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PubMed1998

A membrane-associated protein, FliX, is required for an early step in Caulobacter flagellar assembly.

The ordered assembly of the Caulobacter crescentus flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarchy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene, fliX. A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. The fliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer. Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.

Amino Acid Sequence↗
PubMed2005

Whole-genome transcriptional analysis of heavy metal stresses in Caulobacter crescentus.

The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.

Adaptation, Physiological↗
PubMed2006

The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter.

Bacterial chromosome partitioning and cell division are tightly connected cellular processes. We show here that the Caulobacter crescentus FtsK protein localizes to the division plane, where it mediates multiple functions involved in chromosome segregation and cytokinesis. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. These results suggest that the interdependence between chromosome partitioning and cell division in Caulobacter is mediated, in part, by the FtsK protein.

Bacterial Proteins↗
PubMedDate not supplied

[A psychrotolerant Caulobacter sp. from Russian polar tundra soil].

Strain Z-0024, a psychrotolerant aerobic heterotrophic representative of the prosthecate bacteria of the genus Caulobacter, was isolated from a methanotrophic enrichment obtained from Russian polar tundra soil. The cells of the new isolate are vibrios (0.5-0.6 x 1.3-1.8 microm) with a polar stalk. The organism grows in a temperature range from 5 to 36 degrees C, with an optimum at 20 degrees C. The pH range for growth is from 4.5 to 7.0 with an optimum at pH 6.0. Strain Z-0024 utilizes a wide range of organic compounds: sugars, amino acids, volatile fatty acids, and primary alcohols. It tolerates a NaCl concentration in the medium of up to 15 g/l. The G + C content of DNA is 66.6 mol %. The 16S rRNA gene sequence analysis revealed that strain Z-0024 belongs to the cluster of Caulobacter species, showing a 98.8-99.2% sequence similarity to them. DNA-DNA hybridization revealed a low level of homology (24%) between strain Z-0024 and C. vibrioides ATCC 15252. The new isolate is described as Caulobacter sp. Z-0024.

Base Composition↗
PubMed1983

Caulobacter flagellin mRNA segregates asymmetrically at cell division.

Molecular processes which promote the spatial localization of subcellular components are fundamental to cell development and differentiation. At various stages in development unequal segregation of molecular information must occur to result in the differentiated characteristics which distinguish cell progeny. Biological attributes of the dimorphic bacterium, Caulobacter crescentus, provide an experimental system permitting examination of the generation of asymmetry at the molecular level. When a Caulobacter cell divides, two different daughter cells are produced--a motile swarmer cell with a polar flagellum and a non-motile cell with a static appendage referred to as a stalk. The two cell types are distinct with respect to surface morphology, developmental potential, protein composition and biosynthetic capabilities. One of the more conspicuous manifestations of asymmetric expression of macromolecules in this system, the flagellum, has been studied extensively. We have cloned the flagellin genes of Caulobacter and report here the use of these sequences as probes to demonstrate that (1) the level of flagellin mRNA is regulated during the cell cycle in a pattern coincident with flagellum polypeptide synthesis and (2) flagellin mRNA synthesized before cell division is segregated with progeny swarmer cells. This provides molecular evidence of specific partitioning of an mRNA at the time of cell division.

Bacteria↗
PubMed1979

Caulobacter crescentus nucleoid: analysis of sedimentation behavior and protein composition during the cell cycle.

The envelope-associated nucleoid (EAN) of Caulobacter crescentus has been isolated during major developmental stages in the cell cycle. The sedimentation coefficient of the Caulobacter EAN changes as a function of development and is closely correlated with the periodicity of DNA synthesis in this bacterium. The contribution of proteins to the structure of the Caulobacter nucleoid has been analyzed by using polyacrylamide gel electrophoresis of proteins labeled both for short intervals and continuously throughout the cell cycle. The EAN proteins are derived primarily from membranes and are sequentially associated with the EAN during the cell cycle. Several distinct proteins are isolated with the EAN at specific stages of differentiation. A 26,000 Mr protein, which appears uniquely associated with the swarmer cell EANs, was identified and shown to segregate preferentially with the swarmer cell EAN at cell division.

