1. Differential expression of genes involved in metabolic and regulatory pathways in oxygen-limited M. smegmatis Chris Rhodes, Nicki Harmon Loyola Marymount University Department of Biology BIOL 368, 12/07/11

2. Outline Mycobacteria show an extraordinary ability to survive in extreme environmental conditions Studies by Berney et al. (2010) show M. smeg implements three distinct responses to oxygen limited conditions Further study of the Berney et al. (2010) microarray data was performed using statistical analysis techniques Statistical analysis shows: – Down-regulation of NAD + /NADH dependent pathways – Up-regulation of regulatory pathways – Significant up-regulation of ferredoxin enzymes involved in TCA cycle Differential expression data agree partially with previous conclusions by Berney et al. (2010)

3. Mycobacteria have shown remarkable adaptability to oxygen- and energy-limited environments Mycobacteria are a group of obligate aerobes which require oxygen to grow, but are capable of surviving in anaerobic environments Mycobacteria have also been show to survive in conditions of nutrient deprivation This adaptability is indicative of a high degree of metabolic flexibility within mycobacteria The mechanisms behind this flexibility have not been adequately studied

4. Berney et al. shows M. smeg adopts 3 different responses in order to adapt to low oxygen conditions 1.Oxygen scavenging: – Up-regulation of cytochromes which procure and conserve oxygen in the cell 2.Up-regulation of NAD + /NADH independent enzymes: – Ferredoxin reducing and oxidizing enzymes power TCA cycle independent of NAD + /NADH conserving energy 3.Up-regulation of hydrogenases: – Hydrogenases carry out metabolic functions while conserving and recycling energy and oxygen

5. Statistical analysis was performed using gene expression data from Berney et al. (2010) Microarrays were performed using 5 biological replicates from 2.5% and 50% oxygen level experiment with dye swap chips – 50% Oxygen Level  Cys3 – 2.5% Oxygen Level  Cys5 For each microarray the Average log 2 -value, p-value, and Tstat were calculated This data was run through MAPPFinder in order to find differentially expressed ontologies The most significantly increased and decreased ontologies were found and compared to the results of Berney et al. (2010)

6. Outline Mycobacteria show an extraordinary ability to survive in extreme environmental conditions Studies by Berney et al. (2010) show M. smeg implements three distinct responses to oxygen limited conditions Further study of the Berney et al. (2010) microarray data was performed using statistical analysis techniques Statistical analysis shows: – Down-regulation of NAD + /NADH dependent pathways – Up-regulation of regulatory pathways – Significant up-regulation of ferredoxin enzymes involved in TCA cycle Differential expression data partially agree with previous conclusions by Berney et al. (2010)

7. Significant genes represent a low percentage of the entire genome P<…Number of Genes Under Threshold 0.05215 0.0142 0.0013 Total Number of Genes6947 Number of MAPPFinder Errors221

8. The ten most differentially expressed genes show no discernable pattern in function Protein NameP-ValueFunction Transcriptional Regulator LysR Family Protein0.000589Transcriptional Regulator Linear Gramicidin Synthetase mbtf0.000591Antibiotic Synthesis Putative Carboxylesterase/Lipase0.000659Lipid Hydrolysis Probable Conserved Transmembrane Protein0.001346Transmembrane Protein MmpL Protein0.001619Lipid Transport Protein Taurine Transport System Permease Protein TauC 0.002122Taurine Transport Protein Hypothetical Protein0.002569NA [2Fe-2S] Binding Domain Protein0.002674Ferredoxin enzyme Conserved Hypothetical Protein0.003256NA Putative Oxygen-Independent Coproporhyrinogen III Oxidase 0.003371Oxidase Activity

