Chris Rhodes Week 11

Article

The article we will be working with is Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia Berney et al. 2010

The data sets for the article can be found here M. smeg DNA Arrays

Citation: 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.

10 Terms

Obligate aerobes: A bacteria that which requires oxygen for respirations Obligate Aerobes
Hypoxia: An environment containing insufficient oxygen for normal cell function Hypoxia
Exogenous: Originating from outside the organism being studied Exogenous
Real-time PCR: Real time PCR is a relatively new technique which allows gene sequences to be quantified through fluorescent tags as they are being amplified in PCR eliminating the need for gel electrophoresis of the finished product. Real Time PCR
Chemostat: A bioreactor where new fresh media is constantly added while old culture-containing media is constantly removed at a simultaneous rate allowing for control of bacterial growth rate. Chemostat
Colony forming unit: A measure of cell viability in a culture of bacteria, 1 colony forming unit corresponds to 1 viable cell. CFU
Glyoxylate shunt: An alternative pathway of TCA cycle in which isocitrate is converted to glyoxylate and glyoxylate to malate or through the use of NAD⁺/NADH to glycine Glyoxylate Cycle
Oxidoreductase: A class of enzymes which reduce their substrates through either oxygen addition or hydrogen removal. Oxidoreductase
Sigma factor: A component of RNA polymerase responsible for binding to a DNA sequence at the promoter site Sigma Factor
Ferredoxin: Iron containing proteins used in the facilitation of electron transfer between two enzyme systems or pathways Ferredoxin

Outline

Introduction

Mycobacteria have an extraordinary ability to adapt and survive in extreme conditions, but the exact mechanisms behind these adaptations is still fairly under-researched.
Previous experiments have studied mycobacteria under conditions of low oxygen, nutrient starvation, and extended stationary phase but the accuracy and scope of their results have been limited by their methods of experimentation.
This experiment hopes to better study the effects of extreme environments on mycobacteria by performing experiments using continuous cultures of Mycobacterium smegmatis
The use of continuous cultures allows this experiment to study various environmental conditions while still being able to control and maintain the growth rate of the cultured bacteria.
Previous studies by Beste et al. 2007 and Bacon et al. 2004 have implemented continuous cultures as a means of studying environmental effects on mycobacteria gene expression, but these experiments only studied the effects of a singular limiting factor i.e. low oxygen.
There is yet to be a study that observes the effects that simultaneous conditions of low oxygen and carbon starvation have on the gene expressions of mycobacteria.
The goal of this experiment is to study the effects that decreasing oxygen level has on the transcriptional response of a carbon-limited continuous culture of Mycobacterium smegmatis at varying growth rates.
Since M. tuberculosis and M. smegmatis are closely related and share numerous enzyme homologs, studying M. smeg provides valuable insight into the mechanisms of M. tube and how best to combat its resiliency and pathogenesis.

Methods

M. smegmatis strain mc²155 was used for all experiments
M. smeg was grown in a bioreactor under the various experimental conditions.For each condition, the M. smeg was grown to an OD of around 80% of the determined steady state and then switched to chemostat mode at a dilution rate of 0.01 per hour for slow growth cultures and 0.15 per hour for fast growth cultures. For low oxygen cultures there were 5 chemostats made for 2.5% oxygen and 6 chemostats for 0.6% oxygen.
A final volume of 50 ml of culture was collected from each chemostat but it is not stated how many chemostats were used for fast and slow growth at 50% oxygen. 40 ml of the final collected culture was equally partitioned into 4 sample tubes each containing 20 ml of glycerol saline and centrifuged to pellet, the pellets were resuspended in an equal volume of glycerol saline and snap frozen for future use. It is not stated how many samples were used from each pellet nor how many overall samples were used in the experiments.
Total cell RNA was extracted and purified from M. smeg cultures and 10 μg was used to create cDNA for DNA Microarrays. It was not made clear what was labelled with Cys5 or Cys3
The experiment utilized the use of 15 microarray chips. It is once again unclear exactly how many biological or technical replicants were made. The methods do indicate there were at least 5 biological replicants, but this is never explained. There is also no mention of technical replicants in article.
Microarrays were scanned in a Genepix 4000A scanner and quantified with TIGR Spotfinder. The data was then processed in TIGR MIDAS.
There is little discussion on how they normalized their data only the terms Total Intensity and LOWESS

