and GenBank databases
carried out at NCBI.
(58).
with the Roche
(gagcggataacaatttcacacagg) primers
derived
or
DNA. Both strands of the inserts
(tggcgcaagcgtccgattag)
Nucleotide and amino acid sequences
Navigator
isolated
sequencing grade
using
appropriate
pGEM7-Zf(+) (Promega)
to Birnboim and
Li-Cor 4000L sequencer
kit and IRD41-
was
supplier.
Switzerland.
cut with the
and cloned into
polymerase (Klenow
from Roche Molecular
the
specified by
gels,
Chromosomal DNA
DNA
were
synthesized by Microsynth, Balgach,
pKKPalk (240)(chapter 3).
was
ligase,
using
BLAST
98
PCR. PCR reactions
GeneAmp
1'
PCR
were
System
carried out
9600. The
was
visible after
verified
Cloning
by
hydroxylase
identified
isolating
restriction
fragments
were
Madison, USA)
coli DHIOB
ligated
cloned
using
To construct
the
digested
pPF8
as a
as a
plasmid pPF20, pPFl
fragment,
an
as
the
25x
When
4°C).
To clone the
resulting plasmid,
electroporation,
The
complete
clone
was
called
cultured
fluorescens
of
fragment
An
was
digested
pPFl 10
The
constructed
The DNA
was
were
were
identified and cloned
4.0 kb Pstl
over
pPF32npt2
were
LB until the
was
fragment
kanamycin
the
was
was
ligated
digested
pPF20
with
kanamycin
kanamycin.
resistance
was
A
with
a
was
AspllS
and
was
ligated
resistance. The
introduced in P. fluorescens CHAO
selected with
using
gel.
knockout mutant. Plasmid
the
by
plasmid, pPFl 10.
purified
resulting plasmid
identified
sequenced.
with BamHl and Nsil, and
and
by
and transformed into E.
the target genes
in
fragments
pGEM7-Zf(+) (Promega,
overlapping
pCK217, containing
integrants
on
a
P. fluorescens
restriction
were
Netherlands)
probe, resulting
cut from
religated.
fragment
and
in
fragments
EcoRN, treated with Klenow and religated. This plasmid, digested wiihXbal,
Xbal
product
no
template
preparative gel.
a
sites of
containing
probe.
alkB-praAB-ompPl
with Sail and
to the small
(45" 95°C,
studies the Roche
Both strands of the inserts
probes.
same
the insert of
Construction of
used
Enriched gene banks
appropriate
Transformants
pCHAO (239)
3.3 kb BamHl-Pstl
oo
flanking regions, easy-to-clone
A 4.5 kb Xhol-BamHl chromosomal DNA
of
(4' 95°C,
expression
of the desired size from
between the
(Gibco BRL).
fragment
was
genes for
pZeROl (Invitrogen, Leek,
or
used
Perkin Elmer
used. The sequence of cloned PCR
was
blotting (220).
fragments
colony blotting using
PCR
product
flanking regions.
gene and
Southern
by
kit
was
program
a
sequencing.
of the alkB gene and
CHAO alkane
were
DNA
of
pil
amplify complete
Expand High Fidelity Polymerase
(123) using
Tm), 1' 72°C), 5' 72°C,
first round of PCR, 1
a
second round of PCR. To
was
following
below
annealing temperature (5°C
described in
as
kanamycin
by
resistant
lost, resulting in P.
KOTl and KOT2. The extent of the deletion in these mutants
was
confirmed
99
by
PCR with
Hindfw
primers
(tcgaagcttcaagagcaagttgag)
and mutCrv
(ccgccgtactgcttgggaatgat).
Construction of
containing
pPF20
was
Fragments
alkB
(pPF21), alkB-praAB (pPF22)
cut with M««I and
of the
right
size
(240)(chapter 3), digested
was
expression
and PraBrv, and
with the
Southern and
and
appropriate
with the
same
fragment
used
as
agarose
dUTP
probes
gel,
using
pCHAO (239)
for Southern
1 % agarose
the DIG
gel
as
by
was
as
follows:
praB using primers
digested
fragments
using
to
completion
Molecular
phosphatase coupled
according
to Anti-DIG antibodies
chemiluminescent substrate.
was
called
pPF7 (alkB)
fragments
priming
and
were
was
The 550
method with
(Roche
carried out
using
as
the
were
1%
over a
digoxigeninMolecular
standard
colony
to the manufacturer. Alkaline
used with CSPD
bp
pPF8 (ompPl)
purified
were
with
Diagnostics,
previously (220).
and chemiluminescence detection kit
the Roche DIG-kit
PraBfw
were
were
buffer without formamide at 65°C. Detection of Southern and
earned out
praA
pKKPalk
in TAE buffer. DNA
(Roche
the random
Diagnostics, Rotkreuz, Switzerland). Hybridizations
hybridization
constructed
praAB respectively.
described
All DNA
pCom5
alkB-praAB-ompP 1
resulting plasmids
were
membrane
hybridisation.
labelling
were
and
and the inserts of
electroeluted and labelled
in
alkB-praAB fragment.
enzymes. The
positively charged nylon
from
ligated
and PraBrv. The PCR
blots. Chromosomal DNAs
on an
Xhol, respectively.
restriction enzymes and cloned in
Rotkreuz, Switzerland) by alkaline transfer
PCR
and
PraAfw and PraArv;
primers praAFw
restriction enzymes and loaded
transferred onto
and praAB
praA, praB
pPF107 encoding praA, praB
colony
gels,
with EcoRl and Sail for the alkB and
with
(240)(chapter 3), digested
pPF105, pPF106
M««I and
or
isolated from agarose
pPFl using primers
praAB
purified, digested
Sail, Muni and Pstl,
were
vectors for
from
amplified
alkB-praAB-ompPl (pPF23), plasmid
or
and with EcoRl and Pstl for the
containing fragments,
E. coli
fluorescens expression plasmids
vectors. To construct P.
expression
blots
100
Protein
About 10 pig
protein
StrataClean resin
gel electrophoresis
SDS-PAA
analysis.
was
in
Jolla, USA)
La
(Stratagene,
loading
buffer and loaded
Electrophoresed proteins
transferred to
a
according
loaded per lane. To concentrate supernatant
was
and incubated at 37°C for 30 minutes. The resin
resuspended
done
was
were
on
to Laemmli
samples,
pil
added to 0.5 ml culture supernatant,
was
collected
15 % SDS-PAA
by centrifugation,
gels.
stained with Coomassie Brilliant Blue R-250
PVDF filter membrane for
5
(149).
subsequent
or
N-terminal amino acid sequence
analysis.
For MALDI-TOF
cut from the
Coomassie stained bands
analysis,
proteins
resulting
were
mixture
subjected
was
dihydroxybenzoate
Sequence.
PraA and/or PraB
washed with 100 mM ammonium acetate, then with
gel,
with 50 mM ammonium acetate in
The
containing
as
to
a
50 % acetonitrile
to
mass
for three hours at
37°C, and the
spectrometry analysis (MALDI-TOF) using 2,5-
matrix.
The sequence of the 7.8 kb Xhol-Pstl
determined in this
acetonitrile, and
solution, and subsequently dried.
in-gel trypsin digestion
subjected
were
study
was
deposited
fragment
of P. fluorescens CHAO
in GenBank and received the accession number
AJ009579.
RESULTS AND DISCUSSION
and sequence
Cloning
fluorescens
fragment,
amplify
clone
a
protein,
CHAO
encoding
of
the
a
7.8 kb chromosomal DNA
alkB, praAB
and
ompPl
obtained earlier from P. fluorescens CHAO
internal
fragments
of alkB
4.5 kb Xhol-BamHl
an
sequence
overlapping
was
to the
homologs (239),
fragment.
4.0 kb Pstl
To
incomplete
complete
fragment
found to contain six open
named estFl is
identity
analysis
reading
due to the Xhol
lactone-specific
esterase
was
used
ORF
cloned
frames
cloning site,
(estFl)
genes. A 550
bp
of P.
PCR
using degenerate primers
was
an
fragment
as
as a
probe
encoding
well. The
an
to
which
identify
and
outer membrane
complete 7.8
(ORFs)(figure 1).
kb
The first ORF
and has 69.7 % DNA sequence
of P. fluorescens DSM50106
(135).
The
101
alignment suggests
that the N-terminal 40 amino acids of the P. fluorescens CHAO EstFl
missing.
are
Xhol
Sail
Muni
estFl
y \prafy \praB)
alkB
y
I
I
Muni
Sail
1
Pstl
Sail
Pstl
I
pPF23
%
protein
1
hydroxylase
Xhol
1
1
gene alkB is located 295
identity
sequence
fluorescens
J
1
Figure 1: organisation of the 7.8 kb Xhol-Pstl fragment encoding
the complementation plasmids pPF21, pPF22 and pPF23.
The alkane
yaflT
y
I
Muni
pPF22
ompPl
PjstI
Sail
Muni
pPF21
Xhol
Pstl
DSM50106
to the
(135)
hydroxylases (chapter 4).
partial
downstream of
sequence of
and 40-60 %
Like its
bp
protein
homologs,
the alkB gene and extent of the inserts of
a
putative
sequence
estFl, and has 64.0
alkB gene of P.
identity
to other alkane
the CHAO AlkB sequence contains the
conserved
histidines, which
active site
(233, 239). Six transmembrane helices could be identified and suggest that the
CHAO AlkB has the
CHAO alkane
likely ligands
topology
with other alkane
Comparison
fluorescens
same
are
as
the P.
for the two non-heme iron atoms in the
putida (oleovorans)
hydroxylases (chapters
hydroxylase
has
a
large
4 and
GPol AlkB
(261).
9) showed that the P.
insertion between the third and fourth
transmembrane helices.
Two ORFs located
directly
30.7 and 44.8 % sequence
PraA of P.
and
praB.
including
aeruginosa
downstream of the alkane
identity
PG201
The three Pra
to the
"protein
hydroxylase
have
no
other
homologs
PraA and PraB
aeruginosa
strongly
ORFs
were
in the sequence
the unfinished genome sequences. PraB of CHAO is
related to PraA of P.
proteins
activator of alkane oxidation"
(103) (figure 2). Therefore, these
proteins
encode
clearly
with
protein
named
praA
databases,
more
closely
PG201 than the CHAO PraA. The N-termini of both
resemble
signal peptides (SIGNALP: http://www.cbs.dtu.dk)
102
(187). The predicted cleavage site is indicated with
aeruginosa
%
PG201 PraA and P.
fluorescens
an arrow
CHAO PraB
are
GSTAP) and contain very few charged residues (7 and
2). The
CHAO PraA contains
significantly
more
charged
in
figure
2. The mature P.
rich in small amino acids
4 %
(55
DEKR, respectively) (table
amino acid residues
(18 %
DEKR).
10
20
I
30
I
40
I
fllfA
1
MKSIKSLPSFAALALCLSVSSMAS
1
M-SISPLFTAQLFVVLVGISLMGAAE
1
MKGIKTLVSATAIVACLGAASMAS
M..I..L
aIa
l_
PVNSAFT
PraA
(PG201)
RIE--PANTEF T
PraA
(CHAO)
SIAIDGANPDGPFT
PraB
(CHAO)
TIT
aIa
M..A
50
FT
60
I
70
I
37
APG-TISVSSPASLNLPVT
38
AKG
41
TPGGTITVRSPSSFN
..G
Consensus
80
I
l_
CNITFKGKTAADGSYASI
PraA
(PG201)
PISFAKSIINADCTIQVSGKVSPDGSFASV
PraA
(CHAO)
(CHAO)
P.
QPVT
S
90
CNINFSGNIA
GGVASI
PraB
C.I.
G.
Consensus
..G
100
I
.AS.
110
I
120
I
l_
73
DSVTVSGSNTLCSVPQMTGLPWKLTVSSTTAGKVDGVGFK
PraA
(PG201)
71
EKVDFSGGLK-CGQVEATHLPWKLIAKDETSGAMSGIQVT
PraA
(CHAO)
76
TGATVSGSNALCNLPKITGLPWTLSASSTTAGTVTNVGYT
PraB
(CHAO)
SG....C
T.LPW.L
130
140
I
113
T.G
Consensus
150
I
160
I
l_
SSTCGPSTVNGSWSNATNTLS-ASNQSLAGNCKI
PraA
(PG201)
110VHAPLVGGDCGPSTAEGSWNNSTGKLE-AAQVSLDGGCTI
PraA
(CHAO)
116ISF-FPATNCGPTTINVAWSNVTHSLSLSTPQTLSGGCSV
PraB
(CHAO)
IL
CGP.T....W.N.T..L
L.G.C..
