Functional Analysis Of Three Ftsk Homologoues From S. Coelicolor

The complicated life cycle of streptomyccs is one of the most important reason that attracts researchers on its genetics. A great deal of valuable information on the morphological, physiological differentiation of Streptomyccs coclicolor will emerge after genome sequencing is completed. Based on the S. coelicolor DNA sequences that have been finished and focusing on three ftsk homologous, which may be closely related with cell division, this study tried to find out the functions of three ftsK homologues genes by gene replacement or disruption.Firstly, a vector pllZ1112 for disruption of SC6A9. 34 and a vector pHZ1102 containing the whole SC6A9. 34 were constructed. pllZ1112 was transformed into M145 to disrupt SC6A9. 34. A mutant named HXY1 was obtained and confirmed to be disrupted at correct site by Southern blotting. However there was not significant difference with respect to the antibiotic production and sporulation from the wild type strain when HXY1 was inoculated on MMAsp and R5 medium; pHZ1102 , a vector with copy number about 10 in streptomyces , was transformed into J1702 and J1700, but it can't supress the bald phenotype.Secondly another vector pHZ1117 which for replacement of SC(J11.17/SCD78. 01, was constructed. pHZ1117 was transformed into wild type strain M145 and a gene replacement strain named HXY3 was obtained. HXY3 was confirmed by Southern blotting. No obvious phenotype change from M145 was observed when HXY3 was inoculated on different mediums.Thirdly a vector pllZ1114 for replacement of SC7C7.05 and a vector pHZ1104 including part of SC7C7.04, SC7C7.06 and intact SC7C7. 05 were constructed. Meanwhile, pllZ1124 containing whole SC7C7. 04, SC7C7. 05 , SC7C7. 06 and part of SC7C7. 03 was constructed. pHZ1114 was transformed into the M145 and a gene replacement strain named HXY2 was obtained and testified by Southern blotting. Phenotypic heterogeneity of the progeny of IIXY2 was observed on MMAsp and SFM mediums. pHZ1104, which contained the intact SC7C7. 05, could not complement the phenotypic heterogeneity of HXY2. However, pHZl124, which contain SC7C7.05 and its upstream gene SC7C7. 04, could complement HXY2 with morphologicalheterogeneity. Thus it was supposed that co-transcription occurred between SC7C7.05 and SC7C7.04.The phenotypic restoration by pHZ1124 also indicated that it was the disruption of SC7C7. 05 that caused the morphological heterogeneity of HXY2. The sensitivity to chloramphenicol of those tiny colonies and blue colonies suggested the morphological alteration was due to genome instability, which might be the secondary effect of loss-of-function of SC7C7.05.