We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacilus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC-and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively.The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open readifig frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control. pIM13 is a 2.2-kilobase multicopy, Bacillus subtilis plasmid that confers resistance to the macrolide-lincosamidestreptogramin B (MLS) antibiotics. It was isolated and first described by Mahler and. Halvorson (24). We explored the properties of pIM13 for three reasons. (i) Molecular cloning in B. subtilis is usually attempted with vectors derived from Staphylococcus aureus; however, it is possible that vectors based on an indigenous plasmid might minimize the instability problems that often defeat cloning efforts in B. subtilis.(ii) Our laboratory maintains an interest in the regulation and evolution of MLS resistance. (iii) Although plasmids native to S. aureus have been characterized in some detail (11, 31), little effort has been devoted to studying plasmids from other gram-positive genera, such as the bacilli.In this communication we present a characterization of pIM13, including its base sequence. MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used in this study are listed in Transfer of DNA from agarose gels to nitrocellulose membranes was by the procedure of Southern (44). Membranes were prehybridized for 4 h at 650C in 5 x SSC-(lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) 5x Denhardt reagent (lx Denhardt reagent is 0.02% each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin) (5) containing 200 pug of denatured salmon sperm DNA per ml and then hybridized at 650C overnight with the denatur...

A 1.5-kb genomic fragment isolated from Streptomyces averninlis that directs the synthesis of a brown pigment in Eschenichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototpe of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients.Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitUis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.

A second cluster of genes encoding the E1␣, E1␤, and E2 subunits of branched-chain ␣-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1␣), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1␤) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1␣ subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5 end of bkdF is also described. The mutant exhibited a typical Bkd ؊ phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an ␣-branched-chain fatty acid starter unit, either S(؉)-␣-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(؉)-␣-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.

The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes (acdH) were expressed in Escherichia coli, each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n-butyryl-CoA and n-valeryl-CoA in vitro. NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13 C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoAderived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5 end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.