Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting >100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved

Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria

Expression profiles of direct and indirect targets of CtrA upon CtrA depletion.

<p>Expression profile of genes directly (A) and indirectly (B) controlled by CtrA. Shown are the average log2 expression levels for each gene in control cells (+IPTG) and the average log2 expression levels for each gene across each time point in cells depleted of CtrA (-IPTG). The scale for expression level is at the bottom of figure panel. Genes are grouped by functional classification explained in the legend on the bottom.</p

CtrA regulates the expression of at least 126 <i>S</i>. <i>meliloti</i> genes.

<p>A. Hierarchical clustered expression profiles for 126 genes in cells expressing <i>ctrA</i> (control; +IPTG) and in cells depleted of <i>ctrA</i> (-IPTG) at several time points (t = 0, 1, 2, 4 and 6 hours) following the initiation of the—IPTG or +IPTG treatment. Normalized log2 expression levels are shown for each gene. The scale for expression level is located on the right. B. Fold change in <i>divJ</i> and <i>divK</i> expression in cells after depletion of CtrA (-I, IPTG) for two hours relative to control cells expressing CtrA (+I). Expression of <i>divJ</i> and <i>divK</i> in each sample was normalized to the expression of the control gene <i>smc00128</i>. Shown are data from a representative biological replicate. Error bars indicate standard error. C. Fold change in <i>minC</i> and <i>minD</i> expression in cells after depletion of CtrA (-I, IPTG) for four hours relative to control cells expressing CtrA (+I). Data normalization was performed as in B. Shown are data from a representative biological replicate. Error bars indicate standard error.</p

Model of CtrA network in <i>S</i>. <i>meliloti</i>.

<p>A. Scheme of genes regulated by CtrA. As reported in the legend two kinds of connections are reported: in red those confirmed by both ChIP-Seq and microarray and in yellow those not detected by microarrays but confirmed by other techniques. Phosphorylation of CtrA is essential [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005232#pgen.1005232.ref028" target="_blank">28</a>] and the roles of DivJ, PLeC and CbrA have been previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005232#pgen.1005232.ref033" target="_blank">33</a>]. Despite the representation here, there is no indication of the preferred form of CtrA subjected to proteolysis. CtrA working on the promoters of genes is a simplification to represent of the direct effect of CtrA on transcription of the gene. B. Comparison between the circuit regulating cell cycle in <i>S</i>. <i>meliloti</i> and <i>C</i>. <i>crescentus</i>. Although the two organisms share the same logic of cell cycle regulatory circuit, differences in the factors connected and involved in the regulation of specific functions are present.</p

CtrA plays an essential role in <i>S</i>. <i>meliloti</i>.

<p>A. Optical density (OD₆₀₀) of wild type <i>S</i>. <i>meliloti</i> and the CtrA depletion strain grown with and without IPTG, error bars represent standard errors. Mid-log phase cells depleted of CtrA show a stable OD level suggesting an impairment of normal growth. B. CFU of the experiments in (A) showing that cells without <i>ctrA</i> expression lost viability. C. Morphology of <i>S</i>. <i>meliloti</i> after 7 hours of CtrA depletion compared with wild type and bacteroid <i>S</i>. <i>meliloti</i>; cells appear elongated and enlarged (bar corresponds to 2 μm). D. Immunoblot analysis using anti-CtrA antibodies over a time course of CtrA depletion. E. FACS analysis of <i>S</i>. <i>meliloti</i> CtrA depletion strain after 8 hours +IPTG (control) and—IPTG (CtrA depleted) showing increased DNA content of up to 20 copies per cell in cells depleted of CtrA.</p

Proteolysis of CtrA is essential in <i>S</i>. <i>meliloti</i> and requires at least CpdR, RcdA and the last three amino acids of CtrA.

<p>A. CtrA protein level changes during the cell cycle with a minimum around 120 min that corresponds to the G1-S transition [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005232#pgen.1005232.ref040" target="_blank">40</a>]. Cells were synchronized and samples were collected every 30 minutes. CtrA antibodies were used to detect the protein level, protein levels were normalized for cell number and error bars represent standard error; B. Pulse-chase experiment of showing decrease over time of radiolabeled CtrA in <i>S</i>. <i>meliloti</i> cells. Values are averages from three separate experiments and the error bars represent standard deviation. C. Morphology of CtrA degradation defective mutants. CpdR^- [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005232#pgen.1005232.ref029" target="_blank">29</a>], although barely vital, shows compromised cell morphology. Cell depleted of RcdA for 7 hours also have altered morphology. Over-expression of <i>rcdA</i> for 7 hours causes cell elongation and division defects (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005232#pgen.1005232.g001" target="_blank">Fig 1C</a>). Overexpression of a stable version of CtrA (lacking the last three amino acids) for 7 hours causes altered cell morphology similar to that of the RcdA depletion strain. D. CtrA protein levels (% of CtrA in wild type cells) in the genetic backgrounds described in the panel C. Cell lysates were normalized for protein content, error bars represent standard error of three different replicates.</p

BRIEF REPORT: Utility of a Short Screening Scale for DSM-IV PTSD in Primary Care

OBJECTIVE: To evaluate Breslau's 7-item screen for posttraumatic stress disorder (PTSD) for use in primary care. DESIGN: One hundred and thirty-four patients were recruited from primary care clinics at a large medical center. Participants completed the self-administered 7-item PTSD screen. Later, psychologists blinded to the results of the screen-interviewed patients using the Clinician Administered PTSD Scale (CAPS). Sensitivity, specificity, and likelihood ratios (LR) were calculated using the CAPS as the criterion for PTSD. RESULTS: The screen appears to have test-retest reliability (r=.84), and LRs range from 0.04 to 13.4. CONCLUSIONS: Screening for PTSD in primary care is time efficient and has the potential to increase the detection of previously unrecognized PTSD