Understanding Microbial Persistence and Adaptation in the Environment
A Case Study of Sulfate-Reducing Bacteria
Qiang He
Assistant Professor
Department of Civil and Environmental Engineering
Center for Environmental Biotechnology
The University of Tennessee
Knoxville, TN, USA
Stress Response as Key to Survival and Adaptation
Ecosystem
Identify key factors (i.e., stresses) that drive community structure and composition and
impact the survival and efficacy of microorganisms
Ecosystem
Community
How do communities respond to stress?
Ecology
Computational
Ecology
Geochemistry
Computational
Community
Population
Populations
Determine the impact of stress on organisms
(Desulfovibrio vulgaris)
Cell
Protein
RNA
Cell
Infer key stress response pathways and how gene networks
interplay under different stress conditions
DNA
Genomic
Proteomic
Metabolomic
Computational
Steps to Study Stress Responses
1. Identify key factors (biotic and abiotic) that might control nutrient flow, stress, and
survival
2. Determine impact and stress response pathways in key microorganisms
3. Construct conceptual models for stress and survival for chosen microorganisms
4. Demonstrate how stress conditions impact biochemical capacity (e.g., metal
reduction) and cellular survival
Definition of Stress
No Universal Definition for Stress
Highly dependent upon the individual cell
Working definitions
•
Any deviation from optimal growth conditions
that results in reduced growth rate
•
An environmental situation that results in
damage of cellular components in the absence
of a cellular response
Any situation that stimulates expression of
known stress-response genes
•
IMPORTANCE OF SULFATE-REDUCING BACTERIA
Why study SRB?
Producers of toxic compounds
Ecological areas
ex: hydrogen sulfide
Industrial areas
Reduction of heavy metals
and radioactive compounds
Anaerobic corrosion of underground
buried ferrous metals
ex: tanks and pipelines
Human Health
Ulcerative Colitis; Anaerobic Abscess
Processes
Metabolism of environmental
polutants
Bioremediation
Sulfate removal from the medium
SULFATE REDUCING BACTERIA
•Anaerobic bacteria that respire sulfate:
SO42-
8e- S2Found in a large variety of environments
Electron acceptors - sulfate, sulfite,
thiosulfate, sulfur, nitrate e fumarate.
Electron donors - lactate, pyruvate,
hydrogen, fumarate, malate, ethanol.
•
Can use molecular oxygen to sustain life, but not to grow.*
*Lemos et al. (2001) FEBS Lett 496:40-43
Frazão et al. (2000) Nature Struct.Biol. 7:1041-1045
Cypionka (2000) Annu.Rev.Microbiol. 54:827-848
LeGall and Xavier (1996) Anaerobes 2:1-9
Santos et al. (1993) Biochem.Biophys.Res.Commun. 195:551-557
Why Sulfate-Reducing Bacteria?
SO42SO32organic S
S0
H 2S
Global S Cycle
Global C Cycle
Souring of oil
reservoirs
Microbial-induced corrosion
Pathogenic Desulfovibrio
Interspecies Genomic Hybridization:
RMA 14127: 96.6%
61
RMA 14567
90
RMA 16092
82
RMA 15168: 97.7%
93
Desulfovibrio fairfieldensis
Desulfovibrio sp D4
99
RMA 10276
96
Desulfovibrio sp. oral clone BB161
RMA 8703
100
100
89
RMA 16470
Desulfovibrio pigra
Desulfovibrio vulgaris Hildenborough
100
100
100
78
RMA 14127
RMA 15168
Desulfovibrio desulfuricans G20
Desulfotalea psychrophila LSv54
Myxococcus xanthus DK 1622
E. coli K12
0.02
In collaboration with Goldstein lab, UCLA
General Scheme: Stress Response in SRB
What strains?
Which stress/ Stimuli?
Biomass
Knockouts/mutants
How do environmental stimuli affect individual microorganisms?
How do these responses affect microbial communities?
Which stimuli are the
most informative?
APPLICATION/ MODELS
Functional Genomics: Stress Response in SRB
Wild-type
Available mutants
Environmental isolates
Knockouts/mutants
Stress
Physiology
Transcriptomics
Proteomics
Metabolomics
Computation
Single mutations
Multiple mutations
Decision?
Physiology
Core Stressors
•
•
•
•
•
•
•
Temperature change
Nitrate, Nitrite
Osmotic – NaCl, KCl
Oxygen, Air
pH – high, low
Chromate
fur mutant
LS4D
+ 250mM NaCl
A Systems Biology Approach
Growth (OD)
C1
control
baseline
T0
V1
stress
0.3
0
Transcriptomics
2
Time (hours)
Leu
Proteomics
Lys
Ile
Arg Val
5
10
15
Phe
Pro Met Asp
His
0
Glu
20
25
Metabolomics
Time [min]
Nitrate as a stressor
•NaNO3 is more inhibitory
8 8
Cell
CellDensity,
cells/ml
Density,1010cells/ml
•Indicative of responses in
addition to osmotic stress
8 8
Cell
cells/ml
Density,
1010
Cell
cells/ml
Density,
Phenotype Array Results
He et al., 2010. ISME J.
