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At least 343 records · Page 19Linked to original sources
PubMed1976

Regulation of polar surface structures in Caulobacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors.

A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages. The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile. Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25 degrees C. However, at a higher temperature (35 degrees C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35 degrees C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects. It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.

Bacteria↗
PubMed1987

Separation of temporal control and trans-acting modulation of flagellin and chemotaxis genes in Caulobacter.

The genes involved in the biogenesis of the flagellum and the chemotaxis machinery are temporally regulated during the Caulobacter crescentus cell cycle. Using plasmid complementation, we have mapped the extent of the flaY and flaE genes. These genes function in trans to regulate the expression of the flagellin genes and the chemotaxis genes. We have found that the trans regulation that modulates the amount of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. This conclusion is based on two observations: the low level of synthesis of flagellins and chemotaxis proteins in flaY and flaE mutant strains occurred at the correct time in the cell cycle, and complementation with plasmids containing intact flaY and flaE genes resulted in the synthesis of normal levels of flagellins and chemotaxis gene products with the maintenance of temporal cell cycle control.

Bacteria↗
PubMed1985

Recombination deficient mutant of Caulobacter crescentus.

A recombination-deficient (Rec-) strain of Caulobacter crescentus has been isolated from a collection of mutants sensitive to ultraviolet irradiation. The Rec- mutant fails to give recombinants following phi Cr30-mediated generalized transduction or following RP4-mediated conjugation. The recombination frequency in the Rec- strain is at least 5000-fold lower than in the wild type strains. The Rec- mutant is indistinguishable from wild type in terms of morphology, growth rate, viability, and phage sensitivities, differing only in properties known to be associated with recA-type mutations in other organisms: recombination frequency, ultraviolet sensitivity, and Weigle reactivation. The map location of the rec-526 allele has not been identified, but rec-526 can be cotransferred with the fla-169 mutation by RP4-mediated conjugation at low frequency. This apparent linkage has been used to move the rec mutation to other strains. The Rec- mutant resembles recA strains of other organisms and provides a healthy strain severely deficient in recombination for use in complementation and cloning studies involving C. crescentus.

Conjugation, Genetic↗
PubMed1984

The role of calcium in stalk development and in phosphate acquisition in Caulobacter crescentus.

Calcium was found to stimulate stalk development in Caulobacter crescentus and to relieve the inhibition of development long known to be caused by phosphate. This suggested that phosphate inhibition could be attributed to its interaction with Ca2+, thereby depriving the cells of a factor that promoted development. Calcium was also found to promote phosphate acquisition by the cells, observed as acceleration of growth at extremes of phosphate concentration, as promotion of carbon-source utilization rather than storage, and as support for phosphate-dependent resistance to arsenate inhibition of growth. Cytological studies of dividing cells revealed that stalked siblings had greater access to exogenous phosphate for use in growth or for storage as polyphosphate, and that access of non-stalked siblings to phosphate was dependent on the length of the stalk of the dividing cell. It was concluded that the physiologic role of the stalk is enhancement of phosphate acquisition. The stimulatory role of calcium in this process was attributed to its support of stalk development and to its stabilization of internal membrane/cell envelope association within the cell-stalk juncture.

Bacteria↗
PubMed1988

A phylogenetic survey of budding, and/or prosthecate, non-phototrophic eubacteria: membership of Hyphomicrobium, Hyphomonas, Pedomicrobium, Filomicrobium, Caulobacter and "dichotomicrobium" to the alpha-subdivision of purple non-sulfur bacteria.

