Physical mapping and complementation analysis of transposon Tn5 mutations in Caulobacter crescentus: organization of transcriptional units in the hook gene cluster (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, 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...
Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus
Proceedings of the National Academy of Sciences of the United States of America, 1987
Caulobacter crescentus contains a 25-and a 27-kDa flagellin, which are assembled into the flagellar filament, and a 29-kDa flagellin, which is related in sequence but is of unknown function. We have used DNA sequence analysis and nuclease S1 assays to map the in vivo transcription start sites of the three flagellin genes and to study their regulation. These experiments lead to several conclusions. First, copies of the 29-, 25-, and 27-kDa flagellin genes are organized in a tandem array in the flaEY gene cluster of C. crescentus. Second, flagellin genes are under transcriptional control and each gene is expressed with a characteristic periodicity in the cell cycle. Third, flagellin gene promoters contain conserved nucleotide sequence elements at-13,-24, and-100 that are homologous to thefla genes in the hook gene cluster. The-13 and-24 sequences conform to afla gene promoter consensus sequence (C/TTGGCC/GC-N5-TTGC) that is similar in sequence to the-12,-24 consensus sequence of the Klebsiella pneumonia nif gene promoters. Fourth, the sequence element at approximately-100 in the 25-and the 27-kDa flagellin genes is homologous to a 19-base-pair sequence [designated previously as 11-1; see Chen, L.
Journal of bacteriology, 1990
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 ...
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, 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...
Journal of bacteriology, 1994
The periodic and sequential expression of flagellar (fla) genes in the Caulobacter crescentus cell cycle depends on their organization into levels I to IV of a regulatory hierarchy in which genes at the top of the hierarchy are expressed early in the cell cycle and are required for the later expression of genes below them. In these studies, we have examined the regulatory role of level II fliF operon, which is located near the top of the hierarchy. The last gene in the fliF operon, flbD, encodes a transcriptional factor required for activation of sigma 54-dependent promoters at levels III and IV and negative autoregulation of the level II fliF promoter. We have physically mapped the fliF operon, identified four new genes in the transcription unit, and determined that the organization of these genes is 5'-fliF-fliG-flbE-fliN-flbD-3'. Three of the genes encode homologs of the MS ring protein (FliF) and two switch proteins (FliG and FliN) of enteric bacteria, and the fourth enc...
Journal of bacteriology, 1992
The differentiating bacterium Caulobacter crescentus has been studied extensively to understand how a relatively simple life form can govern the timing of expression of genes needed for the production of stage-specific structures. In this study, a clone containing the 5.3-kb flaP region was shown to contain the flgI, cheL, and flbY genes arranged in an operon with transcription proceeding from flgI to flbY. The predicted flgI polypeptide shows remarkable identity (44%) to the flagellar basal body P-ring protein encoded by the flgI gene of Salmonella typhimurium. flgI mutations case a reduction in the levels of flagellin production and the overproduction of the hook proteins. Therefore, the flgI-encoded P-ring protein is required for normal flagellin and hook protein synthesis, suggesting that basal body assembly may play a role in the regulation of flagellar gene expression. The flbY gene probably is a basal body component as well, since flbY mutants have flagellin and hook protein ...