Results

Figure 1
- A: Shows a graph of OD₆₀₀ representing bacterial growth and the level of glycerol in culture versus time. This shows that the rate at which glycerol levels decline (consumed by the cells) is proportional to the rate of cell growth in the culture. Glycerol is used as the carbon limiting agent meaning that glycerol levels represent the remaining the food supply of the culture. During steady state shown in the graph there is no longer cellular growth because the glycerol has been depleted.
- B and C: Shows the fluorescent microscopy of the fast growing M. smeg (B) and the slow growing M. smeg (C). In the fast growing sample, the M. smeg cells are significantly longer (7X) and are fewer in number than the slow growing sample cells.
Table 1: Shows a comparison of various conditions of each of the two culture growth rates including dilution rates, steady state OD₆₀₀, concentration of viable cells in culture, steady state glycerol concentrations, membrane potential, and proton motive force. The fast growing culture is represented by the dilution rate of 0.15 and the slow growing culture by the dilution rate of 0.01. The OD₆₀₀ and CFU respectively indicate the fast growing culture produces a larger amount of cells than does the slow growing culture, but the fast growing culture contains a smaller percentage of viable cells. The membrane potential and proton motive force indicate that despite the differences in cell viability and cell size between the two cultures, the resulting cells are energetically comparable.
From the DNA Microarray data collected it was found that there were 1294 genes that were differentially expressed in the low dilution rate culture when compared to the high dilution rate culture. Of these genes, 691 were induced and 603 were repressed.
There was also differential expression of genes between different oxygen levels of low dilution rate cultures. At 2.5% oxygen the culture contained 68 induced and 91 repressed genes compared to the low dilution rate culture at 50% oxygen. At 0.6% oxygen the culture contained 429 induced and 441 repressed genes compared to the 50% oxygen culture.
Figure 2: Shows a pictorial representation of the proposed scheme of enzymes preferentially used by M. smeg for metabolism under energy-limited and oxygen-limited conditions. Each enzyme included in each scheme was shown to be up-regulated during the experiment by a certain numerical factor which is shown in red next to each enzyme. The up-regulation of each of the enzymes indicates the increased usage of the cellular pathways that utilize those enzymes. This means that up-regulation of an enzyme can be considered as the up-regulation of the pathway the enzyme is involved with.
Table 2: Shows the differential expression of certain genes coding for hydrogenases and assembly proteins between cultures of various dilution rates and oxygen levels. The culture comparisons include 0.01 dilution rate at 50% oxygen versus 0.15 dilution rate at 50% oxygen, 0.01 dilution rate at 50% oxygen versus 0.01 dilution rate at 2.5% oxygen, and 0.01 dilution rate at 50% oxygen versus 0.01 dilution rate at 0.6%. A description of each gene product is also also shown which includes the name and function of the gene product.
Based on large differential gene expression between slow growth cultures in differing oxygen levels it can be seen that M. smeg cells use distinct enzymatic pathways when dealing with carbon-starvation versus hypoxia. This also shows the importance of hydrogenases in the new hypoxia adapted pathway.
Figure 3
- A:Shows a pictorial representation of the up-regulated enzyme pathways of M. smeg under low oxygen conditions along with the factor of up-regulation of each enzyme when compared to cells grown in a 50% oxygen environment.
- B and C: Shows the change in pigmentation of M. smeg cultures from a 50% oxygen environment (B) to a 0.6% oxygen environment (C). The culture in the lower oxygen conditions shows a significantly darker redish-brown color when compared to the culture in the higher oxygen conditions.
There were very few TCA cycle and glyoxylate shunt genes whose expression changed between fast growth and slow growth cultures. Three genes involved in pyruvate dehydrogenase increased 11 fold in the slow growth culture.
There were multiple TCA cycle and glyoxylate shunt genes up-regulated in low oxygen conditions. Malic enzyme, succinate dehydrogenase, pyruvate kinase, putative pyruvate dehydrogenase and succinic semialdehyde dehydrogenase were all down-regulated in low oxygen conditions.
Figure 4: TCA cycle and glyoxylate shunt showing the enzymes differentially expressed in low oxygen conditions versus high oxygen conditions with factors of regulation shown in red.
In 2.5% and .6% oxygen levels of 0.01 dilution rate cultures there was up-regulation of two gene clusters coding for enzymes utilized in the Dos regulon. Additionally in 0.6% oxygen levels there was further up-regulation of three two-component regulatory systems.
In slow growth cultures there is a large amount of up-regulatied sigma factors and anti-sigma factors. 17 of 29 sigma factors and 3 of 6 anti-sigma factors were shown to have significant up-regulation (p<0.05)
There are three hydrogenase clusters located in the M. smeg genome two of which are up-reglated during energy-limitation. Hydrogenase seems to play a role in energy-limited cell growth. To test this, the researchers inactivated the most up-regulated hydrogenase from the genome to test its effects on cell growth. It was found that the mutant genome contained 20% less bio-mass than the wild type. In the experimental conditions, low dilution rate, 50% oxygen, this difference in biomass increased to 40%.
Figure 5
- A:Shows the graph of mutant and wild type culture growth based on OD₆₀₀ versus time and the final weights of the two cultures. The growth of the mutant strain was severely limited when compared to the wild type and the mutant strain contained 20% less biomass than the wild type.
- B:Shows the graph of mutant, complemented mutant, and wild type culture growth based on OD₆₀₀ versus time. While the growth of the mutant strain was still limited compared to the wild type strain, the complemented mutant growth was very close to wild type levels. Indicating that the effects experienced by the mutant strain were indeed caused by the inactivated hydrogenase gene.
- C:Shows the graph of mutant and wild type culture growth under experimental conditions of 50% oxygen and slow growth dilution based on OD₆₀₀ versus time. The mutant culture once again shows a large decrease of 40% in final biomass when compared to the wild type.

