The glycopeptide vancomycin is a drug of last resort for infection with gram-positive organisms, and three genes are vital to resistance:
vanH, vanA, and vanX. These genes are found in a vanHAXcluster, which is conserved across pathogenic bacteria, glycopeptide antibiotic producers, and other environmental bacteria. The genome sequence of the anaerobic, gram-positive, dehalogenating bacterium Desulfitobacterium hafnienseY51 revealed a predicted vanAhomolog; however, it exists in a vanAWK-murFXcluster, unlike those of other vancomycin-resistant organisms. Using purified recombinant VanA from D. hafnienseY51, we determined its substrate specificity and found it to have a 42-fold preference for d-lactate over d-alanine, confirming its activity as a d-Ala- d-Lac ligase and its annotation as VanA. Furthermore, we showed that D. hafnienseY51 is highly resistant to vancomycin, with a MIC for growth of 64 μg/ml. Finally, vanA Dhis expressed during growth in vancomycin, as demonstrated by reverse transcription-PCR. This finding represents a new glycopeptide antibiotic resistance gene cluster and expands the genetic diversity of resistance to this important class of antibiotic.