Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter

Abstract

A number of mutations have been described with pleiotropic effects on the expression of genes for degradative enzymes in Bacillus subtilis. The sacU32(Hy) and sacQ36(Hy) mutations increase the expression of a wide variety of enzymes that degrade biological polymers. The phenotypes caused by mutations at the hpr locus are more restricted; they are known to increase expression of the alkaline and neutral proteases. The alkaline protease (aprE) promoter was analyzed to determine the target site for stimulation by these loci. Deletion of upstream regions of the aprE promoter could abolish or greatly reduce stimulation by mutations at these loci. A region upstream of -200 was necessary for full stimulation by an hpr-97 mutation, whereas a region between -141 and -164 was necessary for full stimulation by the sacU32(Hy) and sacQ36(Hy) mutations. Northern analyses of mRNA preparations showed that the levels of aprE mRNA were increased in strains carrying the sacU32(Hy) or hpr-97 mutation. Moreover, primer extension analysis of these mRNA preparations revealed that the transcription start point was identical to that in a wild-type strain. We hypothesize that upstream activation of the subtilisin promoter mediated by these genes is a mechanism for global responses to a variety of nutritional conditions.