Fig 1.
CtrA plays an essential role in S. meliloti.
A. Optical density (OD600) of wild type S. meliloti 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 ctrA expression lost viability. C. Morphology of S. meliloti after 7 hours of CtrA depletion compared with wild type and bacteroid S. meliloti; 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 S. meliloti 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.
Fig 2.
CtrA regulates the expression of at least 126 S. meliloti genes.
A. Hierarchical clustered expression profiles for 126 genes in cells expressing ctrA (control; +IPTG) and in cells depleted of ctrA (-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 divJ and divK expression in cells after depletion of CtrA (-I, IPTG) for two hours relative to control cells expressing CtrA (+I). Expression of divJ and divK in each sample was normalized to the expression of the control gene smc00128. Shown are data from a representative biological replicate. Error bars indicate standard error. C. Fold change in minC and minD 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.
Fig 3.
ChIP-Seq analysis reveals direct targets of CtrA.
Genes directly regulated by CtrA. A. Representation of all CtrA binding sites in the three circular replicons of S. meliloti (here represented as linear starting from the origin of replication). B. Promoter region of several genes detected by microarrays. Transcriptional start sites, previously defined [39], are represented as orange arrows (numbers between brackets represent the distance form ATG). In blue the plot of reads per nucleotide measured by ChIP-Seq analysis in a 600bp long region including the beginning of the coding sequence (in red). Green lines represent predicted CtrA binding site [11]. C. ChIP-Seq of the ccrM promoter region. In blue the plot of reads per nucleotide measured by ChIP-Seq analysis. Green lines represent predicted CtrA binding site. D. Beta-galactosidase activity assay using a LacZ fusion of the ccrM promoter in cells (BM249) after depletion of CtrA (no IPTG) for two hours relative to control cells expressing CtrA (+IPTG). Error bars indicate standard error.
Fig 4.
Expression profiles of direct and indirect targets of CtrA upon CtrA depletion.
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.
Fig 5.
Structure of the ctrA promoter in S. meliloti.
A. Promoters of ctrA in C. crescentus and S. meliloti. Both promoters have two transcriptional sites (P1 and P2). In C. crescentus P2 is activated by CtrA-P while P1 is activated by GcrA and repressed by CtrA-P [20,64,72]. In S. meliloti the P1 and P2 transcriptional start sites have been previously defined by primer extension [28]. ChIP-Seq results identified 4 binding sites of CtrA in S. meliloti upstream P1 and P2 while previously a fifth one was discovered by DNase I footprinting [28]. The presence of CtrA binding sites suggests a potential control of transcription by CtrA; B. Details of the ChIP-Seq using antibodies against CtrA (blue) of the ctrA promoter region. C. Promoters P1 and P2 were fused to lacZ measuring the beta—galactosidase activity depleting CtrA for 4 hours.
Fig 6.
Proteolysis of CtrA is essential in S. meliloti and requires at least CpdR, RcdA and the last three amino acids of CtrA.
A. CtrA protein level changes during the cell cycle with a minimum around 120 min that corresponds to the G1-S transition [40]. 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 S. meliloti cells. Values are averages from three separate experiments and the error bars represent standard deviation. C. Morphology of CtrA degradation defective mutants. CpdR- [29], although barely vital, shows compromised cell morphology. Cell depleted of RcdA for 7 hours also have altered morphology. Over-expression of rcdA for 7 hours causes cell elongation and division defects (Fig 1C). 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.
Fig 7.
Model of CtrA network in S. meliloti.
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 [28] and the roles of DivJ, PLeC and CbrA have been previously described [33]. 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 S. meliloti and C. crescentus. 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.