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At least 271 records · Page 15Linked to original sources
PubMed1997

Transcriptional and mutational analyses of the rpoN operon in Caulobacter crescentus.

The alternative sigma factor sigma54 is required for the biogenesis of both the flagellum and the stalk in Caulobacter crescentus. The DNA sequence downstream of the sigma54 gene (rpoN) has been determined, revealing three open reading frames (ORFs) encoding peptides of 203, 208, and 159 amino acids. ORF208 and ORF159 are homologous to ORFs found downstream of rpoN in other microorganisms. The organization of this region in C. crescentus is similar to that in other bacteria, with the exception of an additional ORF, ORF203, immediately downstream from rpoN. There is a single temporally regulated promoter that drives the expression of both rpoN and ORF203. Promoter probe analysis indicates the presence of another promoter downstream from ORF203 which exhibits a temporal control that is different from that of the rpoN promoter. Mutational analysis was used to address the function of the proteins encoded by these three downstream ORFs. The mutations have no effect on the transcription of previously known sigma54-dependent flagellar promoters except for a slight effect of an ORF159 mutation on transcription of fljK.

Amino Acid Sequence↗
PubMed1997

Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system.

Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly.

Amino Acid Sequence↗
PubMed1997

A gene coding for a putative sigma 54 activator is developmentally regulated in Caulobacter crescentus.

In Caulobacter crescentus, the alternative sigma factor sigma54 plays an important role in the expression of late flagellar genes. Sigma54-dependent genes are temporally and spatially controlled, being expressed only in the swarmer pole of the predivisional cell. The only sigma54 activator described so far is the FlbD protein, which is involved in activation of the class III and IV flagellar genes and repression of the fliF promoter. To identify new roles for sigma54 in the metabolism and differentiation of C. crescentus, we cloned and characterized a gene encoding a putative sigma54 activator, named tacA. The deduced amino acid sequence from tacA has high similarity to the proteins from the NtrC family of transcriptional activators, including the aspartate residues that are phosphorylated by histidine kinases in other activators. The promoter region of the tacA gene contains a conserved sequence element present in the promoters of class II flagellar genes, and tacA shows a temporal pattern of expression similar to the patterns of these genes. We constructed an insertional mutant that is disrupted in tacA (strain SP2016), and an analysis of this strain showed that it has all polar structures, such as pili, stalk, and flagellum, and displays a motile phenotype, indicating that tacA is not involved in the flagellar biogenesis pathway. However, this strain has a high percentage of filamentous cells and shows a clear-plaque phenotype when infected with phage phiCb5. These results suggest that the TacA protein could mediate the effect of sigma54 on a different pathway in C. crescentus.

Amino Acid Sequence↗
PubMed1997

Roles of the histidine protein kinase pleC in Caulobacter crescentus motility and chemotaxis.

The Caulobacter crescentus histidine kinase genes pleC and divJ have been implicated in the regulation of polar morphogenesis and cell division, respectively. Mutations in pleC also potentiate the cell division phenotype of divJ mutations. To investigate the involvement of the PleC kinase in motility and cell cycle regulation, we carried out a pseudoreversion analysis of the divJ332 allele, which confers a temperature-sensitive motility (Mot-) phenotype. All cold-sensitive pseudorevertants with a Mot+ phenotype at 37 degrees C and a cold-sensitive swarm phenotype in soft agar at 24 degrees C contained extragenic suppressors that were null mutations mapping to pleC. Instead of a cell division defect at the nonpermissive temperature, however, revertants displayed a cold-sensitive defect in chemotaxis (Che-). In addition, the mutant cells were also supermotile, a phenotype previously associated only with mutations in the response regulator gene pleD that block the loss of motility. We also found that the Mot- defect of pleC mutants is suppressed by a pleD301/pleD+ merodiploid and results in a similar, supermotile, cold-sensitive Che- phenotype. These results implicate signal transduction pathways mediated by PleC-DivK and DivJ-PleD in the regulation of chemotaxis as well as motility. We discuss these findings and the observation that although the PleC kinase does not play an indispensable role in cell division, a temperature-sensitive allele of pleC (pleC319) has severely reduced viability under stringent growth conditions.

Bacterial Proteins↗
PubMed1997

The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus.

