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

Abstract

dGlycopeptides and ␤-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. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the ␤-lactamsensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanY n , the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanY n gene dosage and expressed vanH at A at X at from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanY n , complementing a vanY n mutant, or duplicating vanY n had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanY n activity. The heterologous expression of vanH at A at X at increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanY n -based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

Substrate Inhibition of VanA by d -Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

“…This suggests that its mode of action and the mechanism of resistance are different from those of vancomycin, despite the fact that heterologous expression of the vanHAX cluster increases resistance to A40926 in Nonomuraea spp. (77).…”

New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in Nonomuraea gerenzanensis ATCC 39727

“…Working cell banks (WCB) of Nonomuraea spp. were prepared as described previously (Marcone et al, 2014). Escherichia coli DH5α was used as a routine cloning host and E. coli ET12567 pUZ8002 as a donor for intergeneric conjugations.…”

Classification of Nonomuraea sp. ATCC 39727, an actinomycete that produces the glycopeptide antibiotic A40926, as Nonomuraea gerenzanensis sp. nov.

“…The A40926-producing strain was originally classified as a member of the genus Actinomadura (Goldstein et al, 1987) on the basis of morphology and cell-wall composition, and later assigned to the genus Nonomuraea on the basis of 16S rRNA gene sequence analysis (Monciardini & Sosio, 2004). According to a recent review on the genus Nonomuraea (Sungthong & Nakaew, 2015), strain ATCC 39727 is still considered a poorly taxonomically characterized strain, notwithstanding its pharmaceutical relevance as the producer of A40926 Marcone et al, 2010Marcone et al, , 2014. In this study, we performed a polyphasic taxonomic analysis of strain ATCC 39727, and propose that it represents a novel species of the genus Nonomuraea.…”

“…Samples were then centrifuged (16 000 g for 15 min) and incubated for 1 h at 55 8C. The glycopeptide-containing supernatant was filtered through a Durapore membrane filter (0.45 mm) (Millipore) and analysed by HPLC as reported previously (Beltrametti et al, 2004;Marcone et al, 2010Marcone et al, , 2014.…”

“…For a comparative analyses of antibiotic production, strain ATCC 39727 and the closest strains N. angiospora DSM 43173 T and N. jabiensis DSM 45507 T were cultivated in E26 and FM2 media, which were previously optimized for glycopeptide production (Beltrametti et al, 2004;Marcone et al, 2010Marcone et al, , 2014. The strains were grown for 168 h on a rotary shaker at 200 r.p.m.…”