Living Large: Elucidation of
the Frankia EAN1pec Genome
Sequence Shows Gene
Expansion and Metabolic
Versatility
Louis S Tisa1, David R Benson2, Gary B. Smejkal4, Pascal
Lapierre2, J. Peter Gogarten2, Philippe Normand5, M. Pilar
Francino3, and Paul Richardson3
1Dept.
Microbiology , U New Hampshire, Durham, NH, USA; 2Dept. Mol.
Cell Biol., U Connecticut , Storrs, CT, USA; 3 JGI, Walnut Creek, CA, USA,
4 Pressure Biosciences, Inc, Bridgewater, MA, USA, 5 Ecologie
Microbienne UMR CNRS 5557, Université Lyon, Villeurbanne, France
Actinorhizal
Symbiosis
• Symbiotic association
between Frankia and
woody dicotyledonous
plants
– results in formation of
root nodules
• over 250 species of
Actinorhizal plants
Frankia
• Member of the
Actinomycetales
• Hyphal bacteria
– 67-72% GC
– generation time 24-48 h
• Structures
– vesicles
– spores
in planta vesicle
clusters
Bar = 10 µm
Three Frankia genotypes
Betulaceae
Myricaceae
Casuarinacee
Elaeagnuaceae
Rhamnaceae
Myricaceae
Gymnostoma
Coriaiaceae
Datiscaceae
Rosaceae
Ceanothus
Why Sequence CcI3 and EAN1pec?
CcI3
•
•
•
•
Metabolism
Member of Group I
Narrow Host range
Markers:
KanR,GenR,KasR,NalR
AsO43-
EAN1pec
• Diverse metabolism
• Member of Group III
(globally distributed)
• Broader Host range
Markers:
NovR, LinR,KasR,NalR,
AsO43-,Pb2+ and
CrO42+
• Limited genetics
Surprise One: Three different genome sizes
ACN 6783 CDS 2 rRNA 72.8% GC
CcI3 4515 CDS
2 rRNA 70.1% GC
EAN 7492 CDS
3 rRNA 71.0% GC
Circular
Topology
Comparison of the CDS
Frankia ACN14a
reciprocal
blast search
with a cutoff
of 10-4.
2730
1190
630
2291
Frankia
CcI3
1333
587
3725
Frankia
Ean1pec
Comparative distribution of ORF function
Frankia sp. CcI3
1400
COG Functional
Groups
1200
Frankia sp. EAN1pec
Acidothermus cellulolyticus 11B
Arthrobacter sp. FB24
1000
Kineococcus radiotolerans SRS30216
800
Streptomyces coelicolor A3(2)
600
400
200
AA
M
et
ab
Ca
rb
M
et
ab
Ce
ll C
yc
le
Ce
ll M
ot
Ce
ll W
Ch
al
ro
l
m
a
St
ru
Co
c
en
z
Tr
an
s
De
fe
ns
e
En
er
gy
Fu
nc
Un
k
G
en
Fu
nc
Io
n
Tr
an
In
tra
s
ce
ll T
ra
ff
Li
pid
M
et
ab
Nu
c
St
ru
c
Nu
c
M
Po
et
st
ab
Tr
an
sM
od
RN
A
Pr
Re
oc
pl
ic
Re
Se
pa
co
ir
nd
M
et
ab
Si
gn
al
Tr
an
s
Tr
an
sc
r
Tr
an
sl
0
The EAN Genome is Expanding
1500
CcI3
ACN
EaN
1355
Gene Duplication Level is
higher in EAN (18.5% of the
ORFs) than ACN (7.5 %) or
CcI3 (9.8 %)
1000
500
0
T+I
Duplicates
ORFans
-500
CcI3 has an accelerated
rate of gene loss
compared to EAN and
ACN
-1000
-1500
1054
What are the Major families of duplicated
genes in these Frankia strains?
BlastClust (NCBI) analysis 25% identity over
at least 40% of the length (30% identity /52%
length same result)
An analysis of the Top 20 duplicated gene
families showed major differences in
functional groups
CcI3 (165/444)
• 116 out 165 (70%) duplicated genes belonged to
several classes of transposases and genes associated
with prophage and plasmids
EAN (406/1355)
• Transport proteins,
Dioxygenases, Short chain
dehydrogenases/reductases
(SDR), Regulatory proteins,
cytochrome P450,
monooxygenases
• also like CcI3 132 out of 406
(32.5%) genes associated
with integrases, transposases
ACN (151/512)
• Transport proteins,
SDR,serine-threonine
protein kinases,
methyltransferases,
endonucleases, & a
variety of
dehydrogenases
• no transposases in 151
genes of the top 20
families
CcI3
•loss of genes
associated with
transport and
metabolism
Present day native distribution of actinorhizal plant hosts.
a, ACN: Betulaceae (orange) Myricaceae
(green) and their overlap (khaki).
c, CcI3: Casuarina and Allocasuarina
of the Casuarinaceae (light blue).
b, EAN :Elaeagnaceae (pink), Myricaceae
(green) Rhamnaceae (blue, Tribe Colletieae
in South America, Australia and New
Zealand). Areas of overlap are brown and
dark blue).
