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At least 73 records · Page 4Linked to original sources
PubMed2002

The transcription termination factor Rho is required for oxidative stress survival in Caulobacter crescentus.

A transposon Tn5 mutagenesis library was generated from Caulobacter crescentus strain NA1000, and clones with deficiency in survival in a high concentration of NaCl were selected. One of these clones, 37G10, has the Tn5 integrated within the coding region of the transcription termination factor Rho. Analysis of this mutant phenotype showed that the cells are motile and present a normal cell cycle, but have a longer generation time. This strain is sensitive to acidic pH, to the presence of different salts and to heat shock, but it responds well to UV light and alkaline pH. The most striking phenotype of the rho mutant is that it is extremely sensitive to oxidative stress, in both exponential and stationary phases. Experiments using a transcriptional fusion of the rho promoter region to the lacZ gene showed that rho gene expression varies during the cell cycle, showing very low expression levels at the swarmer cell stage and presenting maximum levels in early predivisional cells. Transcription of the rho gene is increased in the rho mutant strain, which is indicative of an autoregulatory circuit, and there is a small variation in the cell cycle pattern of expression. Several peptides have their synthesis altered in the mutant strain, as analysed by two-dimensional gel electrophoresis, most of which show a reduction in expression. These results indicate that the Rho factor is essential for an efficient response to certain stresses in Caulobacter.

Amino Acid Sequence↗
PubMed2002

DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus.

Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division. Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z-rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle. Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell. Z-rings only assembled at the proper mid-cell location if DNA replication had initiated. Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.

Bacterial Proteins↗
PubMed2004

Visualization of the movement of single histidine kinase molecules in live Caulobacter cells.

The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.

Bacterial Proteins↗
PubMed2005

Two independent spiral structures control cell shape in Caulobacter.

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.

Bacterial Proteins↗
PubMed2001

Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle.

We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.

Bacterial Proteins↗
PubMed2003

Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter.

Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 +/- 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.

Bacterial Proteins↗
PubMed1996

Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter.

Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins. It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria. We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus. Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked. After cell division, only the stalked cell contains FtsZ. FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division. Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell. Thus, the variation of FtsZ concentration parallels the pattern of DNA synthesis. Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division.

Amino Acid Sequence↗
PubMed1999

The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation.

The highly conserved SMC (Structural Maintenance of Chromosomes) proteins function in chromosome condensation, segregation, and other aspects of chromosome dynamics in both eukaryotes and prokaryotes. A null mutation in the Caulobacter crescentus smc gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. Chromosome segregation in wild-type and smc null mutant cells was examined by monitoring the intracellular localization of the replication origin and terminus by using fluorescence in situ hybridization. In wild-type cells, the origin is located at the flagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. The terminus moves from the end of the swarmer cell opposite the origin to midcell. A subpopulation of the smc null mutant cells had mislocalized origins or termini, showing that the smc null mutation gives DNA segregation defects. Nucleoid morphology was also abnormal. Thus, we propose that the Caulobacter chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. The specific cell cycle arrest in the smc null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation.

Bacterial Proteins↗
PubMed1992

A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.

The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

Amino Acid Sequence↗
PubMed2000

Identification and cell cycle control of a novel pilus system in Caulobacter crescentus.

Pilus assembly in CAULOBACTER: crescentus occurs during a short period of the cell cycle and pili are only present at the flagellar pole of the swarmer cell. Here we report a novel assay to visualize pili by light microscopy that led to the purification of CAULOBACTER: pili and the isolation of a cluster of seven genes, including the major pilin subunit gene pilA. This gene cluster encodes a novel group of pilus assembly proteins. We have shown that the pilA promoter is activated late in the cell cycle and that transcription of the pilin subunit plays an important role in the timing of pilus assembly. pilA transcription is regulated by the global two-component response regulator CtrA, which is essential for the expression of multiple cell cycle events, providing a direct link between assembly of the pilus organelle and bacterial cell cycle control.

