Genetic dissection of an inhibitor of the sporulation sigma factor sigma(G)

Sporulation in Bacillus subtilis is controlled by a cascade of four sigma factors that are held into inactive form until the proper stage of development. The Gin protein, encoded by csfB, is able to strongly inhibit the activity of one of these factors, sigma(G), in vivo. The csfB gene is present in a large number of endospore formers, but the various Gin orthologues show little conservation, in striking contrast to their sigma(G) counterparts. We have carried out a mutagenesis analysis of the Gin protein in order to understand its inhibitory properties. By measuring sigma(G) inhibition in the presence of Gin in vivo, assessing Gin ability to bind sigma(G) in a yeast two-hybrid assay, and quantifying Gin-sigma(G) interaction in B. subtilis, we have identified specific residues that play an essential role in binding sigma(G) or in preventing sigma(G) transcriptional activity. Two cysteine pairs, conserved in all Gin orthologues, are essential for Gin activity. Mutations in the first pair are partially complemented by mutations in the second pair, suggesting that Gin exists in oligomeric form, at least as a dimer. Dimerisation is consistent with our in vitro analysis of a purified Gin recombinant protein, which shows that Gin contains 0.5 zinc atom per monomer. Altogether, these results indicate that the conserved cysteines play a structural role, whereas another less conserved region of the protein is involved in interacting with sigma(G). Interestingly, some mutants have kept most of their ability to bind sigma(G) but are completely unable to inhibit sigma(G) transcriptional activity, raising the possibility that Gin might act by a mechanism more complex than just sequestration of sigma(G).