A new resistance gene, linB, conferring resistance to lincosamides by nucleotidylation in Enterococcus faecium HM1025

Abstract

Resistance to lincomycin and clindamycin in the clinical isolate Enterococcus faecium HM1025 is due to a ribosomal methylase encoded by an ermAM-like gene and the plasmid-mediated inactivation of these antibiotics. We have cloned and determined the nucleotide sequence of the gene responsible for the inactivation of lincosamides, linB. This gene encodes a 267-amino-acid lincosamide nucleotidyltransferase. The enzyme catalyzes 3(5'-adenylation) (the adenylation of the hydroxyl group in position 3 of the molecules) of lincomycin and clindamycin. Expression of linB was observed in both Escherichia coli and Staphylococcus aureus. The deduced amino acid sequence of the enzyme did not display any significant homology with staphylococcal nucleotidyltransferases encoded by linA and linA' genes. Sequences homologous to linB were found in 14 other clinical isolates of E. faecium, indicating the spread of the resistance trait in this species.

FIG. 1

Analysis of genomic DNA from E. faecium HM1025 and HM1025-1, digested with SmaI (A and B, left) and from E. faecalis JH2-2 and a transconjugant digested with SfiI (A and B, right) by pulsed-field gel electrophoresis and hybridization. (A) Lanes 1, E. faecium HM1025; lanes 2, E. faecium HM1025-1. (B) Lanes 1, E. faecalis JH2-2; lanes 2, E. faecalis JH2-2/L1 (transconjugant resistant to erythromycin, lincomycin, gentamicin, streptomycin, and tetracycline). The digested fragments were transferred to a nylon sheet and hybridized to an in vitro digoxigenin-labeled linB probe. Numbers on the left of the gels are molecular sizes in kilobases.

FIG. 2

Mechanism whereby clindamycin and lincomycin are converted to lincomycin and clindamycin 3-(5′-adenylate) by LinB in the presence of ATP and MgCl²⁺.

FIG. 3

Autoradiogram of l-[³⁵S]methionine-labeled polypeptide specified in vitro by the amplification product of linB. (A) Protein electrophoresis in a 15% polyacrylamide gel containing sodium dodecyl sulfate. After separation, proteins were stained with Coomassie blue. (B) Visualization of labeled protein bands in the gel by autoradiography. Lanes 1, control without template; lanes 2, protein products synthesized from PCR-generated linB DNA; lanes 3, molecular mass markers (masses on the left are expressed in kilodaltons). The position of LinB (31,195 Da) is indicated.