Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus

Abstract

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.