Analysis of temporal and spatial regulation of protein expression in the genes concerned with DNA translocation within Streptomyces species.

为了分析链霉菌中DNA易位相关基因蛋白表达的时空调控，本研究揭示了spi基因在抑制宿主蓝色链霉菌ATCC14921产孢的接合质粒pSA1.1上的基因表达，以及接合过程中质粒的转移。采用PCR方法构建组氨酸标记的spi，将生成的spi-his- tag基因插入质粒载体pUC118中。重组spi-his-tag基因在大肠杆菌bl21中的表达通过hisag chchcher在约50kDa的不溶性片段中检测到。然后将spi-his-tag基因分别导入高拷贝数链霉菌质粒plJ702和低拷贝数质粒pRES18中。将这些质粒转化为lividans TK24和S. lividans TK24，并携带含有抑制spi的impSA和impSB的质粒pSA1.1。Bennett’s b . More roth中4个转化子的生长曲线表明，大基因剂量的spi对寄主的促生生长有抑制作用，但spi对细胞释放的impS操纵子有抑制作用。在琼脂培养基上，含有plJ702（spi-his-tag）的S. lividans TK24生长迟缓，产孢能力下降。然而，含有pRES18（spi-his-tag）的S. lividans TK24的生长并没有延迟。上述结果表明，大豆中存在spi的表达，其基因产物spi对产孢有抑制作用。少量spi基因对产孢抑制作用不显著。使用抗his /tag碱性磷酸酶偶联物对每个转化中提取的蛋白进行Western blotting。然而，无法分析his标记蛋白的明显定位。为了研究spi表达的时间调控，我们对spi - egfp（增强型绿色荧光蛋白）融合蛋白进行了表征。利用Zweiss Axioscope相衬荧光显微镜观察，底物菌丝中随处可见Spi-EGFP蛋白。因此，spi的表达可能发生在链霉菌形态分化的早期。少

To analyze temporal and spatial regulation of protein expression in the genes concerned with DNA translocation within Streptomyces species, this research project revealed that the gene expression of spi gene on conjugative plasmid pSA1.1 which function for sporulation-inhibition of its host, Streptomyces azureus ATCC14921, and plasmid transfer during conjugation. The histidine tagged-spi was constructed by PCR, and generated spi-his- tag gene was inserted into plasmid vector pUC118. Expression of recombinant spi-his-tag gene in Escherichia coliBL21 could be detected in insoluble fraction as ca. 50kDa by using of HISTAG Chatcher.The spi-his-tag gene was then was introduced to high-copy number Streptomyces plasmid, plJ702, and low-copy number plasmid, pRES18, respectively. These plasmids were transformed into Streptomyces lividans TK24 and S. lividans TK24 carrying plasmid pSA1.1 which contain the repressors, impSA and impSB, for spi. The growth curve of four transformants in Bennett's b … More roth showed that large gene dosage of spi caused inhibition of vesitative growth of host, but coexsition of impS operon of cell release for growth inhibitory effect from spi. On agar plate, the growth of S. lividans TK24 containing plJ702(spi-his-tag) was delayed and generated lown defected its sporulation ability. However, the growth of S. lividans TK24 containing pRES18(spi-his-tag) was not delayed. These results indicated that expression of spi occurred, and the gene product of spi, Spi, inhibited sporulation. Small number of spi gene showed insignificant inhibitory effect for sporulation. Western blotting for extracted proteins from each transformant using anti-His/tag Alkaline phosphatase conjugant was performed. However, distinct localization of His-tagged protein could not be analyzed.For research of temporal regulation of spi expression, characterization of Spi-EGFP (enhanced green fluorescence protein) fused protein was done. By a phase-contrast fluorescence microscopic observation using Zweiss Axioscope, Spi-EGFP protein was detected everywhere in substrate mycelium. So, expression of spi might be occurred in early stage of morphological differentiation of Streptomyces. Less