Expression of <i>cso</i> genes during copper stress in the absence of <i>csoR</i>.

<p>qRT-PCR was used to analyze expression levels of <i>csoR</i>, Rv0968, <i>ctpV</i>, and Rv0970. A schematic of the operon (not drawn to scale) is shown above the graph and the order of the genes in the operon corresponds to the order in which expression levels are graphed for each gene. <b>(A)</b> <i>M</i>. <i>tuberculosis</i> strains H37Rv (black), Δ<i>csoR</i> (striped), or Δ<i>csoR</i>::<i>csoR</i> (white) were exposed to CuCl₂ at 50 or 500μM or left untreated (0μM) for 3 hours. Values shown are the mean fold change between each gene and its untreated wild type counterpart after normalization to <i>sigA</i> expression levels. <b>(B)</b> A second comparison of the same samples was done showing the mean fold change between each gene after normalization to <i>sigA</i> expression levels at 50μM (striped) or 500μM (white) and its untreated counterpart (black) from the same strain. Data represent one of two similar biological replicates. Error bars represent the standard error of the mean from three technical replicates. <b>(C)</b> Expression levels of <i>mmcO</i> as determined by qRT-PCR analysis of the same samples–H37Rv (black), Δ<i>csoR</i> (striped), or Δ<i>csoR</i>::<i>csoR</i> (white)–left untreated (0μM) or stressed with 50μM or 500μM CuCl₂. Fold change is shown as expression levels of each gene relative to expression levels in untreated wild type culture after normalization to <i>sigA</i> expression levels. Data represent one of two similar biological replicates. Error bars represent the standard error of the mean from two technical replicates.</p

Growth of Δ<i>csoR</i> in the lungs during mouse infection.

<p><b>(A)</b> Groups of BALB/c mice were infected by aerosol route with either <i>M</i>. <i>tuberculosis</i> H37Rv (filled circles) or Δ<i>csoR</i> (open squares). Shown are CFU per lung for each individual mouse over the course of 25 weeks across two independent experiments. Asterisks indicate significance with the following <i>P</i>-values: * <i>P</i> < 0.015; ** <i>P</i> < 0.005. <b>(B)</b> Histopathology for mice infected with H37Rv or Δ<i>csoR</i> at 4 and 8 weeks. Mouse lung sections were stained with H&E and are shown at 40× magnification (scale bar = 500μm). Insets show Ziehl-Neelson stained sections of lung tissue with pink bacilli indicated by black arrows at 1000× magnification (scale bar = 20μm).</p

Growth kinetics of Δ<i>csoR</i> under copper stress.

<p><b>(A)</b> Growth of <i>M</i>. <i>tuberculosis</i> H37Rv (circles) and Δ<i>csoR</i> (squares) over the course of 15 days in Sauton’s media left untreated (filled), or <b>(B)</b> treated with 500μM CuCl₂ (open). <b>(C)</b> Growth of stationary phase <i>M</i>. <i>tuberculosis</i> H37Rv (circles) and Δ<i>csoR</i> (squares) inocula over the course of 15 days in Sauton’s media left untreated (filled), or <b>(D)</b> treated with 500μM CuCl₂ (open). The dashed line indicates the limit of detection. Shown are one of two similar biological replicates with error bars representing standard deviation.</p

Potential consequences of Δ<i>csoR</i> disrupted copper homeostasis.

<p>Overexpression of the <i>cso</i> and the proteins it codes for after deletion of <i>csoR</i> (dark red) may result in excessive levels of copper export by CtpV (red). <b>(A)</b> As a result, pools of copper (orange) for metalloenzyme usage may be depleted, reducing the levels of functional proteins such as cytochrome c oxidase (yellow), as seen in the panel on the right. This could potentially slow the electron transport chain leading to the induction of the DosR (blue) regulon through the redox sensor, DosS (blue) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151816#pone.0151816.ref038" target="_blank">38</a>]. The wild type strain is shown in the panel on the left for comparison. <b>(B)</b> Alternatively, the increased cytoplasmic export of copper may lead to a buildup of copper in the mycobacterial periplasmic space where MmcO (green) usually assists in copper detoxification. The increased copper stress in this region may be magnified, however, by the down regulation of <i>mmcO</i> in the mutant strain. Free copper can lead to the production of reactive nitrogen species, such as NO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151816#pone.0151816.ref039" target="_blank">39</a>]. NO can trigger expression of the DosR regulon [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151816#pone.0151816.ref025" target="_blank">25</a>]. The wild type strain is shown in the panel on the left for comparison. CM; cytoplasmic membrane. MM; mycomembrane. Cu; copper. NO; nitric oxide. COX; cytochrome c oxidase. P; phosphoryl group.</p

Analysis of the Δ<i>csoR</i> transcriptome.

<p><b>(A)</b> Counts per million (CPM) of each gene detected in our RNA-Seq study, plotted for <i>M</i>. <i>tuberculosis</i> Δ<i>csoR</i> versus H37Rv wild type. Highlighted are members of the <i>cso</i> (yellow), members of the RicR regulon (orange), genes responsive to copper, but not part of the <i>cso</i> or RicR regulon (green), members of the DosR regulon (blue), and significantly differentially expressed genes as determined by an FDR ≥ 0.05 not included in the above groups (red). Two parallel, black lines demarcate the region outside of which differential expression values exceed our cutoff of a 2.0 fold difference between strains. Genes not meeting both cutoff values are shown as semi-transparent grey diamonds. Data points show the mean of two biological replicates. <b>(B)</b> Overlap of the induced <i>csoR</i> regulon (red) with 500 CuCl₂ inducible genes (green) and genes induced under the control of <i>dosR</i> (blue). Of the 152 genes induced in the Δ<i>csoR</i> strain compared to H37Rv wild type, only 4 overlapped with the 24 genes induced in H37Rv wild type when exposed to copper stress at 500μM CuCl₂. DosR inducible genes showed substantial overlap with Δ<i>csoR</i> with 44 out of 48 overlapping. The diagram is area-proportional.</p

Selected genes differentially expressed in <i>M</i>. <i>tuberculosis</i> Δ<i>csoR</i> as compared to wild type.

<p>Selected genes differentially expressed in <i>M</i>. <i>tuberculosis</i> Δ<i>csoR</i> as compared to wild type.</p

PCR detection of four GIs in 70 soil and 64 clinical <i>B. pseudomallei</i> isolates.

<p>PCR detection of four GIs in 70 soil and 64 clinical <i>B. pseudomallei</i> isolates.</p

Comparative genome analyses of <i>B. pseudomallei</i> K96243 with <i>B. pseudomallei</i> from environmental and clinical isolates.

<p>The comparison between K96243 and environmental isolate (BP45s) was shown in (A) and clinical isolate (H307) in (B). The log₂ hybridization ratio of the tested isolate over the K96243 was plotted on the Y axis and positions of genes on the X axis. Low values of the Log₂ hybridization ratio, seen as peaks, imply absence of genes designated as GIs. The slide averaging window of 6 genes, one gene per step, was applied to the normalized data and smoothed out the fluctuations of the data. Each GI contains at least 6 CDS that have values less than −2SD.</p

The GIs determined by DNA microarrays and their functional classification.

<p>GIs present (+) and absent (−).</p>*<p>Functional classification data obtained by <i>Burkholderia pseudomallei</i> K96243 genome annotation from Pathema Bioinformatics Resource Center <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037762#pone.0037762-Pathema1" target="_blank">[28]</a>.</p

<i>B. pseudomallei</i> isolates used for DNA microarray.

<p><i>B. pseudomallei</i> isolates used for DNA microarray.</p