A sigma 54 promoter and downstream sequence elements ftr2 and ftr3 are required for regulated expression of divergent transcription units flaN and flbG in Caulobacter crescentus

Abstract

In this study, we investigated the cis-acting sequences required for transcription of the divergent, cell cycle-regulated flaN and flbG operons of Caulobacter crescentus. Previous work showed that transcription of flbG in vivo depends on a cr& promoter and a sequence element calledftrl that is located about 100 bp upstream from the transcription start site (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). We now show that regulation offlaN transcription in vivo depends on a o54 promoter and two ftr elements located downstream of the transcription start site at +86 (ftr2) and +120 (ftr3). Mutations in or between the conserved elements at -24 and -12 in this or4 promoter reduced or abolishedflaN transcription, and one mutation that eliminated flaN expression led to an increased level offlbG transcript. Mutations in ftr2 resulted in greatly reduced levels offlaN transcript but had no noticeable effect on flbG transcript levels. All three mutations constructed inftr3 resulted in elevated flaN and flbG transcript levels. We conclude thatftr2 is required for positive regulation offlaN, whereasftr3 appears to play a negative regulatory role inflaN andflbG expression.To explain the coordinated positive activation and negative autoregulation of these two transcription units and the effect of mutations on gene expression, we propose a model in which theflaN andflbG promoters interact through alternative DNA looping to form structures that are transcriptionally active or inactive.Flagellum biosynthesis in Caulobacter crescentus requires more than 50 flagellar (fla) genes, and about half of these are organized in transcription units that are located in three major clusters on the chromosome. These fla genes are under strict temporal regulation, as indicated by the observation that they are transcribed periodically in the cell cycle at about the time that the gene products assemble to form the flagellum. The precisely ordered sequence of fla gene expression during flagellum biosynthesis and the availability of powerful biochemical and genetic tools have made C. crescentus an attractive model for studying the mechanism of temporal gene expression during cell differentiation. A central focus of these studies has been to explain, on the molecular level, how genes are turned on and off in a defined sequence during the cell division cycle (for a review, see reference 26).The role of trans-acting genes in the expression of C. crescentus fla genes was indicated first by epistasis experiments (3, 31) and later examined in detail by experiments that measured the levels of specific fla gene transcripts in different nonmotile mutants (27). These studies suggested that the C. crescentus fla genes, like those in Eschenchia coli (14, 15) and Salmonella typhimurium (18), are organized into a regulatory hierarchy in which the expression of fla genes at each level depends on the expression of genes above them in the hierarchy (27,38 identified, but there is evidence that a step in the DNA synthesis pathway is required for the...

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

“…100 bp downstream of the start site (15,30,31). The exact spacing between centers of adjacent ftr sites varies from promoter to promoter and ranges from 33 to 34 bp for the flbG and flgI promoters to 12 to 15 bp for the fljL and flgH promoters ( Table 1).…”

“…100 bp from the transcription start sites of the class III flbG and flgK promoters and of the class IV fljK and fljL flagellin promoters (26,28). The requirement of these sequence elements for transcription from the class III flbG and flgK genes was subsequently demonstrated both in vivo (15,30,31) and in vitro (3).…”

A mutation that uncouples flagellum assembly from transcription alters the temporal pattern of flagellar gene expression in Caulobacter crescentus

“…This enhancer sequence is also located upstream of the flgI gene, which encodes the P ring of the basal body (33). The hook operon, fljL, and fljK possess a different conserved enhancer element called ftr (24,42,44,45). Late in the cell cycle, the transcription of ftrcontaining promoters is restricted to the swarmer cell compartment of the predivisional cell (22,23,66).…”

Mutations of the Act Promoter in Myxococcus xanthus

“…This defect exemplifies a requirement for proper spacing between the Ϫ12 and Ϫ24 elements, as has been observed elsewhere (3,29,(43)(44)(45). Strict spacing between promoter elements helps to distinguish sigma-54 promoters from sigma-70 promoters (2,18).…”