Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillinits Enterococcus faecium

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and i mmunologically related proteins that are characterized by their low affinit y for penicillin. For this reason, they are mainly involved in penicillin r esistance, due essentially to their ability to take over the function of al l other PBPs already bound and inhibited by the beta-lactam. It has been de monstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 cr by the presence of specific al-nino acid sequence s in the protein that further decrease the affinity for penicillin. In part icular, a specific amino acid box (ANNGA) previously identified in Enteroco ccus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box A NNGA in Enterococcus faecalis, an enterococcal species usually more sensiti ve to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-int roduced into a pbp5 mutant of E. faecalis obtained by insertion of transpos on Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradox ically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is conc erned, differences exist in their three-dimensional structure that affect p enicillin affinity.