Nucleotide sequence and transcriptional analysis of the nosiheptide-resistance gene from Streptomyces actuosus

Abstract

The nucleotide (nt) sequence of a 2326-bp BamHI-PstI DNA fragment previously isolated from Streptomyces actuosus ATCC25421 that confers resistance to the thiopeptide antibiotics, nosiheptide (Nh) and thiostrepton (Ts) upon Streptomyces lividans 1326 was determined. Two open reading frames (ORFs) were found in this 2326-bp sequence; one containing 699 nt and another of 822 nt, both reading in the same direction. The Nh-resistance gene determinant (nsh) is encoded by orf822, as determined by the 74% identity of the deduced amino acid sequence of its gene product to that of the 23S rRNA methylase encoded by the Ts-resistance gene (tsr) of Streptomyces azureus. (The respective sequences had a 72% homology.) ORF699, encoded by a gene situated upstream from orf822, contained an apparent alpha-helix-beta-turn-alpha-helix configuration which is common to DNA-binding proteins and suggests that ORF699 may be a regulatory protein. Two transcription start points (tsp) were found upstream from orf699 as demonstrated by high-resolution S1 nuclease mapping. There was also a weak tsp for the nsh gene at the first nt of ORF. Moreover, transcription was observed to read through a stem-loop structure separating the orf699 and nsh genes, as demonstrated by S1 nuclease mapping of the 3' terminus of the orf699 gene, suggesting an antitermination mechanism for regulation of nsh transcription.