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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, sigma(D). The ylxL gene was disrupted by insertional mutagenesis, and the resultant mutant strain was found to be compromised for sigma(D)-dependent functions.

Expression of the sigma(D)-dependent flagellin gene, hag, is repressed by the CodY protein in nutrient-rich environments. Analysis of a codY mutant bearing a hag-lacZ reporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression. Further, hag-lacZ expression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine. This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response. However, the levels of hag-lacZ measured upon the addition of subsets of amino acids suggest an additional mechanism(s). CodY is a DNA binding protein that could repress flagellin expression directly by binding to the hag promoter region, or indirectly by binding to the fla/che promoter region that governs expression of the sigma(D) transcriptional activator required for hag gene expression. Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both the hag and fla/che promoter fragments. Additionally, CodY acts as a nutritional repressor of transcription from the fla/che promoter region that contains two functional promoters. CodY binds to both the sigma(D)- and sigma(A)-dependent promoters in this region, as demonstrated by DNase I footprint analyses. Footprint analyses of the hag gene demonstrated that CodY binds downstream of its sigma(D)-dependent promoter. Taken together, these results identify new members of the CodY regulon that encode motility functions in Bacillus subtilis and are controlled by the sigma(D) alternate sigma factor.

The sigma(D) regulon of Bacillus subtilis is composed of genes encoding proteins for flagellar synthesis, motility, and chemotaxis. Concurrent analyses of sigma(D) protein levels and flagellin mRNA demonstrate that sigD expression and sigma(D) activity are tightly coupled during growth in both complex and minimal media, although they exhibit different patterns of expression. We therefore used the sigma(D)-dependent flagellin gene (hag) as a model gene to study the effects of different nutritional environments on sigma(D)-dependent gene expression. In complex medium, the level of expression of a hag-lacZ fusion increased exponentially during the exponential growth phase and peaked early in the transition state. In contrast, the level of expression of this reporter remained constant and high throughout growth in minimal medium. These results suggest the existence of a nutritional signal(s) that affects sigD expression and/or sigma(D) activity. This signal(s) allows for nutritional repression early in growth and, based on reconstitution studies, resides in the complex components of sporulation medium, as well as in a mixture of mono-amino acids. However, the addition of Casamino Acids to minimal medium results in a dose-dependent decrease in hag-lacZ expression throughout growth and the postexponential growth phase. In work by others, CodY has been implicated in the nutritional repression of several genes. Analysis of a codY mutant bearing a hag-lacZ reporter revealed that flagellin expression is released from nutritional repression in this strain, whereas mutations in the transition state preventor genes abrB, hpr, and sinR failed to elicit a similar effect during growth in complex medium. Therefore, the CodY protein appears to be the physiologically relevant regulator of hag nutritional repression in B. 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 sigmaD factor, sigD. Sequence and primer extension data demonstrate that a PA promoter immediately upstream of flgB, henceforth referred to as the fla/che PA, and the PD-3 promoter are active in vivo. Transcription from the PD-3 element is dependent on sigmaD activity and is regulated by the flagellum-specific negative regulator, FlgM. In a strain containing a deletion of fla/che PA (PADelta), sigmaD protein was not detected, demonstrating that the fla/che PA 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 sigmaD protein, the PADelta strain grows as long filaments and does not express a sigmaD-dependent hag::lacZ reporter construct. These phenotypes are indicative of a lack of sigD expression or complete inhibition of sigmaD activity by FlgM. However, sigmaD activity is found in a double mutant containing the PADelta 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 sigmaD activity does exist in the PADelta 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.

The sigma D factor of Bacillus subtilis is required for the transcription of the flagellin and motility genes as well as for wild-type chemotaxis. Southern blot and sequence analyses demonstrate that the structural gene for sigma D, sigD, is located immediately downstream of a region of DNA originally identified as the chemotaxis (che) locus and now renamed the fla/che region. In fact, sigD appears to be part of a very large operon (> 26 kb) containing genes which encode structural proteins that form the hook-basal body complex as well as regulatory proteins required for chemotaxis. Transposon insertions up to 24 kb upstream of sigD, within several of the genes for the hook-basal body components, give rise to only a moderate decrease in sigD expression. The transposon insertions, however, block sigma D activity as demonstrated by the lack of flagellin expression in strains bearing these insertions. These effects appear to arise from two types of regulation. In cis the transposon insertions appear to introduce a partial block to transcription of sigD from upstream promoter elements; in trans they disrupt genes whose gene products are required for sigma D activity. It appears that sigD transcription is initiated, at least in part, by a promoter many kilobases upstream of its translation start site and that transcription of the flagellin gene by sigma D is dependent on the formation of a functional hook-basal body complex. The possibility that sigD is part of the fla/che operon was further tested by the integration of an insertion plasmid, containing strong transcription terminators, 1.6 and 24 kb upstream of the sigD gene. In both cases, the introduction of the terminators resulted in a greater decrease of sigD expression than was caused by the plasmid sequences alone. These results indicate that wild-type transcription of sigD is dependent on promoter sequences > 24kb upstream of its structural gene and that the entire fla/che region forms a single operon.

Expression of sigma D protein and of the hag gene, which is transcribed by the sigma D holoenzyme, is not dependent on spo0, abrB, or sin gene products in Bacillus subtilis. Preliminary results, however, suggest that a signal mediated by the spo0K locus may be responsible for the inhibition of sigma D activity during the stationary phase.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.