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
0
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
0
A
A
0mM
0mM
30mM
30mM
50mM
50mM
70mM
70mM
90mM
90mM
100mM
100mM
120mM
120mM
140mM
140mM
160mM
160mM
180mM
180mM
200mM
200mM
NaNO3
20
20
0
0
40
40
Hour
Hour
80
80
60
60
0mM
0mM
50mM
50mM
100mM
100mM
150mM
150mM
200mM
200mM
250mM
250mM
300mM
300mM
350mM
350mM
400mM
400mM
450mM
450mM
500mM
500mM
B
B
NaCl
0
0
20
20
40
40
Hour
Hour
60
60
80
80
Effect of Osmo-protectant
1.4
A
1.2
1.4
OD600 OD
600
1.2
A
1
0.8
1
0.6
0.8
0.4
0.6
0.2
0.4
0.2
0
0
50
1.2
1
0.8
1.2
0.6
1
0.4
0.8
0.2
0.6 0
0.4 0
0.2
0
100
0
50
100
150
150
50
200
100
150
Hour
0
0
50
100
150
200
Hour
1.4
B
1.2
1.4
OD600 OD
600
1.2
B
1
0.8
1
0.6
0.8
0.4
0.6
0.2
0.4
0.2
He et al., 2010. ISME J.
0
0
0
100
200
Hour
300
400
Osmotic Stress
1.4
1.2
Control
2 mM GB
OD 600 nm
1
0.8
250 mM NaCl
0.6
250 mM KCl
0.4
250 mM NaCl +
2 mM GB
250 mM KCl +
2 mM GB
0.2
0
0
20
40
Time (h)
60
Methyl/SAM Cycle
S-adenosyl-Lhomocysteine
Methyl
acceptor
AhcY
DVU0607
MetE
Methyltransferase
Methyl-THF
DVU3371
DVU0606
S-adenosyl-Lmethionine
L-homocysteine
MetF
MetK
DVU2449
DVU0997
L-methionine
Methylene-THF
PFLA
DVU2825
Pyruvate
PFL
DVU2824
Acetyl-CoA+ H+ + HCOOFDH
LDH
Lactate
Lactate permease
DVU2110
Medium
DVU0586-8
CO2 + 2e-
Methyl/SAM Cycle
Log2 Ratio of Transcriptional Response
NaNO2
NaCl
Δfur
Δfur +
NaCl
Gene ID
NaNO3
TIGR Annotation
Δfur + NaNO3
Methyl metabolism
DVU0606
2.5
2.0
-1.0
-2.1
-3.3
-3.5
regulator/methyltransferase, UbiE/COQ5 family
DVU0607
2.7
2.4
1.1
-2.4
-2.9
-3.0
adenosylhomocysteinase, AhcY
DVU0997
2.9
2.2
0.6
-3.2
-1.9
-2.2
5,10-methylenetetrahydrofolate reductase, MetF
DVU2449
1.7
2.1
-1.3
-0.2
-3.8
-2.7
S-adenosylmethionine synthetase, MetK
DVU3371
2.7
3.8
-1.4
-3.4
-2.5
-2.0
5-methyltetrahydropteroyltriglutamate-homocysteine Smethyltransferase, MetE
Impact of Nitrite on SRB
•
Key Questions:
– How does nitrite impact SRB?
– How do SRB respond to nitrite?
– How can we help SRB do their job?
