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PubMed1974

Stalk formation and its inhibition in Caulobacter crescentus.

Estimates of average rates of stalk formation over several generations of growth in Caulobacter crescentus showed that long-stalked Sk1 mutant and phosphate-starved wild-type cultures produce stalk material at about twice the rate of wild-type C. crescentus grown with adequate nutrients. Thus, the long stalks of Sk1 or phosphate-starved caulobacters are not merely a function of their longer doubling times. Inhibition of cell division of Sk1 418 with mitomycin C (MC) caused production of cellular filaments and resulted in inhibition of stalk formation. There was no appreciable decrease in total cell mass or in rates of ribonucleic acid and protein synthesis in the MC-treated cultures as compared with controls, but stalk formation, which is normally dependent on these processes, was severely retarded. Average stalk lengths in MC-treated Sk1 cultures were 30% of those found in control cultures. MC-produced cellular filaments were also subjected to deoxyribonucleic acid analysis and ultrastructural examination. The deoxyribonucleic acid content of MC-treated bacteria was about 50 to 60% that of untreated bacteria. Hydroxyurea also was found to produce some cellular filaments and shorter stalks, but with accompanying decreases in growth rate and yield.

Adenosine Triphosphate↗
PubMed1977

Caulobacter flagellins.

Flagellins from 17 Caulobacter strains were partially purified. Their molecular weights and immunological properties indicated that a two-component flagellin system is common among Caulobacters.

Bacteria↗
PubMed1978

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus.

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.

Bacteria↗
PubMed1978

Rate of major protein synthesis during the cell cycle of Caulobacter crescentus.

The rate of major protein synthesis was examined during the synchronous differentiation of Caulobacter crescentus. Total cell proteins were pulse-labeled with [35S]methionine at different times in the swarmer cell cycle and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The rates of synthesis of total cell proteins and of about one-half of the individual major proteins examined increased through G1 and S periods but remained nearly constant during G2 period. The rates of synthesis of the other half of the individual major proteins either increased continuously throughout the swarmer cell cycle or doubled during S period. One stage-specific protein was also detected in late S period. For most of the major proteins examined, the rate of synthesis in the swarmer cell was less than that in the stalked cell. It seemed that, before the onset of G2 period, the Caulobacter cell was already able to synthesize each major protein at the additive rate of the two progeny cells. Compared to the stability of cellular proteins, the functional degradation rate of mRNA coding for individual major proteins was rapid, with half-lives of 0.4 to 5.8 min. It thus seems that the rate of major protein synthesis mainly reflects the transcriptional control of gene expression.

Bacteria↗
PubMed1978

Caulobacter flagellar organelle: synthesis, compartmentation, and assembly.

In Caulobacter crescentus biogenesis of the flagellar organelle occurs during one stage of its complex life cycle. Thus in synchronous cultures it is possible to assay the sequential synthesis and assembly of the flagellum and hook in vivo with a combination of biochemical and radioimmunological techniques. The periodicity of synthesis and the subcellular compartmentation of the basal hook and filament subunits were determined by radioimmune assay procedures. Unassembled 27,000-dalton (27K) flagellin was preferentially located in isolated membrane fractions, whereas the 25K flagellin was distributed between the membrane and cytoplasm. The synthesis of hook began before that of flagellin, although appreciable overlap of the two processes occurred. Initiation of filament assembly coincided with the association of newly synthesized hook and flagellin subunits. Caulobacter flagella are unusual in that they contain two different flagellin subunits. Data are presented which suggest that the ratio of the two flagellin subunits changes along the length of the filament. Only the newly synthesized 25K flagellin subunit is detected in filaments assembled during the swarmer cell stage. By monitoring the appearance of flagellar hooks in the culture medium, the time at which flagella are released was determined.

Bacteria↗
PubMed1979

Purification and characterization of a polyhook protein from Caulobacter crescentus.

A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.

Amino Acids↗
PubMed1981

Regulation of polypeptide synthesis during Caulobacter development: two-dimensional gel analysis.

