NUCLEOTIDE-SEQUENCE AND REGULATION OF A NEW PUTATIVE CELL-WALL HYDROLASE GENE, CWLD, WHICH AFFECTS GERMINATION IN BACILLUS-SUBTILIS

DNA sequencing of a region upstream of the mms223 gene of Bacillus sub tilis showed the presence of two open reading frames, orf1 and orf2, w hich may encode 18- and 27-kDa polypeptides, respectively. The predict ed amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, a s well as to other autolysins (CwlC, CwlM, and AmiB). The gene was ten tatively named cwlD. Bright spores produced by a B. subtilis mutant wi th an insertionally inactivated cwlD gene were committed to germinatio n by the addition of L-alanine, and spore darkening, a slow and partia l decrease in A(580), and 72% dipicolinic acid release compared with t hat of the wild-type strain were observed. However, degradation of the cortex was completely blocked, Spore germination of the cwlD mutant m easured by colony formation after heat treatment was less than 3.7 x 1 0(-8). The germination deficiency of the cwlD mutant was only partiall y removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2 ) appearing 3 h after initiation of sporulation may have originated fr om an internal sigma(E)-dependent promoter of the cwlD operon, and a l onger transcript (RNA1) appearing 45 h after sporulation may have orig inated from a sigma(G)-dependent promoter upstream of the orf1 gene. T he cwlD mutant harboring a B. subtilis vector plasmid containing the i ntact cwlD gene recovered germination at a frequency 26% of the wild-t ype level.