Chris Rhodes, Nicki Harmon Loyola Marymount University Department of Biology BIOL 368, 12/07/11

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 • Oxygen scavenging: • Up-regulation of cytochromes which procure and conserve oxygen in the cell • Up-regulation of NAD+/NADH independent enzymes: • Ferredoxin reducing and oxidizing enzymes power TCA cycle independent of NAD+/NADH conserving energy • 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 log2-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

8. The ten most differentially expressed genes show no discernable pattern in function

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.