Dual Promoters Are Responsible for Transcription Initiation of the fla/che Operon in Bacillus subtilis

The fla/che region contains more than 30 genes required for flagellar synthesis and chemotaxis in Bacillus subtilis, including the gene for the flagellum-specific ς(D) factor, sigD. Sequence and primer extension data demonstrate that a P(A) promoter immediately upstream of flgB, henceforth referred to as the fla/che P(A), and the P(D-3) promoter are active in vivo. Transcription from the P(D-3) element is dependent on ς(D) activity and is regulated by the flagellum-specific negative regulator, FlgM. In a strain containing a deletion of fla/che P(A) (P(A)Δ), ς(D) protein was not detected, demonstrating that the fla/che P(A) is necessary for wild-type expression of the sigD gene. Thus, sigD is part of the >26-kb fla/che operon. Consistent with a lack of detectable ς(D) protein, the P(A)Δ strain grows as long filaments and does not express a ς(D)-dependent hag::lacZ reporter construct. These phenotypes are indicative of a lack of sigD expression or complete inhibition of ς(D) activity by FlgM. However, ς(D) activity is found in a double mutant containing the P(A)Δ and a null mutation in flgM. The double mutant no longer grows as long filaments, and expression of hag::lacZ is partially restored. These data demonstrate that a low level of ς(D) activity does exist in the P(A)Δ mutant but can be detected only in the presence of a null mutation in flgM. Therefore, normal expression of sigD may also involve another promoter(s) within the fla/che operon.