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.

RNA 3′-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase

“…Total RNA was prepared from S. coelicolor mycelium as described previously (Hsieh & Jones, 1995) and labelled at the 39-end with [ 32 P]pCp and T4 RNA ligase (Bralley & Jones, 2001. Labelled RNAs were digested with RNases A and T1 and the digestion products were displayed on sequencing gels.…”

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels

“…Poly(A) tail lengths were measured by end-labelling of total RNA (Hsieh & Jones, 1995) and digestion with RNases as described previously (Bralley & Jones, 2001). The presence of heteropolymeric tails at the 39-ends of S. antibioticus RNAs was demonstrated by cDNA cloning as described previously for S. coelicolor (Bralley & Jones, 2002).…”

“…It seemed reasonable to expect that if PNPase overexpression occasioned a shortening of overall tail lengths, the stretches containing only A residues would also be shorter. To test this possibility, the lengths of poly(A) stretches were measured for RNAs isolated from IMRU 3720, 3720/pIJ8600 and 3720/pJSE340 as described previously (Hsieh & Jones, 1995;Bralley & Jones, 2001). The pIJ8600 derivatives were grown for 12 h, 5 mg thiostrepton ml 21 was added and growth was continued for 12 h. RNA was then isolated from these cultures and from a culture of IMRU 3720 that had been grown for 24 h. Results of these analyses are shown in Fig.…”

cDNA cloning confirms the polyadenylation of RNA decay intermediates in Streptomyces coelicolor

“…S. coelicolor M145 was grown at 30 mC for 24 h in SMM medium as described by Kieser et al (2000). Total mycelial RNA was isolated as described by Hsieh & Jones (1995). The quantity and quality of RNA were checked by spectrophotometry and agarose gel electrophoresis.…”