<i>Mtb</i> 30 min post-diamide treatment.

<p>Genes induced and repressed by functional category at 30 minutes post-diamide treatment in wild-type. n = 3. MT # and Rv # denote the CDC1551 and the H37Rv gene IDs, respectively.</p

<i>Mtb</i>:ΔRv2745c (comp) 90 min post-diamide treatment.

<p>Genes induced and repressed by functional category 90 minutes post-diamide treatment in the complemented strain. n = 3. MT # and Rv # denote the CDC1551 and the H37Rv gene IDs, respectively.</p

<i>Mtb</i>:ΔRv2745c (comp) 30 min post-diamide treatment.

<p>Genes induced and repressed by functional category at 30 minutes post-diamide treatment in the complemented strain. n = 3. MT # and Rv # denote the CDC1551 and the H37Rv gene IDs, respectively.</p

Venn Diagrams of Diamide Induced Genes.

<p>Venn diagrams describe the extent of overlap between gene-expression upon diamide treatment in <i>Mtb</i> (blue circles), <i>Mtb</i>:ΔRv2745c (red circles) and <i>Mtb</i>:ΔRv2745c (comp) (green circles). Genes induced at least two-fold at: a.) 30, b.) 60, and c.) 90 min post-diamide treatment are shown. Genes induced at least four-fold at: d.) 30, e.) 60, and f.) 90 min post-diamide treatment. g–j.): Delayed response of <i>Mtb</i>:ΔRv2745c (comp). g.) Genes induced at least two-fold comparing <i>Mtb</i> at 30 min to <i>Mtb</i>:ΔRv2745c (comp) at 60 min and h.) Genes induced at least two-fold comparing <i>Mtb</i> at 60 min to <i>Mtb</i>:ΔRv2745c (comp) at 90 min. i.) Genes induced at least four-fold comparing <i>Mtb</i> at 30 min to <i>Mtb</i>:ΔRv2745c (comp) at 60 min. j.) Genes induced at least four-fold comparing <i>Mtb</i> at 60 min to <i>Mtb</i>:ΔRv2745c (comp) at 90 min. n = 3.</p

Diamide Susceptibility.

<p>a.) Disc diffusion assay was performed using discs containing 20 μmol diamide. The zone of inhibition of <i>Mtb</i>:ΔRv2745c (red bar) is significantly larger when compared to both <i>Mtb</i> (blue bar) and <i>Mtb</i>:ΔRv2745c (comp) (green bar) indicating that the isogenic mutant is more susceptible to redox stress. n = 3. *p<0.001 b.) OD graph of diamide treated cultures. In the initial stages of treatment, there is no significant difference in growth between the different groups.</p

<i>Mtb</i>:ΔRv2745c 30 min post-diamide treatment.

<p>Genes induced and repressed by functional category at 30 minutes post-diamide treatment in the isogenic mutant. n = 3. MT # and Rv # denote the CDC1551 and the H37Rv gene IDs, respectively.</p

RT-PCR Confirmation.

<p>a.) The data shows that there is a small increase in σ^H levels of the isogenic mutant, relative to that of the wild type and complemented strain. However, σ^H levels are not sustained in the isogenic mutant, whereas they increase in the wild-type and complemented strain. b.) σ^E levels remain low in all three strains. c.) There is Rv2745c activation in the wild-type and the complemented strain, whereas there is no Rv2745c expression in the isogenic mutant, as expected.</p

Venn Diagrams of Diamide Repressed Genes.

<p>Venn diagrams describe the extent of overlap between gene-expression upon diamide treatment in <i>Mtb</i> (blue circles), <i>Mtb</i>:ΔRv2745c (red circles) and <i>Mtb</i>:ΔRv2745c (comp) (green circles). Genes repressed at least two-fold at: a.) 30, b.) 60, and c.) 90 min post-diamide treatment are shown. Genes repressed at least four-fold at: d.) 30, e.) 60, and f.) 90 min post-diamide treatment. g–j.): Delayed response of <i>Mtb</i>:ΔRv2745c (comp). g.) Genes repressed at least two-fold comparing <i>Mtb</i> at 30 min to <i>Mtb</i>:ΔRv2745c (comp) at 60 min and h.) Genes repressed at least two-fold comparing <i>Mtb</i> at 60 min to <i>Mtb</i>:ΔRv2745c (comp) at 90 min. i.) Genes repressed at least four-fold comparing <i>Mtb</i> at 30 min to <i>Mtb</i>:ΔRv2745c (comp) at 60 min. j.) Genes repressed at least four-fold comparing <i>Mtb</i> at 60 min to <i>Mtb</i>:ΔRv2745c (comp) at 90 min. n = 3.</p

The <i>Mycobacterium tuberculosis</i> Rv2745c Plays an Important Role in Responding to Redox Stress

<div><p>Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle <i>in vivo</i>, <i>Mtb</i> is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for <i>Mtb</i> to establish an infection. The Rv2745c (<i>clgR</i>) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in <i>Mtb</i> pathogenesis. Our isogenic mutant, <i>Mtb</i>:ΔRv2745c, is significantly more sensitive to <i>in vitro</i> redox stress generated by diamide, relative to wild-type <i>Mtb</i> as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, <i>Mtb</i>:ΔRv2745c. Furthermore, induction of <i>clgR</i> in <i>Mtb</i> and <i>Mtb</i>:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that <i>clgR</i> has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the <i>Mtb</i> Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of <i>Mtb in-vivo</i>, regardless of its induction of the Clp proteolytic pathway.</p></div

Negative Regulators Expressed in <i>Mtb</i> post-diamide treatment.

<p>Probable negative transcriptional regulators induced in <i>Mtb</i> post-diamide at each time point. n = 3. MT # and Rv # denote the CDC1551 and the H37Rv gene IDs, respectively.</p