Mutations in ponA , the Gene Encoding Penicillin-Binding Protein 1, and a Novel Locus, penC , Are Required for High-Level Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae

Abstract

Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were ≥1 μg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation ( k ₂ / K' ) than wild-type PBP 1 with a variety of β-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene ( ponA1 ) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5 ) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC , which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 μg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC , is required along with ponA1 to achieve high-level penicillin resistance.