Hypoxia Is Not a Main Stress When Mycobacterium tuberculosis Is in a Dormancy-Like Long-Chain Fatty Acid Environment.

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.

Complete Genome Sequence of the Clinical Beijing-Like Strain Mycobacterium tuberculosis 323 Using the PacBio Real-Time Sequencing Platform.

We report here the whole-genome sequence of the multidrug-resistant Beijing-like strain Mycobacterium tuberculosis 323, isolated from a 15-year-old female patient who died shortly after the initiation of second-line drug treatment. This strain is representative of the Beijing-like isolates from Colombia, where this lineage is becoming a public health concern.

Rv3134c/devR/devS operon of Mycobacterium bovis BCG is differentially transcribed under "in vitro" stress conditions.

DevR is a transcriptional regulator that mediates the genetic response of Mycobacterium tuberculosis and Mycobacterium bovis to oxygen limitation and nitric oxide exposure. devR is part of an operon that includes the genes devS and Rv3134c, which encode an oxygen sensor protein and a protein that contains a universal stress protein domain, respectively. Here, we report the transcriptional analysis and quantitative expression of Rv3134c/devR/devS under in vitro stress conditions including oxygen limitation, low nutrients and ex vivo macrophage infection. At least three different promoters were found to control Rv3134c/devR/devS expression under the stresses tested. Two promoters were identified upstream of devR, one was active under hypoxia and the other under nutrient starvation. A single promoter was identified upstream of Rv3134c, and transcripts from this promoter were detected only under hypoxia. Rv3134c to devR were found to be co-transcribed only under hypoxia, whereas devR/devS were co-transcribed both in aerobiosis and starvation. RT-qPCR showed an increase in the ratio hypoxia/aerobiosis and in starvation/nutrients in all genes. devR/devS showed transient expression in the first days of macrophage infection. Our results indicate that Rv3134c/devR/devS of M. bovis BCG constitutes an operon with complex regulation that participates in bacterial response against a wide range of stresses.