Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

Abstract

ABSTRACTGlycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens.Nonomuraeasp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonicalvanHAXgenes. Consequently, we investigated the role of the β-lactam-sensitived,d-peptidase/d,d-carboxypeptidase encoded byvanYn, the onlyvan-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic inNonomuraeasp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we variedvanYngene dosage and expressedvanHatAatXatfrom the teicoplanin producerActinoplanes teichomyceticusinNonomuraeasp. ATCC 39727. Knocking outvanYn, complementing avanYnmutant, or duplicatingvanYnhad no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production.Nonomuraeasp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYnactivity. The heterologous expression ofvanHatAatXatincreased A40926 resistance inNonomuraeasp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. ThevanYn-based self-resistance inNonomuraeasp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants ofEnterococcus faeciumselectedin vitrofor high-level resistance to glycopeptides and penicillins.