Consensus
170
I
148
NSLSVKPTPAFVVNP
PraA
(PG201)
149
KTVSIQMPPTFRVAP
PraA
(CHAO)
155
DVLNAKPSPQVSVI
PraB
(CHAO)
P
Figure
alignment
2:
.
.
.
of the P.
V
.
aeruginosa
Arrows indicate the
sequences
predicted cleavage
determined in this study.
The fifth ORF,
family.
A
encoded
PG20f PraA with the P.
site of the
designated ompPl,
to several
identity
Consensus
.
prepeptide.
encodes
Haemophilus influenzae
a
peptide
has weaker sequence
outer membrane
%
downstream of the pra gene in P.
homology
to FadL
CHAO PraA and PraB.
are
the N-terminal
which has 23-25 % sequence
highly homologous hypothetical protein (68
immediately
fluorescens
The underlined sequences
proteins
peptide
of the
sequence
aeruginosa
PAOl
OmpPl
identity)
is
(249). OmpPl
(21 % sequence identity), the long-chain fatty
103
acid
uptake protein
of E. coli
(22), and
to
a
set of
aromatic catabolic opérons, which include TodX
SalD
TbuX
(125, 127, 274).
by
(21 % sequence identity), TbuX and
shown to be involved in
was
encoded
hypothetical proteins
uptake
of toluene into the cell
(127).
Table 2: amino acid
PraA
composition
(P. fluorescens CHAO) and
Basic
Acidic
of the mature
PraB
peptides of PraA (P. aeruginosa PG201),
(P. fluorescens CHAO).
Polar
Apolar
KRDEHNSTYQCGAVLI
MFWP
PraA(PG20I)
7
1
PraA
(CHAO)
PraB
(CHAO)
0
3
0
23
18
1
2
4
11
12
13
9
926724
14
11
0
5
4
15
14
11
692529
2
20
20
1
3
4
15
12
12
8
13
0
The outer membrane of
LPS, and constitutes
CHAO
OmpPl
membrane
alkanes
has
a
0
11
1
12
Gram-negative
barrier for
homology
to
a
product
to the
fatty
Strong
2
5
0
12
and
fluorescens
the outer
over
similar function in the transport of
10
2
phospholipids
As the P.
FadL, which transports fatty acids
of the
long-chain
sixth, incomplete, ORF directly downstream of ompPl, has
hypothetical protein
YafH of E. coli
very-long-chain acyl-CoA dehydrogenases
chain
of
10
4
the outer membrane.
The translation
homology
mainly
hydrophobic compounds (190).
(22), OmpPl may have
over
bacteria consists
6
acids
(24) and
to several mammalian
involved in the metabolism of
very-long-
(7, 52).
inverted repeats
located
are
directly
downstream of estFl and
ompPl
but
are
not
present downstream of alkB, praA, and praB. Therefore, alkB, praA, praB and ompPl
may be transcribed from
We did not succeed in
a
single transcription
cloning
site upstream of alkB.
the P. fluorescens CHAO rubredoxin and rubredoxin
reductase genes, the electron transfer components of the alkane
PCR with
highly degenerate primers
for rubredoxins
(T.
hydroxylase system using
H. M. Smits and J. B.
van
Beilen, unpublished results). However, the genome sequence of another P. fluorescens
strain
(Pf0-1)
though
does contain
it does not possess
homologous (around
80
a
rubredoxin and
an
%)
alkane
a
rubredoxin reductase gene
hydroxylase homolog.
to genes encoded
by
the P.
These genes
aeruginosa
homolog,
are
PAOl
even
highly
genome(249),
104
which
complement
deletions of the P.
rubredoxin reductase genes
GPol rubredoxin and
putida (oleovorans)
(chapter 6).
Alkane-solubilizing compounds
in the
of P. fluorescens CHAO cultures.
supernatant
P. fluorescens CHAO possesses two genes related to the so-called
alkane oxidation" PraA of P.
which is found in
large
strains
aeruginosa
aeruginosa
amounts in the culture
(113).
A 16 kDa band
(figure 3A),
not
shown).
but
was
was
were
protein
is
extracellular
an
fluorescens
concentrated and
not detected when CHAO
was
analysed
grown
Addition of 1 ml culture supernatant of
for the presence of these
a
citrate
on
J
]
40
\
20-
on
or
filter sterilized
«-alkanes
dodecanol
(data
hexadecane-grown
B
100^
60
protein,
CHAO also excretes Pra
visible in supernatants of cultures grown
80
activator of
supernatant of hexadecane-grown P.
To determine whether P.
proteins, supernatant samples
proteins.
PG201. This
"protein
s
s?
!"-
CD
N.
|
j;
ilklilUJuJAj.
1000
! S
2000
60000
mix
C
lf211
50000
40000
30000
20000
-I
-
10000^
1
15000
10000
Figure
3:
Analysis
of supernatant from
A: 15 % SDS-PAGE of extracellular
sample
analysis of the f6
of the protein (panel C).
CHAO:
CHAO grown
B: HPLC-MS
mass
C: Molecular
mass
on
hexadecane-grown P. fluorescens CHAO.
proteins from hexadecane-grown P. fluorescens
hexadecane. The
kDa
protein.
arrow
Asterisks indicate the
determined from the LC-MS spectrum. The
calculated molecular
weight
of PraB after
removing
the
CHAO. M:
marker,
indicates the 16 kDa band.
signal
peaks
peak
used to determine the molecular
at f42f f Da
sequence
corresponds
(14213 Da).
to the
105
culture P. fluorescens CHAO to
time and
an
increased
growth
a
fresh 50 ml culture resulted in
rate
(8
32 hrs.
vs.
lag-time
The addition of culture supernatant from citrate-
or
7
,
clearly
a
10 hrs.
vs.
dodecanol-grown
reduced
lag-
doubling time).
P. fluorescens
CHAO cultures did not have this effect.
The N-terminal sequence of the 16 kDa band
PraB sequence,
after the
sequence. To
investigate
just absent,
HPLC-MS. PraA
molecular
predicted signal peptidase recognition
was
not detectable
weight (14211 Da)
weight
Heterologous expression
were
minimal medium
these methods either
of PraB measured
of the mature
by
(figure
based
on
«-alkanes,
we
constructed
not influenced
lower
by
whether
3D). The
well to the
praA, praB
and
Introduction of these
pKKPalk (240)(chapter 3).
praAB
plasmids
(200) allows expression from the P. putida (oleovorans)
containing glycerol
clearly
3B and
corresponds
HPLC-MS
or
PraA
protein (14213 Da).
as
the carbon
induction with 0.05 % DCPK, E. coli
PraB showed
underlying
growth
were
grown
on
source.
JM101::alkS-Km[pPF106] expressing only
rates. The
growth
rate of the other two recombinants
the addition of DCPK. SDS-PAGE
analysis
of the culture
supernatants of all recombinants showed that in cultures expressing the praA gene and
the
praAB
genes,
a
16 kDa
protein
The N-terminal sequence of this
was
prominent (up
protein
PraA at the
predicted position (sequence
fluorescens
CHAO
to 10 % of
indicating lysis
supernatant proteins).
confirmed the
cleavage
of
underlined in
figure 2).
In contrast to the P.
supernatant, many additional proteins
culture supernatants,
in
MALDI-TOF and
analysed by
promoter after induction with DCPK. All three strains
GPol alkB
(underlined
of PraA and PraB in E. coli JM101::alkS-Km. To determine
in E. coli JM101::alkS-Km
was
samples
by
whether PraA and/or PraB solubilize
expression plasmids
an
site
whether the N-terminal sequence of PraA is blocked
culture supernatant
calculated molecular
Upon
to amino acids 26 to 55 of the
The N-terminal sequence data did not show evidence of
figure 2).
PraA is
exactly
corresponds
of induced cells.
were
a
signal
sequence in
observed in the E. coli
106
A
praA
praB
praAB
+
B
Pure
M PraB
+
227516
t
t
1922 78
2291 21
2027 86
25^
I
|«ppüi|
2366 25
^VMw*UwWv'
*mm
c
2027 54
^W^to^wwww»
WvV*
Figure
4:
expression
A: f 5 % SDS-PAGE
of PraA, PraB and PraAB in E. coli JM101 : :alkS-Km
gel
of supernatant
samples. Induced (+) and not-induced (-) samples were applied.
respectively. M: marker
after trypsin treatment. Peaks indicated by an arrow correspond to
Boxes indicate PraA, PraB and PraAB
analysis of PraA
fragments expected from PraA.
C: MALDÎ-TOF analysis of PraAB after trypsin treatment. Peaks indicated by an arrow correspond to
fragments expected from PraA. The peak indicated by an asterisk correspond to a fragment expected from
B: MALDI-TOF
PraB.
D: Emulsification test of
supernatants containing PraA, PraB and PraAB. Negative control is
a
supernatant
of non-induced cells.
In
supernatant of cultures expressing PraB only
but N-terminal
MALDI-TOF
sequencing
in E. coli
faint band could be
and MALDI-TOF confirmed the
analysis (figure 4C)
simultaneously
a
identity
showed that PraA and PraB
JM101::a/fcS'-Km[pPF107]. However,
were
seen
(figure 4A),
of this
protein.
expressed
the relative amounts
107
could not be determined. These results show that E. coli processes and exports PraA
well
PraB,
as
as was
In emulsification
determined.
found for PraA of P.
a
Supernatants containing
fast
PG201
(103).
experiments (59), the emulsification ability of the protein solutions
stable emulsification of
showed
aeruginosa
either PraA, PraB
or
sites
(145),
hexadecane, while negative controls (non-induced samples)
phase separation (figure 4D).
was
in
was
both PraA and PraB all showed
Construction of alkB knockout mutants. A knockout mutant of P.
(chapter 4)
as
constructed
by inserting
a
kanamycin
fluorescens
resistance gene, flanked
the alkB gene. This mutant, named KOB2, did not grow
on
CHAO
by
two
C12-C16
res
«-
alkanes.
—O— CHAO
—A—KOB2
o
—A—
KOB2[pPF21
—•—
KOB2[pPF22]
——
KOB2[pPF23]
5
in
t
Q
O
4
3
-
300
Figure 5 growth curves
andKOB2[pPF23]
of P. fluorescens CHAO, KOB2 and recombinants
KOB2[pPF2f ], KOB2[pPF22]
108
To test whether the knockout mutant could be
KOB2
was
AlkB and
(encoding
plasmids
equipped
restored
containing pPF21
pPF22
or
with the
pCom5
growth
was
«-alkanes. However, the
on
This is
probably
(145)
genes, which suggests that the
observation that KOB2 recombinants
growth
least under these
lag-time,
growth
rates
are
is in accordance with the results of
of
CHAO and
alkB-praAB
gene
were
experiments
from the
polar
on
the
resulting
and
compared
a
polar
effect of the
and
containing pPF22
play
not
a
effect
were
on
the
affected
et
al.
region
in
transcriptional
alkB-parAB
operon. The
have almost identical
role in alkane
degradation,
expression
of the
as
only
praAB
at
of the pra-genes
bp upstream
132
expression
of the alkB
incomplete.
genes for
the
the resolvase gene
using
expression
could be due to limited
may therefore be
alkB deletion mutant KOB2A1
with CHAO and
60-100
hours),
168 after
(estimated
containing
(103). The difference between the
KOB2[pPF23]
or
was
KOB2[pPF21].
complementation
kanamycin
resistance gene
(145), expressed
transformed with
The
200
the
h).
polar
As the
lag-time
growth
from the lac-
inoculation, but
to be tenfold less
KOB2[pPF21] only
at t
=
still is
not in
was
longer
than that of CHAO
completely.
at time
PraB
was
points
t
=
120
A very weak PraB band
strong than for CHAO and KOB2Al[pPF21])
216 and later
were
much shorter than that
KOB2Al[pPF21]
KOB2[pPF21].
pPF21 (chapter 4),
rates of the three strains
effect may not be eliminated
detected in the culture supernatant of CHAO and
=
an
pPF23
the reduced
by
hydroxylases (chapter 4),
removed
KOB2[pPF21] (around
and t
All three
of the downstream encoded
part of
are
genes
similar, but the lag-time of KOB2Al[pPF21] (around 100 h)
(around
OmpPl).
plasmid pUCPParA.