Disscussion

Energy Limitation Conclusions
- In energy-limited conditions M. smeg cells begin using less oxygen for cell respiration and consequently down-regulate normal respiratory chain enzymes while up-regulating low-oxygen respiratory chain enzymes.
- M. smeg also up-regulates alternative primary dehydrogenases which use carbon sources more efficiently in energy production. This energy goes towards powering cell respiration and maintaining membrane potential.
- Few TCA cycle genes were differentially expressed during energy limitation indicating that overall TCA cycle use is not affected by carbon-limited environments
- Succinate dehydrogenase is proposed to act similarly to fumarate reductase which is absent in M. smeg. Up-regulation of succinate dehydrogenase could indicate the use of fumarate as an electron acceptor in oxygen-limited conditions.
Oxygen Limitation Conclusions
- Data indicates M. smeg adapt to hypoxia using 3 strategies.
1. Oxygen scavenging: Shown by the up-regulation of high-affinity cytochrome bd oxidase and genes involved with its assembly and synthesis.
2. The use of NAD⁺ independent enzymes: Shown by down-regulation of enzymes that are dependent on NAD⁺ to function and up-regulation of ferredoxin-reducing and -oxidizing enzymes under hypoxic conditions.
3. Up-regulation of hydrogenases: Shown by the up-regulation of all 3 hydrogenases of the M. smeg genome in hypoxic conditions.
- During hypoxia the M. smeg pigmentation changed from yellow to red. This could be caused by the up-regulation of cytochromes which can contain heme molecules inducing the red color.
The disruption of msmeg_2719 caused a large decrease in cell yield under low energy conditions indicating the importance of hydrogenases for cellular growth in M. smeg.
In summary, M. smeg adapts to conditions of energy limitation and hypoxia by switching to pathways that that require less carbon and oxygen to function and through the usage of dehydrogenases and hydrogenases which conserve and recycle carbon and oxygen in the cell.
The researchers based many of their conclusions on supporting information from past experiments citing almost 29 papers in the Discussion section. Of these 29 cited, though many studied mycobacteria, none specifically studied M. smeg so the article never makes any direct agreements or disagreements to any of these papers, but rather uses them as a means of suggesting or supporting conclusions.

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Chris Rhodes Week 11

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10 Terms

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