Understanding Microbial Persistence and Adaptation in the Environment

1. 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

2. Ecosystem Community Population Cell Protein RNA DNA 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 Community How 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 Ecology Geochemistry Computational Ecology Computational Genomic Proteomic Metabolomic Computational

3. 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

4. Definition of Stress No Universal Definition for StressHighly dependent upon the individual cellWorking 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

5. 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 and radioactive compounds Industrial areas Anaerobic corrosion of underground buried ferrous metals ex: tanks and pipelines Human Health Ulcerative Colitis; Anaerobic Abscess

6. Anaerobic bacteria that respire sulfate: 8e- SO42- S2- Found in a large variety of environments Electron acceptors - sulfate, sulfite, thiosulfate, sulfur, nitratee 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 SULFATE REDUCING BACTERIA

7. SO42- SO32- organic S S0 H2S Global S Cycle Why Sulfate-Reducing Bacteria? Global C Cycle Souring of oil reservoirs Microbial-induced corrosion

8. Pathogenic Desulfovibrio Interspecies Genomic Hybridization: RMA 14127: 96.6% RMA 15168: 97.7% In collaboration with Goldstein lab, UCLA

9. 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

10. Functional Genomics: Stress Response in SRB Wild-type Available mutants Environmental isolates Stress Knockouts/mutants Physiology Transcriptomics Proteomics Metabolomics Computation Decision? Single mutations Multiple mutations Physiology

11. Core Stressors • Temperature change • Nitrate, Nitrite • Osmotic – NaCl, KCl • Oxygen, Air • pH – high, low • Chromate • fur mutant LS4D + 250mM NaCl

12. C1 control baseline T0 V1 Growth (OD) stress 0.3 0 2 Time (hours) Leu Lys Ile Glu Arg Val Phe Pro Met Asp His 10 0 5 15 20 25 Time [min] A Systems Biology Approach Transcriptomics Proteomics Metabolomics

13. Nitrate as a stressor • Phenotype Array Results • NaNO3 is more inhibitory • Indicative of responses in addition to osmotic stress NaNO3 NaCl He et al., 2010. ISME J.

14. Effect of Osmo-protectant He et al., 2010. ISME J.

15. Osmotic Stress

16. Methyl/SAM Cycle

17. Methyl/SAM Cycle

18. 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? Growth inhibition by nitrite Nitrite reduction by D. vulgaris

19. 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 Global Transcriptional Analysis • 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

20. D D C C B B T1 T2 T3 T4 T5 A A Hierarchical clustering analysis Nitrite Reduction vs Gene Expression • Nitrite reductase gene and genes in the Fur regulon were highly up-regulated in nitrite stress. • Genes in protein biosynthesis and energy conservation were severely down-regulated. • An apparent correlation between the dynamics of transcriptional response and the reduction of nitrite. • Nitrite reduction was indicated as the main detoxification mechanism. • Electron flow was shifted from oxidative phosphorylation to nitrite reduction.

21. Up-regulation of Genes of Fe-Proteins Response of Fur Regulon to Nitrite Stress • 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. Response of Per Regulon to Nitrite Stress

22. Responses to Nitrite in Energy Metabolism N Metabolism Hierarchical clustering analysis • A coordinated cascade of responses to nitrite in pathways of energy metabolism, nitrogen metabolism, oxidative stress response, and iron homeostasis.

23. Nitrate vs Nitrite

24. PerR Regulon in Nitrate Stress Response of Per Regulon to Nitrite Stress

25. Cellular Model of NitrateStress Response Cellular Model of NitriteStress 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

26. 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.

27. Practical Implications?

28. Thank you!