9. Up-regulated ontologies are involved in cell regulation pathways RNA Metabolic Process – Regulation of metabolic pathways involving RNA Regulation of Translation – Modulation of DNA dependent transcription Regulation of Nitrogen Compound Metabolic Processes – Modulation of chemical pathways involving nitrogen compounds Regulation of Nucleic Acid Metabolism – Modulation of chemical reactions involving nucleic acids Regulation of Macromolecule Biosynthetic Process – Modulation of the processes of formation of macromolecules

10. Up-regulated ontologies are involved in cell regulation pathways Regulation of Gene Expression – Modulation of processes involved in gene expression Regulation of Cellular Macromolecule Biosynthetic Process – Modulation of the cellular macromolecule biosynthetic process Regulation of Cellular Biosynthetic Process – Modulation of the formation of cellular substances Regulation of Biosynthetic Process – Modulation of the formation of substances Regulation of Macromolecule Metabolic Process – Modulation of the macromolecule metabolic process

11. Down-regulated ontologies are involved in NAD + /NADH dependent pathways Fructuronate Reductase Activity – Catalyses: D-mannonate + NAD + = D-fructuronate + H + + NADH Precorrin-3B C17-methyltransferase Activity – Catalyses: S-adenosyl-L-methionine + precorrin-3B = S-adenosyl-L- homocysteine + precorrin Precorrin-2 C20-methyltransferase Activity – Catalyses: S-adenosyl-L-methionine + precorrin-2 = S-adenosyl-L- homocysteine + H + + precorrin-3A Coniferyl-aldehyde Dehydrogenase Activity – Catlyses coniferyl aldehyde + H2O + NADP + = ferulate + NADPH + H + Inositol-Monophosphatase Activity – Catalyses: myo-inositol phosphate + H2O = myo-inositol + phosphate

12. Down-regulated ontologies are involved in NAD + /NADH dependent pathways Trans-2-enoyl-CoA Reductase NADPH Activity – Catalyses: acyl-CoA + NADP + = trans-2,3-dehydroacyl-CoA + NADPH + H + Aldehyde Dehydrogenase [NADP + ] Activity – Catalyses: aldehyde + NADP + + H2O = an acid + NADPH + H + Oxidoreductase Activity – Catalysis of redox reactions relating to energy functions such as NAD+/NADH Inositol Phosphatase Activity – Inositol phosphate(n) + H2O = inositol phosphate(n-1) + phosphate Cellular Aldehyde Metabolic Process – Cellular chemical reactions and pathways involving aldehydes

13. Outline Mycobacteria show an extraordinary ability to survive in extreme environmental conditions Studies by Berney et al. (2010) show M. smeg implements three distinct responses to oxygen limited conditions Further study of the Berney et al. (2010) microarray data was performed using statistical analysis techniques Statistical analysis shows: – Down-regulation of NAD + /NADH dependent pathways – Up-regulation of regulatory pathways – Significant up-regulation of ferredoxin enzymes involved in TCA cycle Differential expression data partially agrees with previous conclusions of Berney et al. (2010)

14. Analysis of gene expression data agrees with certain findings of Berney et al. (2010) Berney et al. (2010) shows up-regulation of NAD+/NADH independent enzymes coupled with down regulation of NAD+/NADH dependent enzymes Berney et al. (2010) shows differential regulation of ferredoxin usage and synthesis Berney et al. (2010) shows up-regulation of numerous regulatory pathways Analysis shows no up-regulation of hydrogenases or oxygen scavengers which conflicts with conclusions made by Berney et al. (2010)

15. Summary Mycobacteria show a high degree of metabolic plasticity under environmental stress Studies by Berney et al. (2010) show differential gene expression in different oxygen-limited conditions Statistical analysis was performed to further study the Berney et al. data Analysis showed up-regulation of regulatory pathway genes and down-regulation of NAD+/NADH dependent enzymes Results correlate to similar conclusions made Bernie et al. (2010)

16. Acknowledgments Kam D. Dahlquist, Ph. D Berney, Michael, and Gregory M. Cook. "Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia." Ed. David M. Ojcius. PLoS ONE 5.1 (2010): E8614. Print.