CHARACTERIZATION OF AN ENTEROCOCCUS-HIRAE PENICILLIN-BINDING PROTEIN-3 WITH LOW PENICILLIN AFFINITY

Enterococcus hirae S185, a clinical isolate from swine intestine, exhibits a relatively high resistance to penicillin and contains two 77-kDa penicillin-binding proteins 3 of high (PBP 3(s)) and low (PBP 3(r)) affinity to penicillin, respectively. A laboratory mutant S185(r) has been obtained which overproduces PBP 3(r) and has a highly increased resistance to penicillin. Peptide fragment specifically produced by tripsin and SV8 protease digestions of PBP 3(r) were isolated, and the amino acid sequences of their amino terminal regions were determined. On the basis of these sequences, oligonucleotides were synthesized and used as primers to generate, by polymerization chain reaction, a 233-bp DNA fragment the sequence of which translated into a 73-amino-acid peptide segment of PBP 3(r). These structural data led to the conclusion that the E. hirae PBP 3(r) and the methicillin-resistant staphylococcal PBP 2' are members of the same class of high-M(r) PBPs. As shown by immunological tests, PBP 3(r) is not related to PBP 3(s) but, in contrast, is related to the 71-kDa PBP 5 of low penicillin affinity which is responsible for penicillin resistance in E. hirae ATCC 9790 and R40.