Bacteria↗
PubMed1984

Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.

Bacteria↗
PubMed1976

Structure of Caulobacter deoxyribonucleic acid.

The deoxyribonucleic acid of the dimorphic bacterium Caulobacter crescentus contains a component that renatures with rapid, unimolecular kinetics. This component was present in both swarmer and stalked cells and exhibited the sensitivity to endonuclease S1 expected for hairpin loops. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. No extrachromosomal elements were found in spite of systematic attempts to detect their presence. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria.

Bacteria↗
PubMed1977

Chromosome replication in Caulobacter crescentus growing in a nutrient broth.

The pattern of chromosome replication in the Caulobacter crescentus cell cycle was studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G1), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.

Bacteria↗
PubMed1981

Periodic surface array in Caulobacter crescentus: fine structure and chemical analysis.

A periodic array structure on the cell surface of Caulobacter crescentus CB15 was revealed by electron microscopy of the cell envelope, using negative staining, thin-sectioning, and freeze-etching. This structural layer has been isolated from liquid cultures, in which large pieces of the two-dimensional array are shed by cells grown to high density. Often areas of intact array corresponding to the entire cell surface could be found. The hexagonally arranged structure was highly ordered and had an unusual degree of complexity, as determined by optical diffraction and computer processing of micrographs of negatively stained, isolated surface array. Filtered, reconstructed images were obtained from both normal and low-electron-dose micrographs demonstrating resolutions of 2.9 and 25 nm, respectively. Comparison by optical diffraction and image filtering of micrographs recorded by using either normal or minimal beam exposure techniques suggested that the lower-resolution features of the image are very stable to electron exposure. Gel electrophoresis indicated that isolated array preparations contain a number of polypeptides. It appears likely that more than one of these proteins are structural components of the array, in contrast to a single protein found in many bacterial surface arrays. The Caulobacter surface array is also unusual in that the repeated units are widely spaced with no apparent direct connection. Computer spatial averaging provided information about the shape and complexity of the connecting elements, and this was compared with some additional electron microscopic evidence of linking structures. Thin-sectioning studies confirmed the image features seen by other techniques, but the addition of tannic acid in the fixation procedure was required to visualize the structure. A comparison of these results with out current knowledge of the Caulobacter cell envelope suggests interesting questions about the biogenesis of this membrane structure and its involvement in the cell development process of this organism.

Bacteria↗
PubMed1988

Transcriptional analysis of the major surface array gene of Caulobacter crescentus.

The major component of the paracrystalline surface array of Caulobacter crescentus CB15 and one of the most abundant cellular proteins is a protein designated 130K. We have determined the DNA sequence of the 5' portion of the 130K gene, including the N-terminal one-third of the protein coding region, and analyzed the transcription of the gene. The site of transcription initiation was determined by S1 mapping of Caulobacter RNA. Although the DNA sequence upstream from the transcription start site showed significant homology to the consensus promoter sequences of Escherichia coli, S1 analysis of RNA from E. coli carrying the 130K gene on a plasmid indicated that the 130K promoter was not transcribed by E. coli RNA polymerase in vivo. Quantitative S1 analysis of RNA isolated from synchronously growing Caulobacter cells suggested that this promoter was not under developmental regulation; the amount of 130K transcript varied no more than 1.5-fold during the cell cycle. The length of the 130K mRNA was determined to be 3.3 kilobases by Northern (RNA blot) analysis, indicating that the 130K mRNA is not part of a polycistron. The amino acid sequence predicted from the DNA sequence agreed well with the N-terminal amino acid sequence determined by sequencing of the 130K protein. The 130K protein appears to be synthesized without an N-terminal leader sequence, but the N-terminal 20 amino acids are relatively hydrophobic and may function like a signal sequence during transmembrane translocation.

Amino Acid Sequence↗
PubMed2004

Comparison of S-layer secretion genes in freshwater caulobacters.

