The alternate sigma factor, sigma-D, initiates transcription of the hag gene and is encoded in the fla/che operon. The fla/che operon is a >26 kb transcription unit that encodes the majority of the gene products that form the hook basal-body complex (the multi-component structure that tethers the flagellar filament to the bacterial cell), as well as several chemotaxis proteins. We have characterized the promoter region of this large operon by sequence, primer extension, and genetic analyses, as well as identified at least two additional promoters involved in sigD expression. Current work is aimed at determining the relative roles of these promoters in fla/che and sigD expression, as well as determining the transcriptional architecture of this very large transcription unit.

Structural regulation of flagellin gene expression is mediated by the flgM gene product. This gene product appears to act as an anti-sigma factor to inhibit initiation of hag gene expression by sigma-D. Its activity in the bacterial cell is dependent on the functional assembly of the hook basal-body complex. We are interested in analyzing the molecular interaction between FlgM and sigma-D proteins. We have therefore initiated a couple of approaches, including allele specific suppression analyses, as well as site-directed mutagenesis to better understand the molecular nature of the sequestration of sigma-D by FlgM.

B. subtilis is a developmental organism that sporulates upon nutrient deprivation. Interestingly, flagellin protein expression increases coincidentally with the initiation of sporulation. We therefore postulate that hag gene expression is dependent on some of the same transcriptional regulators and signal-transducing proteins that initiate sporulation, and furthermore predict that flagellin gene expression is nutritionally regulated. We have constructed a hag::lacZ reporter construct. The expression of this construct can be easily monitored in mutant strains lacking the proteins required for the initiation of sporulation, and nutrient regulation. We have demonstrated the importance of several regulators in this way, and are currently conducting biochemical experiments to determine the molecular mechanisms employed by these regulators.

Due to recent funding, we have initiated a completely different line of investigation. The sequence of the B. subtilis genome was completed last year and seven ECF paralogs have been identified by sequence analyses. The Helmann laboratory at Cornell University has characterized two of these sigmas; sigma-W has an apparent role in the detoxification of antibiotics that affect peptidoglycan biosynthesis, and sigma-X appears to be important for increased viability at elevated temperatures. Furthermore, members of the Helmann laboratory have constructed a null mutation in the gene for another ECF sigma factor (sigY). Characterization of the sigY mutant has yielded a preliminary phenotype. We are currently working to determine the role of the sigma-Y ECF factor of B. subtilis in collaboration with the Helmann and Gross laboratories. Study of the newly-defined ECF family of sigma factors is intriguing because of their apparent role in adaptive responses, including heat shock and exposure to antibiotics. The proposed work joins the research laboratories of Dr. Gross at UCSF, and Dr. Helmann at Cornell that pioneered work on ECF sigmas of E. coli and B. subtilis, respectively.

Undergraduate students interested in the projects described above may participate at many levels. They may choose to search for and characterize the additional promoters that govern fla/che and sigD expression by using promoter probe plasmids, or to analyze the transcriptional architecture by RNA protection assay. Alternatively, they may be interested in analyzing the molecular interaction between sigma-D and its anti-sigma factor, or investigate how the environment affects hag::lacZ expression. Finally, they may choose to participate in the new sigma-Y project since collaboration with the Gross lab at UCSF, and the Helmann lab at Cornell U. will provide the opportunity for an outstanding research program in which to train underrepresented minority students. Taken together these studies will increase our knowledge of the molecular mechanisms that regulate flagellar gene expression in B. subtilis, as well as begin to analyze the sigma-Y regulon.

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Last modified July 10, 2002