ABSTRACT
The sequence of a 4.8-kbp DNA fragment adjacent to the right-hand end of the actinorhodin biosynthetic (act) cluster downstream of actVB-orf6 from Streptomyces coelicolor A3(2) reveals six complete open reading frames, named orf7 to orf12. The deduced amino acid sequences from orf7, orf10, and orf11 show significant similarities with the following products in the databases: a putative protein from the S. coelicolor SCP3 plasmid, LysR-type transcriptional regulators, and proteins belonging to the family of short-chain dehydrogenases/reductases, respectively. The deduced product of orf8 reveals low similarities with several methyltransferases from different sources, while orf9 and orf12 products show no similarities with other known proteins. Disruptions of orf10 and orf11 genes in S. coelicolor appear to have no significant effect on the production of actinorhodin. Nevertheless, disruption or deletion of orf10 in Streptomyces lividans causes actinorhodin overproduction. The introduction of extra copies of orf10 and orf11 genes in an S. coelicolor actIII mutant restores the ability to produce actinorhodin. Transcriptional analysis and DNA footprinting indicate that Orf10 represses its own transcription and regulates orf11 transcription, expression of which might require the presence of an unknown inducer. No DNA target for Orf10 protein was found within the act cluster.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Regulator , Streptomyces/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Anthraquinones/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , DNA Footprinting , Genes, Bacterial , Molecular Sequence Data , Open Reading Frames/genetics , Plasmids , Sequence Alignment , Sequence Analysis, DNA , Streptomyces/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription, GeneticABSTRACT
Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.
