Organization of the flaFG gene cluster and identification of two additional genes involved in flagellum biogenesis in Caulobacter crescentus (original) (raw)
Related papers
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, 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 ...
FlbT couples flagellum assembly to gene expression in Caulobacter crescentus
Journal of bacteriology, 1999
The biogenesis of the polar flagellum of Caulobacter crescentus is regulated by the cell cycle as well as by a trans-acting regulatory hierarchy that functions to couple flagellum assembly to gene expression. The assembly of early flagellar structures (MS ring, switch, and flagellum-specific secretory system) is required for the transcription of class III genes, which encode the remainder of the basal body and the external hook structure. Similarly, the assembly of class III gene-encoded structures is required for the expression of the class IV flagellins, which are incorporated into the flagellar filament. Here, we demonstrate that mutations in flbT, a flagellar gene of unknown function, can restore flagellin protein synthesis and the expression of fljK::lacZ (25-kDa flagellin) protein fusions in class III flagellar mutants. These results suggest that FlbT functions to negatively regulate flagellin expression in the absence of flagellum assembly. Deletion analysis shows that sequen...
Molecular microbiology, 2001
Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a transacting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a transacting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.
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...
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, 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...
Molecular microbiology, 2002
Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus expression and their capacity to reinitiate chromosomal DNA replication (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). For example, the newly formed stalked cell initiates DNA replication almost immediately after cell division, whereas replication is repressed for a period of time in the swarmer cell. Following this period of repression, the flagellum is shed, a stalk is synthesized in its place and DNA replication initiates (Fig. 1). This programme of cellular differentiation is directed, in part, by both cell cycle and spatial transcription which is regulated by members of the large family of bacterial two-component regulatory systems. The basic regulatory paradigm common to these signal transduction systems consists of a stimulatory cue, often environmental, which is sensed by and, in turn, activates autophosphorylation of a sensor histidine kinase (reviewed in Parkinson and Kofoid, 1992). The phosphate from this kinase is then transferred to a conserved receiver domain of a response regulator protein that, very often, is a transcription factor. One hallmark of the C. crescentus programme of cellular differentiation is the temporal, and spatial, biogenesis of a single polar flagellum at the pole opposite the stalk (Fig. 1). The synthesis of this flagellum is regulated by two distinct global-response regulator transcription factors, CtrA and FlbD, that function at specific times and locations in the pre-divisional cell (reviewed in Brun et al., 1994; Gober and Marques, 1995; Wu and Newton, 1997; Gober and England, 2000). The biogenesis of the polar flagellum requires at least 50 gene products and is regulated by a complex transacting hierarchy that is influenced both by progression of the cell cycle and flagellum assembly. The earliest synthesized flagellar components consist of those that encode the MS-ring (fliF), the flagellar switch and components of the flagellum-specific secretory system. These early, class II genes share a conserved promoter sequence that contains a binding site for the transcription factor, CtrA (Quon et al., 1996; Domian et al., 1997; Reisenauer et al., 1999). CtrA is activated by a cell cycle cue, which is presumably linked to the initiation of DNA replication. Following the expression and assembly of the early, class II-encoded flagellar structure, the genes encoding Molecular Microbiology (2002) 43(3), 597-615
Molecular microbiology, 2001
The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a transacting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a transacting factor. Furthermore, extragenic motile suppressors which arise in DfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. b-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.