Overexpression of DevR in Mtb DKO.

<p><b>(a)</b> Vector map of integrating plasmid overexpressing DevR. <b>(b)</b> Western blotting of lysates from aerobic Mtb cultures. A representative blot is shown. <b>(c)</b> Total DevR levels (DevR + DevR-Myc) in DKO-complemented strains. <b>(d)</b> DevR (endo) level in DKO-complemented strains. <b>(e)</b> RT-qPCR analysis of select DevR regulon genes in DKO-complemented strains. <b>(f)</b> RT-qPCR analysis of additional DevR regulon genes in DKO-P_msp12DevR. Data is Mean ± SD of 2 biological replicates. SigA was used as a loading control in (b). In panels (c) to (f), protein and transcript levels are shown relative to that in DKO (considered as 1).</p

Overexpression of DevR_Δα10 in Mtb RKO.

<p><b>(a)</b> Vector map of integrating plasmid employed to overexpress DevR_α10 protein. <b>(b)</b> RT-qPCR analysis of aerobic DevR regulon genes expression. WT/mutant DevR expression in RKO-P_msp12DevR /RKO-P_msp12DevR_α10 strains is supported by <i>msp12</i> promoter and WT DevR in RKO-P_OperonDevR by its native promoter. <b>(c)</b> RT-qPCR analysis of DevR regulon genes under hypoxia. RT-qPCR data is Mean ± SD of 2 biological replicates.</p

Mtb strains used in the study.

<p>Mtb strains used in the study.</p

<i>Mycobacterium tuberculosis</i> DevR/DosR Dormancy Regulator Activation Mechanism: Dispensability of Phosphorylation, Cooperativity and Essentiality of α10 Helix

<div><p>DevR/DosR is a well-characterized regulator in <i>Mycobacterium tuberculosis</i> which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be ‘pre-adapted’ to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under ‘non-inducing’ conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the α10 helix, establishing the α10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of α10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to the mechanism of DevR overexpression-mediated induction of the regulon in the absence of the normal environmental cue and establishes the α10 helix as an universal and pivotal targeting interface for DevR inhibitor development.</p></div

Overexpression of WT/mutant DevR in Mtb RKO.

<p><b>(a)</b> Vector map of integrating plasmid overexpressing WT/mutant DevR proteins. <b>(b)</b> Mtb RKO- P_msp12DevR, RKO-P_msp12DevR D54E, RKO-P_msp12DevR D54V and RKO-P_msp12DevR T82A strains overexpress DevR variants from <i>msp12</i> promoter. RKO-P_OperonDevR strain expresses DevR from its native promoter. Western blotting of aerobic (Aer) and 5 day hypoxic (Hyp) cultures lysates. A representative blot is shown. <b>(c)</b> RT-qPCR analysis of regulon genes in aerobic cultures. <b>(d)</b> RT-qPCR analysis of DevR regulon genes under hypoxia. RT-qPCR data is Mean ± SD of 2 biological replicates. SigA was used as a loading control in (b).</p

Strains used in this study.

<p>Strains used in this study.</p

DevR regulon expression in DevR_C-expressing cultures declines during hypoxia.

<p>(<b>A</b>) <i>M. tb</i> lysates (15 µg protein) were immunoblotted using rabbit anti-HspX or anti-SigA polyclonal sera and the blots were analyzed using Quantity One software (Bio-Rad, USA). The normalised intensities of the HspX-derived signals (with respect to those of SigA) are denoted as Arbitrary Signal Intensities (ASI) with respect to those obtained in 5 days hypoxic WT cultures (H5). ‘Aer’, aerobic; H1 H3 and H5 refer to 1, 3 and 5 days hypoxic cultures, respectively; ND, not detected. (<b>B</b>) Relative Quantity (RQ) of <i>devR_C</i> transcripts in different Comp strains determined by real time RT-PCR analysis. (<b>C</b>) Real time RT-PCR analysis of DevR regulon transcripts. Fold change in the relative quantity of transcripts under ‘hypoxic’ vs. ‘aerobic’ conditions (<b>fold decrease</b> in Comp5 and <b>fold increase</b> in Comp13) is shown.</p

Primers used in this study.

<p>NdeI, XbaI, and BstBI restriction enzyme sites are underlined.</p

EMSA analysis.

<p>Interaction of DevR_C (<b>A</b>) and full-length DevR (<b>B</b>), with <i>tgs1-Rv3131</i> promoter DNA. Double-stranded oligonucleotides having P+S box sequences belonging to the <i>tgs1-Rv3131</i> divergent promoters were incubated with increasing concentrations of DevR_C or DevR. Arrow, DNA-protein complex; F, free oligonucleotides, arrowheads indicate molecular weight markers in kilobase pairs (lane M), (<b>C</b>) Fraction of bound DNA (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016500#pone-0016500-g001" target="_blank">Fig. 1A, B</a>) plotted against protein concentration.</p

Plasmids used in this study.

a<p>The coordinates of the promoters (in parentheses) are with reference to the transcription start point (TSP) of <i>tgs1</i>;</p><p>Hyg^r, hygromycin resistance;</p><p>Kan^r, kanamycin resistance.</p