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

Abstract

ylxL was found to be the last gene of the fla/che operon in Bacillus subtilis and is cotranscribed with the gene for the flagellum-specific alternate sigma factor, D . The ylxL gene was disrupted by insertional mutagenesis, and the resultant mutant strain was found to be compromised for D -dependent functions.In Bacillus subtilis, the structural genes that encode the hook-basal body complex (HBB), several genes controlling chemotaxis, and the gene for the alternate sigma factor, D , are found adjacent to one another in a 26-kb region of the bacterial chromosome called the fla/che region (15). 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.Genetic organization of ylxL. An open reading frame, originally referred to as orfC and later renamed ylxL as a result of the B. subtilis genome project (3), was identified immediately downstream of the structural gene for D , sigD ( 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). Additionally, sequence analysis of this intergenic region failed to identify a transcriptional terminator, leading to the speculation that ylxL is part of the fla/che operon. Furthermore, the rpsB gene immediately downstream of ylxL is known to encode a ribosomal protein (5) and appears to be monocistronic with its own promoter (http://genolist.pasteur.fr/SubtiList), suggesting that ylxL may be the final gene of the fla/che operon.RPA of ylxL. To determine whether ylxL is the last gene of the fla/che operon, RNase protection assays (RPA) were performed using the RPA II kit from Ambion. Riboprobes were synthesized for this analysis by first cloning the intergenic regions highlighted in Fig. 1 into plasmid pGEM-Zf7(ϩ) from Stratagene. These intergenic regions were amplified by PCR and cloned into the plasmid in the orientation that allows for production of antisense RNA in an in vitro transcription reaction. T7 RNA polymerase and [␣-32 P]UTP were used in such a reaction to yield body-labeled riboprobes. Full-length protection of the riboprobes generated from plasmids pHW1 and pHW2 was obtained (data not shown), confirming that sigD is part of the fla/che operon and demonstrating that ylxL is cotranscribed with sigD. The data obtained using the riboprobe generated from pHW3 were less straightforward.The 416-base riboprobes synthesized from pHW3 include nonhomologous sequence from the vector and 368 bases of sequence comp...

Cell population heterogeneity during growth of Bacillus subtilis

“…Likewise, the swimming behavior seen by the authors might have resulted from selection under conditions that favor swimming for swrA + revertants. In any event, our results agree with those of Werhane et al (2004) …”

“…Like SwrA, SwrB does not resemble other proteins in the databases, and its mode of action in augmenting D activity, and whether it does so in a direct or indirect fashion, are unknown. Werhane et al (2004) have reported that a swrB (ylxL) mutation alone caused a severe reduction in D -directed gene expression (in contrast to the subtle effect we observed) but oddly only a modest defect in motility. This work was, however, conducted with a laboratory strain in which the allelic state of swrA was not assessed.…”

“…As noted above, laboratory strains frequently contain a frameshift mutation in swrA that reverts at high frequency (Kearns et al 2004;Calvio et al 2005). Thus, the strong dependence of D -directed gene expression on swrB observed by Werhane et al (2004) may have been seen with cells that were mutant for swrA. Likewise, the swimming behavior seen by the authors might have resulted from selection under conditions that favor swimming for swrA + revertants.…”

The Structure and Regulation of Flagella inBacillus subtilis

“…σ D protein levels are low in chains due to a failure to express the sigD gene, the penultimate gene in the 27 kb fla/che transcript (1, 96, 147). Transcript abundance decreases along the fla/che operon such that the position of the sigD gene is critical for bias and the number of ON cells in the population may be increased simply by moving the sigD gene forward in the operon, closer to the promoter (Figure 1B) (31).…”

“…SwrB is a single pass transmembrane protein expressed immediately downstream of σ D in the fla/che operon (Figure 1B) (147). Mutation of SwrB specifically abolishes swarming motility and appears to reduce late class flagellar gene expression (72, 147).…”