Urdamycin A, the principal product of Streptomyces fradiae Tu $ 2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked D-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 335 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG and urdH) on an 805 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C-C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, the C-glycosyltransfer of one NDP-D-olivose.

A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae Tü2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C.

Fluorescence labelling of bacteria in human root dentine gives valuable additional information about their vitality status compared to the parameter CFU. The method may be suitable for following the fate of bacteria in dentinal tubules, for example in the presence of intracanal dressings.

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The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence oftcmP is not significantly similar to those of any known proteins in the available data-bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression oftcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis.Polyketides represent a large family of natural products produced by bacteria, fungi, and plants (29). Considerable interest has been directed into analyzing the biosynthesis of these compounds because of their importance as antibiotics, chemotherapeutics, flavoring agents, and pigments. Understanding the biochemical basis of antibiotic production, in particular, is crucial to establishing rational means for engineering hybrid antibiotic producers (32) or performing tasks like targeted gene disruption that yield a specific and desired product (62). It has been demonstrated that aromatic polyketides like tetracenomycin (TCM) C are synthesized by a type II polyketide synthase (33) that consists of a multiprotein complex (6,22,56). The polyketide backbone of such molecules is subsequently modified by a number of different enzymes, resulting in a vast array of structurally diverse metabolites (29). As a part of the study of the genetics and biochemistry of the formation of the anthracycline antibiotic TCM C (63), we analyzed a region of the tcm cluster (Fig. 1A) characterized by three phenotypically different types of mutants (42,43,45). On the basis of the structure of the compounds accumulated by the Streptomyces glaucescens GLA.0 type III and type VII mutants, it was concluded that this region codes for enzymes that catalyze the last two steps of the TCM C pathway (45). Type III mutants appeared to be blocked in the hydroxylation of TCM A2, whereas type VII mutants were unable to methylate the C-9 carboxy group of TCM E (Fig. 1B) (43), and pTrc99c (1) plasmids are described elsewhere. The ermE* promoter (24) was isolated from pIJ4070 (5). pUC19 was purchased from New England Biolabs (Beverly, Mass.). The Escherichia coli strains used were JM105 and DH5ac (54). The construction of the plasmids used in this study is described in Table 1.Culture conditions and chemicals and other materials. R2YENG liquid medium (pH 6) and agar plates (45, 57) were used for production of TCM C and elloramycin as well as its biosynthetic intermediates. S. glaucescens spores wer...