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Title: Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti}
Article:
Abstract:
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.
Introduction:
The alphaproteobacterium Sinorhizobium meliloti can thrive in the soil as a free-living organism or as a nitrogen-fixing symbiotic partner with compatible legume hosts[1]. The S. meliloti-legume symbiosis involves multiple developmental stages, during which the bacteria coordinate their cell proliferation with the development of the host plant cells[2, 3]. A key step in this symbiosis is the striking differentiation of S. meliloti cells into enlarged, polyploid( 16–32 copies of the genome) nitrogen-fixing bacteroids within the specialized host cells that comprise the developing nodule[4]. Differentiation of bacteroids in S. meliloti-legume symbiosis is driven in part by nodule specific cysteine-rich peptides( NCRs) that are produced by the host legume[5, 6]. These peptides, such as NCR247, can provoke in free-living cells many of the changes associated with bacteroid differentiation including the increase in cell size and endoreduplication of the genome[7]. The uncoupling of DNA replication from cell division in S. meliloti during symbiosis stands in stark contrast to the cell cycle of free-living S. meliloti, where DNA replication is tightly coupled to cell division[8]. The involvement of the cell cycle regulatory network in cellular differentiation programs, such as cyst formation in Rhodospirillum centenum and the asymmetric division of Caulobacter crescentus, is a common theme in Alphaproteobacteria[9, 10]. In C. crescentus and presumably in other alphaproteobacteria, cellular differentiation is largely governed by the response regulator CtrA[10–14]. C. crescentus divides asymmetrically to produce two morphologically different cells, a motile swarmer cell and a sessile stalked cell[15]. The two cell types are also distinct in their replicative capacities. The stalked cell, which lacks active CtrA, can immediately initiate DNA replication and re-enter the cell cycle, while in the swarmer cell, the origin of replication is bound and inhibited by phosphorylated CtrA resulting in a G1 arrest[16, 17]. As a transcription factor, phosphorylated CtrA binds ca. 200 promoter regions and controls the transcription of about 95 genes over the course of the cell cycle, thereby modulating diverse processes including polar morphogenesis and cell division[18]. The expression, activity and stability of C. crescentus CtrA are highly regulated during the cell cycle through transcriptional regulation, phosphorylation and regulated proteolysis[19–27]. An essential, functional homolog of C. crescentus CtrA is present in S. meliloti and has been implicated in the symbiotic cellular differentiation program[28]. The genetic circuit controlling CtrA in S. meliloti at the transcriptional and posttranslational levels has been predicted using bioinformatics and all the regulatory factors identified in C. crescentus are conserved on the sequence level in S. meliloti[11]. Genetic experiments on a few of these putative regulators of CtrA have revealed a striking link between symbiosis and cell cycle regulation[29–33]. In addition, gene expression profiling of S. meliloti at different stages of the symbiosis indicated that expression of ctrA is strongly down-regulated in bacteroids once differentiation begins[34], and Western blot analysis of purified bacteroids revealed that CtrA protein levels are very low in mature bacteroids[33]. More specifically, down regulation of CtrA during symbiosis may be caused by exposure to NCR peptides, as in vitro treatment of S. meliloti with a sub-lethal dose of the NCR peptide, NCR247, significantly attenuates ctrA expression[35]. Collectively, these observations suggest that NCR peptides and perhaps other plant factors modulate the cell cycle in part by affecting the level of CtrA activity. It is thus crucial to gain a deeper understanding of the factors governing the S. meliloti cell cycle, especially of the cell processes governed by CtrA and the regulatory mechanisms controlling CtrA activity. In this study, we sought to understand the mechanisms regulating cell cycle regulation in S. meliloti by analyzing the effects of CtrA depletion in S. meliloti free-living cells. We aimed to define the direct and indirect transcriptional regulons of S. meliloti CtrA and probing regulatory mechanisms, such as regulated proteolysis that possibly govern CtrA levels during the S. meliloti cell cycle. As global analysis of the CtrA transcriptional network has not been performed in detail in an alphaproteobacterium other than C. crescentus, this work provides the first insight into how this highly conserved genetic network can evolve to fit the distinct lifestyles of this diverse group of bacteria. Furthermore, the model of CtrA cell cycle regulation in S. meliloti developed in this work will be pivotal in the future elucidation of how the bacterial cell cycle is modulated by plant factors during the symbiosis.