The
of
«-alkanes,
of the recombinant
lag-time
expression
pCom5-derived plasmids,
with other alkane
and its terminator
promoter
praAB
Hardegger
KOB2[pPF22]
cloned. The promoter
To reduce the
on
on
circumstances, and for the tested alkane, hexadecane. The fact that the
but not the
wild-type
due to
suggests that the ompPl gene does
curves
PraAB and
than that of the recombinants
significantly longer
pPF23 (figure 5).
growth
plasmids pPF21 (encoding AlkB), pPF22
PraAB) and pPF23 (encoding AlkB,
terminator in the res-npt-res cassette
praAB
derived
for
complemented
timepoints (data
not
shown).
was
observed
109
Figure 6: growth rates
fluorescens CHAO and
0 180 !
0 160
_
é.
S
KOB2Af
0 140
on
of P.
alkanes.
Upper panel: growth rates
long-chain alkanes. Cl2:
0 120
0 100
0 080
on
ICHAO
dodecane; C16: hexadecane;
I K0B2A1
TX100: Triton X-100.
c
I
panel: growth rates
very-long-chain alkanes,
Lower
0 060
(3
0 040
on
-
added
as
10 % solution in
0 020
dioctylphthalate (DOPh).
0 000
C18:
octadecane; C20:
eicosane; C24: tetracosane;
C28: octacosane.
Substrate
ICHAO
I K0B2A1
C18
in
DOPh
C20inDOPh
C24inDOPh
C28
in
DOPh
Substrate
Characterization of P. fluorescens KOB2A1:
the absence of additives to
not grow
on
improve growth
any of the alkanes
a
second alkane
which
tested, However, when KOB2A1
rate ten times lower than that of CHAO
are
solid at
supported growth
dioctylphthalate.
with
increasing
data
we
30°C, do
not
some
grown
was
observed, with
alkanes
on
a
(C18-C28),
support growth of CHAO. However, these alkanes
The difference in
length,
growth
was
(figure 6). Longer
of both KOB2A1 and CHAO when
chain
In
«-alkanes, P. fluorescens KOB2A1 does
on
hexadecane in the presence of 0.1 % Triton X-100,
growth
hydroxylase system?
and
growth
they
were
dissolved to 10 % in
rates between CHAO and KOB2A1 decreased
disappeared
for C24 and C28
conclude that P. fluorescens CHAO possesses
a
(figure 6).
From these
second alkane oxidation system,
110
which oxidizes
they
are
alkanes. These alkanes
made accessible to CHAO, for
organic phase.
alkanes,
very-long-chain
or
Attempts
soil, they may be accessible because they
In
to clone the second alkane
alkane
hydroxylase primers
signals
other than that
% sequence
analysis (239),
identity
CHAO alkB gene
(data
not
C-sources if
as
the alkanes in
occur
using
gene
(239)
were
inert
an
with shorter
together
hydroxylase
PCR with the
degenerate
not successful. As
to the cloned CHAO
the second alkane
to the alkane
be used
solubilizing compounds.
hydroxylase
described before
corresponding
only
example,by dissolving
because CHAO excretes different
Southern blot
can
alkB gene
hydroxylase
genes used
as
were
specific
no
found
during
gene must have less than 70
the cloned
probes, including
shown).
ACKNOWLEDGEMENTS
The authors wish to thank Martina
sequencing,
Armengaud,
Stefanie
IBS
Röthlisberger,
Balada, ETH Zürich, Switzerland for technical assistance, Jean
Grenoble, France for MALDI-TOF, Michael Kertesz, ETH Zürich,
Switzerland for his
gift
of P. fluorescens CHAO, Rene
Switzerland for N-terminal
Switzerland for his
help
sequencing
with LC-MS.
and Vittorio
Special
037023.
in
Biotechnology
Brunisholz, ETH Zürich,
Ravellino, Hewlard Packard,
thanks for
White, NRC, Montreal, Canada. This research project
Priority Project
Zürich, Switzerland for DNA
ETH
helpful
was
of the Swiss National
discussion to
supported by
Lyle
the Swiss
Foundation, project
nr.
5002-
Ill
Appendix
to
chapter
7:
Influence of alkB knockout mutations
fluorescens
Theo H.M.
CHAO:
initial
an
on
disease
suppression by Pseudomonas
study
Smits, Stefanie B. Baiada, Monika Maurhofer, Geneviève Défago, Bernard
Witholt and Jan B.
van
Beilen
INTRODUCTION
Biocontrol is the
suppression
rhizobacteria which
disease
(at the
protection,
root
tips),
effectively
Soils
compounds (99).
colonize roots and
containing
right
root diseases
fungal pathogens causing plant
produce
such rhizobacteria
are
the biocontrol strain must be present
at the
sufficient numbers
of
time
(before
the
(105-106 colony forming
pathogen
extracellular
called
on
units per gram
antifungal
suppressive
the roots at the
causes
certain
by
too much
soils. For
right
location
damage)
and in
soil).
One of the best characterized biocontrol strains is Pseudomonas fluorescens CHAO
This
strain, isolated from suppressive soil in Morens (Switzerland), is
colonizer and
such
as
produces
a
wide range of
secondary
extracellular metabolites have
fluorescent
fluorescens
CHAO
which lost the
The
to grow
were
proteins
on
are
able to grow
gene
Alkane
alkanes
still able to grow
encoded
and references
antibiotic function
hydroxylase
(chapter 4, 7).
ability
(C16), while they
an
pseudomonads
characterized the alkane
longer).
effective root
antifungal properties,
2,4-di acetyl phioroglucinol, pyoluteorin, pyrrolnitrin, hydrogen cyanide, and
phenazines (e.g. phenazine-l-carboxylate)((99)
Many
metabolites with
an
(250).
by praAB
(alkB)
as
on
on
and
(183, 188).
flanking regions
from dodecane
ompPl,
from P.
knockout mutants
very-long-chain
and
but many other
«-alkanes. We isolated and
hydroxylase
ranging
well
therein),
which
(C12)
alkanes
are
were
generated,
to hexadecane
(octadecane
and
located downstream of
112
alkB, could play
a
role in solubilization and
been proven
unequivocally (chapter 7).
Alkanes
ubiquitous
waxes
are
in natural
environments,
(212). These alkanes could be
involved in biocontrol. To test this
and the alkane
ability.
Here
hydroxylase
hypothesis,
hydroxylase negative
we
possible
a
of
uptake
alkanes, although their role has
they
as
carbon
are
for
source
assayed
we
natural components of
not
plant
bacteria
rhizosphere
P. fluorescens strains CHAO
mutants KOB2 and KOB2A1 for their biocontrol
report first results, which indicate that inactivation of the alkane
gene has
significant
a
effect
on
the biocontrol
ability
of P. fluorescens
CHAO.
MATERIAL AND METHODS
P. fluorescens strains CHAO, KOB2 and KOB2A1
7). Tobacco and wheat plants
containing
or
artificial soil
inoculated with the
percentage of
measured
as
a
fungus
which
plants
causes
were
(133). After harvesting, the plants
the average amount of
infected; 2
main root
stem
strongly
(134) in the presence
pathogen
(134) and wheat plants
tritici, which
var.
were
rating
colony forming
assayed
for wheat and G.
graminis
var.
=
10-25% of roots
infected; 4
base; 6
=
=
>
infected; 3
50% of roots
black,
Lesions all around stem and
chlorotic and
severely stunted;
8
=
=
tritici
0
25-50%
or
=
as
No
or
weight,
Plant dead
described in Keel et
=
<
10%
roots black and at least
chlorosis of
or
was
experiment.
symptoms; 1
all main roots
starting
were
take-
causes
for fresh
units per gram soil per
(133) has been used for scoring the Ggt disease index:
one
gnotobiotic system
inoculated with the
black root rot
(250)(Chapter
root infection and bacterial root colonization. Root colonization
A modified disease
of roots
a
of quartz sand and vermiculite
pathogens Gaeumannomyces graminis
all desease in wheat
on
composed
described before
grown for three weeks in
absence of biocontrol strains. Tobacco
Thielaviopsis basicola,
al.
were
were
nearly
infected; 5
leaves; 7
dead.
=
=
Lesion
Plant
113
Table 1: effect of alkB knockout
pathogen
T. basicola
Microorganisms
on
biocontrol
P. fluorescens
ability by
against
tobacco
.
added
Plant fresh
Root infection
weight
Bacterial root
colonization
P.
T. basicola
fluorescens
(mg)
537
(%)
(x 108 cfu/g soil)
0
a
0
none
+
126 d
89
a
0
CHAO
+
391b
17 b
4.3
KOB2
+
215
c
53
11.5
KOB2A1
+
253
c
46c
none
-
c
4.9
a
a
a
Data of two
experiments were pooled. The means of twenty repetitions (ten répétions per experiment,
plant per repetition) are presented. Means in the same column followed by the same letter are not
significantly different at P 0.05 (Fisher's protected LSD test, Systat).
one
=
RESULTS AND DISCUSSION
Two
gnotobiotic
by
systems
were
used to assay the biocontrol
strains CHAO, KOB2 and KOB2A1
fluorescens
caused
test
the
pathogen Thielaviopsis
Geaumannomyces graminis
var.
wild-type
show
significantly
knockout
on
reduced
the
clearly
plant
protection.
mutation, which would point
fresh
on
separately.
average is
In table
wheat
the soil of 10
As this results in
able to reduce the amount of infected roots
weight,
Root colonization
to
a
not to be affected
role of the alkB gene in
plants,
two data
seems
are
based
on a
in
shown. Here, similar results
mutant KOB2 has
KOB2A1.
the
biocontrol, but it has
single point
and is not measured for each
points
by
total, the standard
per
plant
error
of this
quite large.
assay: both mutants have
polar
only
while both alkB knockout mutants
2, the results of the P. fluorescens biocontrol experiments
are
by
black root rot of tobacco is shown in
to be noted that the root colonization measurements
experiment, by pooling
root rot of tobacco
tritici.
strain CHAO is
and the effect of infection
(133, 134): black
of the P.
basicola and take-all disease of wheat caused
The effectiveness of P. fluorescens biocontrol
table 1. The
ability
a
a
can
be observed
lower biocontrol
significant
ability
as
take-all disease of
with the tobacco biocontrol
than the
lower biocontrol
on
wildtype. Interestingly,
ability
than the
non-polar
the
mutant
114
Table 2: effect of alkB knockout
graminis
var.
tritici
added
Microorganisms
P.
fluorescens
G.
by
on
biocontrol
ability against
the wheat
pathogen
G.
P. fluorescens.
graminis
Plant fresh
weight
Disease index
Bacterial root
(see M&M)
colonization
(x 108 cfu/g soil)
(mg)
\ar.
tritici
a
Ö
none
+
1930 d
4.2
CHAO
+
KOB2
KOB2A1
none
2550
-
Ö
a
0
2435 ab
3.2 d
1.7
a
+
2103 cd
3.9 b
2.0
a
+
2259 be
3.6
2.6
a
c
Data of three
per
experiments were pooled. The means of f 2 repetitions (4 repetitions per experiment, 5 plants
repetition) are presented. Means in the same column followed by the same letter are not significantly
different at P
0.05
=
(Fisher's protected
LSD test,
Systat).
The behaviour of the P. fluorescens strains in the biocontrol assays
several
•
can
be
explained by
hypotheses:
Plants excrete
exudates
can
example,
a
number of
compounds,
known
as
root exudates
be divided in water-soluble and water-insoluble
excretes around 20 % of its total assimilated
(174). These
root
compounds. Wheat,
for
carbon, 75 % of which is
water-extractable, and 25 % water-insoluble. The water-extractable fraction contains
carbohydrates (sugars), organic
fraction
not characterized. In the
was
the bacteria
added.
acids and amino acids
during
Possibly,
degraded by
root colonization
gnotobiotic system,
are
root
lost the
the bacteria. However,
plant
wax
seems
ability
to grow
on
the
not to be affected
same
order of
magnitude.
