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

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89

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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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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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control

baseline

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Asp

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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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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

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-15

-10

-5

00

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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

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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.

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all iron-binding fur-regulated

Gene ID TIGR Annotation

Fold Change (Treatment/Control)b

0.5h 1.0h1.5h

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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!