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.antibiotics ͉ eDNA ͉ metagenomics ͉ natural products ͉ uncultured bacteria

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Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K m value of 2.5 ؎ 0.4 mM at pH 7.5 and 37°C. The enzyme was shown to be specific for L-cysteine oxidation, whereas homocysteine inhibited CDO activity.

A detailed bioinformatics analysis of six glycopeptide biosynthetic gene clusters isolated from soil environmental DNA (eDNA) mega-libraries indicates that a subset of these gene clusters contains collections of tailoring enzymes that are predicted to result in the production of new glycopeptide congeners. In particular, sulfotransferases appear in eDNA derived gene clusters at a much higher frequency than would be predicted from the characterization of glycopeptides from cultured Actinomycetes. Enzymes found on tailoring enzyme-rich eDNA clones associated with these six gene clusters were used to produce a series of new sulfated glycopeptide derivatives in both in vitro and in vivo derivatization studies. The derivatization of known natural products with eDNA derived tailoring enzymes is likely to be a broadly applicable strategy for generating libraries of new natural product variants.

The cloning of DNA directly from environmental samples provides a means to functionally access biosynthetic gene clusters present in the genomes of the large fraction of bacteria that remains recalcitrant to growth in the laboratory. Herein we demonstrate a method by which complementation of phosphopantetheine transferase deletion mutants can be used to restore siderophore biosynthesis and to therefore selectively enrich eDNA libraries for nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences to unprecedented levels. The common use of NRPS/PKS-derived siderophores across bacterial taxa makes this method generalizable and should allow for the facile selective enrichment of NRPS/PKS-containing biosynthetic gene clusters from large environmental DNA libraries using a wide variety of phylogenetically diverse bacterial hosts.

The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, 13, and 14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures including: an apo structure of Teg12, Teg12 bound to the desulfated co-substrate 3'-phosphoadenosine 5'-phosphate and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions.During the 20 th century, widespread use of antibiotics significantly reduced the threat of many once lethal infectious diseases. However, the success of these wonder drugs may soon become their Achilles' heel. Bacterial pathogens that have developed resistance to most widely used antibiotics are now regularly seen in clinical settings. Vancomycin and teicoplanin are glycopeptide antibiotics used in the treatment of many gram-positive bacterial infections, including methicillin-resistant Staphylococcus aureus (MRSA). With the appearance of vancomycin resistant Enterococci in the late 1980s and resistant Staphylococci in the early 1990s, these traditional antibiotics of last resort are in danger of becoming clinically compromised (1-3). As with many bacterial natural products, the discovery of additional glycopeptide congeners that might combat the growing problem of antibiotic resistance has slowed as it has become increasingly difficult to identify new biodiversity from which novel molecules might be characterized.The vast majority of bacteria present in the environment remain recalcitrant to culturing (4). This uncultured majority no doubt contains previously inaccessible glycopeptide biosynthetic gene clusters, many of which could encode the biosynthesis of novel glycopeptide congeners. Although metabolites produced by bacteria that are difficult to culture in the laboratory cannot be characterized using standard microbiological methods, it is possible to extract DNA directly from environmental samples and then analyze this DNA for sequences that might encode the biosynthesis of new natural products. In a recent analysis of DNA extracted directly from desert soil, we uncovered a new glycopeptide biosynthetic gene cluster (the TEG gene cluster) that is predicted to encode the biosynthesis of the first polysulfated glycopeptide congeners (5). The TEG gene cluster contains three closely related 3'-phosphoadenosine 5'-phosphosulfate (PAPS) dependent sulfotransfera...

The TEG gene cluster was recently isolated from an environmental DNA library and is predicted to encode the biosynthesis of a polysulfated glycopeptide congener. Three closely related sulfotransferases found in the TEG gene cluster (Teg12, Teg13 and Teg14) have been shown to sulfate the teicoplanin aglycone at three unique sites. Crystal structures of the first sulfotransferase from the TEG cluster, Teg12, in complex with the teicoplanin aglycone and its desulfated cosubstrate PAP have recently been reported [Bick et al. (2010), Biochemistry, 49, 4159-4168]. Here, the 2.7 Å resolution crystal structure of the apo form of Teg14 is reported. Teg14 sulfates the hydroxyphenylglycine at position 4 in the teicoplanin aglycone. The Teg14 structure is discussed and is compared with those of other bacterial 3 0 -phosphoadenosine 5 0 -phosphosulfate-dependent sulfotransferases.

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