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
Ecology
Ecosystem Geochemistry
Computational
Community Ecology
How do communities respond to stress? Computational Community

Population
Populations Cell
Determine the impact of stress on organisms
Protein
(Desulfovibrio vulgaris)
RNA Genomic
Proteomic
Metabolomic
Computational
Cell DNA
Infer key stress response pathways and how gene networks
interplay under different stress conditions
 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

ex: hydrogen sulfide
Ecological areas
Sulfate removal from the medium

Bioremediation
Processes
Metabolism of environmental
polutants

Reduction of heavy metals

Industrial areas and radioactive compounds

Anaerobic corrosion of underground

buried ferrous metals
ex: tanks and pipelines

Human Health Ulcerative Colitis; Anaerobic Abscess

 SULFATE REDUCING BACTERIA
•Anaerobic bacteria that respire sulfate:
SO42- 8e- S2-

Found 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?

SO42-
SO32-
organic S
S0
H 2S
Global S Cycle
Global C Cycle

Souring of oil
reservoirs

Microbial-induced corrosion
 Pathogenic Desulfovibrio

Interspecies Genomic Hybridization: 61 RMA 14567

90 RMA 16092
RMA 14127: 96.6% 82
Desulfovibrio fairfieldensis
RMA 15168: 97.7% 93
Desulfovibrio sp D4
99
RMA 10276
96 Desulfovibrio sp. oral clone BB161

100 RMA 8703

100 RMA 16470
89
Desulfovibrio pigra
Desulfovibrio vulgaris Hildenborough
100
100 RMA 14127
100 RMA 15168
78

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? Knockouts/mutants
Biomass

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
LS4D
• Chromate
• fur mutant

+ 250mM NaCl
 A Systems Biology Approach

C1
control

baseline
Growth (OD)

T0
V1
stress
0.3
0 2 Transcriptomics
Time (hours)
Leu

Proteomics Lys

Ile Glu
Arg Val
Phe

His Pro Met Asp

0 5 10 15 20 25 Time [min]

Metabolomics
 Nitrate as a stressor

35
Phenotype Array Results 35 A 0mM
•NaNO3 is more inhibitory 30 A 0mM
30mM

cells/ml
30 30mM

cells/ml
50mM
•Indicative of responses in 25 50mM
25 70mM
addition to osmotic stress 70mM

8 8
90mM

1010
20
20 90mM
100mM

Density,
100mM

Density,
15 120mM
15 120mM
140mM
10 140mM
160mM
Cell
10
160mM
Cell

5 180mM
5 180mM
NaNO3 200mM
0 200mM
0
0 20 40 60 80
0 20 40 60 80
Hour
Hour
35
35 0mM
B 0mM
30 B 50mM
cells/ml

30 50mM
Density,1010cells/ml

100mM
25 100mM
25 150mM
150mM
8 8

20 200mM
20 200mM
250mM
250mM
CellDensity,

15 300mM
15 300mM
350mM
10 350mM
10 400mM
Cell

400mM
5 450mM
5 NaCl 450mM
500mM
0 500mM
0
0 20 40 60 80
0 20 40 60 80
He et al., 2010. ISME J. Hour
Hour
 1.4 Effect of Osmo-protectant
A
1.2
1.4
1 A
1.2
0.8

600
1 1.2

OD600 OD
1
0.6
0.8 0.8
1.2
0.6
0.4 1
0.6 0.4
0.8
0.2
0.2 0.6 0
0.4
0.4 0 50 100 150
0 0.2
0.2 0
0 50 100 150 200
0 50 100 150
0 Hour
0 50 100 150 200

Hour
1.4
B
1.2
1.4
1 B
1.2
0.8
600

1
OD600 OD

0.6
0.8
0.4
0.6
0.2
0.4
0
0.2
0 100 200 300 400
He et al., 2010. ISME J. 0 Hour
 Osmotic Stress

1.4

1.2 Control

1 2 mM GB
OD 600 nm

0.8 250 mM NaCl

0.6 250 mM KCl

0.4 250 mM NaCl +

2 mM GB
250 mM KCl +
0.2 2 mM GB
0
0 20 40 60
Time (h)
 Methyl/SAM Cycle

AhcY
S-adenosyl-L- DVU0607
L-homocysteine
homocysteine
Methyl Methyltransferase MetE
Methyl-THF
DVU3371
acceptor DVU0606

