Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia

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

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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

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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
• Hydrogenquinone oxidoreductase plays an
  important role in cell growth under energy- and
  oxygen limited conditions

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Cultures at different growth rates show different
steady state OD600 levels and physiologies

• Steady state No growth
• Fluorescent microscopy
• B Fast growth cells
• C Slow growth cells

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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
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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

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Certain genes show significant (plt0.05)
up-regulation in energy- and oxygen-limited
conditions
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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

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Cultures show pigmentation change from 50 to
0.06 oxygen levels
50 Oxygen Culture
0.6 Oxygen Culture
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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

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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

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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

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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

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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

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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