Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

Abstract

Glycopeptide antibiotics have long served as drugs of last resort for the treatment of antibiotic-resistant gram-positive bacterial infections. Resistance to the clinically relevant glycopeptides, vancomycin and teicoplanin, threatens to undermine the usefulness of this important class of antibiotics. DNA extracted from a geographically diverse collection of soil samples was screened by PCR for the presence of sequences related to OxyC, an oxidative coupling enzyme found in glycopeptide biosynthetic gene clusters. Every soil sample examined contained at least 1 unique OxyC gene sequence. In an attempt to access the biosynthetic gene clusters associated with these OxyC sequences, a 10,000,000-membered environmental DNA (eDNA) megalibrary was created from a single soil sample. Two unique glycopeptide gene clusters were recovered from this eDNA megalibrary. Using the teicoplanin aglycone and the 3 sulfotransferases found in one of these gene clusters, mono-, di-, and trisulfated glycopeptide congeners were produced. The high frequency with which OxyC genes were found in environmental samples indicates that soil eDNA libraries are likely to be a rewarding source of glycopeptide gene clusters. Enzymes found in these gene clusters should be useful for generating new glycopeptides analogs. Environmental DNA megalibraries, like the one constructed for this study, can provide access to many of the natural product biosynthetic gene clusters that are predicted to be present in soil microbiomes.

Fig. 1.

The phylogenetic tree contains OxyC sequences from cultured bacteria and a subset of the eDNA OxyC sequences amplified from a geographically diverse collection of soils. Blue boxes show OxyC sequences from the Utah library and the red box highlights sequences that are closely related to those present in glycopeptide gene clusters known to contain sulfotransferase genes.

Fig. 2.

The VEG and TEG gene clusters have been color coded to denote the general biosynthetic transformation of each ORF. In those cases where the predicted function of the ORF has a standard abbreviation in glycopeptide biosynthesis, the ORF has also been labeled with this abbreviation. The predicted core structure of each glycopeptide congener is shown.

Fig. 3.

HPLC traces and observed ESI-HRMS m/z data for the compounds that are produced by all possible combinations of the TEG sulfotransferases (m/z [M]⁺ calcd for (mono-) C58H46Cl2N7O21S, 1278.1839, [M]⁺ calcd for (di-) C58H46Cl2N7O24S2, 1358.1407, [M]⁺ calcd for (tri-) C58H46Cl2N7O27S3, 1438.0975).

Fig. 4.

The sulfo-teicoplanin aglycones are produced by sulfation of the teicoplanin aglycone with combinations of Teg12, 13, and 14. Minimum inhibitory concentrations (MIC, μg/mL) were determined against S. aureus USA 300 (methicillin-resistant) and JH2 (vancomycin-intermediate-resistant).