Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: Importance of flagellin a for flagellar filament structure and transcriptional regulation

Complex flagellar filaments are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their rigidity, w hich prevents a switch of handedness. The complex filaments of Rhizobium lu pini H13-3 and those of Sinorhizobium meliloti are composed of three and fo ur flagellin (Fla) subunits, respectively. The Fla-encoding genes, named fl aA through flaD, are separately transcribed from sigma (28)-Specific promot ers. Mutational analysis of the fla genes revealed that, in both species, F la-A is the principal flagellin and that FlaB, FlaC, and FlaD are secondary . Fla-A and at least one secondary Fla protein are required for assembling a functional flagellar filament. Western analysis revealed a ratio close to 1 of FlaA to the secondary Fla proteins (= FlaX) present in wild-type extr acts, suggesting that the complex filament is assembled from FlaA-FlaX hete rodimers. Whenever a given mutant combination of Fla prevented the assembla ge of an intact filament, the biosynthesis of flagellin decreased dramatica lly. As shown in S. meliloti by reporter gene analysis, it is the transcrip tion of flaA, but not of flaB,flaC, orflaD, that was down-regulated by such abortive combinations of Fla proteins. This autoregulation of flaA is unus ual. We propose that any combination of Fla subunits incapable of assemblin g an intact filament jams the flagellar export channel and thus prevents th e escape of an (as yet unidentified) anti-sigma (28) factor that antagonize s the sigma (28)-dependent transcription of flaA.