What about genes identified as
potentially involved in Symbiosis?
•
•
•
•
•
Nitrogenase components
Hopanoid biosynthesis
Uptake Hydrogenase biosynthesis
Hemoglobin
Nodulation
Nitrogenase Cluster for EAN1pec
Synteny
hypothetical proteins
3 Fd genes
NifS
NifB NifZ NifW
NifX, NifN, NifE
NifV (homocitrate synthase) is located in
another region of the chromosome
NifK, NifD, NifH
NifV
MaGe site
Frankia Vesicles
• Laminated hopanoid lipids
• Restrict oxygen diffusion
• N2 fixation can occur “freeliving”
Parsons et al 1987
Berry et al. PNAS 1993
Cluster I: shcI
TetR
putative phytoene DH
Polyprenyl
synthetase
Squalene/phytoene
synthase
shcI
EAN & ACN extra shcI gene
amine oxidase
http://img.jgi.doe.gov/
cgi-bin/pub/main.cgi
Surprise Two:
Potential
symbiosis genes
are not clustered
katA
•
•
katG
•
•
•
HbO
shc2
sodF
nifV
HbN
•
•
•
•
•
•
•
cluster I: shc1
FRAEA6946-6954
cluster II: hup2*
FRAEA4081-4086
cluster III: hup1
FRAEA2955-2965
cluster IV: nif,
FRAEA8447-8463
HbO, FRAEA6420
HbN, FRAEA4419
shc2, FRAEA5736
katA,FRAEA8358
sodF,FRAEA4204
nodB-like
FRAEA6279,
NifV, FRAEA4890
Transcription Analysis of Two
Frankia hemoglobins
HboO expression is up-regulated
under hypoxic conditions
HboN expression is up-regulated by
NO release
Nitrogen status did not significantly
affect expression
Why the large genome (9.1 Mb) for
Frankia EAN1pec?
• many soil dwellers have large genomes
(Streptomyces, Bradyrhizobium,
Burkholderia, etc.
• these “boy-scouts” are always prepared
for changing conditions of the soil
environment
– wide array of substrates (uptake systems)
– need for tight regulation
Why the large genome (9.1 Mb) for
Frankia EAN1pec?
• many soil dwellers have large genomes
(Streptomyces, Bradyrhizobium,
Burkholderia, etc.
• these “boy-scouts” are always prepared
for changing conditions of the soil
environment
– wide array of substrates (uptake systems)
– need for tight regulation
Metabolism
• Complete Embden-Meyerhof, TCA and Pentose
Phosphate pathways
• wide arsenal of transport genes
• large numbers of genes for short chain
dehydrogenase/reductase, dioxygenase, etc.
Regulatory mechanisms
•
•
•
•
•
Large number of DNA binding proteins
Two-component systems
Sigma Factors
Anti-sigma Factors
Anti-sigma Factor Antagonists
Is Frankia EAN1pec versatile?
Quercetin
Catechol
DNA Regulatory Proteins
160
Frankia sp. EAN1pec
Frankia sp. CcI3
Acidothermus cellulolyticus 11B
Arthrobacter sp. FB24
Kineococcus radiotolerans SRS30216
Streptomyces coelicolor A3(2)
140
120
100
80
60
40
20
0
ArsR
DeoR
AraC
AsnC
GntR
IclR
LacI
LuxR
LysR
Lrp
MerR
TetR
Vesicle development
is influenced by:
a. N status
b. Oxygen
c. Mo & Fe
d. Calcium
e. Temperature
f. host plant
Proteome profiles of
Frankia CcI3 grown
under N2 or NH4Cl
conditions. Arrow point
out N2-grown specific
proteins.
Search for VesicleSpecific Proteins
Two-dimensional gel electrophoresis of vesicle
proteins isolated by Pressure Cycling
Technology
Purified
Vesicles
Perspectives
• Frankia genome expansion and
contraction reflects biogeographic history
of symbioses
• No “symbiosis islands”
• The time is right for functional genetics
– Proteomic Profiles
– Transcriptome Profiles (DNA arrays)
– Genetics
Acknowledgements
This work was supported by:
USDA Hatch grant 486; USDA 2003-01127;
NSF EF-0333173; DOE Microbial Genome Program
TISA LAB: Tania Rawnsley, James Niemann, Teal Furnholm, Nick
Beauchemin, Joanne Coulburn, Anna Myers
Arnab Sen (U. North Bengal)
UConn
David Benson
Peter Gogarten
UMaine
John Tjepkema
ULyon
Philippe Normand
JGI
Pilar Francino, Alla Lapidus
Paul Richardson, Chris Detter,
UNH CSB
Vern Rienhold
PCT
Gary Smejkal
All of the Frankia
community
The PULSE Tube used in Pressure Cycling Technology facilitates
high efficiency lysis of cells and subcellular components
Isolation of proteins from Frankia mycelium and vesicles by PCT