The phylogenetic position of various budding and/or or prosthecate Gram-negative eubacteria was determined by different methods. Members of the genera Hyphomicrobium, Filomicrobium, Pedomicrobium were investigated by 16S rRNA cataloguing, a 1373 nucleotide long portion of the 16S rRNA was sequenced from Hyphomicrobium vulgare and the 5S rRNAs were analyzed from two Hyphomicrobium strains, Hyphomonas polymorpha and Caulobacter crescentus. Comparison with published sequences indicated a membership of all of these organisms to the alpha subdivision of purple bacteria. While C. crescentus and Hyphomonas polymorpha constitute separate individual lines of descent, the position of a coherent cluster embracing Hyphomicrobium, Pedomicrobium and Filomicrobium is not yet settled. 16S rRNA cataloguing indicate the presence of a distinct line equivalent to other subgroups in its phylogenetic depth. 5S rRNA analysis, on the other hand, groups Hyphomicrobium vulgare and strain IFAM 1761 with members of subgroup alpha-2 (Rhodopseudomonas palustris, Nitrobacter winogradskyi and relatives). In contrast to the present classification, Pedomicrobium ferrugineum and Filimicrobium fusiforme are more closely related to certain Hyphomicrobium strains than these are related among each other. Budding mode of reproduction and prosthecate morphology are dominating morphological features of members of the alpha subdivision. These characteristics may gain diagnostic significance in a future formal description of this subdivision and its subgroups as a higher rank.

Base Sequence↗
PubMed1982

Localization of proteins in the inner and outer membranes of Caulobacter crescentus.

Cytoplasmic and outer membranes of Caulobacter crescentus were separated by isopycnic sucrose gradient centrifugation into two peaks with buoyant densities 1.22 and 1.14 g/cm3. These peaks were identified as outer and cytoplasmic membranes by the enrichment of malate dehydrogenase and NADH oxidase in the lower density peak and the presence of flagellin, a cell surface protein, in the heavier peak. The identity of the heavier peak as outer membrane was confirmed by labeling of cells with diazotized [35S]sulfanilic acid, a reagent that does not penetrate intact cells. Under these conditions only outer membrane proteins were substituted by the sulfanilic acid. The distribution of proteins between the cytoplasmic and outer membranes were examined by the analysis of [35S]methionine-labeled membranes by SDS-polyacrylamide and two-dimensional gel electrophoresis. These results showed that the inner and outer membranes contain approximately equal numbers of proteins, and that the distribution of these proteins between the two layers is highly asymmetric. Although many of the proteins could be assigned to one or the other membrane fraction, a number of the outer membrane proteins in the 32 000-100 000 molecular weight range frequently contaminate the inner membrane fractions. The implications of these results for membrane isolation and separation in C. crescentus are discussed.

Bacteria↗
PubMed1978

Characterization of unstable poly (A)-RNA in Caulobacter crescentus.

A significant amount of poly(A)-RNA in Caulobacter crescentus is located on polysomes and the size distribution of this polysomal poly(A)-RNA is small compared to the total pulse-labeled RNA in these cells. These observations suggest that the poly (A)-RNA represents a subset of small messenger RNA molecules. Poly (A)-RNA, and presumably the poly (A) portion of these molecules is extremely unstable: as assayed by binding to oligo (dT)-cellulose the poly (A)-RNA turns over with a chemical half-life of 15--20 s compared to a half-life of approx. 2 min for total cellular messenger RNA. The presence of adenosine in hydrolysates of poly(A) tracts showed that these sequences are located at the 3'-OH end of the RNA. The ratios of AMP/adenosine in the samples confirmed that the length of the A-tracts is approx. 13-17 nucleotides (Ohta, N., Sanders, M. and Newton, A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2343--2346)

Bacteria↗
PubMed1981

Purification and some properties of ATP-dependent deoxyribonuclease of Caulobacter crescentus.

An ATP-dependent deoxyribonuclease has been partially purified from extracts of Caulobacter crescentus cells in a procedure involving ion-exchange and affinity chromatography. The enzyme was purified approximately 350-fold and was free of contaminating nucleolytic and ATPase activity. The nuclease hydrolyzes linear, double-stranded DNA with subsequent release of short oligonucleotides, mostly from one to four bases in length. The release of nucleotides is accompanied by hydrolysis of ATP, 7.6 nmol ATP being consumed for each nmol of acid-soluble products of DNA degradation. The enzyme shows an absolute requirement for divalent cations and in most active at pH 7.6 to 8.8. The molecular weight of the nuclease, estimated by gel filtration and sucrose density gradient centrifugation, is 280 000.