The gram-negative bacterium Caulobacter crescentus progresses through three distinct morphological transitions, including both motile and nonmotile cell types, during its cell cycle. Assessment of the extent of regulation of polypeptide synthesis during these transitions was carried out with two-dimensional gel electrophoresis of whole-cell extracts. Synchronous cells were pulse-labeled with 14C-amino acids for 10-min intervals throughout the entire 2-h cell cycle. The radioactively labeled polypeptides were analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms resulting from fluorography of the second dimension provided the detection of approximately 1,000 unique spots. The 600 predominant polypeptide spots, representing approximately 40% of the coding capacity of Caulobacter deoxyribonucleic acid, were analyzed for major changes in their synthetic rates. Quantitation by densitometric scanning of individual polypeptide spots represented on the sequential fluorograms demonstrated significant changes in the temporal synthesis of 6% of the polypeptides. Extracts from asynchronous cells were fractionated to obtain total-membrane and deoxyribonucleic acid-binding polypeptide fractions. Subsequent electrophoresis of these cellular fractions revealed approximately 100 membrane polypeptides and 25 deoxyribonucleic acid-binding polypeptides. Eight of the regulated polypeptides were identified as membrane or deoxyribonucleic acid-binding proteins. The regulated polypeptides can be grouped into three main categories based on their interval of synthesis. The three categories are in direct correlation with the three distinct cell cycle stages. This analysis has also revealed a unique transition period in the cell cycle in which a significant proportion of gene expression is regulated.

Bacteria↗
PubMed1981

Improved generalized transducing bacteriophage for Caulobacter crescentus.

Caulobacter phage phi Cr30T, a temperature-sensitive derivative of the lytic, generalized transducing phage phi Cr30, was isolated as a double temperature-sensitive recombinant in a cross between phi Cr30ts1 and phi Cr30ts2, phi Cr30T mediated generalized transduction of Caulobacter crescentus at frequencies comparable to those of phi Cr30 and eliminated the requirement for irradiation of transducing lysates to prevent killing of transductants on the plate.

Bacteria↗
PubMed1984

Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays.

A precisely ordered crystalline array is found on the surface of the bacterium Caulobacter crescentus CB15. Using an immunological assay, we identified recombinant bacteriophage clones expressing the predominant protein of this structure from a lambda 1059 library of C. crescentus CB15 DNA. A single 4.4-kilobase HindIII fragment encoded a polypeptide whose antigenic determinants, molecular weight, and peculiar solubilization properties were identical with those of the authentic predominant polypeptide (130K) of the surface array. The 130K protein was produced as a discrete product as a result of gene transcription initiated from a lambda promoter; several experiments suggested that the Caulobacter promoter for this gene is not efficiently recognized by the Escherichia coli transcription machinery. Genomic Southern analysis revealed a single copy of the 130K protein gene per genome. The 130K protein gene was hybridized with DNA of two closely related laboratory strains of C. crescentus which have lost their ability to produce a surface array. One of these strains, CB2, possesses an homologous copy of the 130K gene, whereas DNA from the other strain, CB13B1a, showed a lesser degree of hybridization to the 130K gene probe; genomic fragments which did hybridize were of different sizes in CB13 as compared with those of CB15. These findings are discussed in relation to studies of the surface array function and its role in cellular morphogenesis in this stalk-forming bacterium.

Bacterial Proteins↗
PubMed1989

Role of the flagellum in cell-cycle-dependent expression of bacteriophage receptor activity in Caulobacter crescentus.

The rate of adsorption of Caulobacter bacteriophage phi CbK to Caulobacter crescentus is dependent on the structural integrity of the flagellum. Cells lacking part or all of the flagellum because of either mutation or mechanical shear were defective in adsorption, and the extent of the defect in adsorption reflected the amount of flagellar structure missing. Maximal adsorption rates were also dependent on cellular motility and energy metabolism, since adsorption to cells with paralyzed flagella was slower than adsorption to motile cells and inhibition of cellular energy metabolism with azide also reduced adsorption rates, even for nonmotile cells. Nevertheless, the flagellum is not the receptor for phage phi CbK, since flagellumless mutants adsorbed phi CbK at detectable rates. While some portion of the fluctuation in the phi CbK receptor activity during the C. crescentus cell cycle can be ascribed to the periodicity of flagellar loss and reappearance, the phage receptor activity remaining in flagellumless mutants was periodic in the cell cycle. Therefore, the periodic expression of phage receptor activity is an intrinsic property of the C. crescentus cell cycle, although the amplitude of the oscillation may be altered by the periodic expression of flagellar motility.

