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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. Ecosystem. - PowerPoint PPT Presentation
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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
EcosystemIdentify key factors (i.e., stresses) that drive community structure and composition and impact the survival and efficacy of microorganisms
CommunityHow do communities respond to stress?
Populations
Determine the impact of stress on organisms
(Desulfovibrio vulgaris)
Cell
Infer key stress response pathways and how gene networks
interplay under different stress conditions
DNA
RNA
Protein
Population
Community
Ecosystem
Cell
GenomicProteomicMetabolomicComputational
EcologyComputational
EcologyGeochemistryComputational
Stress Response as Key to Survival and Adaptation
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
Steps to Study Stress Responses
• 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
No Universal Definition for Stress
Highly dependent upon the individual cell
Working definitions
Definition of Stress
IMPORTANCE OF SULFATE-REDUCING BACTERIA
Why study SRB?
Ecological areasProducers of toxic compounds ex: hydrogen sulfide
Sulfate removal from the medium
Metabolism of environmental polutants
Reduction of heavy metals and radioactive compounds
Industrial areas
Anaerobic corrosion of underground buried ferrous metalsex: tanks and pipelines
Bio
rem
ed
iatio
n
Pro
cesse
s
Human Health Ulcerative Colitis; Anaerobic Abscess
SULFATE REDUCING BACTERIA
Found in a large variety of environments
8e-SO4
2- S2-
•Anaerobic bacteria that respire sulfate:
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 496:40-43Frazão et al. (2000) Nature Struct.Biol. 7:1041-1045Cypionka (2000) Annu.Rev.Microbiol. 54:827-848LeGall and Xavier (1996) Anaerobes 2:1-9Santos et al. (1993) Biochem.Biophys.Res.Commun. 195:551-557
Global C Cycle
Microbial-induced corrosion
Souring of oilreservoirs
Souring of oilreservoirs
SO42-
SO32-
S0
H2S
organic S
Global S Cycle
Why Sulfate-Reducing Bacteria?
Pathogenic Desulfovibrio
RMA 14567
RMA 16092
Desulfovibrio fairfieldensis
Desulfovibrio sp D4
RMA 10276
Desulfovibrio sp. oral clone BB161
RMA 8703
RMA 16470
Desulfovibrio pigra
Desulfovibrio vulgaris Hildenborough
RMA 14127
RMA 15168
Desulfovibrio desulfuricans G20
Desulfotalea psychrophila LSv54
Myxococcus xanthus DK 1622
E. coli K12
100
100
78
100
89
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100
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93
82
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RMA 14567
RMA 16092
Desulfovibrio fairfieldensis
Desulfovibrio sp D4
RMA 10276
Desulfovibrio sp. oral clone BB161
RMA 8703
RMA 16470
Desulfovibrio pigra
Desulfovibrio vulgaris Hildenborough
RMA 14127
RMA 15168
Desulfovibrio desulfuricans G20
Desulfotalea psychrophila LSv54
Myxococcus xanthus DK 1622
E. coli K12
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89
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In collaboration with Goldstein lab, UCLA
Interspecies Genomic Hybridization:
RMA 14127: 96.6%
RMA 15168: 97.7%
Which stimuli are the most informative?
Knockouts/mutants
APPLICATION/ MODELS
What strains? Which stress/ Stimuli?
Biomass
How do environmental stimuli affect individual microorganisms?
How do these responses affect microbial communities?
General Scheme: Stress Response in SRB
Transcriptomics Proteomics Metabolomics
Computation
Knockouts/mutants
Physiology
Decision?
Physiology
Single mutationsMultiple mutations
Wild-typeAvailable mutants
Environmental isolates Stress
Functional Genomics: Stress Response in SRB
• Temperature change• Nitrate, Nitrite• Osmotic – NaCl, KCl• Oxygen, Air• pH – high, low• Chromate• fur mutant
LS4D
+ 250mM NaCl
Core Stressors
T0
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baseline
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Asp
Phe
Glu
Pro
Ile
Leu
Lys
Arg Val
His Met
Metabolomics
Proteomics
Transcriptomics
A Systems Biology Approach
Nitrate as a stressor
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A
B NaNO3
NaCl
He et al., 2010. ISME J.
