Identification of new sigma K-dependent promoters using an in vitro transcription system derived from Bacillus subtilis.

Abstract

In Bacillus subtilis, the genes that depend on sigma K-RNA polymerase for their transcription are expressed in the mother cell compartment at later stages of sporulation. More than a dozen genes belonging to the sigma K regulon have been identified. Here I describe the identification of two additional promoters under the control of sigma K-RNA polymerase. Using a set of histidine-tagged RNA polymerases prepared from cells harvested at various times during the course of growth and sporulation (Fujita, M., Sadaie, Y., 1998. Gene 221, 185-190), transcription initiated from putative promoter sequences on a number of DNA fragments, as inferred from genome sequencing, was examined in vitro. One of these showed sigma K-dependent transcription. For further characterization of transcription initiated from this site, in vitro transcription analysis was performed using RNA polymerase holoenzyme reconstituted from purified sigma K and core RNA polymerase. Two sigma K-dependent promoters, yfhP P1 and yfhP P2, separated by a distance of about 15 bp, were thereby identified. These promoters are located immediately upstream of the yfhP gene that encodes a protein of unknown function consisting of 327 amino acids residues. The promoter strength, the rate of open complex formation and the RNA polymerase binding affinity were examined for these two promoters in comparison with other known sigma K-dependent promoters, gerE and cotD. The promoter strength displayed was in the order of gerE > cotD > yfhP P2 > yfhP P1.