The Disruption And Primary Functional Analysis Of Some TTA-containing Genes In Streptomyces Coelicolor

The rarest codon in the high G+C genome of Streptomyces coelicolor is TTA(UUA), which is recognised by a tRNA(Leu)UUA encoded by the bldA gene. The bldA gene and TTA-containing gene are not essential for vegetative growth in most S.coelicolor strains. However, it was discovered and confirmed that bldA gene in S. coelicolor M145 could not be knocked out unless another copy of bldA gene existed. The dependence of Ml 45 on bldA suggested that there is at least one indispensable gene containing a TTA codon. The aim of this study is trying to identify the essential gene which containing a TTA codon.6 genes containing TTA, SCD69.13, 2SCG1.17, SC10.27, SCK7.13c, SC1C3.22 and SCD95A.34c, most of which had orthologues in S.avermitilis, were chosen to inactivate. Corresponding gene disruptive structures, pHL502, pHL503, pHL505, pHL506,pHL507 and pHL518 were constructed by PCR targeting respectively, then passaged through the non-methylating E.coli ET12567 before their reintroduction into S.coelicolor M145 by conjugation. Screening for double crossover resulted in WJ11, WJ12, WJ13, WJ14, WJ15 and WJ17 respectively. None of them showed apparent phenotype change compared to the wild type strain M145. Indicating: 1). six genes are not essential for M145; 2). they are not target gene for bldA to regulate morphological differentiation and secondary metabolism.Interestingly, when the junction region of SC1C3.21 and SC1C3.22 including 42 nucleotides of 3'-terminal of SC1C3.21 and 740 nucleotides of 5'-terminal of SC1C3.22 was replaced, the resulting WJ1 delayed and reduced sporulation and antibiotics production. This phenotype was reproducible. However, when SC1C3.21 and SC1C3.22 were replaced separately or together, neither respective mutant behaved like wild type strain. The underlined mechanism was to be elucidated.Polyamine is a family of low-molecular weight aliphatic alkaloid showing various activities towards microorganisms and animal cells. Added-polyamine affects the morphological differentiation of bldA mutant strain J1700 of S.coelicolor. At the same contraction, spermine showed stronger effect than spermidine did.