Amino Acid Sequence↗
PubMed1992

A temporally controlled sigma-factor is required for polar morphogenesis and normal cell division in Caulobacter.

The transcription of many spatially and temporally controlled flagellar structural genes in Caulobacter requires the RNA polymerase sigma 54 subunit. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. Surprisingly, the transcription of rpoN is temporally regulated during the cell cycle; it increases 10-fold commensurate with stalk formation and just before the onset of flagellar gene expression. These results suggest that sigma 54 abundance responds to cell cycle cues and is involved in the global timing of the central events of Caulobacter development, whereas the transcriptional activators of sigma 54-dependent promoters are responsible for the refined control of the expression of individual or small groups of genes required for each specific event.

Amino Acid Sequence↗
PubMed2004

MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus.

The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex.

Actins↗
PubMed1994

The Caulobacter crescentus holdfast: identification of holdfast attachment complex genes.

Caulobacters are biofilm bacteria that attach to surfaces via a holdfast, an adhesive expressed at discrete cell surface sites. We have described a cluster of at least three genes involved in the adhesive attachment of the holdfast of Caulobacter crescentus CB2A to the cell, analyzing the sequence of two genes, hfaAB. Here we report hfaC and a fourth open reading frame, hfaD. hfaC predicts a protein of 41 kDa homologous to ATP-binding transport-related proteins, with ChvD of Agrobacterium tumefaciens as best match. HfaD is predicted to be 28 kDa with three membrane spanning regions. hfaA, hfaC, and hfaD were expressed in Escherichia coli; Western analysis with antisera against a holdfast-enriched preparation indicated HfaA was likely holdfast-associated. Cumulative findings predict HfaA and HfaB are developmentally regulated and one or both enhance hfaC transcription, HfaA is a mediator of adhesion, possibly between holdfast and a membrane-bound HfaD, and HfaC mediates export of an unidentified component required for holdfast attachment.

Amino Acid Sequence↗
PubMed1992

Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.

Animals↗
PubMed1991

The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis.

We have characterized flbF, a key locus located at the top of the flagellar gene hierarchy of Caulobacter crescentus. This gene is required for transcription from sigma 54 promoters of fla genes expressed late in the cell cycle. We have determined the nucleotide sequence of the gene, mapped the 5' end of the flbF RNA, and examined the pattern of expression in the cell cycle. Our results show that flbF is expressed earlier in the cell cycle than other fla genes, that it is expressed at a low level throughout the stalked cell cycle, and that its 5' regulatory region contains sequences that can be aligned with the sigma 28 promoter consensus reported for enteric bacteria. flbF contains an open reading frame of 700 residues with an amino-terminal half rich in hydrophobic residues that could correspond to six to eight transmembrane domains. The translated flbF sequence is very similar to LcrD (low calcium response) encoded by virulence plasmids of pathogenic Yersinia spp. (G. Plano, S. Barve, and S. Straley, J. Bacteriol. 173:7293-7303, 1991). LcrD and FlbF can be aligned over the entire length of the proteins with the greatest degree of sequence identity (45%) in the hydrophobic amino-terminal region. The high degree of sequence homology of proteins derived from widely differing organisms, including Caulobacter and Yersinia species, suggests that FlbF and LcrD may be representatives of a larger family of regulatory proteins with a common sensor mechanism for modifying responses to appropriate stimuli.

Amino Acid Sequence↗
PubMed1992

Analysis of a Caulobacter crescentus gene cluster involved in attachment of the holdfast to the cell.