Bacteria↗
PubMed1990

Identification, distribution, and sequence analysis of new insertion elements in Caulobacter crescentus.

We describe two insertion elements isolated from Caulobacter crescentus that are designated IS298 and IS511. These insertion elements were cloned from spontaneous flagellar (fla) gene mutants SC298 and SC511 derived from the wild-type strain CB15 (ATCC 19089), in which they were originally identified as insertions in the flbG operon of the hook gene cluster (N. Ohta, E. Swanson, B. Ely, and A. Newton, J. Bacteriol. 158:897-904, 1984). IS298 and IS511 were each present in C. crescentus CB2 and CB15 in at least four different positions, but neither was present in strain CB13 or in several Caulobacter species examined, including C. vibrioides, C. leidyia, and C. henricii. Nucleotide sequence analysis across the chromosome-insertion element junctions showed that IS298 is located 152 base pairs (bp) upstream from the ATG translation start of the hook protein gene flaK, where it is bounded by a 4-bp direct repeat derived from the site of insertion, and that IS511 is inserted at codon 186 of the flaK coding sequence, where it is also bounded by a 4-bp direct repeat duplicated from the site of insertion. The ilvB102 mutation in strain SC125 was also shown to result from insertion sequence IS511, but no duplication of the genomic sequence was present at the insertion element junctions. IS298 contains an imperfect terminal inverted repeat 16 bp long, and IS511 contains a 32-bp inverted repeat at the termini. IS298 and IS511 are the first insertion elements described in C. crescentus.

Bacteria↗
PubMed1990

Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2.

Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.

Bacterial Adhesion↗
PubMed1991

Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus.

Caulobacters are adherent prosthecate bacteria that are members of bacterial biofouling communities in many environments. Investigation of the cell surface carbohydrates produced by two strains of the freshwater Caulobacter crescentus, CB2A and CB15A, revealed a hitherto undetected extracellular polysaccharide (EPS) or capsule. Isolation and characterization of the EPS fractions showed that each strain produced a unique neutral EPS which could not be readily removed from the cell surface by washing. Monosaccharide analysis showed that the main CB2A EPS contained D-glucose, D-gulose, and D-fucose in a ratio of 3:1:1, whereas the CB15A EPS fraction contained D-galactose, D-glucose, D-mannose, and D-fucose in approximately equal amounts. Methylation analysis of the main CB2A EPS showed the presence of terminal glucose and gulose groups, 3-linked fucosyl, and two 3,4-linked glucosyl units, thus confirming the pentasaccharide repeating unit indicated by 1H nuclear magnetic resonance analysis. Similar studies of the CB15A EPS revealed a tetrasaccharide repeating unit consisting of terminal galactose, 4-linked fucosyl, 3-linked glucosyl, and 3,4-linked mannosyl residues. EPS was not detectable by thin-section electron microscopy techniques, including some methods designed to preserve or enhance capsules, nor was the EPS readily detected on the cell surface by scanning electron microscopy when conventional fixation techniques were used; however, a structure consistent with EPS was revealed when samples were prepared by cryofixation and freeze-substitution methods.

Bacterial Adhesion↗
PubMed1980

Pilus-dependent, double-stranded DNA bacteriophage for Caulobacter.

Caulobacter phage phi 6, previously reported to adsorb specifically to bacterial flagella, was shown here to attach to pili more frequently than to flagella. Phage phi 6 was shown to contain double-stranded DNA by circular dichroism spectroscopy and thermal denaturation accompanied by a hyperchromic shift at 260 nm. Morphologically, phage phi 6 fits group B2 (H.-W. Ackermann, in A. I. Laskin and H. A. Lechevalier, ed., Handbook of Microbiology, vol. 1, p. 638-643, 1973) with a long, noncontractile tail and an elongate head. Pilus-less mutants of the host Caulobacter vibrioides CV6 are phage phi 6 resistant, whereas flagellum-less mutants, which produce pili, are phage susceptible. Treatments of susceptible cells which remove or immobilize pili and flagella, e.g., blending or cyanide, inhibited phage phi 6 infection. Our evidence suggests that phage of phi 6 initiates infection in a manner similar to the pilus-specific phages for Pseudomonas described previously (D. E. Bradley, Virology 51:489-492, 1973; D. E. Bradley and T. L. Pitt, J. Gen. Virol. 24:1-15, 1974).

