Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genesbgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20 -30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.

Glycopeptides are important clinical emergency antibiotics consisting of a glycosylated and chlorinated heptapeptide backbone. The understanding of the biosynthesis is crucial for development of new glycopeptides. With balhimycin as a model system, this work focuses on the investigation of the putative halogenase gene (bhaA) and the putative haloperoxidase/perhydrolase gene (bhp) of the balhimycin biosynthesis gene cluster. An in-frame deletion mutant in the haloperoxidase/perhydrolase gene bhp (OP696) did not produce balhimycin. Feeding experiments revealed that bhp is involved in the biosynthesis of beta-hydroxytyrosine, a precursor of balhimycin. A bhaA in-frame deletion mutant (PH4) accumulated glycosylated but nonchlorinated balhimycin variants. The mutants indicated that only the halogenase BhaA is required for chlorination of balhimycin. Nonglycosylated and/or nonhalogenated metabolites can serve as starting points for combinatorial approaches for novel glycopeptides.

An infectious Shiga toxin (Stx) 2e-converting bacteriophage (P27) was isolated from Stx2e-producing Escherichia coli ONT:H ؊ isolate 2771/97 originating from a patient with diarrhea. The phage could be transduced to E. coli laboratory strain DH5␣, and we could show that lysogens were able to produce biologically active toxin in a recA-dependent manner. By DNA sequence analysis of a 6,388-bp HindIII restriction fragment of P27, we demonstrated that the stx 2e gene was located directly downstream of ileZ and argO tRNA genes. Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of Pseudomonas aeruginosa phage CTX and a gene homologous to the endolysin gene gp19 of phage PS3 were detected. The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a worldwide cause of diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS) (8). Stx identified in human STEC isolates comprise Stx1, Stx2 and variants of Stx2, including Stx2c, Stx2d, and Stx2e (21,31,35). STEC strains associated with diarrhea and HUS usually produce Stx1, Stx2, and Stx2c, either alone or in various combinations (6,24). In contrast, Stx2d was frequently identified in STEC isolates from asymptomatic carriers (21). Stx2e is typically produced by STEC strains that cause pig edema disease and belong to serogroups O138, O139, and O141 (15). However, Stx2e-producing STEC strains have also been isolated, albeit rarely, from patients with diarrhea (20) and HUS (35). These human isolates belonged to serogroups O101 and O9 that have not been reported in STEC strains associated with pig edema disease. Interestingly, Stx2e-producing STEC belonging to serogroup O101 have been isolated from slaughtered healthy pigs (2), suggesting this animal species as a potential reservoir of human infections. Several stx 2e genes have been cloned and sequenced from STEC O101 isolates originating from a patient with diarrhea and from healthy pigs, respectively (4), and were demonstrated to be identical or almost identical to stx 2e present in an STEC O139 isolate from a pig with edema disease (36).Stx1 and Stx2 are encoded in the genome of temperate bacteriophages (11,32,33). Phages that contain the structural genes for Stx1 and Stx2 have been isolated from STEC O157 and O26 strains, and their morphology, genome sizes, and restriction fragment length polymorphism patterns have been characterized (23,32,38). Moreover, the Stx1-converting phage H-19B isolated from STEC O26:H11 strain H19 and the Stx2-converting phage 933W originating from STEC O157:H7 strain EDL 933 (19) have been characterized by nucleotide sequencing and shown to have a genetic structure related to that of bacteriophage (18,22). Analysis of a 17-kb region of the genome of phage H-19B demonstrated that the stx 1 gene was located downstream of a gene encoding...

During analysis of the recently identified gene cluster for the glycopeptide antibiotic balhimycin, produced by Amycolatopsis mediterranei DSM 5908, novel genes were identified and characterized in detail. The gene products of four of the identified genes (bpsA, bpsB, bpsC and bpsD) are nonribosomal peptide synthetases (NRPSs) ; one (Orf1-protein) shows similarities to small proteins associated with several NRPSs without an assigned function. BpsA and BpsB are composed of three modules each (modules 1-6), BpsC of one module (module 7) and BpsD of a minimal module (module 8). Thus, the balhimycin gene cluster encodes eight modules, whereas its biosynthetic product is a heptapeptide. Non-producing mutants were created by a gene disruption of bpsB, an in-frame deletion of bpsC and a gene replacement of bpsD. After establishment of a gene complementation system for Amycolatopsis strains, the replacement mutant of bpsD was complemented, demonstrating for the first time that BpsD, encoding the eighth module, is indeed involved in balhimycin biosynthesis. After feeding with β-hydroxytyrosine the capability of the bpsD mutant to produce balhimycin was restored, demonstrating the participation of BpsD in the biosynthesis of this amino acid. The specificity of four of the eight adenylation domains was determined by ATP/PP i exchange assays : modules 4 and 5 activated L-4-hydroxyphenylglycine, module 6 activated β-hydroxytyrosine and module 7 activated L-3,5-dihydroxyphenylglycine, which is in accordance with the sequence of the non-proteogenic amino acids 4 to 7 of the balhimycin backbone.