ABSTRACT An open reading frame located 230 nucleotides downstream from the stop codon of vanS c and in the opposite direction relative to the other genes of the vanC cluster was identified in Enterococcus gallinarum BM4174. This gene (designated ddl2 ) encoded a protein of 343 amino acids that had significant predicted structural similarity to d -Ala: d -Ala ligases and displayed 33 and 35% amino acid identity to VanC-1 and the previously reported partial sequence of Ddl from E. gallinarum , respectively. Biochemical characterization by thin-layer chromatography confirmed that Ddl2 is a d -Ala: d -Ala ligase with no detectable d -Ala: d -Ser ligase activity. The vancomycin dependence of Enterococcus faecalis BM4320 ( ddl mutant) was lost on electroporation of a plasmid construct expressing ddl2 constitutively. The latter strain was able to grow in the absence of vancomycin, and peptidoglycan precursor analysis under the same conditions indicated the synthesis of pentapeptide[ d -Ala] as the main precursor, confirming the activity of Ddl2 in vivo. Expression of ddl and ddl2 in BM4174 was tested by reverse transcription-PCR: results suggested that both d -Ala: d -Ala ligases were expressed concomitantly. Our findings indicate that vancomycin-resistant E. gallinarum BM4174 is likely to express one d -Ala: d -Ser and two d -Ala: d -Ala ligase genes.