Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Caulobacter”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed2002

Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry.

Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.

2-Propanol↗
PubMed1995

An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints.

Amino Acid Sequence↗
PubMed2003

Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server.

Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus. The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C. crescentus cell cycle. Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of [(35)S]methionine incorporation. Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit. Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface (http://proteom.biomed.cas.cz), which allows the database records to be searched and displays the recovered information. A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy. The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines. Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented. In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C. crescentus proteome.

Bacterial Proteins↗
PubMed1994

A Caulobacter DNA methyltransferase that functions only in the predivisional cell.

Caulobacter crescentus was found to have a DNA methyltransferase, CcrM, that methylates the adenine base of the HinfI recognition sequence, GANTC. The ccrM gene was cloned, and DNA sequence analysis revealed that the predicted amino acid sequence has 49% identity with the Haemophilus influenzae methyltransferase HinfM. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. The time of methyltransferase expression coincides with the time of methylation of these three chromosomal sites and of plasmid DNA in the predivisional cell. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology.

Amino Acid Sequence↗
PubMed1995

Use of flow cytometry to identify a Caulobacter 4.5 S RNA temperature-sensitive mutant defective in the cell cycle.

Flow cytometry was used to screen a collection of temperature-sensitive mutants for those blocked at discrete points in the cell cycle with respect to the replicative status of the chromosome. At the non-permissive temperature, one such mutant, LS439, could not initiate new rounds of DNA replication and arrested primarily as cells with two completed chromosomes Extended incubation at the restrictive temperature resulted in filament formation. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. A 0.2 kb fragment of DNA located immediately upstream of the Caulobacter homolog of the Escherichia coli dnaX gene was able to completely rescue the temperature-sensitive phenotype of LS439. The 0.2 kb fragment contained a homolog of the bacterial gene encoding 4.5 S RNA. The original point mutation is predicted to disrupt the stem structure in the 4.5 S RNA thus providing a rationale for the genetic basis of the LS439 phenotype.

Amino Acid Sequence↗
PubMed1996

A novel promoter motif for Caulobacter cell cycle-controlled DNA replication genes.

Caulobacter crescentus contains a single chromosome that is replicated once during a defined period in the cell cycle. The onset of replication coincides with the stimulation of transcription of several genes involved in the replication process. Analysis of the C. crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, identified the dnaX transcription start site and showed that activity from the dnaX promoter is stimulated fourfold at the onset of DNA replication. We have identified a conserved sequence motif that is present in the promoter of dnaX and several other genes involved in the replication of DNA, all of which show an induction of transcription at the onset of chromosome replication. Independent mutations in the conserved sequence that lies between the -10 and -35 regions increased transcription, suggesting that a repressor may bind at this site. We propose that the coincident transcriptional activation of several dna genes at the swarmer to stalked cell transition occurs in response to cell cycle regulatory factors, in a manner analogous to the transient transcriptional regulation of flagellar and DNA methylation genes later in the cell cycle.

Amino Acid Sequence↗
PubMed2001

Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus.

Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host. The vectors are based on a narrow-host-range colE1-replicon-based plasmid commonly used in Escherichia coli, to which was added the replication origin of the IncQ plasmid RSF1010. C. crescentus strains were modified to enable plasmid replication by introduction of the RSF1010 repBAC genes at the recA locus. The small (4.0-4.5 kb) plasmids were in high copy numbers in both C. crescentus and E. coli and amenable to rapid methods for plasmid isolation and DNA sequencing. The method for introducing repBAC is suitable for other C. crescentus strains or any bacterium with an adequately homologous recA gene. Application of the vector for protein expression, based on the type I secretion system of the S-layer protein, when compared to constructs in broad-host-range plasmids, resulted in reduced time and steps required from clone construction to recombinant protein recovery and increased protein yield.

Bacterial Proteins↗
PubMed1995

Regulation of the Caulobacter crescentus rpoN gene and function of the purified sigma 54 in flagellar gene transcription.

The sequential transcription of flagellar (fla) genes in the Caulobacter crescentus cell cycle is controlled by the organization of these genes in a regulatory hierarchy of four levels (I-IV). Level III and level IV genes at the bottom of the hierarchy are dependent on level II genes and are transcribed late in the cell cycle from sigma 54-dependent promoters. To study the regulation of genes at levels III and IV, we have isolated and sequenced the rpoN gene in order to analyze its expression, purified the rpoN gene product, and examined the role of the RpoN protein in initiation of transcription from sigma 54-dependent promoters. We report here epistasis experiments that show rpoN is required for transcription of level III genes, but that the expression of the rpoN gene itself is not dependent on any of the fla genes examined; these results place rpoN at level II near the top of the hierarchy. Consistent with this conclusion were nuclease S1 assays that mapped the rpoN transcription start site and identified a sequence centered at -24, GTTA/TACCA/TT, which is similar to the core consensus sequence of the level IIB fliF, fliL, and fliQ promoters. We purified the full-length rpoN gene product to near homogeneity and demonstrated that the RpoN protein is required for transcription from the well-characterized sigma 54-dependent glnAp2 promoter of Escherichia coli and specifically recognizes the level III flbG gene promoter of C. crescentus. These last results confirm that rpoN encodes the C. crescentus sigma 54 factor and opens the way for the biochemical analysis of transcriptional regulation of level III and IV fla genes.