Discussion:
In this work we present significant progress towards the understanding of S. meliloti cell cycle regulation and differentiation in bacteroids, specifically involving the conserved master cell cycle regulator CtrA( Fig 7A). We showed that CtrA-deprived cells are unable to divide, exhibit cell elongation, branching and a sharp increase in DNA content. These combined phenotypes result in a clear loss of viability. Through microarray-based gene expression analysis coupled with ChIP-Seq analysis we defined the direct and indirect regulons of CtrA and discovered that, in agreement with the loss of function phenotypes, CtrA acts as a transcriptional regulator controlling essential functions that are required for proper cell cycle progression, such as cell division, chromosome methylation, motility and cell envelope biogenesis. Similarly to C. crescentus, CtrA is subjected to several regulatory mechanisms. We showed that the concentration of CtrA drops at the moment of the G1 to S transition similarly to C. crescentus[22]. Taken together with the striking genome amplification observed during CtrA depletion, these data suggest that CtrA is a repressor of DNA replication initiation that must be inactivated at the G1-S transition and perhaps during bacteroid differentiation. Furthermore, perturbations of the last three C-terminal residues of CtrA results in a marked accumulation of CtrA protein, suggesting that like in C. crescentus, S. meliloti CtrA levels are regulated by proteolysis, likely by the ClpXP protease[49]. We also found that levels of CtrA are higher in mutants lacking functional CpdR and RcdA, which are key regulators of CtrA proteolysis in C. crescentus[26, 51]. Surprisingly, however, unlike C. crescentus, the disruption of CtrA proteolysis is lethal in S. meliloti as both deletion of rcdA and expression of ctrAΔ3 are lethal. The comparison between the S. meliloti CtrA circuit architecture with C. crescentus helped us to understand several general principles of the alphaproteobacterial cell cycle( Fig 7B). First, CtrA plays a crucial role in directly regulating genes involved in motility, chemotaxis and cell division. Motility was identified as an ancestral functional of this regulator in Magnetospirillum magnetotacticum and other alphaproteobacteria[13]. In most cases, however, the regulation of these similar processes is achieved through the control of different target genes. For example, CtrA controls cell division through ftsAQ in C. crescentus, while in S. meliloti this control is likely carried out by other means, such as CtrA repression of the Min system together with other unknown mechanisms, including for example nucleoid occlusion. This suggests that the CtrA regulon has changed significantly during the evolution of alphaproteobacteria and that different regulatory networks have evolved in the same phylogenetic group. A second general feature of the CtrA circuit in alphaproteobacteria concerns the link between CtrA and the DivK/DivJ module, which directly controls the phosphorylation status and indirectly controls the stability of CtrA. In both C. crescentus and S. meliloti, this module is controlled by CtrA via transcriptional regulation. In C. crescentus this important feedback loop is routed through the divK promoter, which is directly activated by CtrA[20]. In S. meliloti, divK transcription is only indirectly affected by CtrA depletion, and instead, CtrA feedback regulation of this module happens directly through the transcriptional activation of divJ by CtrA( Fig 7). The gene encoding the DivJ cognate kinase, cbrA, may also regulated directly by CtrA as its promoter was bound in the CtrA ChIP-Seq experiment, but significant differential expression of cbrA under conditions of CtrA depletion was only detected by lacZ-fusions and not by microarrays( S6 Fig). Previous work has found that altering the native promoter of divK in C. crescentus causes severe cell cycle defects[27], while in S. meliloti altering divJ transcription leads to an uncoordinated progression through the cell cycle, in particular, its overexpression causes a G2 block[33]. These and other interesting divergences in the wiring of the S. meliloti and C. crescentus cell cycle network are just the tip of the iceberg in uncovering how alphaproteobacteria have evolved species-specific wiring of these highly conserved cell cycle components to fit their unique lifestyles and diverse cellular differentiation programs. Perhaps most importantly towards the understanding of symbiotic interactions with legume hosts, our findings provide insight into how S. meliloti cell cycle regulation may be altered during bacteroid differentiation to produce the bacteroid phenotypes of cell elongation and endoreduplication. Within the host nodule cells, S. meliloti is exposed to a microaerobic environment, which activates the FixJ/FixL two-component system. Activation of this system elicits a significant transcriptional response including expression of genes coding for the nitrogen fixation machinery[52–55]. Another important trigger of the bacteroid differentiation process is a large class of nodule-specific cysteine rich( NCR) peptides produced by the host legume[4, 7, 35]. The specific cellular targets of these peptides are unknown, although recent work has provided strong evidence that at least one of these peptides, NCR247, targets diverse cellular processes in S. meliloti[7, 35, 56], including the cell cycle. Furthermore, treatment of S. meliloti with NCR247 directly affects the transcription of CtrA and many other genes included in the direct and indirect CtrA regulons established in this work[35]. The observations that ctrA transcriptional downregulation coincides with bacteroid differentiation within the nodule[34], that CtrA is largely absent from mature bacteroids[33], and that an NCR peptide specifically perturbs the CtrA transcriptional regulon[35] all point to CtrA as an important regulatory node through which the legume host manipulates the bacterial cell cycle. Interestingly the dissection of gene expression changes in the differentiation region of the nodule revealed that the formation of bacteroids is associated increased concentration of rcdA transcript[34], which correlates with our observation that rcdA overexpression enhances CtrA degradation( Fig 6D). This work provides the first global overview of the CtrA cell cycle regulon in S. meliloti, which it will facilitate the exploration how NCR peptides and other plant factors affect the function of CtrA and other important cell cycle regulators to drive bacteroid differentiation in S. meliloti.
In order to propagate , all living cells must ensure that their genetic material is faithfully copied and properly partitioned into the daughter cells before division . These coordinated processes of DNA replication and cell division are termed the “cell cycle” and are controlled by a complex network of regulatory proteins in all organisms . In the class Alphaproteobacteria , the regulation of the cell cycle is closely linked to cellular differentiation processes that are vital for survival in the environment . In these bacteria , the cell cycle regulator CtrA is thought to serve as the primary link between the coordination of the cell cycle and cellular differentiation . The alphaproteobacterium , Sinorhizobium meliloti , an important model symbiont of alfalfa plants , undergoes a striking cellular differentiation that is vital to the formation of an efficient symbiosis dedicated to the conversion of atmospheric nitrogen to biologically available organic nitrogen . However , the link between cellular differentiation and cell cycle control in S . meliloti has not been made . In this study , we showed that S . meliloti cells without CtrA are similar to the symbiotic form . By the identification of the genes whose expression is directly and indirectly controlled by CtrA , we found that CtrA regulates vital cell cycle processes , including DNA replication and cell division , but through different genetic pathways than in other alphaproteobacteria . We importantly show that the levels of CtrA protein are governed by an essential cell cycle regulated proteolysis , which may also be an important mode of CtrA down-regulation during symbiosis to drive cellular differentiation .