Relative Roles of the fla/che P _A , P _D-3 , and P _sigD Promoters in Regulating Motility and sigD Expression in Bacillus subtilis

Abstract

Three promoters have been identified as having potentially important regulatory roles in governing expression of the fla/che operon and of sigD, a gene that lies near the 3 end of the operon. Two of these promoters, fla/che P A and P D-3 , lie upstream of the >26-kb fla/che operon. The third promoter, P sigD , lies within the operon, immediately upstream of sigD. fla/che P A , transcribed by E A , lies >24 kb upstream of sigD and appears to be largely responsible for sigD expression. P D-3 , transcribed by E D , has been proposed to participate in an autoregulatory positive feedback loop. P sigD , a minor A -dependent promoter, has been implicated as essential for normal expression of the fla/che operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of fla/che P A , P D-3 , or P sigD . Our analysis of these strains indicates that fla/che P A is absolutely essential for motility, that P D-3 does not function in positive feedback regulation of sigD expression, and that P sigD is not essential for normal fla/che expression. Further, our results suggest that an additional promoter(s) contributes to sigD expression.Motility and chemotaxis functions in Bacillus subtilis are encoded within the fla/che operon. This large (Ͼ26-kb) operon includes both structural and regulatory components required for motility (6,19,32). The proximal region of the operon includes genes that encode the hook and basal body (HBB) complex, a structure that is required for tethering the flagellar filament to the cell. The distal-most region of the fla/che operon encodes the flagellum-specific sigma factor, D (19). D activity is required for transcription of the genes encoding flagellin (hag) and for the motA and motB genes, which encode the motor proteins that drive flagellar rotation (21,22 (11,15). It is believed that FlgM activity is also controlled by export through the HBB in B. subtilis (5,21,24). Expression of the fla/che operon thus controls motility in a complex manner. First, HBB components are expressed concurrently with D . Subsequent assembly of the HBB structure allows export of FlgM. This activates D to promote transcription of D -dependent motility genes. Recent studies have implicated three promoters in the expression of the fla/che operon and the sigD gene (1, 6) (see schematic, Fig. 1A). One of these promoters, P sigD , is located within the fla/che operon, immediately upstream of the sigD gene. Transcription from P sigD is dependent upon A , the major, vegetative sigma factor in B. subtilis. Previous studies indicated that P sigD contributed only weakly to overall expression of the sigD gene (1). However, genetic data suggested that this slight level of expression might be required to control temporal regulation of the entire fla/che operon (1). This requirement would presumably be indirect, since the location of P sigD precludes it from directly promoting transcription of the fla/che operon (see schematic in Fig. 1A).Two additional promoters, fla/ch...

The Last Gene of the fla/che Operon in Bacillus subtilis , ylxL , Is Required for Maximal σ D Function

“…These results were confirmed and extended by antiflagellin immunoblot analysis performed as described previously (16). The level of flagellin protein was significantly reduced in the ylxL mutant LMB231 when compared to levels found in the wild-type strain (Fig.…”

“…1). While sigD, located 26 kb from the major promoter for this operon, has been shown to be part of fla/che, (2,16), it was not known if ylxL is part of the fla/che transcription unit. Previous studies demonstrated that insertions between sigD and ylxL resulted in defects in chemotaxis (9,18), swimming (9), and motility (10).…”

“…pHW4 contains a 169-bp insert complementary to the 5Ј end of the ylxL open reading frame immediately downstream of its translational start codon. Insertion of this plasmid disrupts ylxL without affecting expression of sigD as determined by anti-D immunoblot analysis, described previously (16).…”

“…Several studies have demonstrated that these genes comprise a single operon (2,10,16,18), while genes encoding these functions in the enteric bacterium are found in at least seven operons located throughout the bacterial chromosome (7). Many of the genes within the fla/che operon have been cloned, sequenced, and characterized (1, 15); however, the 3Ј end of the operon has yet to be mapped, and the function of the ylxL gene product has not been analyzed.…”

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

“…In B. subtilis, the genes are cotranscribed from a common promoter under the control of RNA polymerase and the vegetative 70 homolog A . The genetic organization of the F3 locus of C. difficile suggests that a similar cotranscription of the F3 region genes from a common promoter may be occurring (45). Included in this group of early genes are fliF (MS ring), fliG, and fliM (C ring motor switch), flhB and fliR (export apparatus membrane proteins), and the fliA ( 28 homolog) transcriptional regulator of the F1 locus.…”