Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

Gopinath, Vipin; Raghunandanan, Sajith; Gomez, Roshna Lawrence; Jose, Leny; Surendran, Arun; Ramachandran, Ranjit; Pushparajan, Akhil Raj; Mundayoor, Sathish; Jaleel, Abdul; Kumar, Ram Mohan Ram

doi:10.1074/mcp.m115.051151

Cited by 81 publications

(123 citation statements)

References 72 publications

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“…For inducing hypoxia, MTB was grown in an in vitro dormancy model as described by Gopinath et al . . Briefly, log phase MTB culture grown in Dubos medium (Difco) was transferred to glass tubes at a head space ratio of 0.6.…”

Section: Methodsmentioning

confidence: 99%

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The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR

et al. 2016

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Rv3334 protein of Mycobacterium tuberculosis belongs to the MerR family of transcriptional regulators and is upregulated during hypoxia and other stress conditions. Employing GFP reporter constructs, mobility shift assays and ChIP assays, we demonstrate that Rv3334 binds to its own promoter and acts as an autorepressor. We were able to locate a 22 bp palindrome in its promoter that we show to be the cognate binding sequence of Rv3334. Using chase experiments, we could conclusively prove the requirement of this palindrome for Rv3334 binding. Recombinant Rv3334 readily formed homodimers in vitro, which could be necessary for its transcriptional regulatory role in vivo. Although the DNA‐binding activity of the protein was abrogated by the presence of certain divalent metal cations, the homodimer formation remained unaffected. In silico predictions and subsequent assays using GFP reporter constructs and mobility shift assays revealed that the expression of ketosteroid regulator gene (kstR), involved in lipid catabolism, is positively regulated by Rv3334. ChIP assays with aerobically grown M. tuberculosis as well as dormant bacteria unambiguously prove that Rv3334 specifically upregulates expression of kstR during dormancy. Our study throws light on the possible role of Rv3334 as a master regulator of lipid catabolism during hypoxia‐induced dormancy.

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Section: Methodsmentioning

confidence: 99%

“…Dormancy was induced as reported by Gopinath et al . . DNA·protein crosslinking was carried out by the addition of formaldehyde to a final concentration of 1% to 20 mL of the bacterial culture, and incubated at 25 °C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR

et al. 2016

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“…Because the number of spectral counts correlates with the abundance of the protein, the method can be used to compare protein abundances between biological samples that are prepared and processed in parallel (29)(30)(31). This method is widely accepted in the proteomics community and has been used to identify differences in protein levels in Shewanella oneidensis grown under oxic and anoxic conditions (32), the relative levels of 3,274 mouse proteins in the brain, heart, kidney, liver, lung, and placenta (28), and changes in protein levels in Mycobacterium tuberculosis during reactivation from a dormant state (33). We found that M3 results in a 2-fold increase in the level of ProA*, whereas the level of ProB is unaffected.…”

Section: Discussionmentioning

confidence: 99%

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate

et al. 2016

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When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA*. This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCEThe high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported. Metabolic enzymes, although prodigious catalysts, are not perfectly specific for their physiological substrates. They typically possess secondary activities as a consequence of the assemblage of highly reactive functional groups, metal ions, and cofactors in their active sites. Secondary activities that are physiologically irrelevant, either because they are too inefficient to contribute to fitness or because the enzyme never encounters the substrate, are termed promiscuous activities (1).Promiscuous activities are important from an evolutionary standpoint because they provide a reservoir of catalytic potential within a proteome that can be drawn upon when the environment changes. A promiscuous activity may become important for fitness when a new source of carbon, nitrogen, or phosphorous appears in the environment or when a previously available compound, such as an amino acid or cofactor, becomes unavailable. A promiscuous activity may also become critical when the organism is exposed to a novel toxin, such as an antibiotic or pesticide.A newly recruited pro...

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“…Immunization, serum collection, antibody purification and western blot against MTB cell-free extract were carried out as described by Gopinath et al [16]. All the guidelines of the Institute Animal Ethics Committee, Rajiv Gandhi Centre for Biotechnology, were followed for animal handling.…”

Section: Antibody Preparationmentioning

confidence: 99%

“…RNA isolation, cDNA synthesis and quantitative PCR RNA isolation from MTB was performed following Gopinath et al [16] using Trizol (Sigma-Aldrich) reagent. The RNA preparation was resuspended in nuclease-free water and treated with DNaseI (Sigma-Aldrich) at room temperature for 30 min.…”

Section: Reporter Constructs and Spectrofluorimetrymentioning

confidence: 99%

Rv0474 is a copper‐responsive transcriptional regulator that negatively regulates expression of RNA polymerase β subunit in Mycobacterium tuberculosis

et al. 2018

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We characterize Rv0474, a putative transcriptional regulatory protein of Mycobacterium tuberculosis, which is found to function as a copper-responsive transcriptional regulator at toxic levels of copper. It is an autorepressor, but at elevated levels (10-250 μm) of copper ions the repression is relieved resulting in an increase in Rv0474 expression. Copper-bound Rv0474 is recruited to the rpoB promoter leading to its repression resulting in the growth arrest of the bacterium. Mutational analysis showed that the helix-turn-helix and leucine zipper domains of Rv0474 are essential for its binding to Rv0474 and rpoB promoters, respectively. The mechanism of Rv0474-mediated rpoB regulation seems to be operational only in pathogenic mycobacteria that can persist inside the host.

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Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

Cited by 81 publications

References 72 publications

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate

Rv0474 is a copper‐responsive transcriptional regulator that negatively regulates expression of RNA polymerase β subunit in Mycobacterium tuberculosis

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