The Caulobacter crescentus DNA methyltransferase CcrM (M.CcrMI) methylates the adenine residue in the sequence GANTC. The CcrM DNA methyltransferase is essential for viability, but it does not appear to be part of a DNA restriction-modification system. CcrM homologs are widespread in the alpha subdivision of gram-negative bacteria. We have amplified and sequenced a 258-bp region of the cerM gene from several of these bacteria, including Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens, and Rhodobacter capsulatus. Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family. Isolation of the full-length ccrM genes from the aquatic bacterium C. crescentus, the soil bacterium R. meliloti, and the intracellular pathogen B. abortus showed that this sequence conservation extends over the entire protein. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrM-mediated methylation has important cellular functions. In both organisms, CcrM is essential for viability. Overexpression of CcrM in either bacterium results in defects in cell division and cell morphology and in the initiation of DNA replication. Finally, the C. crescentus and R. meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria.

Amino Acid Sequence↗
PubMed1997

The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a sigma32-dependent promoter.

Sigma factor sigma32, encoded by rpoH, is required for the recognition of heat shock genes during normal growth conditions and in response to heat shock and other stresses. Unlike the well-studied Escherichia coli rpoH gene, which is transcribed from four promoters recognized by either a sigma70 (sigmaD)- or sigma24 (sigmaE)-containing RNA polymerase, the Caulobacter crescentus rpoH gene is transcribed from two promoters, P1 and P2. In this study, we have examined the structure and expression of these promoters and shown that the rpoH P2 promoter is sigma32 dependent. We present evidence here that P2 is specifically recognized and transcribed by the reconstituted C. crescentus Esigma32 RNA polymerase holoenzyme. We show that site-directed mutations within either the -10 or the -35 regions of P2 have substantial effects on the levels of transcription by the Esigma32 polymerase predicted from the sigma32 promoter consensus sequence. The mutations have similar effects in vivo as assayed with rpoH-lacZ transcription fusions. Analysis of the rpoH P1 promoter provided evidence that it is sigma70 dependent. S1 nuclease protection assays of rpoH P1- and P2-specific expression after heat shock at 42 or 50 degrees C and during synchronous cell division cycles under normal growth conditions showed that the two promoters are differentially regulated. Mutations within the rpoH P2 promoter consensus sequences abolished the response to heat shock induction in C. crescentus. We conclude from these results that, unlike rpoH genes studied previously in other bacteria, the major transcriptional response of the C. crescentus rpoH gene to heat shock depends on positive autoregulation of the sigma32-dependent promoter.

Bacterial Proteins↗
PubMed1997

Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins.

We have identified an essential Caulobacter crescentus gene (cgtA) that encodes a member of a recently identified subfamily of GTPases (the Obg family) conserved from Bacteria to Archaea to humans. This evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins possesses a fundamental, yet unknown, cellular role. In this report, we describe the isolation and sequence of the cgtA gene. The predicted CgtA protein displays striking similarity to the Obg family of small, monomeric GTP-binding proteins, both in the conserved guanine nucleotide-binding domains and throughout the N-terminal glycine-rich domain that is found in many members of the Obg family. Disruption of the cgtA gene was lethal, demonstrating that this gene is essential for cell growth. Immunoblot analysis revealed that CgtA protein levels remained constant throughout the C. crescentus cell cycle.

Alleles↗
PubMed1997

Catalase-peroxidase of Caulobacter crescentus: function and role in stationary-phase survival.

Caulobacter crescentus is an obligate aerobe which is exposed to high concentrations of photosynthetic oxygen and low levels of nutrients in its aquatic environment. Physiological studies of oxidative and starvation stresses in C. crescentus were undertaken through a study of lacZ fusion and null mutant strains constructed from the cloned 5' end of katG, encoding a catalase-peroxidase. The katG gene was shown to be solely responsible for catalase and peroxidase activity in C. crescentus. Like the katG of Escherichia coli, C. crescentus katG is induced by hydrogen peroxide and is important in sustaining the exponential growth rate. However, dramatic differences are seen in growth stage induction. E. coli KatE catalase and KatG catalase-peroxidase activities are induced 15- to 20-fold during exponential growth and then approximately halved in the stationary phase. In contrast, C. crescentus KatG activity is constant throughout exponential growth and is induced 50-fold in the stationary phase. Moreover, the survival of a C. crescentus katG null mutant is reduced by more than 3 orders of magnitude after 24 h in stationary phase and more than 6 orders of magnitude after 48 h, a phenotype not seen for E. coli katE and katG null mutants. These results indicate a major role for C. crescentus catalase-peroxidase in stationary-phase survival and raise questions about whether the peroxidatic activity as well as the protective catalatic activity of the dual-function enzyme is important in the response to starvation stress.

