• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.356-361
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• 1991

- To investigate the possibility of genetic development for a multi-purpose strain of Bacillus subtilis YBL-7 against Fusat-iurn solani causing root rot of many impotant corps, the plasmid pGU66 inserting urease gene of Bacillus pasteurii had been introduced into Bacillus subtilis YBL-7 by PEG-induced protoplast (PIP) transformation system. Protoplasts of B. subtilis YBL-7 were prepared by treating the cells with lysozyme (200 $\mu g$/ml) in hypertonic buffer (SMMP). The highest transformation frequency was achieved when cells of the strain with lysozyme at $42^{\circ}C$ for 90 minutes. Optimal transformation was obtained using polyethylene glycol (MW 4000) at final concentration of 30% (V/V). The transformation frequency was increased proportionally to 1.2 $\mu g$ of plasmid DNA. At best condition, the transformation frequency (transformants/ regenerants/$\mu g$ of DNA) for pGU66 was appoximately $4 \times 10^{-3}$. Also, the urease gene was strongly expressed in the transformants of B. subtilis YBL-7 and maintained steadily. The antifungal ability of transformant was very similar to that of B. ssubtilis YBL-7.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.