Transcript CRNHMicroJournal2011

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
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
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
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
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
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
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
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
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
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%
Certain genes show significant (p<0.05) up-regulation
in energy- and oxygen-limited conditions
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
Cultures show pigmentation change from 50%
to 0.06% oxygen levels
50% Oxygen Culture
0.6% Oxygen Culture
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%
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
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
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
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
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