Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis

Abstract

A specialized transducing phage carrying a sporulation gene (spo0B) was constructed from B. subtilis temperate phage .rho.11 by in vitro and in vivo recombinations. Transformation experiments showed that the spo0B gene resides on a 1.4 megadalton [mD] fragment generated by EcoRI endonuclease treatment of the phage DNA. Mutants of this phage which lost transducing activity were isolated and used for genetic complementation tests and the analysis of protein(s) coded by the 1.4 mD fragment. The spo0B locus was shown to be composed of 1 cistron. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins synthesized in UV-irradiated cells infected with these phages showed that the 1.4 mD fragment codes at least 1 protein, of MW 39,000, which is synthesized in vegetative and sporulating cells. A cleavage map of the phage DNA was constructed by use of restriction endonucleases, EcoRI, BamHI and SalI, and the site of integration of the 1.4 mD fragment was determined. Expression and function of the spo0B gene are discussed.