一个枯草杆菌启动子(P_(28-1))对链霉菌分化的影响

THE EFFECT OF A PROMOTER P_(28-1) OF BACILLUS SUBTILIS ON STREPTOMYCES DIFFERENTIATION

亚克隆1.1kb的枯草杆菌启动子P_(28-1)到pUC19上,再亚克隆到以儿茶酚加氧酶为报告基因的链霉菌启动子探测质粒pIJ4083上,构建的重组质粒命名为pIJ4498.用pIJ4498转化天蓝色链霉菌J1501的原生质体,得到了相对于灰色野生型的白色转化子,而用载体pIJ4083转化J1501后得到的转化子是正常的深灰色菌落.经限制性内切酶验证了重组质粒的结构,测定了质粒的稳定性.当pIJ4498转化天蓝色链霉菌的WhiG突变株(C71)后,未观察到任何表型的变化.通过超声波破碎细胞得到的J1501/pIJ4498菌体的蛋白提取液,可使无色的儿茶酚氧化成黄色的2-羟粘糠酸半醛(HMS).而对照株J1501/pIJ4083及C71/pIJ4498菌株的蛋白提取液不能使儿茶酚氧化成黄色的HMS产物.结果表明枯草杆菌的启动子P_(28-1)被天蓝色链霉菌J1501的σ^(whiG) RNA聚合酶所识别,在启动儿茶酚加氧酶报告基因表达的同时,影响了天蓝色链霉菌J1501分化中的孢子形成.

A 1. 1kb promoter P28-1 was inserted into pUCl9. After then, the P28-1 was subcloned into the HindIII-EcoRI sites of the high copy number Streptomyces promoter probe plasmid pIJ4083 containing xy1E reporter gene. This recombinant plasmid was designated as pIJ4498. When pIJ4498 was introduced into Streptomyces coelicolor J1501 protoplasts ,transformants conferred a white phenotype,whereas the vector pIJ4083 gave rise to colonies of normal, dark grey appearance which is the same as that of J1501 itself. After confirming pIJ4498 structure with some restriction enzymes, it was also introduced into whiG mutant (C71). Crude enzyme extracts were isolated from J1501/ pIJ4498, J1501/pIJ4083 and C71/pIJ4498 respectively, the crude enzyme extract from J1501/pIJ4498 could oxidize catechol (colourless) to 2-hydroxy muconic semialdehyde (yellow colour),but crude enzyme extracts from J1501/pIJ4083 and C71/pIJ4498 could not oxidize catechol to 2-hydroxymuconic semialdehyde. The results indicated that P28-1 might be recognised by σwhiG RNA polymerase, and activated the xylE reporter gene expression and reduced J1501 sporulation.