Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium

Abstract

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...

Characterization of the sigD transcription unit of Bacillus subtilis

“…In the enteric bacteria an intact hook-basal body complex must be assembled before flagellin is expressed (21,36), and genetic studies have demonstrated a hierarchy of flagellar assembly, motility, and chemotaxis gene expression responsible for this structural regulation (13,14,22,23). A negative regulator of flagellin expression, designated FlgM, that is responsible for the coupling of hook-basal body assembly to flagellin expression has been identified in S. typhimurium (13,14).…”

“…The cF factor of E. coli displays a promoter specificity similar to that of aD and has been shown to transcribe certain flagellar and chemotaxis genes in that organism (3). The gene for the uF factor was identified in S. typhimurium as being thefliA locus (32), part of a class of fla, che, and mot genes termed class II genes (13,14,22,23).…”

“…Class II gene expression is dependent on the expression of class I gene products, which have been proposed to act as transcription activators (5). Class II gene products are all required for transcription of class III genes (13,14,22,23), and this regulation is based on the need for the functional assembly of class II structural proteins into the hook and basal body and on the expression offliA (13,14,22,23). In the absence of such assembly, the FlgM repressor shuts off synthesis of class III genes by interaction with the fliA u factor (13,14).…”

“…Class II gene products are all required for transcription of class III genes (13,14,22,23), and this regulation is based on the need for the functional assembly of class II structural proteins into the hook and basal body and on the expression offliA (13,14,22,23). In the absence of such assembly, the FlgM repressor shuts off synthesis of class III genes by interaction with the fliA u factor (13,14). In the enteric bacteria, therefore, there appears to be a complex set of transcriptional and structural regulatory mechanisms that ensures that the flagellin gene is expressed and the flagellar filament is assembled only when a functional hook-basal body complex has been formed.…”

FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium

“…It also contains five other genes: flgN,flgM,flgA,flgD, and flgJ. Except for flgM, which codes for an inhibitor of the flagellum-specific sigma factor, u F (2,23), and flgN, which has an unknown function (3), these genes are thought to be related to hook-basal body formation (26,27). Assembly is blocked at the stage of hook protein polymerization inflgD mutants and at the stage of outer-ring formation in flgA mutants (15).…”