MetF
S-adenosyl-L- MetK DVU0997
DVU2449
methionine L-methionine
Methylene-THF
PFLA
DVU2825

Pyruvate PFL Acetyl-CoA+ H+ + HCOO-

DVU2824
FDH
LDH DVU0586-8
CO2 + 2e-

Lactate
Lactate permease
DVU2110

Medium
 Methyl/SAM Cycle

Log2 Ratio of Transcriptional Response

Gene ID Δfur + TIGR Annotation

NaNO3 NaNO2 NaCl 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

5-methyltetrahydropteroyltriglutamate-homocysteine S-
DVU3371 2.7 3.8 -1.4 -3.4 -2.5 -2.0
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?

1 6

0.8

Nitrite, mM
0.6 3
a
OD600

2
0.4
2
1
0 mM
0.2 0.5 mM b
0
1.0 mM
0 5 10 15 20
5.0 mM 0
0
0 2 4 6 8
0 2 4 6 8
hour h

Growth inhibition by nitrite Nitrite reduction by D. vulgaris

 Global Transcriptional Analysis
35
A B C D E F G H I
30 Up-Regulated
T1 T2 T3 T4 T5
25
• Functional categories repressed
20
– (A) Amino acid biosynthesis
– (B) Cofactor biosynthesis
15 – (F) Protein synthesis
– (I) Transport and binding proteins
10
• Functional categories induced
5 – (G) Regulatory functions
– (H) Signal transduction
0 – (E) Energy metabolism

-5

-10
• Normal cell growth stopped
-15
– Consistent with growth curve
-20
Down-Regulated A—Amino acid biosynthesis • Cells in transitional phase
-25 B—Biosynthesis of cofactors
C—Cell envelope

-30
D—Cellular processes • Detoxification mechanism involves electron
E—Energy metabolism transport
F—Protein synthesis
-35 G—Regulatory functions – Nitrite reduction
H—Signal transduction
I—Transport and binding proteins
 Hierarchical clustering analysis Nitrite Reduction vs Gene Expression
DVU0918 ATP synthase F0, A subunit
DVU0917 ATP synthase F0, C subunit
D 350
Up genes
2.5

DVU0777 ATP synthase, F1 alpha subunit 300 Dn genes

DVU0775 ATP synthase, F1 beta subunit Nitrite 2
D DVU0778 ATP synthase, F1 delta subunit 250

Number of ORFs

Nitrite, mM
DVU0776 ATP synthase, F1 gamma subunit 1.5
200
DVU2925 ribosomal protein L1
150
DVU2926 ribosomal protein L10 1
DVU2924 ribosomal protein L11 100
DVU2518 ribosomal protein L13 0.5
C DVU1310 ribosomal protein L16 50
DVU1319 ribosomal protein L18
DVU0927 ribosomal protein L21 0 0
DVU1574 ribosomal protein L25 0.5h 1h 1.5h 2.5h 3.5h
DVU1211 ribosomal protein L28 Shown are genes with > 2 fold change

DVU1303 ribosomal protein L3 C

DVU1074 ribosomal protein L34
DVU2383 tonBribosomal
dependent receptor domain protein
DVU2927 protein L7/L12 1. Nitrite reductase gene and genes in
DVU2571 ferrous iron transport protein B
DVU0958 ribosomal protein L9 the Fur regulon were highly up-
DVU2572 ferrous iron transport protein A, putative
DVU1302 ribosomal protein S10
regulated in nitrite stress.
B DVU2573 hypothetical protein
DVU1298 ribosomal protein S12
DVU2574 ferrous ion transport protein, putative
DVU1316 ribosomal protein S14 2. Genes in protein biosynthesis and
DVU2680 flavodoxin
DVU1312 ribosomal protein S17 B energy conservation were severely
DVU0957
DVU0121 ribosomal
conserved protein S18
hypothetical protein
DVU0122 hypothetical
DVU0874 ribosomal protein
protein S2
down-regulated.
DVU0123 membrane
DVU1896 protein,
ribosomal putative
protein S20 3. An apparent correlation between the
DVU0624 NapC/NirT cytochrome
DVU0956 ribosomal protein S6 c family protein
A DVU0625 cyt c nitrite reductase, catalytic subunit NfrA dynamics of transcriptional response
DVU1299 ribosomal protein S7
DVU0943 membrane protein,
DVU2519 ribosomal protein S9 putative and the reduction of nitrite.
DVU0944 hypothetical protein
DVU1080 iron-sulfur cluster-binding protein 4. Nitrite reduction was indicated as the
DVU1081 iron-sulfur cluster-binding protein main detoxification mechanism.
DVU1419 sigma-54 dependent transcriptional regulator
DVU2132 hypothetical protein 5. Electron flow was shifted from
DVU2133 membrane protein, putative oxidative phosphorylation to nitrite
DVU2543 hybrid cluster protein
T1 T2 T3 T4 T5
DVU2544 iron-sulfur cluster-binding protein A reduction.
 Response of Fur Regulon to Nitrite Stress Up-regulation of Genes of Fe-Proteins

