Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid

Abstract

We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of theflaO operon and the adjacentflbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of theflaO operon transcript by using a nuclease S1 protection assay, and the 5' end of theflbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by C28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from theflbF promoter region, that was predicted to be theflbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYV03 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990).The Caulobacter crescentus flagellum has been used as a model for investigating cell differentiation because its morphogenesis is under strict spatial and temporal control in the cell division cycle (for reviews, see references 35 and 48). Flagellum biosynthesis and function in C. crescentus is a complex process that requires at least 48 genes, and about half of these are contained in three major gene clusters (11). The C. crescentus flagellar genes (6, 7, 36, 38, 55), like those in Escherichia coli (23,24) and Salmonella typhimurium (26), can be arranged in a regulatory hierarchy in which trans-acting genes at one level are required for expression of genes at lower levels. The flbD and flbF genes occupy high levels in the C. crescentus regulatory hierarchy because they are required in trans for the expression of genes needed for the synthesis of the flagellar hook and filament (30,32,36,39). These genes that depend upon flbD and flbF for their transcription contain conserved cis-acting sequences that include ur54-type promoters, a sequence calledftr (30, 32, 33, 37), and another sequence that matches the consensus binding site for E. coli integration host factor protein (15). flbD is a member of the flaO operon (39), whose transcription in vivo depends upon a sequence similar to that of the -10 box of E. coli promoters that are transcribed by cr32 RNA polymerase (34,52). It is evident that the promot...

New members of the ctrA regulon: the major chemotaxis operon in Caulobacter is CtrA dependent

“…2d and Fig. 5 (Van Way et al, 1993), fliQp (Zhuang & Shapiro, 1995), flhAp (Ramakrishnan et al, 1991 ;Sanders et al, 1992), fliXp (Mohr et al, 1998) and fliLp (Yu & Shapiro, 1992 that the mcpA promoter is different from other CtrAdependent class II promoters is provided by the finding that transcription of mcpA is inhibited when membrane synthesis is blocked (Brassinga et al, 2000), whilst other flagellar class II promoters are not affected. Finally, although we have been able to obtain a DNase I footprint of the fliQ promoter with CtrA we have been unsuccessful in binding CtrA to the mcpA promoter (S. E. Jones, unpublished).…”

“…6). The flhA promoter should be classified as IIA since it is expressed early in the cell cycle New members of the ctrA regulon (Ramakrishnan et al, 1991 ;Sanders et al, 1992) and is negatively regulated by CtrA (data not shown). The archetypal class II promoters fliQp, fliLp and fliFp should be classified as IIB.…”

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ 28 RNA Polymerase

“…All functionally studied 28 promoters in E. coli, Salmonella, and Bacillus have had an 11-nt spacer, although the list is relatively small (3,11,12). The Caulobacter flbF promoter was reported to have a 10-nt spacer (17), but the strength of this promoter compared to the highly transcribed E. coli promoters has not been determined. 28 promoters with different spacer lengths have been predicted but not validated in Agrobacterium (2).…”

Global regulation of a sigma 54-dependent flagellar gene family in Caulobacter crescentus by the transcriptional activator FlbD

“…Class IIB genes, including fliF (54), fliQ (13), fliL (52,62), and rpoN (2), share a common promoter consensus that is unlike that of other bacterial promoters and may be recognized by a specialized activator or form of RNA polymerase (3,45,52). The sequence of the class IIA promoter of the flhA is not similar to the class IIB promoter consensus (44,48), and it is presumably recognized by another specialized form of RNA polymerase (3,45).…”