CLONING AND SEQUENCING OF THE LOW-AFFINITY PENICILLIN-BINDING PROTEIN3(R)-ENCODING GENE OF ENTEROCOCCUS-HIRAE S185 - MODULAR DESIGN AND STRUCTURAL ORGANIZATION OF THE PROTEIN

The clinical isolate Enterococcus hirae S185 has a peculiar mode of re sistance to penicillin in that it possesses two low-affinity penicilli n-binding proteins (PBPs): the 71-kDa PBP5, also found in other entero cocci, and the 77-kDa PBP3r. The two PBPs have the same low affinity f or the drug and are immunochemically related to each other. The PBP3r- encoding gene has been cloned and sequenced, and the derived amino aci d sequence has been compared by computer-assisted hydrophobic cluster analysis with that of the low-affinity PBP5 of E. hirae R40, the low-a ffinity PBP2' of Staphylococcus aureus, and the PBP2 of Escherichia co li used as the standard of reference of the high-M(r) PBPs of class B. On the basis of the shapes, sizes, and distributions of the hydrophob ic and nonhydrophobic clusters along the sequences and the linear amin o acid alignments derived from this analysis, the dyad PBP3r-PBP5 has an identity index of 78.5%, the triad PBP3r-PBP5-PBP2' has an identity index of 29%, and the tetrad PBP3r-PBP5-PBP2'-PBP2 (of E. coli) has a n identity index of 13%. In spite of this divergence, the low-affinity PBPs are of identical modular design and possess the nine amino acid groupings (boxes) typical of the N-terminal and C-terminal domains of the high-M(r) PBPs of class B. At variance with the latter PBPs, howev er, the low-affinity PBPs have an additional almost-equal-to 110-amino -acid polypeptide stretch that is inserted between the amino end of th e N-terminal domain and the carboxy end of the membrane anchor. While the enterococcal PBP5 gene is chromosome borne, the PBP3r gene appears to be physically linked to the erm gene, which confers resistance to erythromycin and is known to be plasmid borne in almost all the Strept ococcus spp. examined.