Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis

Abstract

Plasmids carrying different portions of the polycistronic spoIIA locus, and unable to replicate autonomously in Bacillus subtilis, were able to transform a Spo+ B. subtilis strain, BR151, for the plasmid-determined chloramphenicol resistance by Campbell-like insertion into the region of homology on the chromosome. Two such plasmids, pPP35 and pPP36, yielded Spo- transformants, indicating that the cloned regions of these plasmids were entirely within the chromosomal spoIIA transcriptional unit. The cloned regions overlapped the end of a known spoIIA cistron, so that the transcriptional unit was larger than this cistron, and was polycistronic. This is the first demonstration of such a polycistronic sporulation transcriptional unit. The DNA sequence of the region has now been determined (given in an accompanying paper) and suggests a transcriptional unit with three open reading frames. Two other plasmids yielded Spo+ transformants of BR151, and these define the outer limits of the transcriptional unit. The adjacent sporulation locus identified by the spoV A89 mutation was not part of the same transcriptional unit.