Conserved response regulator CtrA and IHF binding sites in the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii chromosomal replication origins.
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CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA. CzcR expression partially compensates for developmental defects in ctrA mutant C. crescentus cells, and CzcR binds to all five CtrA binding sites in the C. crescentus replication origin. Conversely, CtrA binds to five similar sites in the putative R. prowazekii replication origin (oriRp). Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp. Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R. prowazekii. (+info)
Identification of a localization factor for the polar positioning of bacterial structural and regulatory proteins.
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Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a DeltapodJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system. (+info)
Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus.
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The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle. (+info)
Degradation of a Caulobacter soluble cytoplasmic chemoreceptor is ClpX dependent.
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In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of Caulobacter crescentus, we have characterized a soluble cytoplasmic chemoreceptor, McpB. The mcpB gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA. Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated. The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis. Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites. A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility. (+info)
TmRNA is required for correct timing of DNA replication in Caulobacter crescentus.
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SsrA, or tmRNA, is a small RNA that interacts with selected translating ribosomes to target the nascent polypeptides for degradation. Here we report that SsrA activity is required for normal timing of the G(1)-to-S transition in Caulobacter crescentus. A deletion of the ssrA gene, or of the gene encoding SmpB, a protein required for SsrA activity, results in a specific delay in the cell cycle during the G(1)-to-S transition. The ssrA deletion phenotype is not due to accumulation of stalled ribosomes, because the deletion is not complemented by a mutated version of SsrA that releases ribosomes but does not target proteins for degradation. Degradation of the CtrA response regulator normally coincides with initiation of DNA replication, but in strains lacking SsrA activity there is a 40-min delay between the degradation of CtrA and replication initiation. This uncoupling of initiation of replication from CtrA degradation indicates that there is an SsrA-dependent pathway required for correct timing of DNA replication. (+info)
Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus.
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Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the "holdfast," which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export. (+info)
Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation.
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Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5' region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein. (+info)
tmRNA in Caulobacter crescentus is cell cycle regulated by temporally controlled transcription and RNA degradation.
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SsrA, or tmRNA, is a small RNA found in all bacteria that intervenes in selected translation reactions to target the nascent polypeptide for rapid proteolysis. We have found that the abundance of SsrA RNA in Caulobacter crescentus is regulated with respect to the cell cycle. SsrA RNA abundance increases in late G(1) phase, peaks during the G(1)-S transition, and declines in early S phase, in keeping with the reported role for SsrA in the timing of DNA replication initiation. Cell cycle regulation of SsrA RNA is accomplished by a combination of temporally controlled transcription and regulated RNA degradation. Transcription from the ssrA promoter peaks late in G(1), just before the peak in SsrA RNA abundance. SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase. This degradation is surprising, since SsrA RNA is both highly structured and highly abundant. This is the first observation of a structural RNA that is cell cycle regulated. (+info)