Use of a versatile lacZ vector to analyze the upstream region of the Bacillus subtilis spoOF gene

We have constructed a versatile vector, pIS112, in which lacZ translational fusions can be made in Escherichia coli and then analyzed in Bacillus subtilis in three contexts, without recloning: in multicopy during propagation of the plasmid, in single copy integrated via a Campbell-type mechanism into the wild-type locus of the cloned fragment, or in single copy integrated into a heterologous locus. Upstream regions are reconstituted in the integration into the wild-type locus, but not into the heterologous locus, allowing the identification of upstream regulatory sequences. We have used this vector to analyze the expression of the early sporulation gene, spoOF, which, during early stationary phase, is induced 10-fold from a basal vegetative level. When a region, -50 to -150 bp relative to the transcriptional start site, is removed in the spoOF-lacZ gene, stationary phase induction of beta-galactosidase is lost. The same deletion in the upstream region of the functional spoOF gene results in cells which sporulate very poorly, although they are not blocked at the onset of sporulation, as in an spoOF null mutant. This suggests induction of spoOF expression during the beginning of stationary phase is necessary for wild-type sporulation.