Caulobacter crescentus firmly adheres to surfaces with a structure known as the holdfast, which is located at the flagellar pole of swarmer cells and at the stalk tip in stalked cells. A three-gene cluster (hfaAB and hfaC) is involved in attachment of the holdfast to the cell. Deletion and complementation analysis of the hfaAB locus revealed two genes in a single operon; both were required for holdfast attachment to the cell. Sequence analysis of the hfaAB locus showed two open reading frames with the potential to encode proteins of 15,000 and 26,000 Da, respectively. A protein migrating with an apparent size of 21 kDa in gel electrophoresis was encoded by the hfaA region when expressed in Escherichia coli under the control of the lac promoter, but no protein synthesis could be detected from the hfaB region. S1 nuclease analysis indicated that transcription of the hfaAB locus was initiated from a region containing a sequence nearly identical to the consensus for C. crescentus sigma 54-dependent promoters. In addition, a sequence with some similarity to ftr sequences (a consensus sequence associated with other Caulobacter sigma 54-dependent genes) was identified upstream of the hypothesized sigma 54 promoter. At least one of the hfaAB gene products was required for maximal transcription of hfaC. The sequence of hfaB showed some similarity to that of transcriptional activators of other bacteria. The C-terminal region of the putative gene product HfaA was found to be homologous to PapG and SmfG, which are adhesin molecules of enteropathogenic E. coli and Serratia marcescens, respectively. This information suggests that the protein encoded by the hfaA locus may have a direct role in the attachment of the holdfast to the cell, whereas hfaB may be involved in the positive regulation of hfaC.

Amino Acid Sequence↗
PubMed1995

Temporal and spatial regulation of fliP, an early flagellar gene of Caulobacter crescentus that is required for motility and normal cell division.

In Caulobacter crescentus, the genes encoding a single polar flagellum are expressed under cell cycle control. In this report, we describe the characterization of two early class II flagellar genes contained in the orfX-fliP locus. Strains containing mutations in this locus exhibit a filamentous growth phenotype and fail to express class III and IV flagellar genes. A complementing DNA fragment was sequenced and found to contain two potential open reading frames. The first, orfX, is predicted to encode a 105-amino-acid polypeptide that is similar to MopB, a protein which is required for both motility and virulence in Erwinia carotovora. The deduced amino acid sequence of the second open reading frame, fliP, is 264 amino acids in length and shows significant sequence identity with the FliP protein of Escherichia coli as well as virulence proteins of several plant and mammalian pathogens. The FliP homolog in pathogenic organisms has been implicated in the secretion of virulence factors, suggesting that the export of virulence proteins and some flagellar proteins share a common mechanism. The 5' end of orfX-fliP mRNA was determined and revealed an upstream promoter sequence that shares few conserved features with that of other early Caulobacter flagellar genes, suggesting that transcription of orfX-fliP may require a different complement of trans-acting factors. In C. crescentus, orfX-fliP is transcribed under cell cycle control, with a peak of transcriptional activity in the middle portion of the cell cycle. Later in the cell cycle, orfX-fliP expression occurs in both poles of the predivisional cell. Protein fusions to a lacZ reporter gene indicate that FliP is specifically targeted to the swarmer compartment of the predivisional cell.

Amino Acid Sequence↗
PubMed1995

Caulobacter FliQ and FliR membrane proteins, required for flagellar biogenesis and cell division, belong to a family of virulence factor export proteins.

The Caulobacter crescentus fliQ and fliR genes encode membrane proteins that have a role in an early step of flagellar biogenesis and belong to a family of proteins implicated in the export of virulence factors. These include the MopD and MopE proteins from Erwinia carotovora, the Spa9 and Spa29 proteins from Shigella flexneri, and the YscS protein from Yersinia pestis. Inclusion in this family of proteins suggests that FliQ and FliR may participate in an export pathway required for flagellum assembly. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. fliQ and fliR are class II flagellar genes residing near the top of the regulatory hierarchy that determines the order of flagellar gene transcription. The promoter sequence of the fliQR operon differs from most known bacterial promoter sequences but is similar to other Caulobacter class II flagellar gene promoter sequences. The conserved nucleotides in the promoter region are clustered in the -10, -20 to -30, and -35 regions. The importance of the conserved bases for promoter activity was demonstrated by mutational analysis. Transcription of the fliQR operon is initiated at a specific time in the cell cycle, and deletion analysis revealed that the minimal sequence required for transcriptional activation resides within 59 bp of the start site.

Amino Acid Sequence↗