Adenosine Triphosphate↗
PubMed1981

The chromosome structure and the cell cycle of Caulobacter crescentus. Isolation and analysis of envelope-free nucleoids.

Envelope-free nucleoids were isolated from an asymmetrically dividing bacterium, Caulobacter crescentus. In the nucleoid fraction, most of the DNA and nascent RNA in the cell and about 2% of the total cellular proteins were recovered. The sedimentation coefficient of the nucleoid was constant (1260 S) during the G1 period of the swarmer cell cycle and increased to 1940 S during the S period. Since both replicating (S period) and non-replicating (G1 period) chromosomes had a similar superhelical concentration, the increase in the sedimentation coefficient was simply explained by duplication of the nucleoid structure. The duplicated nucleoid was shown to segregate prior to the cell division. The pulse-labeled proteins recovered in the nucleoid fraction contained several stage-specific species, most of which were detected at the beginning of S period.

Bacterial Proteins↗
PubMed1984

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter.

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.

Autoradiography↗
PubMed1984

Caulobacter crescentus flagellar filament has a right-handed helical form.

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.

Biopolymers↗
PubMed1985

Transcriptional regulation of a periodically controlled flagellar gene operon in Caulobacter crescentus.

Temporal regulation of flagellar gene expression in Caulobacter crescentus has been examined by a detailed analysis of the flbG-flaJ-flbH-flaK hook operon. The approximate location of the promoter for this 4.4 X 10(3) base-pair transcriptional unit was determined by deletion mapping, and the flaK gene was shown by nucleotide sequencing to code for the hook protein. flaK messenger RNA was quantified by S1 nuclease mapping with an internal restriction fragment of the gene as the 5'-labeled DNA probe. The results of these assays provide the first direct evidence that periodic expression of a flagellar gene in the C. crescentus cell cycle is regulated at the transcriptional level. The effect of altering the time of gene duplication in the cell cycle was examined by subcloning the complete hook operon on a plasmid that replicates throughout the S phase. The normal periodicity of flaK transcription and translation was maintained in this merodiploid strain, which suggests that replication alone is not sufficient to initiate flagellar gene expression. We also show that the three adjacent transcriptional units III, IV and V are required in trans for transcription of the book operon, and we discuss the possible role of these genes in the hierarchical regulation of the flagellar gene expression.

Bacterial Proteins↗
PubMed1986

Differential localization of membrane receptor chemotaxis proteins in the Caulobacter predivisional cell.

The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Both the flagellum and the MCPs are partitioned to only one daughter cell, the swarmer cell, upon division. We report the results of experiments designed to determine the distribution of these MCPs within swarmer cells and predivisional cells. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum.

Autoradiography↗
PubMed1987

Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus.

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.

Autoradiography↗
PubMed1987

A set of positively regulated flagellar gene promoters in Caulobacter crescentus with sequence homology to the nif gene promoters of Klebsiella pneumoniae.

The study reported here describes nuclease S1 mapping of the in-vivo transcription start sites of transcription units I and III of the hook gene cluster of Caulobacter crescentus. We show that transcription units I and II of this flagellar (fla) gene cluster, which have divergent promoters with transcription start sites separated by 218 nucleotides, are under positive transcriptional control by genes in transcription unit III. The promoters of transcription units I, II, and III were compared with flagellin gene promoters P25, P27 and P29 recently identified in C. crescentus. Promoters PII, P25, and P27, which are under positive regulation by transcription units III to V have strongly conserved sequence elements at -13 and -24 with the consensus sequence (C/T)TGGC(C/G)C-N5-TTGC. The -13, -24 sequence elements are not well conserved in promoter PI, but the promoter does contain a copy of the -13 and -24 consensus sequence 23 base-pairs upstream (PI). The C. crescentus fla gene promoters are not homologous to the canonical Escherichia coli -10, -35 promoter sequence, but they are very similar to the -12, -24 nif gene promoter sequence reported for Klebsiella pneumoniae and Rhizobium sp. The four positively regulated fla gene promoters examined here also share a third conserved element designated II-1, with the consensus sequence C-C-CGGC--AAA--GC-G, located at approximately -100. We speculate that the conserved sequence elements mapping at -13, -24 and -100 are cis-acting regulatory elements required for the transcription and periodic regulation of these fla genes in the C. crescentus cell cycle.