Bacterial Proteins↗
PubMed1997

Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus.

An inducible promoter is a useful tool for the controlled expression of a given gene. Accordingly, we identified, cloned, and sequenced a chromosomal locus, xylX, from Caulobacter crescentus which is required for growth on xylose as the sole carbon source and showed that transcription from a single site is dependent on the presence of xylose in the growth medium. P(xylX) promoter activity was determined as a function of the composition of the growth medium both in single copy and on a plasmid using different reporter genes. One hundred micromolar exogenously added xylose was required for maximal induction of P(xylX) in a strain that is unable to metabolize xylose. P(xylX) activity was induced immediately after the addition of xylose and repressed almost completely when xylose was removed from the growth medium. In addition to the strong transcriptional control, the expression of xylX is also regulated on the translational level.

Amino Acid Sequence↗
PubMed1997

Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion.

Linker insertion mutagenesis was used to modify the paracrystalline surface layer (S-layer) protein (RsaA) of the gram-negative bacterium Caulobacter crescentus. Eleven unique BamHI linker insertions in the cloned rsaA gene were identified; at the protein level, these linker insertions introduced 4 to 6 amino acids at positions ranging from the extreme N terminus to the extreme C terminus of the 1,026-amino-acid RsaA protein. All linker-peptide insertions in the RsaA N terminus caused the secreted protein to be shed into the growth medium, suggesting that the RsaA N terminus is involved in cell surface anchoring. One linker-peptide insertion in the RsaA C terminus (amino acid 784) had no effect on S-layer biogenesis, while another (amino acid 907) disrupted secretion of the protein, suggesting that RsaA possesses a secretion signal lying C terminal to amino acid 784, near or including amino acid 907. Unlike extreme N- or C-terminal linker-peptide insertions, those more centrally located in the RsaA primary sequence had no apparent effect on S-layer biogenesis. By using a newly introduced linker-encoded restriction site, a 3' fragment of the rsaA gene encoding the last 242 C-terminal amino acids of the S-layer protein was expressed in C. crescentus from heterologous Escherichia coli lacZ transcription and translation initiation information. This C-terminal portion of RsaA was secreted into the growth medium, confirming the presence of a C-terminal secretion signal. The use of the RsaA C terminus for the secretion of heterologous proteins in C. crescentus was explored by fusing 109 amino acids of an envelope glycoprotein from infectious hematopoietic necrosis virus, a pathogen of salmonid fish, to the last 242 amino acids of the RsaA C terminus. The resulting hybrid protein was successfully secreted into the growth medium and accounted for 10% of total protein in a stationary-phase culture. Based on these results and features of the RsaA primary sequence, we propose that the C. crescentus S-layer protein is secreted by a type I secretion system, relying on a stable C-terminal secretion signal in a manner analogous to E. coli alpha-hemolysin, the first example of an S-layer protein secreted by such a pathway.

Bacterial Outer Membrane Proteins↗
PubMed1997

Identification, characterization, and chromosomal organization of cell division cycle genes in Caulobacter crescentus.

We report a detailed characterization of cell division cycle (cdc) genes in the differentiating gram-negative bacterium Caulobacter crescentus. A large set of temperature-sensitive cdc mutations was isolated after treatment with the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Analysis of independently isolated mutants at the nonpermissive temperature identified a variety of well-defined terminal phenotypes, including long filamentous cells blocked at various stages of the cell division cycle and two unusual classes of mutants with defects in both cell growth and division. The latter strains are uniformly arrested as either short bagel-shaped coils or large predivisional cells. The polar morphology of these cdc mutants supports the hypothesis that normal cell cycle progression is directly responsible for developmental regulation in C. crescentus. Genetic and physical mapping of the conditional cdc mutations and the previously characterized dna and div mutations identified at least 21 genes that are required for normal cell cycle progression. Although most of these genes are widely scattered, the genetically linked divA, divB, and divE genes were shown by genetic complementation and physical mapping to be organized in one gene cluster at 3200 units on the chromosome. DNA sequence analysis and marker rescue experiments demonstrated that divE is the C. crescentus ftsA homolog and that the ftsZ gene maps immediately adjacent to ftsA. On the basis of these results, we suggest that the C. crescentus divA-divB-divE(ftsA)-ftsZ gene cluster corresponds to the 2-min fts gene cluster of Escherichia coli.

