The ability of a regulatory protein to sense the integrity of the bacterial flagellar structure was investigated. In response to a defective hook-basal body complex, the anti-sigma 28 FlgM protein inhibits flagellin transcription. In cells with a functional hook-basal body complex, the flagellin genes are transcribed normally and the FlgM protein is expelled into the growth medium. In strains with a defective hook-basal body structure, FlgM is absent from the media. The presence of flagellin protein in the media is substantially reduced in strains carrying a FlgM-LacZ protein fusion, suggesting that the fusion is blocking the flagellar export apparatus. These results suggest that the FlgM protein assesses the integrity of the flagellar hook-basal body complex by itself being a substrate for export by the flagellar-specific export apparatus.

The complex regulation of flagellin gene expression in Salmonella typhimurium was characterized in vivo by using lac transcriptional fusions to the two flagellin structural genes (fliC [Hl] andfljB [H2]). Phase variation was measured as the rate of switching of flagellin gene expression. Switching frequencies varied from 1/500 per cell per generation to 1/10,000 per cell per generation depending on the particular insertion and the direction of switching. There is a 4-to 20-fold bias in favor of switching from thefljB(On) to thefljB(Of) orientation. Random TnlOdTc insertions were isolated which failed to express flagellin. While most of these insertions mapped to loci known to be required for flagellin expression, several new loci were identified. The presence of functional copies of all of the genes responsible for complete flagellar assembly, except the hook-associated proteins (flgK,flgL, andfliD gene products), were required for expression of thefliC orfljB flagellin genes. Two novel loci involved in negative regulation offliC andfljB infla mutant backgrounds were identified. One of these loci, designated the flgR locus, mapped to the flg operon at 23 min on the Salmonella linkage map. An flgR insertion mutation resulted in relief of repression of thefliC andfljB genes in allfla mutant backgrounds except for mutants in the positive regulatory loci (flhC, flhD, and fli4 genes).Flagellar phase variation in Salmonella typhimurium results from the reversible inversion of a 996-bp chromosomal segment of DNA ( rium and with the homologous genes in Escherichia coli has uncovered a complex hierarchy of regulation for these genes (26,31,32,53). In S. typhimurium one and presumably also the other of the flagellin genes are at the bottom of this regulatory scheme and are grouped in the class of late genes (Fig. 2) (32). The members of this class are dependent on the early and middle gene classes for their expression. If any of the genes in the early and middle classes do not express functional proteins, the genes in the late class are not expressed.Similarly, the genes in the middle class are dependent upon the early class for their expression but are regulated independently of any defects in the late genes (32). The early genes include the flhD and flhC genes, which are believed to encode positive activators of flagellar expression (29). These early genes are required for expression of the middle genes.The fliA gene, shown recently to encode a flagellar-specific sigma factor (38), is included among the middle genes. The fliA gene product is thought to be required for the expression of the late genes, including flagellin.At the top of this regulatory scheme is the cAMP-Catabolite gene activator protein complex, which acts at the early genes (flhDC operon) (29, 42). In cya or crp mutant strains or in wild-type cells grown in the presence of glucose, no flagella are synthesized (1, 54). These proposed classes or levels of regulation of flagellar assembly in S. typhimurium are similar to those proposed for E. coli by Kome...

The expression of flagellin in Salmonella typhimurium is coupled to the assembly of complete flagella.Mutations which disrupt this coupling define a gene,flgM, which represses the expression of the flagellin genes in strains with mutations in the basal body, switch, or hook flagellar gene (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:2301Bacteriol. 173: -2310Bacteriol. 173: , 1991. Complementation studies demonstrated that theflgM gene is contained within a 600-bp cloned DNA fragment. Sequence analysis revealed that this fragment carries a small open reading frame corresponding to a 97-amino-acid protein. The FlgM protein observed in a T7-mediated expression system showed an apparent molecular mass of 9.5 kDa, similar to the predicted size of 10.6 kDa. Upstream of the flgM coding region is a putative promoter sequence which shows strong homology to that thought to be recognized by the flagellin-specific sigma factor (FliA). Consistent with the use of this promoter in vivo, promoter mapping by primer extension demonstrated a transcriptional start site 11 bases downstream from the center of the putative -10 promoter element, which was dependent on functional FliA for full expression.Motility and chemotaxis in peritrichously flagellated Salmonella typhimurium require the expression of over 50 genes and subsequent ordered assembly of these gene products into about 7 to 10 complete flagellar structures (for a review, see references 27 and 28). The flagellum is a complicated structure consisting of a filament, the external element composed of flagellin which is rotated for motility and chemotaxis; the basal body, a group of membranebound rod and ring proteins of the flagellar motor; and the hook, a flexible joint linking the filament and the basal body. Additional associated components include the switch complex and chemotaxis proteins, which change the direction of filament rotation (20,45,46); three hook-associated proteins incorporated into the external structure (16); and proteins required for energy transduction (2,8,19).Expression of flagellar components is highly regulated (21,22,24,25). Cells defective for basal body, hook, or switch proteins do not transcribe either of the flagellin genes,fliC or fljB, alternately expressed by S. typhimurium (22,25,40 MATERIALS AND METHODSMedia. Nutrient broth (Difco) supplemented with NaCl (5 g/liter) was used as a rich medium for growing strains, and Bacto-agar (Difco) was added to a final concentration of 1.5% for solid medium. LB (9) supplemented with E-salts (43) and 0.2% glucose was used for growth of P22 phage. MacConkey-lactose (Difco) and TTC-lactose plates were used as indicator media. TTC-lactose agar (26, 44) contained tryptone (10 g/liter), yeast extract (1 g/liter), NaCl (5 g/liter), lactose (10 g/liter), Bacto-agar (15 g/liter), and triphenyl tetrazolium chloride (TTC; 50 ,jig/ml). The same medium without TTC, lactose, or agar was used for growth of strains in primer extension assays. E-medium (43) was used as a minimal medium and contained 0.2% glucose; t...

The flgM gene product has been shown to be a negative regulator of flagellin transcription in Salmonella typhimurium (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:2301-2310, 6453-6459, 1991; K. Ohnishi, K. Kutsukake, H. Suzuki, and T. Iino, Mol. Microbiol. 6:3149-3157, 1992). Mud-lac fusions to the flgM gene were isolated and used to characterize the regulation of flgM gene expression. Transcription of the flgM gene was decreased more than 30-fold in strains with the flagellar master regulatory genes, flhC and flhD, deleted. A class 2 flagellar defect caused a slight increase of flgM gene transcription unless a wild-type copy of the flgM gene was present, in which case transcription was decreased threefold. A deletion in the gene for the alternative sigma factor sigma 28 (FliA) caused a fourfold decrease in flgM expression. Insertional inactivation of a gene upstream of the flgM gene (flgA) in a fliA mutant strain caused transcription of the flgM gene to be decreased to a basal level. Northern (RNA) blot analysis confirmed the presence of two transcripts through the flgM gene, one which initiates upstream of the flgM gene and a second which initiates upstream of the flgA gene.