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 polymyxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (Mr of 53,707 Da). The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183 aa, Mr of 20,050 Da) of the cwlV ORF. Sequencing of the flanking region revealed 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% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous 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 as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (VdeltaS and UdeltaS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that VdeltaS and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but UdeltaS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively.
Ishikawa
S
S
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
Kawahara
S
S
Sekiguchi
J
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics*; D001483:Base Sequence; D020134:Catalytic Domain; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D010802:Phylogeny
D004269:DNA, Bacterial; D009238:N-Acetylmuramoyl-L-alanine Amidase