Lincomycin resistance gene lnu(D) in Streptococcus uberis

Abstract

Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 microg/ml) while remaining susceptible to clindamycin (MIC = 0.06 microg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinA(N2), and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2'') was identified.

FIG. 1.

Partial alignment of the N termini of the deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A) (GenBank accession no. M14039), Lnu(C) (GenBank accession no. AY928180), LinA_N2 (GenBank accession no. AAF74724), and Lnu(D) (GenBank accession no. EF452177) and aminoglycoside nucleotidyltransferase ANT(2′′) (GenBank accession no. CAA87464). The conserved domain is boxed. *, identical residues; :, conserved substitutions; ., semiconserved substitutions.

FIG. 2.

Phylogenetic relationship among lincosamide and aminoglycoside nucleotidyltransferases. The tree was constructed using the neighbor-joining method with the closely related amino acid sequences from S. uberis [Lnu(D)] (GenBank accession no. EF452177), S. haemolyticus [Lnu(A)] (GenBank accession no. M14039), S. aureus [Lnu(A′)] (GenBank accession no. J03947), S. agalactiae [Lnu(C)] (GenBank accession no. AY928180), and B. fragilis (LinA_N2) (GenBank accession no. AAF74724). More distantly related sequences from Enterococcus faecium [Lnu(B)] (GenBank accession no. AF110130), E. coli (LinF) (GenBank accession no. AJ561197), and E. coli [ANT(2′′)] (GenBank accession no. CAA87464) were also included.

FIG. 3.

HPLC-electrospray ionization MS analysis of inactivated clindamycin. (A) HPLC chromatogram showing a major peak eluting at 10.8 min. (B) Full MS analysis of the 10.8-min-eluted fraction reveals the dominance of a 754.1-amu compound and its Na adduct (+22 amu), which is 329 Da heavier than the original clindamycin. (C) Adenylylation of clindamycin was ascertained by the CID-MS2 analysis. Results are shown for fragments observed after CID. Fragments indicated by arrows resulted from leakage of the adenine or of the adenosine moiety of the compound.