Amino Acid Sequence↗
PubMed1997

Posttranscriptional regulation of Caulobacter flagellin genes by a late flagellum assembly checkpoint.

Flagellum formation in Caulobacter crescentus requires ca. 50 flagellar genes, most of which belong to one of three classes (II, III, or IV). Epistasis experiments suggest that flagellar gene expression is coordinated with flagellum biosynthesis by two assembly checkpoints. Completion of the M/S ring-switch complex is required for the transition from class II to class III gene expression, and completion of the basal body-hook structure is required for the transition from class III to class IV gene expression. In studies focused on regulation of the class IV flagellin genes, we have examined fljK and fljL expression in a large number of flagellar mutants by using transcription and translation fusions to lacZ, nuclease S1 assays, and measurements of protein stability. The fljK-lacZ and fljL-lacZ transcription fusions were expressed in all class III flagellar mutants, although these strains do not make detectable 25- or 27-kDa flagellins. The finding that the fljK-lacZ translation fusion was not expressed in the same collection of class III mutants confirmed that fljK is regulated posttranscriptionally. The requirement of multiple class III genes for expression of the fljK-lacZ fusion suggests that completion of the basal body-hook is an assembly checkpoint for the posttranscriptional regulation of this flagellin gene. Deletion analysis within the 5' untranslated region of fljK identified a sequence between +24 and +38 required for regulation of the fljK-lacZ fusion by class III genes, which implicates an imperfect 14-bp direct repeat in the posttranscriptional regulation of fljK. Our results show that fljL is also regulated posttranscriptionally by class III and unclassified flagellar genes, apparently by a mechanism different from the one regulating fljK.

Bacterial Proteins↗
PubMed1997

Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus.

DNA replication in the dimorphic bacterium Caulobacter crescentus is tightly linked to its developmental cell cycle. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell. To identify the signals responsible for the cell cycle control of DNA replication initiation, we have characterized a region of the C. crescentus chromosome containing genes that are all involved in DNA replication or recombination, including dnaN, recF, and gyrB. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. It is transcribed from three promoters; one is heat inducible, and the other two are induced at the transition from swarmer to stalked cell, coincident with the initiation of DNA replication. The single gyrB promoter is induced at the same time point in the cell cycle. These promoters, as well as those for several other genes encoding DNA replication proteins that are induced at the same time in the cell cycle, share two sequence motifs, suggesting that they represent a family whose transcription is coordinately regulated.

Amino Acid Sequence↗
PubMed1998

The Caulobacter crescentus paracrystalline S-layer protein is secreted by an ABC transporter (type I) secretion apparatus.

Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3' of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system of Pseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. The prtB and aprA genes were introduced into C. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.

ATP-Binding Cassette Transporters↗
PubMed1998

A new class of Caulobacter crescentus flagellar genes.

Eight Caulobacter crescentus flagellar genes, flmA, flmB, flmC, flmD, flmE, flmF, flmG, and flmH, have been cloned and characterized. These eight genes are clustered in pairs (flmAB, flmCD, flmEF, and flmGH) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the flm genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the flmAB, flmEF, and flmGH operons was shown to occur primarily in predivisional cells. In contrast, the flmCD operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the flm genes. Furthermore, the flm genes did not affect the expression of class II or class III flagellar genes. However, mutations in the flm genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that the flm operons belong to a new class of flagellar genes.

Bacterial Proteins↗
PubMed1998

Genetic analysis of mecillinam-resistant mutants of Caulobacter crescentus deficient in stalk biosynthesis.

Stalk synthesis in Caulobacter crescentus is a developmentally controlled and spatially restricted event that requires the synthesis of peptidoglycan at the stalk-cell body junction. We show that the beta-lactam antibiotic mecillinam prevents stalk synthesis by inhibiting stalk elongation. In addition, mecillinam causes an increase in the diameter of the stalk at the stalk-cell body junction. We describe two mutations that confer resistance to mecillinam and that prevent stalk elongation. These mutations are probably allelic, and they map to a locus previously not associated with stalk synthesis.

Amdinocillin↗
PubMed1999

Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease.

The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the C. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX from E. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. The clpP and clpX genes are separated on the C. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5' end of the gene and a terminator structure following its 3' end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for clpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP and clpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Regulation of podJ expression during the Caulobacter crescentus cell cycle.

The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJ results in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors.

Bacterial Proteins↗