The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gylDNA

Summary Glycerol-3-phosphate dehydrogenase (gylB) mutations (which cause glycerol sensitivity), and presumed gylcerol kinase (gylA) and/or regulatory mutations eliminating both glycerol-3-phosphate dehydrogenase and glcerol kinase activities, map close to the argA locus of Streptomyces coelicolor A3(2). Using the plasmid vector pIJ702 and restriction enzymes Bg/II and SstI, extensively overlapping S. coelicolor DNA fragments of 2.74 kb and 2.84 kb were isolated, either of which could restore the wild-type phenotype to gylB and some gylA mutants. Genetic and biochemical analyses of mutants carrying the cloned gylDNA suggested that a functional gyl promoter had not been cloned, and that restoration of the Gyl⁺ phenotype was achieved by recombination between the cloned and chromosomal gyl DNA sequences. After subcloning parts of this DNA into the phage vector C31 KC400, gene disruption analysis was carried out, which confirmed the absence of the gyl promoter, and indicated that a polycistronic mRNA traverses gylA and then gylB.