Phenotype Array Results
•NaNO3 is more inhibitory
•Indicative of responses in addition to osmotic stress
Effect of Osmo-protectant
He et al., 2010. ISME J.
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Control
2 mM GB
250 mM NaCl
250 mM KCl
250 mM NaCl +2 mM GB250 mM KCl +2 mM GB
Osmotic Stress
Methyl/SAM Cycle
Medium
Lactate permeaseDVU2110
Lactate
LDH
Pyruvate
CO2 + 2e-
Acetyl-CoA+ H+ + HCOO-
S-adenosyl-L-homocysteine
AhcYDVU0607 L-homocysteine
MetEDVU3371
L-methionineMetK
DVU2449S-adenosyl-L-methionine
Methyl-THFMethyltransferaseDVU0606
Methyl acceptor
MetFDVU0997
Methylene-THFPFLA
DVU2825
PFLDVU2824
FDHDVU0586-8
Medium
Lactate permeaseDVU2110
Lactate
LDH
Pyruvate
CO2 + 2e-
Acetyl-CoA+ H+ + HCOO-
S-adenosyl-L-homocysteine
AhcYDVU0607 L-homocysteine
MetEDVU3371
L-methionineMetK
DVU2449S-adenosyl-L-methionine
Methyl-THFMethyltransferaseDVU0606
Methyl acceptor
MetFDVU0997
Methylene-THFPFLA
DVU2825
PFLDVU2824
FDHDVU0586-8
Methyl/SAM Cycle
Gene ID
Log2 Ratio of Transcriptional Response
TIGR Annotation
NaNO3 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 -2.05-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase, 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?
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ite, m
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b
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ite, m
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b
Growth inhibition by nitrite Nitrite reduction by D. vulgaris
• Functional categories repressed– (A) Amino acid biosynthesis– (B) Cofactor biosynthesis– (F) Protein synthesis– (I) Transport and binding proteins
• Functional categories induced– (G) Regulatory functions– (H) Signal transduction– (E) Energy metabolism
• Normal cell growth stopped– Consistent with growth curve
• Cells in transitional phase
• Detoxification mechanism involves electron transport
– Nitrite reduction
Global Transcriptional Analysis
-35
-30
-25
-20
-15
-10
-5
00
5
10
15
20
25
30
35
T1 T2 T3 T4 T5
A B C D E F G H I Up-Regulated
Down-Regulated 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
T1 T2 T3 T4 T5
A
B
C
D
DVU0121 conserved hypothetical protein DVU0122 hypothetical protein DVU0123 membrane protein, putative DVU0624 NapC/NirT cytochrome c family protein DVU0625 cyt c nitrite reductase, catalytic subunit NfrA DVU0943 membrane protein, putative 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
A
DVU2383 tonB dependent receptor domain protein DVU2571 ferrous iron transport protein B DVU2572 ferrous iron transport protein A, putative DVU2573 hypothetical protein DVU2574 ferrous ion transport protein, putative DVU2680 flavodoxin
B
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 DVU2927 ribosomal protein L7/L12 DVU0958 ribosomal protein L9 DVU1302 ribosomal protein S10 DVU1298 ribosomal protein S12 DVU1316 ribosomal protein S14 DVU1312 ribosomal protein S17 DVU0957 ribosomal protein S18 DVU0874 ribosomal protein S2 DVU1896 ribosomal protein S20 DVU0956 ribosomal protein S6 DVU1299 ribosomal protein S7 DVU2519 ribosomal protein S9
C
DVU0918 ATP synthase F0, A subunit DVU0917 ATP synthase F0, C subunit DVU0777 ATP synthase, F1 alpha subunit DVU0775 ATP synthase, F1 beta subunit DVU0778 ATP synthase, F1 delta subunit DVU0776 ATP synthase, F1 gamma subunit
D
1. Nitrite reductase gene and genes in the Fur regulon were highly up-regulated 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.
