The Study On Genes Related To The Translational Fidelity Of Rare UUA Codon In Streptomyces Coelicolor A3(2)

Streptomyces coelicolor M145 is derived from A3(2) and has been sequenced in the S.coelicolor whole genome sequencing project. M145 was a bad (bldA-dependent) mutant strain since bldA could not be disrupted, therefore bldA was essential for its vegetative growth. bldA encodes the only tRNA that can efficiently translate the rare UUA leucine codon in S. coelicolor. It plays an important role in the morphological differentiation and physiological differentiation by controlling the expression of target genes with a TTA codon. The aim of this study is trying to raveal the mechanism by which bldA became essential in M145.The dependence of M145 on bldA suggested that there is at least one essential gene containing a TTA codon in M145. The TTA containing gene SCD95A.45c was chosen to study. pHL352, a gene replacement plasmid was constructed and used to replace SC95A.45c. A null mutant suggested that SCD95A.45c is not essential in M145. The null mutant also shown no apparent phenotype change compared to the wild type strain.pHL351, a plasmid containing bldA_a (homeologous gene of bldA in S. avermitilis) and its flanking sequence, was obtained by screening a genome sub-library of S. avermitilis NRRL8165. Based on this pHL358, the bldA_a replacement plasmid, was constructed and conjugated into NRRL8165. bldA_a-inactivated strain TW10 was obtained, indicating that NRRL8165 was bad~+. In addition, TW10 lost avermectin production and failed to develop aerial mycilium and spores, suggesting that bldA_a controlled morphological differentiation and avermectin biosynthesis in S. avermitilis.Shotgun method was used to clone wild bad gene from bad~+ strain S. coelicolor M600. 7 bald shotgun clones were picked out among 21,819 transformants in which bldA was replaced by an antibiotic resistant gene cassette. However, none of these clones could confer bldA -independence to M145::SCE25AT when they retransformed, suggesting that the phenotypes were not caused by shotgun clones but some mutations of the strains.