Bacteria↗
PubMed1989

Positioning of gene products during Caulobacter cell differentiation.

Caulobacter crescentus has one of the simplest known developmental programs that exhibits both temporal and spatial organization. A hallmark of the Caulobacter cell cycle is that the progeny cells that result from each cell division differ from one another with respect to structure and developmental program. The process of establishing asymmetry prior to cell division requires that a number of gene products be targeted to a pole of the predivisional cell and consequently segregated to one of the two progeny. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. Evidence suggests that the protein product of some fla and che genes is targeted to the incipient swarmer cell pole. In the case of other flagellar genes, it is the mRNA that is apparently segregated to the swarmer cell. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell.

Bacterial Proteins↗
PubMed1983

Differential template recognition by the Caulobacter crescentus and the escherichia coli RNA polymerases.

Transcription of Escherichia coli and Caulobacter crescentus phage DNAs by their respective host RNA polymerase was examined to determine their ability to recognize specific transcription signals on the heterologous template. Analysis of coliphage T7 in vitro transcripts showed that, like the E. coli enzyme, the C. crescentus RNA polymerase initiated transcription from the three major T7 early promoters and recognized the terminator at the end of the early region. On the other hand, several differences were found between the C. crescentus and E. coli RNA polymerases with respect to their interaction with Caulobacter phage phiCdl DNA. The rates of open complex formation and RNA elongation were slower when phiCdl DNA was transcribed by the E. coli RNA polymerase. In addition, transcription of phiCdl DNA by the E. coli enzyme produced a subset of transcripts not synthesized by the C. crescentus enzyme. The production of these different transcripts by the E. coli enzyme was dependent on salt concentration and, in at least one case, appeared to be the result of differential termination. Although both enzymes protected the same sites on phiCdl DNA from cleavage with HincII, the E. coli enzyme was unable to form stable complexes with some phiCdl restriction fragments that formed stable complexes with the C. crescentus RNA polymerase. These results indicate that although the C. crescentus RNA polymerase can accurately recognize transcription signals on a heterologous phage template, the E. coli enzyme exhibits altered specificity with a heterologous phage template of higher G + C content.

Bacteriophages↗
PubMed1981

Caulobacter crescentus pilin. Purification, chemical characterization, and NH2-terminal amino acid sequence of a structural protein regulated during development.

The pili of Caulobacter crescentus are assembled during swarmer cell development at the differentiating cell pole. Under specific growth conditions, it was found that C. crescentus CB15 will produce insoluble pigmented granules which entrap pili lost during cell growth along with other extracellular proteins. This provided a strategy for protein purification. Pilin was purified from these granules by gel filtration in the presence of high levels of detergent. A protein of the appropriate molecular weight for pilin was isolated by this procedure and demonstrated to be pilin by specific labeling of the intact pilus with ferritin-coupled antibodies. CB15 pilin has an apparent molecular weight of 8,000, is rich in hydrophobic amino acids (greater than 70%), and has a broad isoelectric focusing range centered about a pI of 6.6. Periodic acid-Schiff reagent staining did not demonstrate carbohydrate modification of the monomer. The pilins of both CB15 and a related strain CB13 cross-react with anti-CB15 pilin antibody. Both strains also demonstrate similar RNA bacteriophage sensitivity, although there were significant differences in the amino acid composition, molecular weight, and other physical properties of the pilins from these two related strains. The NH2-terminal amino acid sequence of about 40% of the CB15 pilin molecular has been determined and bears some resemblance to that of some common pili; however, the sequences are not homologous and there is no indications of an unusual NH2-terminal amino acid in Caulobacter pilin.

Amino Acid Sequence↗