CLONING, SEQUENCING AND EXPRESSION IN ESCHERICHIA-COLI OF THE LOW-AFFINITY PENICILLIN-BINDING PROTEIN OF ENTEROCOCCUS-FAECALIS

Low-affinity penicillin binding proteins are particular membrane prote ins, in several Gram-positive bacteria, which are involved in beta-lac tam antibiotic resistance. The structural gene for the low-affinity pe nicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding regi on was identified. From biochemical analysis it emerges that E. faecal is PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48% and 33% of identity with Enterococcus hirae PBP5 a nd Staphylococcus aureus PBP2a, both low-affinity PBPs involved in bet a-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membran e protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thu s suggesting that genes coding for low-affinity PBPs of enterococci ar e not strictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.