|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 1999 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1998 : ¥2,600,000 (Direct Cost : ¥2,600,000)
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
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