1. Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia 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. Chris Rhodes and Nicki Harmon Loyola Marymount University Department of Biology BIOL 368 11/16/11

2. Outline Mycobacteria show an extraordinary ability to survive in extreme environmental conditions Previous experiments studying mycobacteria have been inhibited by an inability to maintain a constant cell growth rate M. smegmatis was grown at varying oxygen levels and constant growth rates in chemostat to study changing gene expression Microarray results show differential expression of gene clusters involved in metabolic and regulatory pathways of M. smeg M. smeg induces various hydrogenases and dehydrogenases as a means of energy recycling and oxygen conservation

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. The results of previous studies have been limited by their experimental methods Conventional experiments provided useful information, but studied multiple experimental factors concurrently By using a continuous culture the growth rate of bacterial cultures can be easily controlled By controlling growth rate, it is possible to study the effects of a singular environmental condition To date (2010) there have been no experiments that studied the effects of both low oxygen and low energy on the mycobacteria transcriptome This study uses continuous cultures to determine the transcriptional effects of oxygen and carbon limitation in M. smegmatis

5. Continuous cultures of M. smeg were used to obtain transcriptional data for experimental environments A chemostat was used to maintain consistent growth rates of M. smeg cultures at a 50% oxygen level – Doubling time of 4.6 hours for fast growth cultures – Doubling time of 69 hours for slow growth cultures 3 Different oxygen levels were studied for slow growth cultures: 50%, 2.5%, and 0.6% Total RNA was extracted and purified from samples of the experimental cultures cDNA was synthesized from 10 μg of experimental RNA and used for DNA microarrays

6. Outline Mycobacteria show an extraordinary ability to survive in extreme environmental conditions Previous experiments studying mycobacteria have been inhibited by an inability to maintain a constant cell growth rate M. smegmatis was grown at varying oxygen levels and constant growth rates in chemostat to study changing gene expression Microarray results show differential expression of gene clusters involved in metabolic and regulatory pathways of M. smeg M. smeg induces various hydrogenases and dehydrogenases as a means of energy recycling and oxygen conservation

7. DNA microarrays were utilized to measure the transcriptional response of the experimental cultures Under oxygen limited conditions: – TCA cycle enzymes are up-regulated – Cytochrome assembly and synthesis is up-regulated – Hydrogenase is up-regulated Under energy limited conditions: – TCA cycle enzymes remain consistent – Dehydrogenases involved in ETC are up-regulated – Hydrogenase is up-regulated Hydrogen:quinone oxidoreductase plays an important role in cell growth under energy- and oxygen –limited conditions

8. Cultures at different growth rates show different steady state OD 600 levels and physiologies Steady state = No growth Fluorescent microscopy B: Fast growth cells C: Slow growth cells

9. Continuous cultures of fast and slow growth rates show different growth capacities but equivalent energetics Data relating to cellular growth Data relating to energetic equivalency

10. Up-regulated respiratory chain enzymes vary between energy- and oxygen-limited environments Ratio of expression of shown in red Energy limitation ratio: slow/fast Oxygen limitation ratio: 2.5%/50%

11. Certain genes show significant (p<0.05) up-regulation in energy- and oxygen-limited conditions

12. M. Smeg shows up-regulation of heme-containing cytochrome pathways under 0.6% oxygen conditions Ratio of expression shown in red Oxygen level ratio: 0.6%/50% Cytochromes are oxygen scavengers

13. Cultures show pigmentation change from 50% to 0.06% oxygen levels 50% Oxygen Culture0.6% Oxygen Culture

14. M. Smeg cultures show up-regulation of TCA cycle enzymes in 0.6% oxygen conditions Ratio of expression shown in red Oxygen level ratio: 0.6%/50%

15. Repression of msmeg_2719 causes a decrease in the final biomass of M. smeg cultures Msmeg_2719 codes for hydrogenase Under batch conditions: 20% reduction in mutant biomass Complementation returns mutant growth to wild type levels Under experimental conditions: 40% reduction in mutant biomass

16. Outline Mycobacteria show an extraordinary ability to survive in extreme environmental conditions Previous experiments studying mycobacteria have been inhibited by an inability to maintain a constant cell growth rate M. smegmatis was grown at varying oxygen levels and constant growth rates in chemostat to study changing gene expression Microarray results show differential expression of gene clusters involved in metabolic and regulatory pathways of M. smeg M. smeg induces various hydrogenases and dehydrogenases as a means of energy recycling and oxygen conservation

17. Energy-limitation induces enzymes involved in energy recycling and oxygen conservation In energy limited conditions M. smeg uses less oxygen causing: – Up-regulation of oxygen conserving enzymes in the respiratory chain – Repression of respiratory chain enzymes used under ideal conditions To conserve energy, dehydrogenases are induced which use carbon sources more efficiently TCA cycle usage is not affected by energy-limitation

18. 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

19. Summary Mycobacteria show a high degree of metabolic plasticity, but have not been thoroughly researched Previous studies have not provided conclusive results due to multiple experimental factors The effects of oxygen- and energy-limited environments on M. smeg were studied separately through DNA microarrays Microarrays indicate differences in metabolic and regulatory enzyme usage in experimental conditions Specifically, the up-regulation of hydrogenases and dehydrogenases as a means of resource recycling and conservation