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

分析链霉菌属物种内与 DNA 易位有关的基因中蛋白质表达的时间和空间调节。

• 批准号: 10660093
• 负责人: DOI Katsumi
• 金额: $ 2.18万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660093/
• 关键词: Streptomyces azureus; Streptomyces lividans TK24; plasmid; gene dosage; sporulation inhibition; His-tag; EGFP; phase-contrast microscopy; Streptomyces; spolllE様遺伝子; 遺伝子ライブラリー; spi遺伝子; His・tag; GFP融合タンパク質; 膜局在性

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

为了分析链霉菌属内DNA易位相关基因蛋白表达的时空调控规律，本研究揭示了具有抑制宿主蓝色链霉菌ATCC 14921产孢功能的接合质粒pSA 1.1上spi基因的表达以及接合过程中质粒的转移。通过PCR方法构建组氨酸标记的spi，将spi-his- tag基因插入质粒载体pUC 118中。重组spi-his-tag基因在大肠杆菌BL 21中的表达可在不溶物中检测到，将spi-his-tag基因分别导入高拷贝链霉菌质粒p1 J702和低拷贝链霉菌质粒pRES 18。将这些质粒分别转化变铅青链霉菌TK 24和S.携带质粒pSA 1.1的变铅青菌TK 24，该质粒含有spi.四个转化子在班尼特氏B中的生长曲线 ...更多信息 Roth的结果表明，spi基因的高剂量对寄主的增殖有抑制作用，而细胞释放的impS操纵子的共表达对spi基因的生长抑制作用无影响。在琼脂平板上，S.含有pI 702（spi-his-tag）的变铅青菌TK 24的产孢延迟，产生的LLOW缺陷其产孢能力。然而，S.含有pRES 18（spi-his-tag）的青丹TK 24没有延迟。这些结果表明，spi基因表达，spi的基因产物，Spi，抑制孢子形成。少量spi基因对产孢的抑制作用不明显。使用抗-His/标签碱性磷酸酶接合物对从每一个细胞中提取的蛋白质进行Western印迹。为了研究spi表达的时间调控，对Spi-EGFP（enhanced绿色fluorescence protein）融合蛋白进行了表征。用Zweiss Axioscope进行相差荧光显微镜观察，在基质菌丝体中到处都能检测到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阶段。少

项目成果

K. Doi, E. Yokoyama and S. Ogata: "Conjugative plasmid of Streptomyces (in Japanese)"Seibutsu - kogaku Kaishi. 76, 2. 66-71 (1998)

K. Doi、E. Yokoyama 和 S. Ogata：“链霉菌接合质粒（日语）”Seibutsu - kogaku Kaishi。

K. Doi, et al: "Whole' Sequence of spolllE-Like, Sporulation-inhibitory, and Transfer Gene (spi) ina Conjugative Plasmid, Psa1.1, of Streptomyces azureus and Detection of spi-Like Gene in the Actinomycete Chromosome"Biosci. Biotech. Biochem.,. 62(8),. 159

K. Doi 等人：“天青链霉菌接合质粒 Psa1.1 中 spollE 样、孢子形成抑制性和转移基因 (spi) 的完整序列以及放线菌染色体中 spi 样基因的检测”Biosci

土居克実 他: "「放線菌の接合性プラスミド」"生物工学会誌,. 76,(2),. 66-71 (1998)

Katsumi Doi 等：“‘放线菌的接合质粒’”日本生物技术学会杂志，76, (2), 66-71 (1998)

E. Yokoyama et al.: "Gene encoding a replication initiator protein and replication origin of conjugative plasmid Psa1.1 of Streptomyces cyaneus ATCC14921"FEMS Microbiol. Lett.,. 169. 103-109 (1998)

E. Yokoyama 等人：“蓝链霉菌 ATCC14921 的接合质粒 Psa1.1 的复制起始蛋白和复制起点的基因编码”FEMS Microbiol。

A.K.Okba,K.Doi et al.: "Effect of Bacitracin and Excess Mg^<2+> on Submerged Mycelial Growth of Streptomyces." J.Fement.Bioeng.86(1). 28-33 (1998)

A.K.Okba、K.Doi 等人：“杆菌肽和过量 Mg^<2> 对链霉菌沉没菌丝生长的影响”。