Berney et. al. 2010

My partner for this presentation is Chris Rhodes, we will be looking at Myobacterim Smegmatis and this is the article that we will be using for Journal Club Berney et. al. 2010.

Vocabulary Terms

Obligate Aerobe-an aerobe that requires oxygen for aerobic respiration. Biology Online
Hypoxia-a condition in which there is oxygen deficiency in a habitat or a body part. Biology Online
Paucity-smallness of quantity. Biology Online
Fluorescence Microscopy-a technique using a light microscope to study properties of organic and inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. Biology Online
Optical Density-the measure of the amount of light absorbed by a suspension of bacterial cells or a solution of an organic molecule with the use of a colorimeter or spectrophotometer. Biology Online
Up-Regulation-increase in expression of a gene; in the narrowest sense, that in which transcription of a specific mRNA is increased, but also used more broadly to refer to an increase in mRNA levels for a particular gene from any cause, such as increased stability of the specific mRNA. The Free Dictionary
Down-Regulation-a decrease in the number of receptors for a chemical or drug on cell surfaces in a given area, usually caused by long-term exposure to the agent. The Free Dictionary
Cytochrome-any of a class of iron-containing proteins important in cell respiration as catalysts of oxidation-reduction reactions. The Free Dictionary
Malate-a salt or ester of malic acid. The Free Dictionary
Redox Signaling-the process wherein free radicals, reactive oxygen species (ROS), and other electronically-activated species act as messengers in biological systems. The Free Dictionary

Outline

Abstract
- Mycobacteria need oxygen in order to grow, but not necessarily for survival.
- Because of this, M. Smegmatis have to adapt to slow growth rate and hypoxia through the use of carbon-limited cultures.
- Slow growing M. Smegmatis is compared with faster growing M. Smegmatis, the former took 69 hours to double in numbers while the latter only took 4.6 hours to double.
- When the M. Smegmatis is undergoing a slower growth rate, more molecular machinery is used to make sure energy is being used efficiently.
- The use of alternative electron donors such as hydrogenase and primary dehydrogenase maintain flow of electron transport chain.
- Slower growing cells that are subjected to hypoxia have adaptations that allow them to cope with changes.
- Ferredoxin-dependent enzymes allow the cell to have a higher carbon flux of the central carbon metabolism.
- Myobacterial cells have a unique metabolic plasticity for survival in low energy conditions.

Introduction
- Microorganisms have a metabolic flexibility that allows them to adapt to environmental changes.
- For adaptation, a bacterium must be able to balance oxidative and reductive reactions.
- It is unknown what mechanisms make it possible for metabolic flexibility.
- Past studies that have tried to understand metabolic flexibility used approaches such as low oxygen levels and nutrient starvation.
- These approaches have provided information but with multiple factors changing, the myobacteria fail to grow and metabolize.
- Continuous culture allows growth rate to stay at a constant value while environmental factors can change.
- This is useful for studying gene expression.
- No studies have been performed that simultaneously look at carbon starvation and oxygen limit.
- This study wanted to determine the molecular response of M. Smegmatis to growth rate change (approximately 15-fold) and decreasing oxygen supply using carbon-limited continuous culture.

Materials and Methods
- M. Smegmatis strain mc² was used for entire experiment.
- PCR-overlap extensions was used for flanked fragment, this was confirmed using Southern Hybridization Analysis.
- Bacteria were inoculated from a cyro-culture (-80°C) and incubated for 3 days at 37°C.
- One colony was incubated on a rotary shaker at 200rpm.
- After inoculation, cultures were switched to chemostat mode to correspond to the doubling times.
- 5 individual chemostats were performed to correspond to low dilution rates, first brought to 50% before being lowered to either 2.5% or 0.6%.
- Bacterial cell samples were withdrawn from chemostat and stained with SYBRGreen1.
- After 15 minutes of incubation, sample was analyzed using epifluorescent microscopy.
- RNA was extracted through Invitrogen.
- Concentrations were determined using a Nanodrop spectrophotometer and checked on 1.2% agarose gel.
- After cDNA synthesis, real-time PCR was conducted.
- Results were normalized to the gene sigA.
- For microarray analysis, RNA was used to create cDNA with aminoallyl.
- Fluoroscent Cy3 and Cy5 dyes were attached to aminoallyl tags.
- These tags were hybridized and buffered and left on microarray slides overnight in a 42°C water bath, the slides were then washed.
- Arrays were scanned and data was processed in TIGR MIDAS.
- In-slide replicate spots were averaged before ratios were calculated.
- Genes with ratios of >2 and <0.5 and p-value of <0.05 were used for data interpretations.
- Gas end products were analyzed by gas chromatography.
- For phylogenetic analysis, amino acid sequences were compared to databases.
- Sequences of interest were aligned using ClustalW.