«-alkanes
C12-C16 n-alkanes
by
units per gram soil for the
forming
as no
only
the alkB
are
carbon
extra carbon
sources
as an
«-alkanes, that
deletion,
wild-type
as
are
mainly very-long-chain
mutants
(chapter 7). Furthermore,
important
for
sources are
(212), while the alkB knockout
colonization of the roots, which is described
(161),
exudates,
the
the insoluble fraction of the root exudate contains
alkanes in the range between C25 and C37
only
(174), but the insoluble
the
factor for biocontrol
the numbers of
colony
and both knockout mutants
are
within
115
•
The alkane
a
role in the
metabolites. However, the
important secondary
metabolites has been elucidated
a
function for the alkane
Degradation products
production
of
hydroxylase
of the
%)(99).
for
biosynthetic pathways
We observed similar and
alkB mutants. However, the
enhanced
larger
pathways
long
time
on
antifungal secondary
KOB2 and KOB2A1 still
as
2,4derive from
antifungal compounds
of the
levels of the
a
block in
metabolites
secondary
ability (generally
up to 30
ability by
the two
and
laboratory experiments,
additional mutation in
an
are
ability.
important
metabolites
produce
be excluded. However, the
highly complex
significant
A
as
general
and not
fully
a
pathway
or
trait of P. fluorescens strains
instability
of the
gacA/gacS regulatory
for the
global regulators
(154). Laboratory experiments
antibiotics that
are
regulatory
production
under control of the
so
that
a
of
showed that both
gacA/gacS
gacA/gacS
cascades of P. fluorescens CHAO
mutation
are
understood.
difference in the effect of the two alkB knockout mutants in the wheat
inserted in the alkB gene. This terminator affects the
downstream of
alkB, such
the solubilization and
as
uptake
the
of
praAB-ompPl
long-chain
expression
genes, which
alkanes
or
cassette
of genes located
are
(chapter 7).
putative very-long-chain acyl-coA dehydrogenase,
further
are even
regulatory
system is correlated with the presence of the terminator in the res-npt-res
of the
can
antifungal compound pyoluteorin
regulatory system (M. Maurhofer, unpublished results),
test
precursors for the
as
losses in biocontrol
rich medium is the
system (65). GacA and GacS
A
unlikely.
(M. Maurhofer, unpublished results).
cultivated for
•
is
It has been described that
not affected in
cascade which affects the biocontrol
can
for the most
((99) and references therein),
loss of the biocontrol
even
are
The mutant strains could have
the
all other
production
production
the alkB knockout mutants
by
in these
((99) and references therein).
partial (often insignificant)
cause a
(some of) the
(some of) the antifungal secondary metabolites, such
other precursors
one
of
biosynthetic pathway
of alkane oxidation could be necessary
diacetylphloroglucinol (75, 133). Nearly
•
biosynthesis
antifungal secondary
and
•
could have
hydroxylase
possibly
Even the
involved in
expression
other genes located
downstream, could be affected by the inactivation of alkB.
116
CONCLUSION
Based
on
the results of this
study,
influences the biocontrol
ability
chemical
plant
composition
of
we can
and/or
yet explain why the mutation in alkB
of P. fluorescens CHAO to the observed extent. The
root exudates needs to be
measurements would have to be made for
fluorescens
not
fungal pathogen.
plants
determined, and similar
grown in the presence of either P.
A role for PraAB
or
OmpPl
can
not be excluded at
this stage, and knockout mutants will be useful to show the effect of these deletions
biocontrol. As
second alkane
a
hydroxylase system
is present in P. fluorescens CHAO, it
will be necessary to clone the gene and construct knockout mutants
wild-type
CHAO
clearer view
biocontrol
on
as
in the mutant KOB2A1. These
experiments
the influence of inactivation of the alkane
ability
of P. fluorescens CHAO.
on
as
well, both in the
may in the end
hydroxylase
genes
give
on
the
a
117
Chapter
8
FACTORS INFLUENCING THE SUBSTRATE RANGE AND
ACTIVITY OF THE ALKANE HYDROXYLASE OF
PSEUDOMONAS PUTIDA
Theo H.M.
(OLEOVORANS) GPol
Smits, Bernard Witholt and Jan
van
Beilen
118
SUMMARY
Intrinsic and extrinsic factors such
of P.
hydroxylase
rhamnolipids,
enhance
induction of the
effect
on
putida (oleovorans)
growth
alk-system by
product
«-dodecane. In
accumulation rates of the
substrates. However,
GPol and
an
expressed,
In five
at
host
strain, substrate
on
to grow
on
«-tetradecane
substrates, but
independent
55 to
a
mutants,
serine
or
in in vivo
(TGG -> TCG)
or
positive
assays with
activity
putida (oleovorans)
hydroxylase
Therefore,
we
C14 and C16
on
mutation
single point
a
are
genes
tested
a
the GPol alkB gene. It also does not
mutants able to grow
a
«-alkanes, and also enable
do not allow P.
«-hexadecane.
containing
as
addition, rhamnolipids have
E. coli recombinant in which the GPol alkane
these
position
Biosurfactants, such
alk-system
rhamnolipids
recombinant P. fluorescens strain
on
GPol.
of all recombinant strains
high logP
grow
regulation, uptake,
and downstream metabolism influence the apparent substrate range of the
solubility
alkane
as
changed
the
cysteine (TGG ->
can
be selected.
tryptophan
TGC
or
residue
TGT) residue.
INTRODUCTION
The substrate range of the P.
putida (oleovorans)
system has been investigated in detail, but it is
determined
the host
of
by
the dimensions of the substrate
(TF4-1L) alkane hydroxylase
not clear to what extent it is
binding
site
or
by
factors
depending
on
strain, substrate solubility, uptake and regulation. Besides the hydroxylation
aliphatic
and
been shown to
alicyclic compounds (173, 259),
catalyse:
epoxidation
of branched
of terminal olefins
Some of these
compounds
putida (oleovorans) GPol,
dodecane,
a
induce the
methyl ethers;
because
they
even
has
corresponding
sulfoxidation of
as
carbon
substrate for the alkane
putida (oleovorans)
hexadecane
sources
do not induce the
alk-genes efficiently (248, 283).
tetradecane, and
hydroxylase system
thioethers, and
(131, 132, 171, 172).
cannot be utilised
reasonably good
substrate for P.
the alkane
the oxidation of terminal alcohols to the
aldehydes; demethylation
growth
GPol
(173), which
same
were
the
alk-genes.
wild-type
For
hydroxylase (150),
GPol because this
The
by
example,
is not
compound
P.
a
does not
could also be true for
oxidised at around 22 and 3
%,
119
respectively,
of the
activity
with «-octane, based
on
the
other cases, the downstream metabolism cannot handle the
oxidation reactions. For
instance, in
contrast to
of the initial
products
ethylbenzene,
substrate, substituted ethylbenzenes and isopropylbenzene
allow
of NADPH. In
disappearance
are
which is
a
growth
oxidised, but do
not
growth (87).
A third factor is the solubilisation and
and E. coli recombinants
(69) do
not
probably
produce
equipped
surfactants
with the P.
because medium-chain alkanes
cytoplasmic
alkanes, which
must be solubilised
studied biosurfactants
are
(reviewed by (193)).
concentration
(around
40
membrane
the
of alkanes. P.
simply
on
rhamnolipids
growth
alk-system
alkanes,
this is not true for
The most
by (bio)surfactants (28).
enhances
GPol
traverse the outer membrane and
(245). However,
Addition of
GPol
medium-chain
rhamnolipids, produced by
mg/1)
putida (oleovorans)
putida (oleovorans)
(223), but grow well
diffuse into the
strains
uptake
of P.
long-chain
thoroughly
Pseudomonas
aeruginosa
above the critical micelle
aeruginosa
on
«-alkanes
(109,
290).
In this
study,
we
test the influence of
rhamnolipids
on
the
growth
rates of the wild-
type P. putida (oleovorans) GPol and the recombinant host strains E. coli GEcl37
and P. fluorescens KOB2A1
(oleovorans) GPol,
on
expressing
the alkane
of P.
putida
to determine the effect of the extrinsic factors mentioned above
the apparent substrate range of the alkane
AlkB mutants with
hydroxylase
expanded
an
hydroxylases.
substrate range
by
In
selection
addition,
on
longer
we
obtained
«-alkanes.
MATERIAL AND METHODS
Bacteria and
growth
conditions. P.
GEcl37[pGEc47] (69)
were
used in this
(220).
12.5
study.
To select for
piglmï
For P.
Recombinants
and P. fluorescens
LB with
pGEc47
appropriate
in E.
were
antibiotics
coli, tetracycline
grown in 50 ml baffled
10
piM CaCl2
of E. coli GEcl37, thiamine
was
GPol
(228), E. coli
KOB2Al[pCom8B (GPol)] (chapter 4)
was
fluorescens KOB2A1, gentamycin
(220) supplemented with
case
putida (oleovorans)
added to
a
precultures
concentration of
used at 100
piglmï
flasks with either M9 medium
piM FeS04
a
used for
added to
was
Erlenmeyer
and 10
was
or
E2 medium
(151).
concentration of 0.001 %
In the
(w/v).
As
120
carbon sources, 1 %
(C14)
hexadecane
or
the medium.
% w/v. For
rpm in
used, supplied via the gas phase
Rhamnolipids
were
added to the medium in
optical density
described before
cell
OD450
was
measured
Methods for
(chapter 4).
experiments.
with 10
supplemented
overnight preculture
alk-system
0.1 %
%
substrate at
For in vivo alkane
were
piM CaCl2
The cell
suspension
and 10
was
sequencing
piM FeS04
in 50 mM
taken out
were
were
was
acidified to
0.01 % 2-octanol
glucose
used.
respectively
a
Subsequently,
harvested three hours
incubated in 10 ml
added to
Pyrex
a
10 mM
tubes with 1
concentration of
after 1, 2, 4 and 21 hours and frozen
pH
as
«
2, and products
internal standard.
were
extracted with 1 ml
Samples
(Chrompack, Middelburg,
separate the extracted compounds,
Gas
was
were
were
an
and incubated at 30°C in
tetracycline,
Na-phosphate buffer, pH 7.0,
were
(220)
inoculated with 2 ml of
was
were
capillary gaschromatograph equipped
column CP-Wax CP52
increment of 10°C per
measurements, E.
extracted.
Fisons HRGC MEGA 2 series
analysed using
hydroxylase activity
source, 0.5 %
Portions of 1 ml
were
were
containing
conditions.
pellet
sequencing pCom8B (GPol)
30°C, 200 rpm. Rhamnolipids
Tubes
samples
capillary
spun down at 15000
was
grown in M9 medium. 200 ml M9 medium
of the strains in LB with
glucose.
until the
hexane
liquid
isolation and DNA
Primers used for
resuspended
MgS04,
(w/v).
end concentration of 0.1
the alkane and alkane-surfactant
including
induced with 0.05 % DCPK. Cells
was
after induction and
0.01 %
added to
(280).
plasmid
rotary shaker for 8 hours. As carbon
(v/v)
directly
or
pKKrev (240).
coli strains GEcl37 and K19
the
an
removed. After addition of 0.5 ml water, the cell
manipulations.
PalkFw3 and
measurements, 1 ml culture
and 0.5 ml supernatant
and the
resuspended
(C8), decane (CIO), dodecane (C12) tetradecane
were
were
Resting
octane
(C16)
microfuge,
a
micelles
DNA
(v/v)
a
The
was
with
a
To
to 200°C with
used. Products
Chromatography-Mass Spectroscopy (GC-MS)
on a
fused
Netherlands).
temperature gradient from 60°C
minute, after 2 minutes 60°C,
analysed
were
under the
same
an
121
Chemicals. All linear
Fluka. DCPK
extraction
alkanes, alkenes,
from Aldrich.
was
procedure
from P.
and /?-cymene
cumene
Rhamnolipids
aeruginosa
gift
were a
PG201
was
obtained from
were
from Dr. Urs Ochsner. The
described before
(97).
RESULTS AND DISCUSSION
Influence of
rhamnolipids
Rhamnolipids
alkanes.
aeruginosa
on
alkanes
are
on
the
growth
biosurfactants which facilitate the
ranging
rhamnolipids promote growth
from C16 to C20
of
a
by
and C12, C14 and C16
on
grown
the addition of
alk-system (96)
to
experiments,
are
whether
study
oxidising
strain
on «-
C8, CIO and C12 supplied via
dicyclopropylketone (DCPK),
guarantee that the alk-genes
To
on
of Pseudomonas
added to the medium
directly
absence of the biosurfactant. In these
or
presence
induced
phase,
was
alkane
length
GPol
growth
(109, 289, 290).
medium-chain
alkanes, P. putida (oleovorans) GPol
the vapour
putida (oleovorans)
of P.
a
the
alk-genes
gratuitous
to the
expressed
(table 1)
in the
were
inducer of the
same
level in all
experiments.