6
1
Nitrite, mM
0.8
OD600
0.6
0.4
4
3
a
2
2
1
0 mM
b
0.5 mM
0.2
0
1.0 mM
5.0 mM
0
0
2
4
6
hour
Growth inhibition by nitrite
0
0
8
0
2
4
5
10
6
h
Nitrite reduction by D. vulgaris
15
20
8
Global Transcriptional Analysis
35
30
A
B
C
Up-Regulated
D
E
F
T1
G
T2
H
T3
T4
I
T5
•
25
Functional categories repressed
–
–
–
–
20
15
•
10
Functional categories induced
–
–
–
5
0
(A) Amino acid biosynthesis
(B) Cofactor biosynthesis
(F) Protein synthesis
(I) Transport and binding proteins
(G) Regulatory functions
(H) Signal transduction
(E) Energy metabolism
-5
-10
•
-20
Down-Regulated
-25
-30
-35
Normal cell growth stopped
–
-15
A—Amino acid biosynthesis
B—Biosynthesis of cofactors
C—Cell envelope
D—Cellular processes
E—Energy metabolism
F—Protein synthesis
G—Regulatory functions
H—Signal transduction
I—Transport and binding proteins
Consistent with growth curve
•
Cells in transitional phase
•
Detoxification mechanism involves electron
transport
–
Nitrite reduction
Hierarchical clustering analysis
C
ATP synthase F0, A subunit
ATP synthase F0, C subunit
ATP synthase, F1 alpha subunit
ATP synthase, F1 beta subunit
ATP synthase, F1 delta subunit
ATP synthase, F1 gamma subunit
D
350
2.5
Up genes
300
Dn genes
Nitrite
2
250
1.5
200
150
1
Nitrite, mM
DVU2925 ribosomal protein L1
DVU2926 ribosomal protein L10
DVU2924 ribosomal protein L11
DVU2518 ribosomal protein L13
DVU1310 ribosomal protein L16
DVU1319 ribosomal protein L18
DVU0927 ribosomal protein L21
DVU1574 ribosomal protein L25
DVU1211 ribosomal protein L28
DVU1303 ribosomal protein L3
DVU1074 ribosomal protein L34
dependent receptor domain protein
DVU2383 tonBribosomal
protein L7/L12
DVU2927
iron transport protein B
DVU2571 ferrous
DVU0958 ribosomal protein L9
iron transport protein A, putative
DVU2572 ferrous
DVU1302 ribosomal protein S10
DVU2573 hypothetical protein
DVU1298 ribosomal protein S12
DVU2574 ferrous ion transport protein, putative
DVU1316 ribosomal protein S14
DVU2680 flavodoxin
DVU1312 ribosomal protein S17
ribosomal
protein S18
DVU0957
DVU0121
conserved
hypothetical
protein
DVU0122
hypothetical
protein
ribosomal
protein S2
DVU0874
DVU0123
membrane
protein,
putative
ribosomal
protein
S20
DVU1896
NapC/NirT
cytochrome
c family protein
DVU0624
DVU0956 ribosomal protein S6
cyt
c
nitrite
reductase,
catalytic
subunit NfrA
DVU0625
DVU1299 ribosomal protein S7
DVU0943
membrane
protein,
putative
DVU2519 ribosomal protein S9
DVU0944 hypothetical protein
DVU1080 iron-sulfur cluster-binding protein
DVU1081 iron-sulfur cluster-binding protein
DVU1419 sigma-54 dependent transcriptional regulator
DVU2132 hypothetical protein
DVU2133 membrane protein, putative
DVU2543 hybrid cluster protein
DVU2544 iron-sulfur cluster-binding protein
Number of ORFs
D
DVU0918
DVU0917
DVU0777
DVU0775
DVU0778
DVU0776
Nitrite Reduction vs Gene Expression
100
0.5
50
0
0
0.5h
1h
1.5h
2.5h
3.5h
Shown are genes with > 2 fold change
C
B
B
A
T1 T2 T3 T4 T5
A
1.
Nitrite reductase gene and genes in
the Fur regulon were highly upregulated in nitrite stress.
2.
Genes in protein biosynthesis and
energy conservation were severely
down-regulated.
3.
An apparent correlation between the
dynamics of transcriptional response
and the reduction of nitrite.
4.
Nitrite reduction was indicated as the
main detoxification mechanism.
5.
Electron flow was shifted from
oxidative phosphorylation to nitrite
reduction.
Up-regulation of Genes of Fe-Proteins
Response of Fur Regulon to Nitrite Stress
9
8
7
Fold Change (Treatment/Control)b
Gene ID
TIGR Annotation
0.5h
1.0h
1.5
h
2.5
h
4.0
h
DVU0763
GGDEF domain protein
+11.9
+2.1
—
—
—
DVU2378
transcriptional regulator, AraC family
+4.3
+4.1
+2.4
—
—
DVU2574
ferrous iron transport protein, putative
FeoA
+3.5
+5.0
+3.9
—
—
DVU2680
Flavodoxin
+27.6
+22.6
+4.9
—
—
DVU3330
conserved hypothetical protein
+2.3
+5.7
+2.3
—
—
DVU0273
conserved hypothetical protein
+15.3
+5.2
+1.8
—
-2.2
DVU0304
hypothetical protein
+34.0
+10.1
+3.7
—
—
Response of Per Regulon to Nitrite Stress
Fold Change (Treatment/Control)b
Gene ID
TIGR Annotation
0.5h
1.0h
1.5h
2.5h
4.0h
DVU0772
hypothetical protein
+1.8
+2.4
+2.6
+2.1
—
DVU2247
antioxidant, AhpC/Tsa family
+3.0
+3.1
+2.1
+1.8
—
DVU2318
rubrerythrin, putative
—
—
+1.5
—
-1.9
DVU3095
Transcriptional regulator, Fur family,
PerR
—
—
—
+2.2
—
DVU3096
hypothetical protein
—
+1.8
—
—
—
fold
6
5
4
3
2
1
0
0
30
60
90
150
240
min
all
iron-binding
fur-regulated
•Nitrite stress led to in the derepression of
the Fur regulon, which was possibly
resulted from iron deficiency.
