THE FLK GENE OF SALMONELLA-TYPHIMURIUM COUPLES FLAGELLAR P-RING AND L-RING ASSEMBLY TO FLAGELLAR MORPHOGENESIS

The flagellum of Salmonella typhimurium is assembled in stages, and th e negative regulatory protein, FlgM, is able to sense the completion o f an intermediate stage of assembly, the basal body-hook (BBH) structu re, Mutations in steps leading to the formation of the BBH structure d o not express the flagellar filament structural genes, fliC and fliB, due to negative regulation by FlgM (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:6453-6459, 1991). We have discovered another novel regu latory gene, flk, which appears to sense the completion of another ass embly stage in the flagellar morphogenic pathway just prior to BBH for mation: the completion of the P- and L-rings. Cells that are unable to assemble the L- or P-rings do not express the flagellin structural ge nes. Mutations by insertional inactivation in either the flk or flgM l ocus allow expression of the fliB flagellin structural gene in strains defective in flagellar P- and L-ring assembly. Mutations in the flgM gene, but not mutations in the flk gene, allow expression of the fliB gene in strains defective in all of the steps leading to BBH formation . The flk gene was mapped to min 52 of the S. typhimurium linkage map between the pdxB and fabB loci. A null allele of flk was complemented in trans by a flk(+) allele present in a multicopy pBR-based plasmid. DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlap ping, divergent promoter with the pdxB gene promoter (P. A. Schoenlein , B. B. Roa, and M. E. Winkler, J. Bacteriol. 174:6256-6263, 1992). An open reading frame of 333 amino acids corresponding to the flk gene p roduct of S. typhimurium and 331 amino acids from the E. coli sequence was identified. The transcriptional start site of the S. typhimurium flk gene was determined and transcription of the flk gene was independ ent of the FlhDC and sigma(28) flagellar transcription factors. The Fl k protein observed in a T7 RNA polymerase-mediated expression system s howed an apparent molecular mass of 35 kDa, slightly smaller than the predicted size of 37 kDa. The predicted structure of Flk is a mostly h ydrophilic protein with a very C-terminal membrane-spanning segment pr eceded by positively charged amino acids. This finding predicts Flk to be inserted into the cytoplasmic membrane facing inside the cytoplasm .