A Streptococcusfaecalis genomic bank was obtained by partial digestion with MboI and cloning into the Sall restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on a medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.Bacteria exhibit several types of enzymes, known as autolysins, that can degrade their own cell walls (31); autolysins are usually active on bacterial-wall peptidoglycans (29) and can be as diverse as N-acetylmuramidases (lysozymes), N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases, and transglycosylases (14). A given bacterial species can possess several autolysins; these can be involved in cell wall turnover (6), cell separation, competence for genetic transformation, formation of flagella, and sporulation (25).Some Streptococcus (Enterococcus) autolysins have been characterized. For example, Streptococcus pneumoniae possesses a well-studied cell wall amidase in addition to an endo-,-1,4-N-acetylglucosaminidase (12). Streptococcus faecium has two autolytic hydrolases: muramidase-1, which is a ,-1,4-N-acetylmuramidase with an 87-kDa active form (5), and muramidase-2, which has a 125-kDa and a 75-kDa active form (4). These two S. faecium autolysins differ in substrate specificity, molecular mass, and probably hydrolysis mechanism. It is interesting that the two active forms of S. faecium muramidase-2 can bind penicillin covalently (4). The lytic enzymes produced by S. faecalis (8) were reported to have molecular masses and substrate specificity similar to those of the two S. faecium peptidoglycan hydrolases just described.In this study, we present the cloning, sequencing, and expression in Escherichia coli of one Streptococcus faecalis autolysin. Although the sequence of an S. pneumoniae amidase has been published elsewhere (11)
