IL 61 790-41 20, USA The authors previously reported the cloning of a lytic-enzyme-encoding gene, lytM, from an autolysis-defective mutant of Staphylococcus aureus. In the present work, the lytM gene was overexpressed in Escherichia coli and the product was purified to homogeneity by affinity chromatography and HPLC. Biochemical analysis of LytM-cleaved peptidoglycan fragments indicated that LytM is a glycylglycine endopeptidase. lmmunoelectron microscopic studies with anti-LytM rabbit IgG showed that LytM is expressed during the early exponential phase and is overexpressed in an autolysis-defective mutant compared with the parent strain. Also, a uniform distribution of gold particles on the surface of actively growing bacterial cells indicates that LytM plays a role in cell growth. Northern blot analyses of /ytM expression in two global regulatory mutants, agr and sar, showed that expression of /ytM is increased about twofold in these mutants as compared with the parents. Protein homology searches revealed that LytM could be a member of the zinc protease family, as it contained a homologous 38-amino-acid motif, Tyr-X-His-X,,-Val-X, , , -Gl y-X, -Hi s.Atomic absorption spectrometric analysis of LytM revealed the presence of 0 9 mol zinc (mol LytM)".Keywords : autolysin, glycylglycine endopeptidase, lytM gene, transmission electron microscopy, Staphylococcus aureus INTRODUCTIONPeptidoglycan hydrolases are enzymes that are ubiquitous in bacteria and which hydrolyse the peptidoglycan of the bacterial cell envelope (Ghuysen et al., 1966). There are five main classes of peptidoglycan hydrolase, the N-acetylmuramidases, N-acetylmuramoyl-L-alanine amidases, endopeptidases, glucosaminidase and transglycosylases (Actor et al., 1988;Holtje & Tuomanen, 1991;Rogers et al., 1980; Tomasz, 1984). Several roles have been proposed for these enzymes in cell wall growth, cell separation, cell wall turnover, lysis initiated by antibiotics affecting the cell wall, competence for genetic transformation, flagellum formation, sporulation arid pathogenicity (Actor et al., 1988;Berry et al., 1989;Rogers et al., 1980;Shockman & Barrett, 1983;Shockman & Holtje, 1994; Ward & Williamson, 1984).Abbreviations: DEPC, diethyl pyrocarbonate; DNP, 2,4-dinitrophenyl; PCW, purified cell wall.Since the first report on Staphylococcus aureus autolysins (Welsch & Salmon, 1950), a variety of intracellular and extracellular lytic enzymes have been reported and characterized in this bacterium (Sugai, 1997). Tipper (1969) demonstrated the presence of amidase, glucosaminidase and endopeptidase activities in isolated cell walls of S. aureus. A bifunctional structural lytic gene encoding the S. aureus N-acetylglucosaminidase and N-acetylmuramoyl-L-alanine amidase was later characterized (Foster, 1995 ;Oshida et al., 1995;Oshida & Tomasz, 1992). Recently, we identified a lytic gene, l y t M , from an autolysis-defective mutant (Lyt-) of S. aureus (Ramadurai & Jayaswal, 1997). The deduced amino acid sequence of LytM showed significant homology to lysostaphin...

We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889–1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureuschromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated α-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.