Genetic Mapping And Rudimental Studies Of Cloning Of BldA-dependent Mutant In Streptomyces Coelicolor A3(2)

bldA encodes the tRNA for the rare UUA codon in Streptomyces coelicolor A3(2). Most Streptomyces coelicolor A3(2) strains do not rely on bldA function for their vegetative growth. Some TTA containing antibiotic resistant genes and regulatory genes still expressed in bldA deficient mutants, suggesting that there is an alternative pathway to translate UUA codon while bldA tRNA is not available. Streptomyces coelicolor A3(2) strain M145 has been subjected to certain mutation resulted in dependence on bldA. The mutation was termed bldA -dependent mutation (bad ~-), and its allele gene was termed bad~+. In this study we tried to localize bad ~ mutation, clone bad ~+ gene, and reveal the alternative mechanism through which UUA is translated instead of bldA tRNA when it is absent.In order to localize bad ~ mutant by genetic mapping, strain LY3 was constructed by several steps of modification or mating. 217 recombinants were selected from the progeny of genetic cross between LY3 and 1258. The genotype of these 217 recombinants was analyzed and scored, resulted in the localization of bad ~ mutation to the region between SCO3934 and SCO4659 on the 758 kb fragment of the Streptomyces coelicolor A3(2) chromosome.Shotgun method was used to clone bad ~+ gene from bad~ + strains, Streptomyces coelicolor A3(2) M600. 21,800 transformants were screened, 2 transformants of "bald" phenotype were picked and their antibiotic resistant markers were tested to be Km~sApr~RThio~R. PCR confirmed that bldA in these 2 transformants were disrupted. However, 2 inserted fragments of recombined plasmids have no overlapped regions. Further more, when these recombined plasmids were retransformted to M145::SCE25AT, no Km~sApr~R and "bald" phenotype transformant were obtained in 5,000 Thio~R transformants. The results imply that bad ~+ maybe existed in a larger operon therefore a vector of larger capacity should be used to clone the bad ~+ gene, and genetic mapping data might be helpful.