From a genomic library of the tyrocidine producer Bacillus brevis ATCC 8185 constructed in the bacteriophage vector EMBL3, a recombinant phage which contains the structural genes coding for tyrocidine synthetases 1 and 2, TycA and TycB, was identified. The location of the tycA gene within the 16-kilobase insert of this clone, EMBL25-1, was mapped by hybridization studies by using the previously isolated tycA DNA as a probe. Restriction analyses, the construction of subclones, and the analysis of proteins encoded by the subclones located the tycB gene at the 3' end of the tycA gene and revealed that the two genes are transcribed in the same direction. Nuclease S1 protection studies and DNA sequencing studies of the intergenic region indicated that tycA and tycB are separated by a 94-base-pair noncoding region and suggested that these genes are organized as an operon.At the beginning of the stationary growth phase, Bacillus brevis ATCC 8185 produces two peptide antibiotics, tyrocidine and linear gramicidin. These compounds are synthesized by a nonribosomal mechanism via the thio-template pathway (18,20) by the action of multifunctional enzymes. The tyrocidine synthetase system has been studied in detail (22), whereas the enzymes involved in the biosynthesis of linear gramicidin have been only partially purified and characterized (1, 15).At least three multifunctional enzymes catalyze the synthesis of the cyclic decapeptide tyrocidine (Fig. 1). The substrate amino acid, which is a constituent of the antibiotic, is activated by the corresponding enzyme by the formation of an aminoacyladenylate. These soluble intermediates are transferred to specific thiol groups on and by the multienzyme (20,24). Mediated by a covalently bound 4'-phosphopantetheine-containing arm (14,22), peptide synthesis occurs via transthiolation and transpeptidation reactions. In contrast to tyrocidine synthetase 1 (TycA), which initiates tyrocidine biosynthesis, tyrocidine synthetases 2 and 3 (TycB and TycC), which incorporate the subsequent three and six amino acids, respectively, into the antibiotic contain 4'-phosphopantetheine as an integral part.Genes involved in the biosynthesis of several antibiotics from the genera Streptomyces (5, 9, 10) and Bacillus (17,28) and from filamentous fungi (4, 33) have been cloned, and some have been sequenced. The genes involved in polyketide (25, 26) and peptide (16) antibiotic production were clustered in the genome of the producer.The structural gene encoding TycA was previously isolated on the recombinant plasmid pBT2 (28); tycA and the 5'-untranslated region were sequenced (37), and the regulation of the tycA expression in the heterologous host Bacillus subtilis was studied (29). tycA was shown to be expressed from a Cr43-like promoter. Its transcription is dependent on the SpoOA, SpoOB, and SpoOE gene products, whereas a mutation in the suppressor locus abrB relieved the inhibition of tycA transcription in spoOA and spoOB backgrounds. This paper describes the isolation and characterization of a recom...
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