Amino Acid Sequence↗
PubMed1995

The control of asymmetric gene expression during Caulobacter cell differentiation.

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Bacterial Proteins↗
PubMed2001

Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar hook.

This paper reports on the organization, expression, and function of the divergently transcribed flbG and flaN operons in the hook gene cluster of Caulobacter crescentus. The transcription initiation site of flbG was determined previously, and in this work the transcription map was completed by locating the 3' end of the mRNA using nuclease S1 protection assays. A previous genetic study had suggested that the flbG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5'-flbG, flbH, and flgE. FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaffolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein. The divergently transcribed flaN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins. Based on the amino acid sequence similarities and phenotypes of mutants, flbG, flbH, and flaN have been renamed fliK, flgD, and flgK, FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identified as part of a 190-kDa FliK-LacZ fusion protein. We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly.

Amino Acid Sequence↗
PubMed2006

Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia.

Ginsenoside Rb1 is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong beta-glucosidase-producing activity, converted ginsenoside Rb(1) to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb1 had been consecutively converted to ginsenoside Rd (1), F2(2) and compound K (3) via the hydrolyses of 20-C beta-(1-->6)-glucoside, 3-C beta-(1-->2)-glucoside, and 3-C beta-glucose of ginsenoside Rb1.

Biotransformation↗
PubMed1992

Organization and ordered expression of Caulobacter genes encoding flagellar basal body rod and ring proteins.

The biogenesis of the polar flagellum in Caulobacter crescentus is limited to a specific time in the cell cycle and to a specific site on the cell. The basal body is the first part of the flagellum to be assembled. In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation. The genes in this cluster form an operon whose expression is controlled temporally. The first two genes encode homologs of FlgF and FlgG, which are the proximal and distal rod proteins, respectively. The sequences of the N and C termini of the Salmonella typhimurium flagellar axial proteins, rod, hook and HAP-1, known to be highly conserved, share a high degree of sequence identity with the FlgF and FlgG rod proteins of the distantly related, C. crescentus. Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences. The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter. Transcription from the flgF promoter initiates prior to initiation at the internal flgH promoter. The internal promoter and its activator site reside within the C-terminal coding sequence of the upstream flaD gene. This type of gene overlap is also observed in bacterial genes involved in cell division. Flagellum biogenesis, like cell division, is a morphogenic event that requires the orderly assembly of component proteins and the overlapping gene organization may affect this "ordering" of assembly. The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription. The use of sigma 54 promoters, known to require cognate binding proteins, could allow the fine-tuning that provides the temporal ordering of flagellar gene transcription. In this context, we have found that the flgF operon and the distal flgI gene encoding the P-ring, share a sigma 54 activator sequence (class IIA) that differs from the flgH L-ring gene sigma 54 activator site (class IIB) and the hook cluster (class IIC) sigma 54 activator site. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum.

Amino Acid Sequence↗
PubMed1992

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus.

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

Bacterial Proteins↗
PubMedDate not supplied

The role of FlbD in regulation of flagellar gene transcription in Caulobacter crescentus.

The flagellar (fla) genes in Caulobacter crescentus are organized into a regulatory hierarchy of four levels (I-IV) in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. We have been particularly interested in understanding the function and regulation of one such transcription factor, FlbD. FlbD belongs to the NtrC family of bacterial response regulators that catalyse the initiation of transcription by sigma 54 RNA polymerase (E sigma 54) and its function is required for transcription of the class III and IV fla genes. Here we show that purified FlbD binds to ftr elements that are required for transcription from the sigma 54-dependent class III flbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4). Dimethylsulphate footprinting assays demonstrated that FlbD makes base-specific contacts at highly conserved guanine nucleotides in each half site of the ftr sequences. In a reconstituted in vitro transcription system using E. coli E sigma 54, we found that FlbD was clearly capable of driving transcriptional initiation from the flbG promoter and that this activity relied on the ftr1 binding site. Several observations suggest that phosphorylation plays a role in the regulation of FlbD activity. First, we found that a mutant form of FlbD (FlbDS140F) corresponding to the substitution found in a constitutively active NtrC protein (NtrCS160F), displayed a greater potential for activating E sigma 54-dependent transcription that the wildtype protein. We also observed that high energy-phosphate-containing molecules stimulate transcription activation by the wild type FlbD. Together, these results suggest that FlbD is responsible for mediating fla gene transcription initiation by E sigma 54 and that covalent modification is likely to play a role in governing FlbD activity during the cell cycle.

Bacterial Proteins↗
PubMed1996

Signal transduction in the cell cycle regulation of Caulobacter differentiation.

Caulobacter crescentus differentiates to form a new cell type during asymmetric cell division. Recent results indicate that signal transduction pathways mediated by protein kinases and essential response regulators play a central role in the regulation of development and cell division in response to cell cycle checkpoints.

Caulobacter crescentus↗
PubMed2005

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase.

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.

Amino Acid Sequence↗
PubMed2004

Cytokinesis monitoring during development; rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter.

For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development.

Bacterial Proteins↗
PubMed2006

MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter.

Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. After replication of the polarly located origin region, one copy moves rapidly to the opposite end of the cell in an MreB-dependent manner. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated.

Adenosine Triphosphatases↗