International Short-Course Series
Bioremediation and Phytoremediation
of Organics and Nutrients
University of Ljubljana
Biotechnical faculty
Vecna pot 111, SI-1000 Ljubljana, Slovenia
Microbial loop and nutrient cycling
David Stopar
October, 2001
Nova Gorica
O2, CO2,
organic
other gases material
hn
GRAZING
CH4
DMS
Phytoplankton
POM
Zooplankton
C, N, P, S, Fe,...
aggregates
0-200 m
Protozoa
DOM
MICROBIAL LOOP
SEDIMENTATION
Viruses
200-11000 m
bentos
Fish
Bacteria
solubilization
Microbial loop
primary producer
protozoa
20 %
50 %
DNA, RNA,
sugars, ions
DNA, RNA,
sugars, ions
10 %
Why bacteria die?
• starvation
CFU
• disease (phages)
• programmed cell death
• predation
• lethal environment
time (h)
Vibrio gazogenes organic carbon sources
Sugars
Fatty acids
Polymers
Organic acids
glucose
acetate
gelatin
succinate
D-fructose
propionate
DNA
DL-malate
D-mannose
butyrate
cellobiose
DL-lactate
maltose
caprate
peptone
citrate
yeast extract
a-ketoglutarate
D-xylose
sucrose
piruvate
trehalose
L-arabinose
D-galactose
D-ribose
Amino acids
Alcoholes
D-manitol
D-sorbitol
glycerol
L-serine
L-glutamat
L-proline
L-aspartate
Vibrio lysate as a source of organic carbon for a
bacterial community
CFU
lysate
9.6 x 108
lysate + mN +mN
7.3 x 108
PYE
8.4 x 108
initial
8.2 x 105
Natural bacterial community
is able to grow on bacterial
lysate
Out of 26 different natural
no growth
bacterial isolates tested, 20
bacterial isolates were able to
growth
use bacteria lysate as a source
of organic carbon.
Why bacteria die?
• starvation
• disease (phages)
• programmed cell death
• predation
• lethal environment
Phage life cycle
Phage abundances
phages are probably the most abundant living
entities in the ecosystem
sea water
106 - 108/ml
fresh water
106 - 108/ml
sediments
108 - 109/g
soil
ND
Phage role in the ecosystem
• phages mediate horizontal gene exchange
• phages mediate community structure
• phages influence the flow of energy and carbon
Impact of lysogenic viruses on nutrient cycling
Bacteriophage induction
experiment
OD
No phages
with TEM
control
t (min)
OD660 = 0.5
OD
Phages
with TEM
mitomycin C
t (min)
In vitro phage induction from bacterial isolates
• 75 % of all tested strains
were lysogenic
• 51 % of all tested strains were
polylysogenic
In situ induction of phages from a sea water samples
20.0
BDC/ml *10(5)
VLP/ml*10(7)
25.0
20.0
15.0
5
15.0
7
BDC/ml*10(5)
VLP/ml *10(7)
Anaerobic incubation
BDC/ml *10 , VLP/ml*10
25.0
5
BDC /ml *10 , VLP/ml *10
7
Aerobic incubation
10.0
5.0
10.0
5.0
0.0
0.0
tot0
control
kontrola
58 % of bacterial
community induced
Mit-C
Mit-c
24h
tot0
kontrola
control
32 % of bacterial
community induced
mit-c
Mit-C
Impact of lytic viruses on nutrient cycling
phage
titer
tG
MFT
Phage titer
Pt  Po B( t / t G )
Burst size
B = (L-E) R
Phage generation time
tG = L + (kN)-1
MFT adsorption
(kN)-1
E
Exponential decay
R
E
L-E
L
B
t (min)
Pt  Po e kNt I
I
Simulating phage production with and without
mean free time simplification
log (PHAGE NUMBER)
40
10
10
9
8
30
10
7
20
10
10
6
0
0
500
1000
MINUTES
1500
2000
Phage growth as a function of host density:
theoretical versus experimental
1
1
1
0
1
0
1
0
B
9
1
0
PHAGE OR HOST DENSITY
PHAGE OR HOST DENSITY
A
6
1
0
9
1
0
8
1
0
5
1
0
7
1
0
4
1
0
8
1
0
6
1
0
3
1
0
7
1
0
5
1
0
6
1
0
4
1
0
2
1
0
5
1
0
3
1
0
1
1
0
4
1
0
2
1
0
0
1
0
1
1
0
0
3
1
0
1
1
0
2
1
0
0
1
0
5
0
1
0
0 1
5
0 2
0
0
M
I
N
U
T
E
S
 host density
o phage titer
 exponential decay
 MFT function
 MFT function, Eqn2
0
C
1
0
1
0
PHAGE OR HOST DENSITY
7
1
0
5
0
1
0
0 1
5
0 2
0
0
M
I
N
U
T
E
S
1
1
0
02
55
07
51
0
0
1
2
5
1
5
0
M
I
N
U
T
E
S
OPTIMAL LATENT PERIOD (min)
Impact of host density on phage latent-period optima
10000
A
5000
3000
2000
1000
500
300
200
C
(Lopt = 281 min)
D
100
50
30
20
B
(Lopt = 48 min)
10
103 104 105 106 107 108 109 1010 1011
HOST-CELL DENSITY (per ml)
Impact of host quality on latent period optima
glucose
4
0
3
0
2
0
A
5
0
4
0
3
0
2
0
1
0
0
9
0
8
0
7
0
6
0
OPTIMAL LATENT PERIOD (min)
5
0
1
0
0
9
0
8
0
7
0
6
0
OPTIMAL LATENT PERIOD (min)
OPTIMAL LATENT PERIOD (min)
1
0
0
9
0
8
0
7
0
6
0
acetate
glycerol
B
C
5
0
4
0
3
0
2
0
1
e
+
5
1
e
+
6
1
e
+
7
1
e
+
8
1
e
+
9
1
e
+
1
0
1
e
+
5
1
e
+
6
1
e
+
7
1
e
+
8
1
e
+
9
1
e
+
1
0
1
e
+
5
1
e
+
6
1
e
+
7
1
e
+
8
1
e
+
9
1
e
+
1
0
H
O
S
T
C
E
L
L
D
E
N
S
I
T
Y
(
p
e
r
m
l
)
H
O
S
T
C
E
L
L
D
E
N
S
I
T
Y
(
p
e
r
m
l
)
H
O
S
T
C
E
L
L
D
E
N
S
I
T
Y
(
p
e
r
m
l
)
 high quality host, control
 E-varied
 k-varied
 R-varied
 E + R + k varied
Why bacteria die?