Our freshwater caulobacter collection contains about 40 strains that are morphologically similar to Caulobacter crescentus. All elaborate a crystalline protein surface (S) layer made up of protein monomers 100-193 kDa in size. We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100-108 kDa), medium (122-151 kDa), and large (181-193 kDa). All contained genes predicted to encode ATP-binding cassette transporters and membrane fusion proteins highly similar to those of C. crescentus, indicating that the S-layer proteins were all secreted by a type I system. The S-layer proteins' C-termini showed unexpectedly low sequence similarity but contained conserved residues and predicted secondary structure features typical of type I secretion signals. Cross-expression studies showed that the 6 strains recognized secretion signals from C. crescentus and Pseudomonas aeruginosa and similarly that C. crescentus was able to secrete the S-layer protein C-terminus of 1 strain examined. Inactivation of the ATP-binding cassette transporter abolished S-layer protein secretion, indicating that the type I transporter is necessary for S-layer protein secretion. Finally, while all of the S-layer proteins of this subset of strains were secreted by type I mechanisms, there were significant differences in genome positions of the transporter genes that correlated with S-layer protein size.

ATP-Binding Cassette Transporters↗
PubMed2006

Cell cycle control by oscillating regulatory proteins in Caulobacter crescentus.

Significant strides have been made in recent years towards understanding the molecular basis of cell cycle progression in the model bacterium Caulobacter crescentus. At the heart of cell cycle regulation is a multicomponent transcriptional feedback loop, governing the production of successive regulatory waves or pulses of at least three master regulatory proteins. These oscillating master regulators direct the execution of phase-specific events and, importantly, through intrinsic genetic switches not only determine the length of a given phase, but also provide the driving force that catapults the cell into the next stage of the cell cycle. The genetic switches act as fail safe mechanisms that prevent the cell cycle from relapsing and thus govern the ordered production and the periodicity of these regulatory waves. Here, we detail how the master regulators CtrA, GcrA and DnaA coordinate cell cycle progression and polar development in Caulobacter.

Bacterial Proteins↗
PubMed1994

Caulobacter flagellar function, but not assembly, requires FliL, a non-polarly localized membrane protein present in all cell types.

Caulobacter crescentus has a single polar flagellum, which is assembled in the predivisional cell. Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function. These genes are organized in several classes which form a transcriptional regulatory hierarchy. A member of the Class II genes, the fliLM operon, encodes homologs of the Escherichia coli flagellar switch protein, FliM, and a protein with a hitherto unknown function, FliL. We report here that flagellar rotation requires the FliL protein. In-frame deletions in the chromosomal copy of the fliL gene result in cells that form a flagellum but are non-motile. The FliL protein was found to be associated with the inner membrane and to be present in all cell types. This is the first report of a Caulobacter crescentus protein that is essential for motility but is not spatially restricted to the region of the flagellar basal body. Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins.

Amino Acid Sequence↗
PubMed2000

Signal transduction mechanisms in Caulobacter crescentus development and cell cycle control.

The life cycle of the aquatic bacterium Caulobacter crescentus includes an asymmetric cell division and an obligate cell differentiation. Each cell division gives rise to a motile but replication inert swarmer cell and a sessile, replication competent stalked cell. While the stalked progeny immediately reinitiates DNA replication and cell division, the swarmer cell remains motile and chemotactically active for a constant period of the cell cycle before it differentiates into a stalked cell. During this process, the cell looses motility by ejecting the flagellum, synthesizes a stalk and eventually initiates chromosome replication and cell division. The link of morphogenic transitions to the replicative cycle of Caulobacter implies that the developmental programs which determine asymmetry and cell differentiation must be tightly connected with cell cycle control. This has been confirmed by the recent identification of signal transduction mechanisms, which are involved in temporal and spatial control of both development and cell cycle. Interestingly, the cell has recruited two-component signal transduction systems for this internal control, a family of regulatory proteins which usually are involved in the information transfer between the environment and the inside of a cell. The response regulator protein CtrA controls several key cell cycle events like the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. The activity of this master regulator is subject to complex temporal and spatial control during the C. crescentus cell cycle, including regulated transcription, phosphorylation and degradation. Three membrane bound sensor kinases have been proposed to control the phosphorylation status of CtrA. Two of these, CckA and DivJ, exhibit specific subcellular localization and, in the case of CckA, dynamic rearrangement in the course of the cell cycle. These findings support the idea that the developmental program of C. crescentus is controlled at least in part by localized cues that act as checkpoints for the control of morphological changes and cell cycle progression.

