Identification, distribution, and sequence analysis of new insertion elements in Caulobacter crescentus (original) (raw)
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Isolation and expression of cloned hook protein gene from Caulobacter crescentus
Proceedings of the National Academy of Sciences of the United States of America, 1982
Previous genetic analysis of Caulobacter crescentus showed that the periodic synthesis of hook protein, flagellin A, and flagellin B, the major flagellar subunits, is coupled in some way to chromosome replication. To examine the regulation of flagellar gene expression at the molecular level, we isolated the gene that codes for the 72,000-dalton hook protein. A specific 125I-labeled anti-hook protein IgG was used to screen a hybrid lambdaL47.1 bank of 4,500 clones and to compare peptide maps of the cloned gene product with purified hook protein. Restriction analysis of DNA from the positive lambda clones and plasmid subclones showed that the structural gene for the hook protein is contained on a 2.3-kilobase (kb) BamHI fragment. The direction of transcription was established by demonstrating the inducibility of hook protein gene in strains with the 2.3-kb fragment fused to the Escherichia coli lipoprotein gene-lactose gene promoter-operator region of pIN-II. Preliminary genomic analy...
Journal of bacteriology, 1984
Using the cloned DNA from the hook protein gene region of Caulobacter crescentus ( Ohta et al., Proc. Natl. Acad. Sci. U.S.A. 79:4863-4867, 1982), we have identified and physically mapped 19 Tn5-induced and 2 spontaneous insertion mutations to this region of the chromosome. These nonmotile mutants define a major cluster of fla genes that covers approximately 17 kilobases on the chromosome (hook gene cluster). Complementation analysis of the mutants using DNA fragments from the region subcloned in the broad host range plasmid pRK290 has shown that these fla genes are organized into at least five transcriptional units (I to V). Transcriptional unit II contains at least one gene in addition to the hook protein gene, which makes this the first operon described in C. crescentus. Expression of the hook protein gene and the genetically unlinked flagellin A and B genes by this set of mutants also furnishes additional insights into the hierarchial regulation of flagellar genes. We have found...
Journal of bacteriology, 1989
In Caulobacter crescentus, mutations have been isolated in more than 30 flagellar genes (fla, flb, and flg) which are required in the cell cycle event of flagellum biogenesis. The flaF and flaG mutations and two newly identified mutations, flbT and flbA (P.V. Schoenlein and B. Ely, J. Bacteriol. 171:000-000, 1989), have been localized to the flaFG region. In this study, the genetic and physical organization of this region was analyzed, using the cloned 4.0-kilobase flaFG region in the recombinant plasmid pPLG727. Plasmid pPLG727 complemented flaF, flaG, flbA, and flbT mutations. Further complementation studies with pPLG727 derivatives indicated that flaF and flbT are unique but overlapping transcription units, whereas flbA and flaG constitute a single transcription unit. To determine the direction of transcription of the putative flbA-flaG operon, the promoterless chloramphenicol transacetylase gene was inserted into various positions in the flbA-flaG region, and merodiploid strains...
Journal of bacteriology, 1989
During the Caulobacter crescentus cell cycle, flagellin synthesis and filament assembly are temporally controlled events which require the products encoded by the contiguous flaF, flbT, and flbA-flaG transcription units (P.V. Schoenlein, L.S. Gallman, and B. Ely, J. Bacteriol. 171:000-000, 1989). To better define the functions of these genes, immunoprecipitation studies, Western blot (immunoblot) analyses, and electron microscopic analyses characterized flagellin synthesis and assembly in mutant and merodiploid strains. Mutations in the flaF or flbA-flaG transcription unit resulted in reduced synthesis of the 25- and 27-kilodalton (kDa) flagellins. In contrast, mutations in flbT resulted in overproduction of these flagellins. The FlbT phenotype is unique, since all other identified C. crescentus fla mutations cause a reduction in the levels of the 25- and 27-kDa flagellins. Furthermore, the flbT mutant showed a chemotaxis deficiency even though it was motile. Thus, the flbT gene pro...
Journal of bacteriology, 2005
In the Caulobacter crescentus predivisional cell, class III and IV flagellar genes, encoding the extracytoplasmic components of the flagellum, are transcribed in the nascent swarmer compartment. This asymmetric expression pattern is attributable to the compartmentalized activity of the σ54-dependent transcriptional activator FlbD. Additionally, these temporally transcribed flagellar promoters possess a consensus sequence for the DNA-binding protein integration host factor (IHF), located between the upstream FlbD binding site and the promoter sequences. Here, we deleted the C. crescentus gene encoding the β-subunit of the IHF, ihfB (himD), and examined the effect on flagellar gene expression. The ΔihfB strain exhibited a mild defect in cell morphology and impaired motility. Using flagellar promoter reporter fusions, we observed that expression levels of a subset of class III flagellar promoters were decreased by the loss of IHF. However, one of these promoters, fliK-lacZ, exhibited a wild-type cell cycle-regulated pattern of expression in the absence of IHF. Thus, IHF is required for maximal transcription of several late flagellar genes. The ΔihfB strain was found to express significantly reduced amounts of the class IV flagellin, FljL, as a consequence of reduced transcriptional activity. Our results indicate that the motility defect exhibited by the ΔihfB strain is most likely attributable to its failure to accumulate the class IV-encoded 27-kDa flagellin subunit, FljL.
Journal of bacteriology, 1992
At a specific time in the Caulobacter crescentus cell cycle, a single flagellar filament and multiple receptor sites for the swarmer-specific phage phi Cbk are assembled at one pole of the predivisional cell. One cluster of genes required for this morphogenesis, the flaYG region, includes the flgJKL genes, which encode structural proteins of the flagellar filament. These flagellin genes are flanked by genes required for filament assembly, the flaYE genes at one end and the flaF-flbT-flbA-flaG genes at the other. In this study, we characterized mutants carrying large chromosomal deletions within this region. Several of these strains are phi CbK resistant and produce a novel 22-kDa flagellin that is not assembled into flagella. Merodiploid strains containing either the entire flaFG region or individual fla transcription units from this region were constructed. These strains were used to correlate the presence or absence of specific gene products to changes in flagellin synthesis, fila...
Flagellar hook and basal complex of Caulobacter crescentus
Journal of bacteriology, 1979
Intact bacterial flagella possessing a membrane-free hook and basal complex were purified from Caulobacter crescentus CB15, as well as from mutants which synthesize incomplete flagella. The basal body consisted of five rings mounted on a rod. Two rings were in the hook-proximal upper set, and three rings (two narrow and one wide) were in the lower set. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. The lower rings were all approximately 21 nm in diameter, although they varied significantly in width. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. The hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component.