Isolation of sigma-28-specific promoters from Bacillus subtilis DNA

Sigma-28-RNA polymerase is a minor form of RNA polymerase found in vegetative cells of Bacillus subtilis which utilizes promoter sites distinct from those recognized by the major RNA polymerase. We have isolated a collection of cloned B. subtilis DNA segments that contain in vitro promoter sites for σ 28-RNA polymerase by screening a bacteriophage λ library of B. subtilis genomic fragments. At least nine new σ 28-specific promoter sites have been identified in this collection, and four have been partially mapped for further study. Our strategy employed a mix of RNA probes prepared by in vitro transcription with σ 28-RNA polymerase of total B. subtilis DNA EcoRI and HindIII fragments. Over 70% of the unique clones identified contain σ 28-specific promoter sites, suggesting that the method may have general application for identification of promoter-containing sequences. The efficiency with which σ 28-specific promoters are detected is consistent with there being a relatively small number of such sites in the B. subtilis genome of which twelve have been cloned.