The bacillus subtilis flagellar regulatory protein σd: overproduction, domain analysis and dna-binding properties

Abstract

Flagellar biosynthesis requires an alternative sigma (σ) subunit of RNA polymerase to allow recognition of the promoters for flagellin and other late genes of the flagellar regulon. We have now overproduced and characterized Bacillus subtilis σD: the prototype of the σ28 family of flagellar σ factors. Limited protease digestion studies indicate that σD contains an amino-terminal domain, comprising conserved regions 1.2 and 2, and a carboxyl-terminal domain containing conserved regions 3.2 and 4. The protease-sensitive region between these two domains correlates with a region of very low sequence conservation among bacterial a factors. Unlike the primary σ factor, σD binds to DNA. In non-denaturing polyacrylamide gel electrophoresis the σD-DNA complex has an apparent equilibrium dissociation constant of 1 μM. Binding of σD to the promoter for flagellin, PD-6, appears to lead to an altered DNA structure near the -35 and -10 recognition elements as detected by DNase I f ootprinting and by the enhanced reactivity of several bases to dimethylsulfate. © 1995 Academic Press Limited.