Overproduction and characterization of the Bacillus subtilis anti-sigma factor FlgM

FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subuni t of RNA polymerase in Bacillus subtilis, and it is responsible of the coup ling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and charac terized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the c ognate sigma(D) factor. The FlgM .sigma(D) complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, na tive polyacrylamide gel electrophoresis, and isoelectric focusing. sigma(D) belongs to the group of sigma factors able to bind to the promoter sequenc e even in the absence of core RNA polymerase. The FlgM .sigma(D) complex ga ve a shift in a DNA mobility shift assay with a probe containing a sigma(D) -dependent promoter sequence. Limited proteolysis studies indicate the pres ence of two structural motifs, corresponding to the N- and C-terminal regio ns, respectively.