Growth of P.
the medium
putida (oleovorans)
was
significantly
GPol
on
C8-C12 vapour and C12 added
faster in the presence of
absence. However, the biosurfactant did not allow P.
grow
on
C14 and C16. As C12-C16 1-alkanols
this may be due to either
The
growth
rates of P.
an
putida (oleovorans)
somewhat lower than the
1.5
uptake problem
growth
factor for
growth
on
higher growth
Effect of
an
strain,
on
both C8 and CIO vapour
mass
as a
second
phase (C8:
transfer of C8 and CIO is
Apparently, rhamnolipids
to the
were
liquid phase
a
facilitate
and/or the
cells, resulting
rate.
rhamnolipids
absence of
phase
GPol to
substrates for this
rates found with C8 and CIO
transfer of alkanes from the vapour
a
GPol
alkane vapour.
than in its
putida (oleovorans)
growth
to
the substrate range of AlkB.
h., CIO: 2.2-2.5 h. (150, 224)) suggesting that
limiting
in
or
are
rhamnolipids
directly
on
the induction of the
inducer. Dodecane is
but it cannot induce the
a
alk-system
in the presence
substrate of the alkane
alk-genes efficiently (96, 283).
or
hydroxylase (150, 259),
This could be due to the
geometry of the inducer binding site of AlkS, but could also be uptake-related: C12
a
:
122
E. coli
+DCPK,
b
+DCPK,
-Rhl
b
+DCPK,
+Rhl
GPol
+DCPK,
1.6(2)
putida (oleovorans)
+DCPK,
n.g.
-Rhl
P.
-DCPK,
+Rhl
GEcl37[pGEc47]
-DCPK,
1.6(2)
+DCPK,
b
+DCPK,
P .fluorescent
[pCom8B (GPol)]
17
(90)
(90)
-
+Rhlc
(290)
17
n.g.
+Rhl
28
(400)
(170)
9.3
(8)d
-
10(16)
88
-
101
(700)
(180)
158
32
46
(500)
(700)
round
hexadecane;
138
-
-
-
-
-Rhl
:
(32)
n.g.
14
11(4)
(2)
3.3(2)
-
n.g.
n.g.
tetradecane; C16
n.g.
2.8
+Rhl
16
-
1.8(2)
(32)
(2)
22(4)
2.7
11(8)
-
17(8)
-
21(8)
19
(8)
n.g.
11(4)
(24)
n.g.
43(4)
dodecane; C14
n.g.
-
decane; C12
n.g.
n.g.
:
-
-
-
octane; C10
:
n.g.
:
107
C8
:
-Rhl
KOB2A1
Table 1: doubling and lag-times (hours) of P.putida (oleovorans) GPol,.E.
GEcl37[pGEc47] and P. fluorescens KOB2Al[pCom8B (GPol)] during
growth on «-alkanes.
coli
Substrate
C8 vapour
CIO vapour
a
C12 vapour
C12 liquid
C14 liquid
liquid
C16
Footnotes:
b
-
: not tested; values are doubling times in hours, values in
: n.g. : no growth;
parenthesis are lag-times in hours;c : these values are growth rates of cultures after one
d
of adaptation;
: growth rate after multiple rounds of adaptation
123
may not reach AlkS.
with and without
allow
growth
a
Therefore,
rhamnolipids
rate
on
we
grew P.
shows that C12, if
directly
Growth of
system
a
solubilised,
can
only
recombinant E. coli
mutant strain of
with «-alkanes
slower
on
poorly
on
cosmid
-
C12
supplied
growth
on
This
(table 1).
significant
extent, and
GPol did not grow
on
to grow
on
GPol alk-
in E. coli
pGEc47
minimal medium
via the vapour
«-alkanes in
is toxic for E. coli
significant
for
phase (69).
liquid cultures,
on
longer
are
highly
in
not grow in
We
with and
in the
Pseudomonas and that
the
Pseudomonas may be the low
toxicity
a
alk-system
supplied
as a
C8
of P.
second
stationary phase (72). Possibly,
C8
while this effect is less
by rhamnolipids,
of the above results is that medium-chain
for
on
the «-alkane.
rhamnolipids
causes
only
cultures
liquid
alkanes. The toxic effect may be neutralised
containing
grew
However, in the
containing
liquid cultures,
traverse the cell membranes of E.
and the
GPol.
sensitive to «-octane, when
glucose, especially
simplest interpretation
Therefore, the main
GEcl37[pGEc47]
putida (oleovorans)
GEcl37[pGEc47]
which form micelles
(245)
E. coli
CIO vapour. E. coli strains
on
GPol
phase during growth
(236).
rate
putida (oleovorans)
the P.
alk-genes
rhamnolipids,
«-alkanes than P.
putida (oleovorans)
coli
a
putida (oleovorans)
biosurfactant, GEcl37[pGEc47] did
vapour and very
simply
to
DHl, enables this strain
from C6
ranging
in the presence of
absence of the
The
rhamnolipids
rhamnolipids.
Generally,
slightly
growth
alk-genes
containing
also tested this recombinant strain for
without
C12 vapour,
added to the medium in the absence of DCPK.
GEcl37, afadR
plates
doubles the
induce the
alkanes. Introduction of the
on
on
C12 that is five times faster than that obtained without
thus is available to the cells and AlkS. P.
C12
GPol
and DCPK. In the absence of DCPK,
The addition of DCPK
rhamnolipids.
putida (oleovorans)
coli
have
a
by
positive
difference in
(nearly)
influence
growth
specific activity
of medium-chain
diffusion
easily
alkanes
as
for
the diffusion rates.
rates between E.
of the alkane
length
on
as
length
coli and
hydroxylase system
in E.
alkanes to the E. coli recombinants
124
The effect of
rhamnolipids
the P.
containing
(oleovorans)
on
growth
putida (oleovorans)
GPol
nor
E. coli
alkanes of
on
alkB-gene.
GPol
GEcl37[pGEc47]
hexadecane. This may be due to
an
the substrate range of the alkane
hydroxylase.
is the alkane
from
strain able to grow
a
KOB2A1
fluorescens
(oleovorans)
alkanes
As the
on
a
grow
mutant P.
hydroxylase
from C8 to C16. No
growth
of these alkanes
uptake
the P.
encoding
for
(pCom8B (GPol))
was
observed
on
which is derived
putida
growth
C8 with
not grow
on «-
rhamnolipids.
C8 to CIO
on
either, the rubredoxin and rubredoxin reductase genes, which encode the other
components of the alkane hydroxylase system necessary for activity, may
time, and
induced.
was
resulted in
Initially, growth
on
C14 and C16 started
significantly higher growth
an
adaptation
rates with shorter
codon
(TGG) into
acid into
TCG
single point
a
a
a
mutation at
cysteine
small residue. In
other
no
changes
change
position
codon
(TGT), thus changing
AVLI
TGG -> TTG
were
not found
(chapter 9), suggesting
plasmid containing
on
48 hours with
after the
same
substrate
was
was
changed
to allow
sequenced. Indeed,
the
a
same
a
tryptophan
large hydrophobic
codon
TGC codon
a/fcB-sequence.
(possible
a
changed
twice to
(cysteine).
Other
a
In all five
possible one-basepair
amino acids
at the
changed
amino
by
a
one-basepair
corresponding
that additional mutations
might
include
(W55L).
KOB2A1, growth
only
found in the
to
the
CSGRL). Amino acids present in other AlkBs
are
When the
hydroxylase
165. This mutation
independent cultures,
were
in the W55 codon
are
position
changes
the
long lag-
adapted KOB2Al[pCom8B (GPol)
codon, encoding serine (W55S) and twice
cases,
on
a
lag-times (table 1),
hexadecane, the alkB gene of pCom8B (GPol)
sequence of the alkB gene isolated from the
showed
after
not be
process.
To check whether the sequence of the GPol alkane
on
only
slow. However, further cultivation of all cultures
presumably by
growth
to
or
We tested P.
(chapter 4)
parent strain of P. fluorescens KOB2A1, CHAO, does
efficiently
of
tetradecane and
fluorescens KOB2A1,
derivative
gene
putida
The ideal host to test these alternatives
«-alkanes
pCom8
Neither P.
on
unknown barrier for
long-chain
containing
GPol alkane
ranging
alkanes
two
hydroxylase negative
recombinant P. fluorescens
a
the W55C mutation
hexadecane in presence of
a
long lag-phase
doubling
time of 56
was
transformed back into
rhamnolipids
hours, similar
in the initial cultures.
Apparently,
started after
to the
growth
a
lag-time
rate observed
the mutation enables the P.
125
putida (oleovorans)
GPol alkane
hydroxylase
to oxidize hexadecane with
is sufficient to allow the P. fluorescens recombinant to grow
Prediction of residues involved in substrate
for the alkane
residue at
of P.
hydroxylase
position
binding.
putida (oleovorans)
GPol
this substrate.
on
Based
rate that
a
on
the
topology
model
(261), the tryptophan
55 is located in the second membrane helix.
Table 1 shows that cultures grown
hexadecane, while cultures
tetradecane need the
on
start to grow
same
dodecane after
on
a
long lagtime
very
short
lagtime.
as
This
suggests that dodecane fits in the substrate binding pocket, while tetradecane and
hexadecane
Supposing
are
too
large,
and need the mutation of W to C
that linear alkanes bind in
a
fully
dodecane molecule is about 17 A. Also
segments of AlkB fold
molecule
corresponds
transmembrane
of the
core
core
a
on
a
dodecane
of the
comparisons
sequence.
Counting
from the
periplasmic
end of the
W55, the
to
are
at
10 and 14 residues from
or
correct distance to
position
position
a
the activated oxygen
extended dodecane molecule.
Transmembrane
(TM) helices often form well-packed bundles.
sequence tend to be
neighbours
In the alkane
antiparallel.
residue,
a-helix. Based
4, and position 24 for helix 6,
position corresponding
an
1.5 A per
sequence of a-helices 4 and 6, the essential histidines
20 and 24 for helix
at the end of
perfect a-helices, rising
an
conformation, the length of
that the six transmembrane
assuming
to 11 amino acids in
S to be able to bind.
stretches, the W is located about 10 residues from the periplasmic end
hydrophobic
hydrophobic
as
extended
or
in the structure
hydroxylases,
(153),
all three
and
are
TM helices
adjacent
in
consequently
proposed periplasmic loops
are
quite
short, which suggests that the integral membrane domain of the AlkBs consist of three
antiparallel
as
far
TM helix
as can
pairs.
All six TM helices have
be estimated from the
approximately
multiple alignments.
This
the
implies
same
that the
the TM helices relative to the membrane surface must be similar and is
length,
angle
probably
of
close
to 90°. The W55C and W55S mutants show that the enzyme accommodates the
highly apolar
alkane
substrates in
hydroxylases,
distantly
related
a
hydrophobic pocket
which is formed
by
inside the membrane-domain of the
residues
on
helix 2 and other helices. In the
desaturases, the segment corresponding
to helices 1 and 2 is not
present. Recent studies with desaturase chimeras of the A6 fatty acid desaturase and A8
sphingolipid
desaturase from
Borago officinalis
indicate that TM helices 1 and 2
126
(corresponding
forming
the substrate
in the alkane
are
to TM helices 3 and 4 in the alkane
active
as
hydroxylases
The
site of these enzymes
may
play
the
same
hydroxylases,
the substrate
involved in
are
helices 3 and 4
(159). Therefore,
role. It is
dimers, and the active site is located
while in the alkane
protein
binding
hydroxylases)
that the desaturases
possible
at the interface of the two
binding pocket
is located in
subunits,
one
unit.
cofactors in the alkane
only
hydroxylases
are
the two irons that
are
bound
by
the
histidine clusters located downstream of TM helices 4 and 6. As the histidine motifs
and W55 delineate the vertical boundaries of the substrate
AlkB, other residues involved in substrate binding may be located
helix 2
as
W55
(residues 58, 59, 62, 66)
89-98), and the last
previous
in vivo
solubility
activity
on
in vivo
study,
we
investigated
same
side of
(residues
(residues 128-137).
activity
assays the effect of substrate
addition of 0.05 % Triton X-100 without
this
the
on
the first 10 amino acids of helix 3
,
10 amino acids of helix 4
Influence of substrate
in the GPol
binding pocket
of the alkane
solubility
knowing why
the link between the
was
hydroxylase.
reduced
this is effective
hydrophobicity
by
In
the
(245, 259).
of alkane
In
hydroxylase
substrates, expressed in logP values (148, 270), and the in vivo activity of the alkane
hydroxylase
in E. coli
The observed
of
activity
of the alkane
rhamnolipids (table 2),
with Triton X-100
was
was
(245, 259).
in the range obtained before in similar
With
of the
some
formed due to further oxidation of the
logP
value of the substrate. The low
converted with
logP
value
high
higher
Rhamnolipids
substrate cumene,
amounts
rates, while
no
a
negative
logP
on
perhaps by increasing
substrates
more
experiments
than
one
product
were
strongly dependent
cumene
and cymene
was
converted at
a
were
lower
oxidises these substrates with lower
cumene
efficiency (259)
cycle
logP
in the membrane to toxic
activity, probable
less efficient. It has to be noted that the ß-oxidation
a
low rate.
accumulation rates with the low
a
on
could be observed for substrates with
the level of
(236). High logP substrates gave
hydroxylase
substrates,
rhamnolipids
activity
effect
rhamnolipids.
primary products (table 2).
than that of octane, which
showed
absence of
or
towards C8 and C10, in the presence
hydroxylase
The accumulation rates in the absence of
the
in the presence
GEcl37[pGEc47],
or
as
the alkane
because
in the strain is still
uptake
fully
is
127
Table 2: in vivo activities of the alkane
(+ Rhl)
or
absence
Substrate
hydroxylase
in
(- Rhl) of rhamnolipids.