Base Sequence↗
PubMed1987

Cascade regulation of Caulobacter flagellar and chemotaxis genes.

The assembly of a functional flagellum in the bacterium Caulobacter crescentus requires the protein products of approximately 30 genes expressed in a temporally discrete and spatially distinct manner. Our current understanding of this system has been limited by the fact that purified protein products are available for only about one-fifth of these genes. A genetically engineered transposon promoter probe, Tn5-VB32, containing a promoterless gene encoding neomycin phosphotransferase II (NPTase II) was used to generate a series of non-motile (fla-), kanamycin resistant strains of C. crescentus. These transcription-fusions allow the expression of NPTase II to be controlled by flagellar promoters, and thus questions of temporal regulation of flagellar genes can be addressed without the need to obtain purified protein products. The flagellar promoters accessed by Tn5-VB32 exhibited temporal regulation analogous to the known flagellar and chemotaxis gene products. The expression of NPTase II in these mutants is read from a chimeric mRNA that initiates in a chromosomal fla promoter and continues through the inserted NPTase II gene. Thus, temporal regulation is controlled by modulating either the initiation of transcription, or transcript turnover, at specific times in the cell cycle. Epistatic interactions between the genes accessed by the promoter probe and other flagellar loci were studied in double fla mutants generated by transducing the promoter-probe mutations into spontaneously derived second-site fla-mutant backgrounds. The synthesis of both natural fla gene products and the accessed NPTase II was assayed in these strains using antisera to purified components of the flagellum and to purified NPTase II. On the basis of these interactions, a trans-acting hierarchy of flagellar and chemotaxis gene expression is proposed.

Chemotaxis↗
PubMed1987

Identification of a gene cluster involved in flagellar basal body biogenesis in Caulobacter crescentus.

The bacterial flagellum is a complex structure composed of a transmembrane basal body, a hook, and a filament. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. To help to define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed. Mutants in the flaD flaB flaC gene cluster were found to be unable to assemble a complete basal body. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. A series of Tn5 insertion mutations in the flaD BC region were mapped. Complementation analysis of the Tn5 insertion mutants indicated the existence of at least four transcriptional units in the region and identified the presence of two new genes designated flbN and flbO. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in the early steps of basal body formation. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein and hook protein precursor, and the level of chemotaxis methylation.

Bacterial Proteins↗
PubMed1988

Three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus. A flagellin lacking the outer domain and its amino acid sequence lacking an internal segment.

We obtained a three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus CB15 from electron micrographs of negatively stained preparations. The C. crescentus filament appears, both in negative stain and in the frozen-hydrated state, significantly smoother and narrower than other filaments. Its helical symmetry, and unit cell size, however, are similar to that of other filaments. Although the molecular weight of the C. crescentus flagellin is about half that of other plain flagellins, there is only one monomer per unit cell as indicated by diffraction studies and by linear mass density measurements with the scanning transmission electron microscope. Alignment of the primary amino acid sequences of Salmonella typhimurium (serotype i) and C. crescentus (29,000 Mr) flagellins shows that whereas there is homology at the amino and carboxyterminal ends of the two sequences, the central segment of the S. typhimurium sequence has no homology to that of C. crescentus. A correlated comparison between the three-dimensional reconstructions of the two filaments and primary amino acid sequences of the two flagellins suggests that: (1) the C. crescentus subunit is missing the outer molecular domain but is, otherwise, similar to that of S. typhimurium; (2) the outer molecular domain in S. typhimurium corresponds, therefore, to a central stretch of the primary amino acid sequence; and (3) the outer molecular domain, missing in C. crescentus, is not obligatory for flagellar motility.

Amino Acid Sequence↗