Caulobacter crescentus↗
PubMed2001

Expression and testing of Pseudomonas aeruginosa vaccine candidate proteins prepared with the Caulobacter crescentus S-layer protein expression system.

A novel bacterial protein secretion system was used to produce vaccine candidates against Pseudomonas aeruginosa. The surface protein (RsaA) of Caulobacter crescentus was adapted to produce recombinant vaccine proteins based on the pilus tip epitope ('adhesintope') of P. aeruginosa. Two versions of the adhesintope, with (PCK) or without (PE) the cysteine residues that flank the epitope were investigated, fused to the C-terminus or inserted into full-length RsaA. When expressed in caulobacter the fusion proteins were secreted as aggregates. Full length RsaA-containing adhesintopes assembled on the cell surface as an S-layer; these were recovered by low pH extraction. Vaccine candidates were evaluated in a mouse challenge model. PCK-containing proteins produced at least 1000-fold higher antibody titers against the adhesintope, compared to the PE version, exceeding titers achievable with any other vaccine preparation method. Immunoglobulin isotyping indicated a balanced IgG1/IgG2 response, though when challenged with P. aeruginosa, the PE- and PCK-containing proteins did not afford mice a significant level of protection. Overall, we describe a new system for vaccine production that shows promise as a fast, economical way to construct, evaluate and produce vaccine proteins.

Animals↗
PubMed1998

Dominant C-terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA.

The cell division protein FtsZ is composed of three regions based on sequence similarity: a highly conserved N-terminal region of approximately 320 amino acids; a variable spacer region; and a conserved C-terminal region of eight amino acids. We show that FtsZ mutants missing different C-terminal fragments have dominant lethal effects because they block cell division in Caulobacter crescentus by two different mechanisms. Removal of the C-terminal conserved region, the linker, and 40 amino acids from the end of the N-terminal conserved region (FtsZdeltaC281) prevents the localization or the polymerization of FtsZ. Because two-hybrid analysis indicates that FtsZdeltaC281 does not interact with FtsZ, we hypothesize that FtsZdeltaC281 blocks cell division by competing with a factor required for FtsZ localization or that it titrates a factor required for the stability of the FtsZ ring. The removal of 24 amino acids from the C-terminus of FtsZ (FtsZdeltaC485) causes a punctate pattern of FtsZ localization and affects its interaction with FtsA. This suggests that the conserved C-terminal region of FtsZ is required for proper polymerization of FtsZ in Caulobacter and for its interaction with FtsA.

Amino Acid Sequence↗
PubMed2001

A set of ftsZ mutants blocked at different stages of cell division in Caulobacter.

FtsZ is required throughout the cell division process in eubacteria and in archaea. We report the isolation of novel mutants of the FtsZ gene in Caulobacter crescentus. Clusters of charged amino acids were changed to alanine to minimize mutations that affect protein folding. Molecular modelling indicated that all the clustered-charged-to-alanine mutations had altered amino acids at the surface of the protein. Of 13 such mutants, four were recessive-lethal, three were dominant-lethal, and six had no discernible phenotype. An FtsZ depletion strain of Caulobacter was constructed to analyse the phenotype of the recessive-lethal mutations and used to show that they blocked cell division at distinct stages. One mutation blocked the initiation of cell division, two mutations blocked cell division randomly, and one mutation blocked both early and late stages of cell division. The effect of the recessive mutations on the subcellular localization of FtsZ was determined. Models to explain the various mutant phenotypes are discussed. This is the first set of recessive alleles of ftsZ blocked at different stages of cell division.

Amino Acid Sequence↗