Substrate
Accumulation rate
of total
logP
Products
Accumulation rate
products
per
(U/g CDW)
+
3.4b
Cumene
+
6.4
8.1
12.1
Effects
ofRhT
product
(U/i;CDW)
-Rhl
7.8
4.5
Octane
Rhl
3.9
4.1
Cymene
in presence
GEcl37[pGEc47]
2.6
Decane
5.6
10.3
<
0.01e
Dodecene
6.2
7.0
<0.01
Rhl
-Rhl
2-phenyl -1 -propanol
0.5
0.8
2-phenyl-1propanoic acid
2-(4-methylphenyl)1-propanol
2-(4-methylphenyl)1-propanoic acid
3.5
5.6
0.4
0.2
7.3
7.9
Octanol
1.6
Octanoic acid
10.5
Decanoic acid
10.3
1,2-epoxydodecane
2.5
11-dodecenoic acid
4.3
11,12-epoxy-
0.2
d
X
+
2.6
+
-
+
-
dodecenoic acid
Dodecane
6.6
5.3
<0.01
Tetradecene
6.7
0.4
<0.01
Dodecanoic acid
5.3
1,2-
0.3
+
-
+
epoxytetradecene
13-tetradecenoic
0.1
-
acid
Tetradecane
Footnotes:a
below
7.6
<
n.d.e
negative effect, x: no effect,
d
detection limit.
: no products.e : not
: -:
active, and fatty acids
In
0.01e
can
+:
No
positive
effect.
b
-
:
estimated value,
n.d.
-
no
literature value.
°
:
determined.
be oxidised
further, which may affect the obtained activity.
addition, epoxides might be oxidised
These results show that
products
rhamnolipids
at the
co-position.
extend the substrate range of the alkane
hydroxylase by making hydrophobic compounds
more
soluble and
thereby
more
accessible for the host strain.
CONCLUDING REMARKS
Rhamnolipids significantly
P.
putida (oleovorans)
experiments
as
well
as
influence the
GPol alkane
the in vivo
availability
of
hydrophobic
hydroxylase system,
activity
assays.
as
shown
Expression
substrates for the
by
the
growth
of the GPol alkane
128
hydroxylase
in P. fluorescens KOB2A1 allowed the selection of alkB mutants that
able to oxidize
hexadecane, while repeated cultivation of
hexadecane resulted in
fluorescens
CHAO
growth
rates that
such
as
an
enzyme,
the alkane
comparable
of these mutants
with that of the
on
wildtype
P.
(chapter 7).
As the host strain and substrate
range of
are
one
are
mapping
by
have
a
large
effect
on
the apparent substrate
and modification of the substrate range of enzymes,
hydroxylase,
the target substrates
solubility
should be
accompanied by
studies
on
the
uptake
of
the host strain.
ACKNOWLEDGEMENTS
We wish to thank Martina
Röthlisberger
technical assistance. This research
Biotechnology
was
for
sequencing
supported by
of the Swiss National Science
and Stefanie Balada for
the Swiss
Priority Program
Foundation, project
nr.
in
5002-037023.
129
Chapter
9
SUMMARY AND CONCLUSIONS
Theo H. M. Smits
130
Although
the
degradation
of medium- and
attention from researchers in the fields of
and
biochemistry, bioremediation,
concentrated
relatively
on a
molecular
microbiology,
biocatalysis,
genetics,
most of these studies have
small number of strains. The most
alkane oxidation system is that of Pseudomonas
in
alkanes has received much
long-chain
described
extensively
putida (oleovorans)
(reviewed
GPol
(263)).
Highly degenerate
(oleovorans)
PCR
GPol
primers
on
five non-heme iron alkane
colony hybridizations,
flanking regions
were
(168)(chapter 4;
J. B.
a
shows that the
full-length
alkane
as
hydroxylase
and
us
to show that
some cases
J. B.
van
up to
Beilen,
in Southern and
probes
Gram-positive
Comparison
divergence,
monooxygenase
sequence
identity,
oxygenases, such
Based
hydroxylases
integral-membrane
sequence
gene sequences and
Gram-negative
strains
indeed
hydroxylase homologs
on
as
the
and other
of the alkane
with
protein
hydroxylases
(251)
acid
as
even
non-
(chapter 4)
have
low
as
35 %. The
substrates, the P. putida
only
have around 25 %
integral-membrane
desaturases, have
some
sequences
hydroxylases
that oxidize other
and related enzymes,
homology, alignments
(table 1, figure 2),
hydroxylase
sequence identities
while the other related
fatty
integral-membrane
non-heme iron alkane
define thirteen different subclasses of the
oxygenases
fragments
showed that all tested alkane
closest relatives of the alkane
identity.
(213), enabled
alkanes.
heme iron oxygenases.
protein
ADPl
putida
Beilen, unpublished results). Functional analysis by
Structural features of alkane
xylene
of the PCR
some
total of 17
van
of P.
hydroxylases
«-alkanes contain at least one, and in
obtained from several
heterologous expression
mt-2
the alkane
hydroxylase homologs (239)(chapter 2;
unpublished results). Using
significant
on
(143) and Acinetobacter sp.
many strains able to grow
hydroxylate
based
non-heme iron
lower levels of sequence
and the function of these enzymes,
integral-membrane
we
could
non-heme iron
structural features of which will be discussed
below.
The
alignment
of the
manually optimized
alkane
complete
based
hydroxylase (261).
spanning segments
on
alkane
the
hydroxylase
topology
sequences shown in
model for the P.
In all AlkB sequences, the six
could be identified
easily.
Most
figure
1
putida (oleovorans)
hydrophobic
hydrophobic
was
GPol
membrane
residues
are
not well
monooxygenases
Xylene
hydroxylases
9
2
C5 steroid oxygenases
Cholesterol
7
6
A9 desaturases
Q-3 desaturases
359-453
355-510
374-443
272/298
VT(Y/f)LHH
H(R/k)THH
representatives
parentheses:
residues not
possible
that of
a
HV(I/a)HH(L/i)F
GEG(F/Y)HN(Y/F)HH
(T/v)H(V/i)XHH(L/i)(F/s)
HHD(L/M)HHS
H(T/ad)XHH
HDXHH
YTPSYHSLHH
certain subclass many
more
are
known.
b: capitalized letters: absolutely
small letters: minor residues.
sequences
sequences of the 4 histidine boxes. The sequences used to determine the consensi
absolutely conserved, capitals: predominant residues,
of each subclass, and it is well
conserved residues; in
are
consensus
GDHCGHG
NS(A/L)AHXXG
HR(I/A)HH(R/K)
HR(L/m)W(S/A)HR
TX(L/v)(Q/H)HT
H(R/d)(R/n)HH
EDHSGY
(f/d)H(D/t)
H(E/D)C(G/a)H(H/k)
H(A/K)XHH
H(R/m)XLH
271-365
H(S/C)GY
HKVHH
YK(Y/kn)(I/v)H(K/s)XHH
HR(L/G)(L/F)H
DF(L/M)RCLGH(C/S)NVEI
YTPS(F/Y)HSLHH
H(H/W)EHH
FGT(W/Y)LPH(K/R)P
DFMNNMGHCNFEL
AHXLHH
AHR(L/M)HHAV
EITNHX(N/D)HH
LQRH(S/A)DHH
H4
YMFVHDGLVH
HLLFffT
YHS(H/F)HHSS
H(R/K)X(L/F)H
524/566
253-406
YHS(H/F)HHSS
H(R/K)X(L/F)H
567-622
HXSHH
HXXHH
H(R/K)A(I/L)WH
HXSHH
H(R/K)(W/Y)fMHG
FfDCMHG
242-320
308-322
NYXQHYG(L/Q)
H(I/V)XXHH
FfELXH
(L/V)HDG(L/V)VH
NYXEHYG(L/M)
EHXXGHH
H3
HE(L/m)XHK
no.
H2
Histidine box
Hlb
non-heme iron oxygenases
Footnotes:a: No seqs: number of sequences used to determine the
9
A12/Ê2-6 desaturases
hydroxylases
8
C4 steroid oxygenases
3
5
5
162-176
369-377
5
6
355-490
Length
16
seqsa
2
CER-like
No.
integral-membrane
Decarbonylases, Glossy-like
Decarbonylases,
Carotene ketolases
Plant carotene
hydroxylases
Bacterial carotene
hydroxylases
subclass
Alkane
Enzyme
Table 1: sequence motifs of
131
50
MNAPVHVDQNE'EEVINAARSMEŒIDR^YIOTIISPALPVIGIGIT^
^ELMPVPASMEŒIDRE^YIMMISPALPVIGIJGILA.
1
GEDFNNPPF^V-VFiaZKFJ^YYRVLTYLTVPMHYAALIVSAMMÏTa
GEDYSNPPESV-VPKLEQDRYYKVLTYLTVPIHYAALIISAWWVS'IQ
GKDASNPTSF£-lKQI^lsDPYYA[lLVKSFIPLQYIAlslVYACYLVSRK
GKDARMTOF£-IKIIZKDPYYSRLVKSFIPIÛYAANIYACYLTSRK
GDDI^NPPEDV-VPHLFJM^YYRLIVYIATLVEWAFFM^VRIVG'IH
GVDSENPPESI-VPRffiSroYYRYVVYIATFWWÄFAGATWIVÄIQ
GEDASNPPESA-VPDl^QPYYKFnArSCÄVLS^
GTXiI^NPPDDV-LEQI^lsDRYYRV^ITAYLPIQYAGFWALWLIMRG
GPK^NPPDEV-TDRIEISDKYYRYCTYIYIPFQYLSVVIJGAYLFTAA
GE^PANPDF£TQTTQIZßQGYYVIIjTLATVPVLIGTLVWAAGVF^
GRDPANPF^^EVPFJŒ^^YYRVI^I^TVPU
MDSVNPTt^FÖ-F^FiiJ^KFFKYITCÄYVPIQITimYGVYIVSTQ
PKDFSARKVSPFFADL
40
72
79
78
64
64
64
79
81
64
64
48
45
M3
TQYIÛLPLMIGLYGLLVFGVElSlGRIE
GEDFNNPPF^V-VFiaZKF^YYRVLTYLTVPMHYAALIVSAMMÏIQ
67
-D. .NP
130
GEDFMsrpPFZV-VPKI^S^YYRVLTYL^
120
67
110
MDTLRYYLIPWTACGLIGFYY
1
100
MDDU^KYSFLLVYLLIPLPFVSLYL
1
90
MFASJ^SAOTOÏU^TCYWIWLIAVrjGIPISYWWS
1
Ml
MFENFSPSIMLAIKKYAYWLWIZZALSMPFNYWMA.
1
•
MIVSVAAPGVWTDSKRHU^nJGTLPMITPLESGIF
MSDDAPAAASRPMASIIPAATGGTEEWTDR^YIWLIGLVVPSLVFIAIGL
1
i
60
J_
P..D...
-IWYGLVLIIWYGLVPLIDTML
-IFYGLVLAVWYGVLPIJXAMF
-IFYGLVLAVWYGVLPIJXAMF
-IFYGLVIXVWYGALPIJXAMF
i
70
GGY
GGW
IfiSDYPN
QDSAHPA
140
^VWIWIGPIVILVWELLDLVA
VFWWSGVLVIFGLIPLIDGIM
VFWWPGP^AFGVIPVLDLLI
^VFWWPGPLFAFGVIPILDTLI
150
cepacia RR10
pseudomallei K96243
P. fluorescens CHAO
D
P
A
D
L
N
TR
QE
aureofaciens
RTWH529
cepaciaRRlO
pseudomallei K96243
P. fluorescens CHAO
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L. pneumophila Philadelphia
P. putida mt-2 XylM
p
p.
