microbial loop and nutrient cycling david stopar october, 2001 nova gorica international...
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Microbial loop and nutrient cycling
David Stopar
October, 2001
Nova Gorica
International Short-Course Series
Bioremediation and Phytoremediation
of Organics and Nutrients
University of LjubljanaBiotechnical faculty
Vecna pot 111, SI-1000 Ljubljana, Slovenia
O2, CO2,
other gases
CH4
DMS
0-200 m
200-11000 m
organic material
Phytoplankton
POM
GRAZING
h
Zooplankton Fish
aggregatesDOM
Viruses Bacteria
bentos
Protozoa
C, N, P, S, Fe,...
solubilization
SEDIMENTATIONMICROBIAL LOOP
Microbial loop
DNA, RNA, sugars, ions
DNA, RNA,
sugars, ions
50 %
20 %
10 %
primary producerprotozoa
Why bacteria die?
• predation
• lethal environment
• starvation
• disease (phages)
• programmed cell death time (h)
CFU
Vibrio gazogenes organic carbon sources
Sugars
glucose
D-fructose
D-mannose
maltose
D-xylose
sucrose
trehalose
L-arabinose
D-galactose
D-ribose
Fatty acids
acetate
propionate
butyrate
caprate
Polymers
gelatin
DNA
cellobiose
peptone
yeast extract
Alcoholes
D-manitol
D-sorbitol
glycerol
Amino acids
L-serine
L-glutamat
L-proline
L-aspartate
Organic acids
succinate
DL-malate
DL-lactate
citrate
-ketoglutarate
piruvate
Vibrio lysate as a source of organic carbon for a bacterial community
Natural bacterial community is able to grow on bacterial lysate
CFU
lysate 9.6 x 108
lysate + mN +N 7.3 x 108
PYE 8.4 x 108
initial 8.2 x 105
Out of 26 different natural
bacterial isolates tested, 20
bacterial isolates were able to
use bacteria lysate as a source
of organic carbon.
no growth
growth
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
• phages mediate horizontal gene exchange
• phages mediate community structure
• phages influence the flow of energy and carbon
Phage role in the ecosystem
Impact of lysogenic viruses on nutrient cycling
OD660 = 0.5 OD
OD
t (min)
t (min)
control
mitomycin C
No phages
with TEM
Phages
with TEM
Bacteriophage induction
experiment
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
0.0
5.0
10.0
15.0
20.0
25.0
t0 kontrola Mit-c 24h
BD
C /m
l *10
5,
VLP
/ml *
107 BDC/ml*10(5)
VLP/ml *10(7)
58 % of bacterial community induced
Aerobic incubation
to control Mit-C
0.0
5.0
10.0
15.0
20.0
25.0
t0 kontrola mit-cB
DC
/ml *
105 ,
VLP
/ml*1
07
BDC/ml *10(5)
VLP/ml*10(7)
32 % of bacterial community induced
Anaerobic incubation
to control Mit-C
Impact of lytic viruses on nutrient cycling
L-E
B
E
R
t (min)
phagetiter
EMFT
tG
L
Phage titer )t/t(BoPtP G
Burst size B = (L-E) R
Phage generation time tG = L + (kN)-1
MFT adsorption (kN)-1
Exponential decay I
I
tNkeoPtP
Simulating phage production with and without
mean free time simplification
MINUTES
0 500 1000 1500 2000
log
(P
HA
GE
NU
MB
ER
)
0
10
20
30
40
106
108
107
109
Phage growth as a function of host density:theoretical versus experimental
MINUTES
0 50 100 150 200
PH
AG
E O
R H
OS
T D
EN
SIT
Y
10-1
100
101
102
103
104
105
106
107
A
MINUTES
0 50 100 150 200
PH
AG
E O
R H
OS
T D
EN
SIT
Y
100
101
102
103
104
105
106
107
108
109
1010
B
MINUTES
0 25 50 75 100 125 150
PH
AG
E O
R H
OS
T D
EN
SIT
Y
101
102
103
104
105
106
107
108
109
1010
1011
C
host density
o phage titer
exponential decay
MFT function
MFT function, Eqn2
Impact of host density on phage latent-period optima
HOST-CELL DENSITY (per ml)
103 104 105 106 107 108 109 1010 1011
OP
TIM
AL
LA
TE
NT
PE
RIO
D (
min
)
203050
200300500
200030005000
10
100
1000
10000
(Lopt = 48 min)
(Lopt = 281 min)
A
B
C
D
Impact of host quality on latent period optima
HOST-CELL DENSITY (per ml)
1e+5 1e+6 1e+7 1e+8 1e+9 1e+10
OP
TIM
AL
LA
TE
NT
PE
RIO
D (
min
)
20
30
40
50
60
708090
100C
acetate
HOST-CELL DENSITY (per ml)
1e+5 1e+6 1e+7 1e+8 1e+9 1e+10
OP
TIM
AL
LA
TE
NT
PE
RIO
D (
min
)20
30
40
50
60
708090
100B
glycerol
HOST-CELL DENSITY (per ml)
1e+5 1e+6 1e+7 1e+8 1e+9 1e+10
OP
TIM
AL
LA
TE
NT
PE
RIO
D (
min
)
20
30
40
50
60
708090
100A
glucose
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
At high cell density in a rich
medium a sub population of
cells commit suicide. In the
lysate viruses are present.
At low host density cell in a
poor medium there are no
viruses present.0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 20 40 60 80 100
time (h)
OD
660
PYE 5
PYE 2
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
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 80 100
time (h)
OD
66
0
rich growth conditions
poor growth
conditions
Pheromones and quorum sensing(a coordinated response to stress environment)
cell producing pheromone
cells attracted by pheromone
cells aggregate
Genetic competence in Bacillus subtilis
• 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
time (h)
Cel
l den
sity
% c
o mpe
ten c
e
Quorum sensing players in Bacillus subtilis
kinase domain
ComP
PComA
DNA
pre-ComX
ComQ
ComX
ATP
comXcomQ comP comA
Response regulator
Receptor kinase
PheromoneprecursorModification
maturation
Pheromone comX specificity test
comQXP
comX
producer strain
comP
srfA-lacZ
tester strain
lacZ activity
Quorum-sensing specificities
* Strains are grouped according to phylogenetic relationship
comQX comPA
producer Tester strain
strain 168 RO-C2 RO-FF1 RO-E2 RO-H1 RO-B2 NAF4
168 ++ ++ +
RO-C2 + ++
RO-FF1 ++
RO-E2 + ++ +
RO-A4 + + +
RO-PP2 + +
RO-H1 ++
RO-B1 ++ +
RO-DD2 ++ +
RO-B2 + ++
NAF4 ++
ComX(s) purification and characterization
srfA-lacZ
1- comQ and comX cloning and expression in E. coli 2- Purification by reverse phase chromatography
ComX(s) characteristics
Strain Sequence Δ
Mass
168 (A)DPITRQWGD + 206
RO-C-2 TREWDG + 206
RO-E-2 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?
• predation
• lethal environment
• starvation
• disease (phages)
• programmed cell death
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