•The primary cause of iron deficiency could
be attributed to increased demand for iron
under nitrite stress.
•Nitrite as an oxidizing agent also induced
oxidative stress exemplified by the up
regulation of the Per regulon.
Hierarchical clustering analysisResponses to Nitrite in Energy Metabolism
•A coordinated cascade of responses to nitrite in pathways of energy
metabolism, nitrogen metabolism, oxidative stress response, and
iron homeostasis.
N Metabolism
Nitrate vs Nitrite
Log2 Ratio of Transcriptional Responseb
Gene ID
NaNO3
TIGR Annotation
NaNO2
NaCl
Δfur
Δfur +
NaCl
Δfur + NaNO3
Methyl metabolism
DVU0606
2.5
2.0
-1.0
-2.1
-3.3
-3.5
regulator/methyltransferase, UbiE/COQ5 family
DVU0607
2.7
2.4
1.1
-2.4
-2.9
-3.0
adenosylhomocysteinase, AhcY
DVU0997
2.9
2.2
0.6
-3.2
-1.9
-2.2
5,10-methylenetetrahydrofolate reductase, MetF
DVU2449
1.7
2.1
-1.3
-0.2
-3.8
-2.7
S-adenosylmethionine synthetase, MetK
DVU3371
2.7
3.8
-1.4
-3.4
-2.5
-2.0
5-methyltetrahydropteroyltriglutamate-homocysteine Smethyltransferase, MetE
Nitrogen metabolism
DVU2543
1.8
5.7
-1.2
1.9
-0.3
1.9
hybrid cluster protein
DVU2544
1.9
6.2
0.5
1.6
1.2
2.4
iron-sulfur cluster-binding protein
DVU0624
0.3
4.4
-1.3
-0.4
-0.6
2.6
NapC/NirT cytochrome c family protein
DVU0625
0.7
4.1
-1.3
0.1
0.5
2.8
cytochrome c nitrite reductase, catalytic subunit NrfA
PerR Regulon in Nitrate Stress
Log2 (Expression Ratio)b
Gene ID
Description
DVU0772
hypothetical protein
DVU2247
alkyl hydroperoxide reductase C, ahpC
DVU2318
Rubrerythrin, putative, rbr2
DVU3093
Rubredoxin-like protein, rdl
DVU3094
Rubrerythrin, rbr
DVU3095
Peroxide-responsive regulator PerR
30 min
60 min
120min
240 min
0.1
0.7
2.0
2.4
0.2
0.5
0.7
1.6
0.4
0.6
0.9
2.2
-0.2
0.0
0.8
1.2
-0.2
-0.1
-0.3
0.7
-0.5
0.0
0.8
1.4
Response of Per Regulon to Nitrite Stress
Fold Change (Treatment/Control)b
Gene ID
TIGR Annotation
0.5h
1.0h
1.5h
2.5h
4.0h
DVU0772
hypothetical protein
+1.8
+2.4
+2.6
+2.1
—
DVU2247
antioxidant, AhpC/Tsa family
+3.0
+3.1
+2.1
+1.8
—
DVU2318
rubrerythrin, putative
—
—
+1.5
—
-1.9
DVU3095
Transcriptional regulator, Fur family,
PerR
—
—
—
+2.2
—
DVU3096
hypothetical protein
—
+1.8
—
—
—
Energy Metabolism
+
FDH
H
2e + 2
HCOOH
BT
Nitrite Stress
G
Hcp
Acetyl-CoA + H+ + HCOO-
Cycle
PerR
AHP
RBR
RDL
PFL
Pyruvate
LDH
SAM/Methyl
PFLA
Cytoplasm
Periplasm
NH3
hi
NO3-
In
NO3-
bi
tio
n
G
B
HCOOH
NH3OH
NO2-
Inhibition
Sa
lt
St
re
ss
-
Cellular Model of Nitrate Stress Response
NO3-
Lactate + ADP + Pi
General
Stress
Response
NO3Lactate
Components of nitrate stress response
•Characteristics of osmotic stress: Glycine betaine transporters
•Characteristics of nitrite stress: Hybrid cluster protein
•General stress response: oxidative stress genes
•SAM/Methyl cycle genes
Cellular Model of Nitrite
Stress Response
Comparative Analysis of Nitrate Stress Responses in D. Vulgaris
Gene expression correlations
•Minimal correlation in gene expression between nitrate, nitrite, NaCl, and other stress conditions
•Nitrate stress response includes components of both nitrite and NaCl stress responses.
•Nitrate stress shares general stress responses with other stressors.
Practical Implications?
Thank you!