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 subtlis. The sacU32(Hy) and sacQ36 (Hy) In Bacillus subtilis, the expression of a variety of degradative enzymes can be increased by mutations at a number of loci. These loci are unlinked to the structural genes for the affected enzymes. Mutations at the sacU and sacQ loci can increase the expression of levansucrase, alkaline protease, neutral protease, xylanase, ,-glucanase, a-amylase, and intracellular senine protease (1, 2, 10-13). Other mutations of the sacU locus decrease the expression of these enzymes.Another locus, designated hpr, has been defined which has a more restricted phenotype. Mutations at this locus lead to increased expression of alkaline and neutral proteases but do not affect the expression of levansucrase or a-amylase (8). Mutation at the hpr locus may have a small effect on the expression of intracellular serine protease (M. Ruppen, personal communication). Another gene, designated prtR, has recently been isolated whose overexpression on a plasmid can stimulate levansucrase and alkaline and neutral protease production (14,21). No chromosomal mutation of this gene has been described.The mechanism by which the sacU32(Hy) and sacQ36 (Hy) mutations act to increase the expression of the levansucrase (sacB) gene has been the focus of a number of recent reports (2, 17). Mutations at both loci can increase the amount of sacB mRNA, probably by increasing the rate of transcription initiation. The mRNA start site was identical in strains that have sacU32(Hy), sacQ36(Hy), or wild-type alleles at these loci (17). How these mutations act to increase transcription of the sacB gene is unknown. Deletion analysis of the sacB promoter suggested that a region near -100 was necessary for complete stimulation of sacB transcription by the sa,cU32(Hy) and sacQ36 (Hy) (1,14,20,21). It is not known whether these polypeptides act directly on their target genes or indirectly by stimulating the production of other regulatory factors. The nature of the sacU gene is unknown, and an initial report of its isolation has subsequently been retracted (G. Rapoport, personal communication). The hpr gene has been recently isolated, and the nature of stimulatory mutations at this site should soon be known (M. Perego and J. A. Hoch, unpublished data).In these studies, the aprE promoter was analyzed to determine the site at which the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations act to stimulate its transcription. We determined that there are at least two regions involved in the stimulation of this promoter. The target site for hpr-97 stimulation is separable from that of sacU32(Hy) and sacQ36(Hy) stimulation, and both target sites lie rather far upstream of the transcription start site. We also demonstrated that the hpr-97 and sacU32(Hy) mutations act by increasing the amount of mRNA initiated at the same start point as in wild-type strains. The generation and characterization of deletions are descri...

A functional Spo0A is required for maximal aprE expression in Bacillus subtilis

“…The same results was obtained in an hpr2 background. Here is important to mention that as previously reported, and by unknown reasons, the effect of the hpr2 mutation on this shorter version of the aprE regulatory region, is lost [12]. Furthermore, PaprE-2o4 lost partially its capacity to respond to the degU32 mutation.…”

“…1; it is known that such regulatory region is insensitive to SinR repression [12,18]. Essentially, this construction is the same as the one reported previously [12] in plasmid pSG35.8 and integrated into the amy locus. However, because we have found that some of the original construct carry an uncharacterized deletion on the aprE regulatory region, we built a new plasmid called pSub-204 carrying the upstream region of the aprE promoter up to the --204 position (PaprE--2o4) (Fig.…”

“…However, in this set of experiments, we used an aprE regulatory region devoided of the SinR binding site as shown in Fig. 1; it is known that such regulatory region is insensitive to SinR repression [12,18]. Essentially, this construction is the same as the one reported previously [12] in plasmid pSG35.8 and integrated into the amy locus.…”

“…The sequence of the primers is the following: 5'GGAATTCGGACTCAGGAGC-ATTTAACCY (Subl); 5'CGGGATCCCGTTAACGCAAACAA-CAAGCY (Sub2). These primers hybridize with the -204 to -185 region of the aprE regulatory region and from the +83 to +100 of the aprE structural gene respectively [12]. The recognition sequence for the EcoRI or BamHI restriction enzymes were introduced in the primers to facilitate the cloning of the DNA fragment into pSG35.1 (see Fig.…”

DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ

“…Expression of degQ was shown to be controlled by DegS-DegU. This Production of a class of degradative enzymes in Bacillus subtilis, including an intracellular protease and several secreted enzymes (levansucrase, alkaline and metalloproteases, a-amylase, 3-glucanase[s], and xylanase) (3,5,11,29), is controlled at the transcriptional level by the DegSDegU two-component system and by at least two additional regulatory genes, degQ and degR, which encode small polypeptides of 46 and 60 amino acids, respectively (3,25,42,48,49).Although the two-component system is required for degradative enzyme production, the products of degQ and degR appear to be dispensable (48,49). Mutations were identified in both degS and degU leading to either deficiency of degradative enzyme production or a pleiotropic (Hy) phenotype, which includes hyperproduction of degradative enzymes, ability to sporulate in the presence of glucose, decreased genetic competence, and loss of flagella (6,12,17,23).…”