Hierarchical clustering analysis
0
50
100
150
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350
0.5h 1h 1.5h 2.5h 3.5h
Num
ber
of O
RF
s
0
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1
1.5
2
2.5
Nit
rite
, mM
Up genes
Dn genes
Nitrite
Shown are genes with > 2 fold change
Nitrite Reduction vs Gene Expression
Up-regulation of Genes of Fe-Proteins
•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.
0
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8
9
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all iron-binding fur-regulated
Gene ID TIGR Annotation
Fold Change (Treatment/Control)b
0.5h 1.0h1.5h
2.5h
4.0h
DVU0763 GGDEF domain protein +11.9 +2.1 — — —
DVU2378 transcriptional regulator, AraC family +4.3 +4.1 +2.4 — —
DVU2574ferrous 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 — —
Gene ID TIGR AnnotationFold Change (Treatment/Control)b
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
DVU3095Transcriptional regulator, Fur family, PerR
— — — +2.2 —
DVU3096 hypothetical protein — +1.8 — — —
Response of Per Regulon to Nitrite Stress
Response of Fur Regulon to Nitrite Stress
Hierarchical clustering analysisResponses to Nitrite in Energy Metabolism
N Metabolism
•A coordinated cascade of responses to nitrite in pathways of energy metabolism, nitrogen metabolism, oxidative stress response, and iron homeostasis.
Nitrate vs Nitrite
Gene ID
Log2 Ratio of Transcriptional Responseb
TIGR AnnotationNaNO3 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 -2.05-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase, 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
Gene ID Description
Log2 (Expression Ratio)b
30 min 60 min 120min 240 min
DVU0772 hypothetical protein0.1 0.7 2.0 2.4
DVU2247 alkyl hydroperoxide reductase C, ahpC0.2 0.5 0.7 1.6
DVU2318 Rubrerythrin, putative, rbr20.4 0.6 0.9 2.2
DVU3093 Rubredoxin-like protein, rdl-0.2 0.0 0.8 1.2
DVU3094 Rubrerythrin, rbr-0.2 -0.1 -0.3 0.7
DVU3095 Peroxide-responsive regulator PerR-0.5 0.0 0.8 1.4
Gene ID TIGR AnnotationFold Change (Treatment/Control)b
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
DVU3095Transcriptional regulator, Fur family, PerR
— — — +2.2 —
DVU3096 hypothetical protein — +1.8 — — —
Response of Per Regulon to Nitrite Stress
Cellular Model of Nitrate Stress Response
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
HCOOH
FDH
2e- + 2H+
PF
L
PF
LA
Acetyl-CoA + H+ + HCOO-
HCOOH
Pyruvate
Lactate + ADP + Pi
LD
H
Lactate
SAM/Methyl
Cycle
GBT
GB
Inhi
bitio
nNH3OH
NH3
Hcp
NO3-
NO2-
NO3-
Periplasm
Cytoplasm
Inh
ibit
ion
NO3-
NO3-
Nitrite Stress
Energy Metabolism
AHP
RBR
RDL
Per
R
General Stress
Response
HCOOH
FDH
2e- + 2H+
PF
L
PF
LA
PF
L
PF
LA
Acetyl-CoA + H+ + HCOO-
HCOOH
Pyruvate
Lactate + ADP + Pi
LD
H
Lactate
SAM/Methyl
Cycle
SAM/Methyl
Cycle
GBT
GB
GBT
GBT
GB
Inhi
bitio
nNH3OH
NH3
Hcp
NO3-
NO2-
NO3-
Periplasm
Cytoplasm
Inh
ibit
ion
NO3-
NO3-
Nitrite Stress
Energy Metabolism
AHP
RBR
RDL
Per
R
General Stress
Response
Cellular Model of Nitrite Stress Response
Comparative Analysis of Nitrate Stress Responses in D. Vulgaris
•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.
Gene expression correlations
Practical Implications?
Thank you!