Results
- M. Smegmatis was grown at diltuion rates of 0.15 h^-1 or 0.01 h^-1 to correspond to the doubling times of 4.6h and 69h, respectively.
- The density of the culture was monitored along with oxygen consumption.
- Carbon limitation was tested by pulsing glycerol into the culture vessel and showing an increase in OD₆₀₀ and respiration rate by measuring residual glycerol. This is shown in Figure 1.
- The saturation level of oxygen used was 50%.
- Optical density was lower at low dilution rate compared to high dilution.
- However, the number of viable cells was higher with the lower dilution rate than at the higher dilution rate, this is shown in Table 1.
- This indicates a difference in cell volume.
- There was a 7-fold difference in cell length between the group, but these groups were energetically equivalent, this is shown in Table 1 and Figure 1.
- When oxygen saturation was lowered from 50% to 2.5%, the slower group maintained doubling at 69h.
- However, when the oxygen saturation rate was changed to 0.6%, it took 86h to double.
- 1294 genes were differentially expressed at low dilution rates compared to high dilution rates.
- 691 genes were up-regulated and 603 were down-regulated.
- 2 clusters, both containing devR which is homolgous to dosR in tuberculosis, were up-regulated.
- They found 870 genes differentially expressed at 0.6% compared to 50%.
- 429 genes above a 2-fold expression ratio and 441 genes below 0.5-fold level.
- RT-PCR results correspond with the microarray data.
- In low dilution rates at 50% oxygen saturation, 128 genes involved in energy metabolism were up-regulated and 62 down-regulated.
- The majority of down-regulated genes are components of Oxidative Phosphorylation Machinery.
- Several primary dehydrogenases considered to be electron donors to Electron Transport Chain were up-regulated at slow growth; this is shown in Figure 2.
- Data for hydrogenases with roles in energy metabolism are shown in Table 2.
- When oxygen saturation was lowered to 2.5%, 8 genes in energy metabolism were up-regulated.
- When oxygen saturation was lowered to 0.6%, a pattern in gene expression emerged.
- Almost all of the dehydrogenases that were up-regulated during slow growth showed lower levels of mRNA during hypoxia. This did not apply to the hydrogenases.
- M. Smegmatis makes a switch, hydrogenases are important for this, from energy starvation to oxygen limitation.
- There is also a change in pigmentation for yellow to red as shown in Figure 3.
- Most of the genes involved in the TCA cycle and glycolysis did not change in gene expression at low growth rates.
- There were 3 genes that are associated with pyruvate dehydrogenase that were up-regulated in response to slow growth rate.
- In the 0.6% saturation, enzymes of the TCA cycle and glyoxylate shunt were compared to cultures grown at 50% oxygen saturation, this is shown in Figure 4.
- Transcriptional response to 2.5% oxygen saturation at slow growth, was mostly in the up-regulation of 2 gene clusters that had 31 and 6 genes, respectively.
- Most of these genes are homologous to tuberculosis.
- The M. Smegmatis genome has 29 sigma factors and 4 anti-sigma factors.
- At slow growth, the expression ratio was 17 sigma factors to 3 anti-sigma factors.
- All but 1 sigma factor and 1 anti-sigma factors were up-regulated.
- There are 3 different hydrogenase gene clusters in the M. Smegmatis genome.
- 2 of the 3 showed strong up-regulation under energy limitation conditions and all 3 were up-regulated under hypoxia.
- Predictions have been made about these hydrogenases, they should be soluble proteins and have a Ni-Fe center.
- A mutation was introduced, to determine roles of these hydrogenases, on the most responsive of the three hydrogenases; this can be seen in Figure 5.
- Differences in growth was greater in 50% oxygen saturation showing the significant role of hydrogenases in M. Smegmatis energy limitation adaptation.

Discussion
- M. Smegmatis can serve as a model for learning about mycobacteria adaptation to low energy conditions and hypoxia.
- Mycobacteria adjust molecular machinery to adjust to physiological demands for energy.
- Even with lower rates of respiration, membrane potential was maintained at same level as fast growing cells.
- Energy-limited cell shift to more efficient mechanisms.
- Primary Dehydrogenases are activated to power respiration when energy is limited.
- Adaptation of M. Smegmatis includes induction of enzymes to transport sugars and a host of other functions.
- 2 putative succinate dehydrogenases were the only TCA enzymes to show induction-repression under energy limitation conditions.
- M. Smegmatis has 3 strategies for metabolizing under hypoxia; cells can induce high-affinity for cytochrome bd oxidase and machinery parts, can switch to NAD+/NADH independent enzymes, and soluble hydrogenases are up-regulated to oxidize/produce hydrogen.
- Role of cytochrome bd in mycobacteria is still unknown.
- Red pigmentation could be because data shows strong up-regulation of heme/porphyrin, cytochromes and cysteine.
- Recycling reducing equivalents, such as NADH, under hypoxia is still a mystery.
- During hypoxia, M. Smegmatis is dependent of ferredoxin.
- Under energy limiting conditions, hydrogenases can scavenge hydrogen as an energy source and it is re-utilized during hypoxia.
- H₂ gas production was undetectable, supports idea that it is recycled.
- Overall, mycobacteria have a number of different strategies to survive low energy conditions and hypoxia.