M. tuberculosis H37Rv
P. rugosa NRRLB-2295.
B.
B.
Acinetobacter calcoaceticus EB 104
Acinetobacter sp. ADPl
A. borkumensis API
p.
P.putida (oleovorans)
putida PI
Consensus
GPol
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L. pneumophila Philadelphia
P. putida mt-2 XylM
P.
P.
M. tuberculosis H37Rv
P. rugosa NRRL B-2295.
B.
B.
Acinetobacter calcoaceticus EB 104
S
K
RTWH529
GPol
Acinetobacter sp. ADPl
P.
P
aureofaciens
A. borkumensis API
P.
P.putida (oleovorans)
putida Pf
P.
Consensus
S
P
NLSWLGFDGA
160
M2
WW-LGAATFPAIWVLDVIL
YSFLPFFIVFAFIPLIDA-F
AWFWLVISWFGLIPILDAIV
FWAFSLVIAVFGIGELLDMLF
GWHAAAfy/PT MTGPTT J ,YOT ,T ,PT ,T ,nT RF
RFATGWG
ALTIGVQ
AtSTGWH
AtSTGFH
GVHFGPRPT .KKWAT GGPT J J .HWTPATnTTT
GYQFSPRPIKKIFALGGPIVLHIIIPVIDTII
VSQTGWS—
ANETGWG--
ANETGWG--
ANETGWG--
Nn^nTG<^PKAPRPPFA7FFMRnKKRYT NT MTX .TAPTAT ,\AMT ,PT .TMnMNIQT
MMQQPVSATJra^Tt^YV^lIZJGALTITLPLVAALL
1
1
MATSDAPAASWSOa^YIWLDGALTITLPIFAAQL
1
-MIMNAPVNVQQ
1
1
1
WPATPMIGIWL
MSENILTEPPI^DADNEGYVDRt^RHUttlSVIMPATPIIGLYL
1
_1_
MNGKSSVIXiSAPEYVDKKKYFWIISTEWPATPMIGIWL
i
40
1
l_
30
MI^^HRVIXiSAPEYVDKKKYIWnT^TEMPÄrPMIGIWL
i
I
1
20
10
180
I
230
Y. .F. .EH. .GHH
I
220
T. .D
I
240
G
F...
l_
250
cepacia RRf 0
pseudomallei K96243
P. fluorescens CHAO
-T^lsELLQGWGMSAAFHASM\Ä3IPGKGTIPYT^T^ÄFYGISLFEIIlsrYIEH^
-WRTSE^LHAWAMTWVWGTATAIGGVWIEFLVIQAVYGASLI^^^^
TVIGSFKSAIEIEKNRIKÏKGKEFWS-
TV\Ä3SVRSAMy^KTRLARTJGRSPWT-
T\mKIASAMy^3^ARLARSGRSÄWS-
T\^^SJ^SAMiLESQRIEKlJGLPTLH-
TV\ÄKLRSAWRLERKRYARRDRHPFR-
SOTGGT RS A\7HT F AQRT RRT ra7SPWNTCWrVT RNTft/T ,NAWT M£\ A/T MGGT ,T A\/FGPAT .TPFCT TQA\7FGFST T F A\7MVT FHYGT T R
209
195
195
232
210
221
IHNNFWWILTVPmiCn^IIKK^^
180
173 AT\Ä3SVKDAIKIEAETLRRKGQSPW
M5
M6
H3
NT .SNKTyQYVAT T T AT .PGT VSYT GGPAT 0[ .OTTAÇMTTAKGT\7FGFMVFQHYnT
WRNELBTOWAISÄLFIJJSFSLAT^^
198 AYKHNFTJslAMy^MmKRKGLPALH
TJJJGSFQSALILEEKRlßHKNYSIWS
^WQNELB^rYIXSLAJJLVGFGWAFGWDGMVFFljGQÄFVAVT^
198 AYKYNFTJslAMy^ÄVRLRKKGLPVPG
-IGKDVLNAWUeAVmSVMIümjSIGV^
-WKNGVLSAWLYSVVOO^TAWDGAAVIEFLVIQGIYGFSLI^^^
^-
-KDlsELLQGWGMSAAI^SSITAIPGKGTIPYLVTQÄFYGISLFEIIlsrYIEHYGT^
TVPGSn^SAJEIETHRIKÏKGKKFWS-
210
-WRISE^LQAWAMTVAWSAATAFGGAKVVEFIJLIQAAYGAST^^
P.
aureofaciens
GPol
RTWH529
cepacia RRf 0
pseudomallei K96243
P. fluorescens CHAO
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L. pneumophila Philadelphia
P. putida mt-2 XylM
P.
P.
M. tuberculosis H37Rv
P. rugosa NRRL B-2295.
B.
B.
Acinetobacter calcoaceticus EB 104
Acinetobacter sp. ADPl
A. borkumensis API
P.
putida (oleovorans)
putida PI
Consensus
-]^NFA^m:iXTWLYAAIXAFFGEr^
A. ..E..R
.R
L
340
-FnNF TT qpmottctt .ytt t t affgpkmt s/ft PTQMAFGwwnr .ts anvtfhygt t r
l
330
171 EIPGAFRRÄWGLEEQRIßRRGQSVWS-
l
320
203 EIPGÄFKRÄWGLEEQRISRCGKSVWS-
G
l
310
P.
.N
l
300
-FFINF TT QPMOTTS A/T YTT J T AFFGPKMT \TVT PTQMAFGWMrj .TS ANVTFHYGT J R
..
l_
290
198 EIPGAFRRÄWGLEEQRIßRRGQSVWS-
l_
280
198 EIPGAFIRÄWGLEEQRIßRRGQSVWS-
l_
270
H2
NY.EHYG.
Q
-FDlsEILQmiITVILYAVIXAIKÏPKMLVFT^lQMAFGWVO^TSANYIEH^
i_
260
Hl
SEPLQVAGCILSLAMjSGVPTLEVSHFIMIRRHWLFRKMAQT^^
M4
LVVflÄnJSFT^LSLISGGVGIIOTAHFM^
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L. pneumophila Philadelphia
P. putida mt-2 XylM
P.
M. tuberculosis H37Rv
P. rugosa NRRL B-2295.
B.
B.
Acinetobacter calcoaceticus EB f 04
P.
88
RTWH529
GPol
Acinetobacter sp. ADPf
113 WnW\7GQT GWTT .<^7GTS7Mr!ATGTTVSHFT .THKnPQT FQMAGGT
95
aureofaciens
putida (oleovorans)
putida PI
A. borkumensis API
P.
P.
P.
Consensus
113 VOŒ^RlJGWILSMGTVMGAVEIVVAHFilHKDSA^
J J .AA\7r.YAGFTa7FH\7RGHH\7H\/RTFFnAq.q.qRVITKr YSFT PH
LSWAGKlJSVÄLSVGVLGG-VGnOT^
LAWYDYIGFÄFSLSAATG-ISIOTAHFJGHKre^
111
137
LAWYDYVGFÄLSLGAATG-ISINTAHF1JGHKTNRFERWLM
111
IGWFTYIfiMAMSTGAATG-IAINIAHFljGHKNRGMAKFLAKT^^
TSFJ^KIIIßlSMGMNG-IAINTAHFlßHKHDRIDHILSHLALVP^
125
T^TVDKVGLMT\fëCVGG-IG]OTA^
TSFIDKIIIßlSMGMNG-mVNTAHFlßffi^ADRIDHILSHLALV^
126
126
IGVFEFIJffALSDSn/NG-L^^
119
148
MSWFEIGALÄLSDSn/NG-L^^
87
L
MSWFEn/ALÄLSDSn/NG-:^^
I
210
114
HE. .HK
I
200
MSW^IGALAT^LGIVNG-LÄLNIGHELGHKKETFDRWMAKIVT^^
G.. .G
I
190
114
_l
EKLPNG
QKRADG
QKKENG
RRLPDG
RQLPTG
QKQPNG
QE^GSEFT^RRYERVDPSHSl^S^TATNVI^YHLQRHSDHH^
QKSANG
RKGEDG
RRLDNG
KMLSNG
VRDLDQ
284
291
290
276
276
313
291
306
279
279
261
252
I
460
M
Acinetobacter sp. ADPl
cepacia RR10
pseudomallei K96243
P. fluorescens CHAO
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L. pneumophila Philadelphia
P. putida mt-2 XylM
VLATjr^DœiTOANIQPGKRFiaT^
VI^HYGGDITRVNLHPRVRTiKALARYGASA
VRAYYAGVEFQLTAEQSERPAAS
VRAYYAGEEYQLTDTQRI
VLAYRIQEiRQEiEQL
LRI»D^YATPGERETJSMAA2s|T^^
387
360
360
342
330
,
.T^Yf^.qNTTnqriTat
375
357
vr,AHY.qmvRT,anmhpatcrttft ,t
VVAJJYRGÏSIMAQSNIKPSIRDKVLAQYPAPA
WAHYGAOlAQSNIKPSIRigcVLAH
357
394
VT^HYKGDKSIK^ISEKRRAKIFKKFGVGG
371
cepacia RR10
pseudomallei K96243
P. fluorescens CHAO
aeruginosa PAOl AlkBl
aeruginosa PAOl AlkB2
L. pneumophila Philadelphia
P. putida mt-2 XylM
P.
P.
M. tuberculosis H37Rv
P. rugosa NRRL B-2295.
B.
B.
Acinetobacter calcoaceticus EB104
Acinetobacter sp. ADPl
A. borkumensis API
putida (oleovorans) GPol
putida PI
aureofaciens RTWH529
VraHYIŒDLTKANIYPKRRAKlTJSKFGLTDFNIENGK
P.
P.
Consensus
P.
P.
M. tuberculosis H37Rv
P. rugosa NRRL B-2295.
B.
B.
Acinetobacter calcoaceticus EB104
372
L
510
putida (oleovorans) GPol
putida PI
aureofaciens RTWH529
A. borkumensis API
P.
P.
P.
Consensus
P.
Rubredoxin domain (57 a.a.)
i_
500
P
420
VMEWABGDINKIQIQPGMREFYEQKFGVK
GSESPDTTVAK
I
490
410
365
I
480
P. .P.GY
400
290
I
470
VVNWANGDLSKIQIEDSMRAEYIKKFTHNVGADDKRGA^
I
450
360
I
I
VVIXmGGDTJslKIQIDDSMRETYTja^GTS-SAGHSSSTSAVAS
440
430
H4
PILLHHAIflNHMGTBZRPLGCEITNH^
QYFiCVNPNHSrotrANHWL^^
RYFTOTTEHSWSNFIiTirrjFIjFH^
RYraTNHfflSWSNFVFTOTLVLF^
RYraOffVHSWSDHBrarr^YHLC^
RYFT^CSERHSWSMRBraJIFljFi^^
RYraCir^HSWSNHWTtTT^YCP^^
RYraCir^HSWSNHWTtTT^YCP^^
QYERTMPEHSWNMSINWI^^
lsrYFT^TMraHSTONMsIN
RYEH^HHSWSNHVMarLILFHLQRHS^^
RYEHQE^HHSlftNSNHIVSNLVLFHLQRHSDHHA
360
V.
EKMADG
252
Q.LR
390
RYEHQE^HHSI^SNHWSDTLVLFHLQRHSDHHATTPTT^YOSIiR^
EKMADG
.LQRH.DHHA
279
N. .L.
RYEH(3ŒHHSWSNHWarLVLFHLQR^
HSTON
380
QKMEDG
370
360
279
YE
350
135
Figure 1: mulitple alignment of full-length alkane hydroxylases, generated by ClustalX and manually
optimized. The consensus is based on the full-length alkane hydroxylases. Membrane-spanning segments
(M1-M6) based on the topology model for the P. putida (oleovorans) GPof alkane hydroxylase are marked
by solid bars, while the histidine boxes (H1-H4) are underlined. The dot in the ruler indicates the position
of the P. putida (oleovorans) GPof AlkB W55 residue.
conserved, but
are
transmembrane
replaced by
similar
residues, while
helices, often glycine and proline,
are
membrane non-heme iron oxygenases contain 3, 4, 5
which
are
below).
a
number of other residues in the
well conserved. The other
or
6 membrane
located between the histidine boxes H2 and H3
A third membrane helix is
always
located
(figure
helices,
2 and
integral-
two of
explanation
of the first histidine
directly upstream
box.
All AlkB sequences contain the
eight previously
residues in three histidine boxes that
are
contain
conserved
The
a
ninth histidine residue in
corresponding
(table 1).
