Coordination of symbiosis and cell cycle functions in Sinorhizobium meliloti

The symbiotic nitrogen fixing species Sinorhizobium meliloti represents a remarkable model system for the class Alphaproteobacteria, which includes genera such as Caulobacter, Agrobacterium and Brucella. It is capable of living free in the soil, and is also able to establish a complex symbiosis with leguminous plants, during which its cell cycle program is completely rewired presumably due, at least in part, to the action of peptides secreted by the plant. Here we will discuss how the cell cycle regulation works in S. meliloti and the kinds of molecular mechanisms that take place during the infection. We will focus on the complex regulation of the master regulator of the S. meliloti cell cycle, the response regulator CtrA, discussing its implication in symbiosis.     

Glutamate at the phosphorylation site of response regulator CtrA provides essential activities without increasing DNA binding

The essential response regulator CtrA controls the Caulobacter crescentus cell cycle and phosphorylated CtrA~P preferentially binds target DNA in vitro. The CtrA aspartate to glutamate (D51E) mutation mimics phosphorylated CtrA~P in vivo and rescues non-viable C.crescentus cells. However, we observe that the CtrA D51E and the unphosphorylated CtrA wild-type proteins have identical DNA affinities and produce identical DNase I protection footprints inside the C.crescentus replication origin. There fore, D51E promotes essential CtrA activities separate from increased DNA binding. Accordingly, we argue that CtrA protein recruitment to target DNA is not sufficient to regulate cell cycle progression.     

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.     

The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection

Cysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids. The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessary for successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a small size, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. Unlike NCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosis in root hairs and nodule primordia. Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensable for nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progression of bacterial invasion with restricting the competence of root hairs for infection.     

Dynamical Modeling of the Cell Cycle and Cell Fate Emergence in Caulobacter crescentus

The division of Caulobacter crescentus, a model organism for studying cell cycle and differentiation in bacteria, generates two cell types: swarmer and stalked. To complete its cycle, C. crescentus must first differentiate from the swarmer to the stalked phenotype. An important regulator involved in this process is CtrA, which operates in a gene regulatory network and coordinates many of the interactions associated to the generation of cellular asymmetry. Gaining insight into how such a differentiation phenomenon arises and how network components interact to bring about cellular behavior and function demands mathematical models and simulations. In this work, we present a dynamical model based on a generalization of the Boolean abstraction of gene expression for a minimal network controlling the cell cycle and asymmetric cell division in C. crescentus. This network was constructed from data obtained from an exhaustive search in the literature. The results of the simulations based on our model show a cyclic attractor whose configurations can be made to correspond with the current knowledge of the activity of the regulators participating in the gene network during the cell cycle. Additionally, we found two point attractors that can be interpreted in terms of the network configurations directing the two cell types. The entire network is shown to be operating close to the critical regime, which means that it is robust enough to perturbations on dynamics of the network, but adaptable to environmental changes.     

The DivJ,CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen‐fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.     

Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

A CtrA homolog affects swarming motility and encystment in <Emphasis Type="Italic">Rhodospirillum centenum</Emphasis>

The α-proteobacterium, Rhodospirillum centenum, has a complex life cycle that allows adaptation to different environments. Transitions between vegetative swim cell and swarmer cell types depend on whether the organism is growing in liquid surroundings or on a solid substrate. Moreover, starvation can induce vegetative cells to differentiate into quiescent cysts. This paper describes the results of our investigation into the role of a putative DNA-binding response regulator that is homologous to CtrA, the cell cycle regulator from Caulobacter crescentus. Deletion of ctrA from the R. centenum genome resulted in a viable strain with impaired swarming motility coupled with an increased tendency to form cysts. Conversely, overexpression of wild type CtrA or a phosphomimetic allele, CtrAD51E, suppressed cyst cell formation, whereas overexpression of a CtrAD51A allele failed to suppress encystment but did prevent swarming motility. Thus, we propose that CtrA participates within a two-component signal transduction pathway that promotes swarming motility while contributing to the suppression of cyst cell formation.     

A quantitative study of the division cycle of Caulobacter crescentus stalked cells

Progression of a cell through the division cycle is tightly controlled at different steps to ensure the integrity of genome replication and partitioning to daughter cells. From published experimental evidence, we propose a molecular mechanism for control of the cell division cycle in Caulobacter crescentus. The mechanism, which is based on the synthesis and degradation of three “master regulator” proteins (CtrA, GcrA, and DnaA), is converted into a quantitative model, in order to study the temporal dynamics of these and other cell cycle proteins. The model accounts for important details of the physiology, biochemistry, and genetics of cell cycle control in stalked C. crescentus cell. It reproduces protein time courses in wild-type cells, mimics correctly the phenotypes of many mutant strains, and predicts the phenotypes of currently uncharacterized mutants. Since many of the proteins involved in regulating the cell cycle of C. crescentus are conserved among many genera of α-proteobacteria, the proposed mechanism may be applicable to other species of importance in agriculture and medicine.     

Sensory domain of the cell cycle kinase CckA regulates the differential DNA binding of the master regulator CtrA in Caulobacter crescentus

Sophisticated signaling mechanisms allow bacterial cells to cope with environmental and intracellular challenges. Activation of specific pathways ameliorates these challenges and thereby warrants integrity. Here, we demonstrate the pliability of the CckA-CtrA two-component signaling system in the freshwater bacterium Caulobacter crescentus. Our forward genetic screen to analyze suppressor mutations that can negate the chromosome segregation block induced by the topoisomerase IV inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA. Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in increased levels of phosphorylated CtrA (CtrA~P), the master cell cycle regulator. Surprisingly, this increase in CtrA~P levels did not translate into a genome-wide increase in the DNA occupancy of CtrA, but specifically enriched its affinity for the chromosomal origin of replication, C_ori, and for a very small sub-set of CtrA regulated promoters. We show that through this enhanced binding of CtrA to the C_ori, cells are able to overcome the toxic defects rendered by stable NstA through a possible slow down in the chromosome replication cycle. Taken together, our work opens up an unexplored and intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis because of the highly conserved nature of the CckA-CtrA pathway in alphaproteobacteria.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens

The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJ homologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.