fold
Fold Change (Treatment/Control)b
Gene ID TIGR Annotation 4
1.5 2.5 4.0
0.5h 1.0h h h h 3
DVU0763 GGDEF domain protein +11.9 +2.1 — — — 2
DVU2378 transcriptional regulator, AraC family +4.3 +4.1 +2.4 — —
1
ferrous iron transport protein, putative
+3.5 +5.0 +3.9 — — 0
DVU2574 FeoA
0 30 60 90 150 240
DVU2680 Flavodoxin +27.6 +22.6 +4.9 — —
min
DVU3330 conserved hypothetical protein +2.3 +5.7 +2.3 — — all iron-binding fur-regulated
DVU0273 conserved hypothetical protein +15.3 +5.2 +1.8 — -2.2
DVU0304 hypothetical protein +34.0 +10.1 +3.7 — —
•Nitrite stress led to in the derepression of
Response of Per Regulon to Nitrite Stress the Fur regulon, which was possibly
resulted from iron deficiency.
Fold Change (Treatment/Control)b
Gene ID TIGR Annotation
0.5h 1.0h 1.5h 2.5h 4.0h •The primary cause of iron deficiency could
DVU0772 hypothetical protein +1.8 +2.4 +2.6 +2.1 — be attributed to increased demand for iron
DVU2247 antioxidant, AhpC/Tsa family +3.0 +3.1 +2.1 +1.8 — under nitrite stress.
DVU2318 rubrerythrin, putative — — +1.5 — -1.9
•Nitrite as an oxidizing agent also induced
Transcriptional regulator, Fur family, oxidative stress exemplified by the up
— — — +2.2 —
DVU3095 PerR
regulation of the Per regulon.
DVU3096 hypothetical protein — +1.8 — — —
 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 Δfur + TIGR Annotation
NaNO3 NaNO2 NaCl 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

5-methyltetrahydropteroyltriglutamate-homocysteine S-
DVU3371 2.7 3.8 -1.4 -3.4 -2.5 -2.0
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

Log2 (Expression Ratio)b

Gene ID Description
30 min 60 min 120min 240 min

0.1 0.7 2.0 2.4

DVU0772 hypothetical protein

0.2 0.5 0.7 1.6

DVU2247 alkyl hydroperoxide reductase C, ahpC

0.4 0.6 0.9 2.2

DVU2318 Rubrerythrin, putative, rbr2

-0.2 0.0 0.8 1.2

DVU3093 Rubredoxin-like protein, rdl

-0.2 -0.1 -0.3 0.7

DVU3094 Rubrerythrin, rbr

-0.5 0.0 0.8 1.4

DVU3095 Peroxide-responsive regulator PerR

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
Transcriptional regulator, Fur family,
— — — +2.2 —
DVU3095 PerR
DVU3096 hypothetical protein — +1.8 — — —
 Energy Metabolism Cellular Model of Nitrate Stress Response
FDH
+
H HCOOH
2e + 2-

ss
re
St
lt
Sa

BT Nitrite Stress
G

HCOOH NH3
B
G

Hcp
n
tio
NO3- NO3- bi
hi
In

Acetyl-CoA + H+ + HCOO- NH3OH

Cytoplasm

Inhibition
PFLA

PFL
SAM/Methyl
NO2-
Cycle
Cellular Model of Nitrite
Pyruvate
AHP NO3- Stress Response
PerR

LDH

RBR
Periplasm
RDL Lactate + ADP + Pi
General
NO3-
Stress
Response
Lactate

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
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!