In the
immediately
essential for
histidine residue of the P.
shown to be essential for the
communication)
a
and has
topology
a
in all
putida
are
of the
as
helps
hydroxylases (CrtZ)
the latter
(239).
was
Wubbolts, personal
non-heme iron oxygenases
this residue is located
surface,
a
position equivalent
role in the
analysis
of
peptide
catalytic
centre
sequences
subgroups (table 1),
newly sequenced
by
directly flanking
and
can
be used
the
as
genomes.
N-terminus, C-terminus, and the regions between the histidine boxes
and membrane helices also
carotene
a
well conserved in each of the
sequences in the functional
length
W.
sequence motif
monooxygenase
hydroxylases (261)
the iron atoms in the active site. The
histidine boxes
The
(M.
xylene
but also
box, which is located immediately after transmembrane helix 4. This
supports the hypothesis that this residue plays
signature
mt-2
after transmembrane helix 6 at the membrane
to the first histidine
coordinating
catalytic activity (233),
integral-membrane
model for the alkane
conserved histidine
highly
NYXEHYG(L/M)
of this enzyme
activity
pendant
identified
typically
can
have very
classification into subclasses. For
be
long
distinguished
from the
N-terminal extensions.
plant
example,
carotene
Similarly,
the bacterial
hydroxylases,
the A9 desaturases
have far fewer residues between histidine box H2 and the third transmembrane helix than
the other desaturases. However,
within each
large
subclass, like the fusion
rugosa NRRL B-2295 alkane
variations in
length
of these
regions
can
also
occur
to the rubredoxin domain at the C-terminus of the P.
hydroxylase.
136
The
alignment
of the alkane
medium-chain alkane
hydroxylases
hydroxylating
does not allow the identification of
enzymes
from the sequence,
solely
sequence identities between
long-chain hydroxylases
and
hydroxylase.
a
medium-chain alkane
W55C)
is
required
hydroxylate longer
are as
addition, only
In
to allow the medium-chain alkane
alkanes such
as
tetradecane
or
transmembrane helices
supported by
the
findings
are
one
as
(figure 1),
we
(159)
transmembrane helix 1 and 2 of desaturases
research is necessary to map the substrate
are
a
or
peptide
long-chain
mutation
(W55S
or
from GPol to
As W55 is
(chapter 8).
involved in the substrate
of Libisch et al.
the
as
between
point
hydroxylase
hexadecane
located in the middle of the second membrane helix
hydrophobic
low
long-
conclude that the
This is
binding.
who found indications that
involved in substrate
binding
site of the alkane
binding.
Further
hydroxylases
in
more
detail.
Structural features of the
transmembrane
are
clearly
integral-membrane
helices, and the localization of the catalytic
connected to the role of these enzymes in
substrates, which
are
soluble in the membrane
In contrast, oxidation of the
alkenes
Ml
can
M2
oxygenases, such
also be
M3
M4H1
more
catalysed by
H2
as
the presence of
centre close to the membrane
oxidizing relatively hydrophobic
bilayer,
but
hardly
dissolve in water
(236).
water-soluble short-chain alkanes and short-chain
soluble non-heme iron monooxygenases
MS M6 H3
(160, 178, 267).
H4
Alkane
hydroxylases (AlkB)
Xylene
monooxygenases
Bacterial carotene
Plant carotene
(XylM)
hydroxylases (CtrZ)
hydroxylases (CrtZ)
Carotene ketolases
(CrtW)
Decarbonylases (CER-type)
Decarbonylases (Glossy type)
C4 steroid oxygenases
C5 steroid oxygenases
Cholesterol
hydroxylases
A12/Q-6 desaturases
A9 desaturases
Q-3 desaturases
Figure
2: linear
representation of integral-membrane
non-heme iron oxygenases. White bars represent the
membrane helices, while grey circles represent histidine boxes.
Drawing
not to scale.
137
Role of substrate
chain alkanes is
strains
uptake
in alkane oxidation. The
largely dependent
yielded growth
rates
on
on
growth
the mode of alkane
hexadecane of
rates of bacteria
uptake.
maximally
0.06
surfactants, growth
h"1 (td
literature values of
h)(chapters 4, 6, 7, 8). However,
10
=
Acinetobacter strains
on
hexadecane
are
All Pseudomonas
h"1 (td
presence of exogenous surfactants. Without
long-
on
=
5
in the
h)
rates did not exceed 0.03
growth
in the order of 0.3-0.4 h"1
(td
by
rates
=
1-1.5
h)(10, 16,
214). The main difference between both genera is the mode of uptake. Acinetobacter
strains take up alkanes via surfactant-mediated direct interfacial
Pseudomonas strains
Both the
growth
production
limited
of biosurfactants and cell surface
a
growth
In contrast,
or
on
cell surface
membrane
second
rather
diffusion of the alkane
vast amounts of biosurfactants
uptake
by
phase
maximum
over
puts
a
(245).
Growth rates of P.
rate of
(td
=
1.5
Pseudomonas strains
alkane
the
degenerate
hydroxylases
PCR
approach,
we were
related to the alkane
(chapter 2). Although
these
organisms
simply
putida (oleovorans)
h)(151, 223),
on
glucose
or
by
to be
substrate
traverse the
GPol
on
octane
as
which is close to the
citrate
(223).
membrane alkane
integral
seem
the bacteria.
these alkanes
hydroxylases.
able to show that many bacteria contain
hydroxylase
were
on
rates
addition, the production of
not to be limited
seems
hydrophobicity. Apparently,
may therefore limit
surface; the growth
metabolic burden
in the order of 0.4 h"1
growth
cell
(28).
Pseudomonas cultures cannot
the outer membrane. In
medium-chain n-alkanes
diffusion
are
hydrophilic
Environmental relevance of non-heme iron
Using
hydrophobicity
rates. The addition of exogenous surfactants to
by
while many
biosurfactants to enable solubilization of the alkanes
produce
compensate for the effect of
uptake,
of P.
isolated from
a
putida (oleovorans)
large variety
GPol
of
environments, the relative importance of strains harboring integral-membrane non-heme
iron alkane
hydroxylases
A few other enzyme
systems
oxidizing cytochrome
systems
are
bacteria
as
in these environments has not been determined yet.
are
P450 enzymes
most often found in
well
known to oxidize n-alkanes. Of
(44, 179).
In
a
were
described in
yeasts (122, 196), but
sandy
soil
some
seem
these, the alkane
detail. These enzyme
to have their
counterparts in
microcosm, alkane assimilating yeasts
were
138
able to overgrow bacteria such
C10/C14 mixture
dependent
on
composition,
important
The
use
on
in the
the
ecology
GPol did not
the
alkane
hydroxylases
tree. An
failed
H. M. Smits and J. B.
can
be used to
attempt
the low
on
van
or
gene families is
yield
a
probe
a
based
on
alkane
hydroxylase
(239) show that due
2
hydroxylase
sequences,
to create
specificity
a
Alkane
alkane
and
hydroxylases
internal
hydroxylase
as
a
some
has
a
been used for the
(50).
analysis,
a
of
of
(98, 243,
specific
derived from
branch in
for all alkane
hydroxylases (T.
and
water
The P.
can
phenylpropanoic
B.
van
and
hybridisation
diversity
of
samples.
putida (oleovorans)
oxidize
GPol
hydrocarbons regio-
interesting
for the
(281). Alkane oxidizing strains have already
of fine-chemicals such
laboratory (J.
hydroxylases,
would reveal the
This makes this class of enzymes
production
our
or
perspective.
A strain collection of around 200 alkane
put together in
of that
fluorescence in situ
broad substrate spectrum and
of fine-chemicals
(56, 89), phenylpropanol
hydroxylase
probe
of the alkane
of the alkane
and contaminated soil
biocatalysts:
stereoselectively (259).
biocatalytic production
developed
biodégradation ability
molecular
hydroxylases
to detect
be
can
single oligonucleotide probe
fragments
restriction
pristine
as
used
to the low sequence
denaturating gradient gel electrophoresis (DGGE)
in
probes
in the environment
(FISH)
hydroxylases
commonly
a
the alkane
correlation between
techniques
alkane
are
organisms (106, 152).
molecular
or
highly
Beilen, unpublished results). However, the degenerate primers
amplify
such
soil
genes
detects alkane
hydroxylase only
phylogenetic
(239)
specific
particular
identities of the individual alkane
single
are
of the contaminant
of n-alkanes in the environment
247). The results described in chapter
a
bioavailability
the available sequences. However,
alkanes and presence of this
experiments
a
studies. To elucidate the role of non-heme iron alkane
degradation
putida (oleovorans)
in the oxidation of
of the soil used for these studies. Soil
biodégradation by
to detect
probes
in microbial
hydroxylases
P.
content, pore size and
influencing
pseudomonads
the results of such
physicochemical properties
water
factors
rhodococci and
(225). Nevertheless,
of molecular
technique
based
the
as
acid
as
octanoic acid
(219), epoxyalkanes
(119) and phenoxy propanoic acids
degrading
strains in microtitre
plate format,
Beilen, W. Duetz, personal communication), has
139
been screened
successfully
benzylpyrrolidines
of the most
contains
a
A
strains in this
alkane
activity
use
more
of
than
a
a
library
product
alkane
hydroxylase (chapter 2).
single wild-type
activities which
of
would
single
alkane
require
are
superior catalytic properties
a
clone
principle
hydroxylase,
can
1-alcohols,
as
some
The alkanesum
of all
where
as a
alcohol
or a
library
or
enantioselectivity,
different
regio-
of recombinant strains
hosts, in which the oxidized
has been proven effective to test the
host
complementation experiments (chapter 4)
Nevertheless, they
hydroxylase
genes would be useful. The setup of such
the construction of suitable
strain has been used
oxidation of n-alkanes to
organism
present under the applied conditions. This could
hydroxylase
substrate range of the GPol alkane
used for the
This
strains thus reflects the
opposite stereo-selectivity
is not further metabolized. This
dehydrogenase negative
pyrrolidones (46).
One
was
strains in the strain collection is that
for the desired reaction. For this reason,
expression
clone
wild-type
one
because the enzymes may have the
for the
sp. HXN-200,
that is not related to the GPol alkane
the detection of enzymes with
selectivity
A^-benzyl-3-hydroxypyrrolidine (158).
Beilen, unpublished results).
measured with
hydroxylase
preclude
van
limitation of the
of these strains contain
inducible
active
study, Sphingomonas
hydroxylase
Z. Li and J. B.
potential
and stereoselective oxidation of N-
used for the oxidation of A^-substituted
soluble alkane
(D. Chang,
regio-
produce optically
to
promising
subsequently
for the
a
medium-chain alcohol
(26, 259). The recombinant hosts
would not be suitable for the
dehydrogenase activity
be used to convert non-metabolizable
is present.
compounds
into
high-value
fine-chemicals.
CONCLUSION
In the last few years,
alkane
These
and
a
large body
of information has been
gathered
on
the
genetics
of
oxidation, and many alkane hydroxylase genes have been cloned and expressed.
experiments
uptake,
and
illustrate the essential roles of electron
regulation
in alkane
transfer, alkane solubilization
degradation. Mutagenesis experiments
have
given
us
140
a
first
insight
structure of
substrate
other
better
This
in the location of the substrate
an
alkane
hydroxylase
binding, catalysis,
proteins.
binding pocket. However, only
would allow
us
to
identify
residues
a
crystal
important
for
electron transfer and interaction with the membrane and
This could also lead to the rational
design
of alkane
hydroxylases
with
catalytic properties.
study
demonstrates the
hydroxylase,
probes
are
which has
necessary to
environments.
an
diversity
impact
study
on
of the class of
integral
membrane alkane
the environmental research in that
the microbial communities in
pristine
more
(complex)
and contaminated
141
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Curriculum vitae
Name:
Smits
First
Theodorus Henrikus Maria
name:
June, 22 1973
Born in
1977-1985
Primary
1985-1991
Gymnasium
St.
1991 -1997
Vlijmen,
school
The Netherlands
Vlijmen,
's
Janslyceum,
The Netherlands
Hertogenbosch,
Landbouwuniversiteit
The Netherlands
Wageningen,
The Netherlands
Study Bioprocess technology
Orientation
M.Sc. in
1997-2001
Molecular-Cellular, suborientation Microbiology
Bioprocess technology
Eidgenössische
Ph.D.
position
Technische Hochschule Zürich
at the Institute of
Since March
Post-doc fellow at the
2001
IATE/Pédologie
(ETHZ):
Biotechnology
École Polytechnique
Fédérale de Lausanne