• starvation
• disease (phages)
• programmed cell death
• predation
• lethal environment
developmental processes (i.e. sporulation)
altruistic suicide
ageing
antibiotics or stress related factors
What is the benefit for unicellular organism of
committing a suicide?
• no obvious reason unless we consider a unicellular organism as
being part of a complex microbial community
• better use of resources
• reduced mutation rate (elimination of DNA damaged cells)
• reducing the impact of infection by pathogens
• lowering the probability of take over mutants
• facilitating genetic exchange
Population of Vibrio committing a suicide after
entering a stationary phase
1.8
At high cell density in a rich
1.6
1.4
PYE 5
1.2
OD660
medium a sub population of
cells commit suicide. In the
1
lysate viruses are present.
0.8
0.6
0.4
At low host density cell in a
PYE 2
poor medium there are no
0.2
0
0
20
40
60
time (h)
80
100
viruses present.
Survival of rare cells in a population
• sensitivity of the whole population to a programme cell death could
eliminate the whole clonal population (a contraproductive strategy)
• experimentally it is known that the entire population is not sensitive
to the external damaging effect (i.e. UV, antbiotics)
• a random variation of regulator molecules can induce or prevent a
suicide program
Survival of rare cells after induction with mitomycin C
1.2
rich growth
conditions
1
OD660
0.8
0.6
0.4
poor growth
conditions
0.2
0
0
20
40
60
time (h)
80
100
Pheromones and quorum sensing
(a coordinated response to stress environment)
cell producing
pheromone
cells attracted
by pheromone
cells aggregate
Genetic competence in Bacillus subtilis
time (h)
• develops during stationary
phase, when 1-10% cells
become competent and
ready to uptake foreign
DNA
• genetic competence is under
nutritional control and cell
density control i.e. quorum
sensing
• it is cell last chance to avoid
sporulation
Quorum sensing players in Bacillus subtilis
Pheromone
Modification precursor
maturation
comQ
comX
Receptor
kinase
comP
ComX
ComP
kinase domain
ComQ
ATP
pre-ComX
P
DNA
ComA
Response
regulator
comA
Pheromone comX specificity test
producer strain
tester strain
comX
comQXP
comP
srfA-lacZ
lacZ activity
Quorum-sensing specificities
comQX
comPA
producer
Tester strain
strain
168
RO-C2
RO-FF1
168
++
++
+
RO-C2
+
++
RO-E2
RO-H1
RO-FF1
++
RO-E2
+
++
+
RO-A4
+
+
+
+
+
RO-PP2
RO-B2
RO-H1
++
RO-B1
++
+
RO-DD2
++
+
RO-B2
+
++
NAF4
* Strains are grouped according to phylogenetic relationship
NAF4
++
ComX(s) purification and characterization
1- comQ and comX cloning and expression in E. coli
2- Purification by reverse phase chromatography
srfA-lacZ
ComX(s) characteristics
Strain
168
RO-C-2
RO-E-2
Sequence
Δ Mass
(A)DPITRQWGD
+ 206
TREWDG
+ 206
GIFWEQ
+ 136
RS-B-1
(M)MDWHY
+ 120
RO-H-1
(M)LDWKY
+ 120
RO-B-2
(Y)TNGNWVPS
+ 136
*Δ Mass = obtained mass - calculated mass
Modification masses are consistent with farnesylation or geranylation of
com X in addition ComQ resembles a farnesyl-geranyl transferase
Why bacteria die?
• starvation
• disease (phages)
• programmed cell death
• predation
• lethal environment
Bacterial and viral loop facilitate nutrient cycling
DNA, RNA,
sugars, ions
DNA, RNA,
sugars, ions
Acknowledgements
Ivan Mahne
Ines Mandič-Mulec
Kaja Gnezda
Aleša Černe
Andrej Žagar
Duško Odič
Dave Dubnaw, New York University, USA
Valentina Turk, National Institute of Biology, SI
Mateja Poljšak-Prijatelj, Institute of Microbiology and Immunology, SI
Stephen T. Abedon, Ohio State University, USA