Cloning and expression of two autolysin genes, cwIU and cwIV, which are tandemly arranged on the chromosome of Bacillus polymyxa var. colistinus

The cwlV gene, which encodes Bacillus poly-myxa var. colistinus autolysin w as cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypep tide of 499 amino acid (aa) residues (M-r of 53,707 Dal. The N-terminal seq uence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermin ed aa); this processed form corresponds to the C-terminal portion (183 aa, M-r of 20,050 Da) of the cwlV ORF. Sequencing of the flanking region reveal ed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% ide ntical to the full-length sequence of CwlV. Downstream of cwlV, the genes f or a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-h omologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed a s a single cistron, but cwlV is transcribed with orfW. The unprocessed form s of CwlV and CwlU (V Delta S and U Delta S, respectively) and their predic ted mature forms (Vcat and Ucat, respectively) were expressed in, and purif ied from, Escherichia coli. Enzyme analysis indicated that V Delta S and Vc at exhibit low and high cell wall hydrolase activities toward B. polymyxa c ell wall, respectively, but U Delta S and Ucat exhibit almost no and low ce ll wall hydrolase activities, respectively.