Exploration Of The Regulation Of BldA Gene In S.Lincolnensis NRRL 2936

In Streptomyces coelicolor,bldA encodes the only tRNA for a rare leucine UUA.Although bldA is unnecessary for growth but required for some aspect of secondary metabolism and morphological development in Streptomyces coelicolor.Mutations in bldA led to a "naked" phenotype due to deficiency of aerobic mycelial development,in addition to the visible effect of bldA expression on the phenotype of S.coelicolor,bldA mutants were also deficient in antibiotic production.It is believed that the effects of bldA mutations on secondary metabolism are mostly attributable to the presence of TTA codons in pathway-specific genes,particularly in transcriptional activator genes.In the Streptomyces lincolnensis NRRL 2936 strain,there are three genes containing TTA codons in the biosynthetic gene cluster of lincomycin:lmbU,lmbB2 and lmbY,this suggets that lmbU,lmbB2 and lmbY in S.lincolnensis NRRL 2936 also were expected to exhibit bldA-dependence.In order to investigate the possible effects of bldA on the expression of lmbU and lmbB2,the bldA of S.lincolnensis NRRL 2936 strain was knocked out by using in-frame in this study.The results showed that the primary metabolism did not be affected obviously,but the ability of aerial mycelium development,spore formation and lincomycin production was interfered in bldA mutant.To investigate the possible regulatory role of bldA in S.lincolnensis NRRL 293 6,the utilization of LmbU-Flag and LmbB2-Flag fusions led us to investigate their mechanism of gene expression respectively.The results showed that the wild type of the lmbU and lmbB2 gene is not expressed in the S.lincolnensis NRRL 2936 bldA mutant strain,but the replacement of TTA with leucine degenerate codon CTA and CTG in lmbU and lmbB2 restore its expression.In order to rule out any effect of elevated levels of the lmbU and lmbB2 mRNA,transcript analysis was carried out in this study,the results showed,in the bldA mutant strains,there is no difference in the levels of lmbB2 transcripts between the replacement of TTA with CTA and CTG and no replacement in lmbB2,and the same phenomenon occur in lmbU;at the same time,the bldA mutant strains,which had the replacement of TTA with CTA and CTG in lmbU and lmbB2,did not restore the synthesis of antibiotics and the formation of spores.These results suggest that bldA is necessary for lmbU and lmbB2 translation,but does not affect its transcription,and the replacement of TTA for a single gene is not sufficient to restore the synthesis of antibiotics and the recovery of morphological differentiation of the bldA knockout strain.This study suggests that the effect of bldA on the secondary metabolism of S. lincolnensis NRRL 2936,especially antibiotic biosynthesis,may take other pathways or mechanisms independent of the function of lmbU and lmbB2.