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KMID : 0903519970400030173
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
1997 Volume.40 No. 3 p.173 ~ p.177
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Transformation and Mutation of Bacillus licheniformis 9945a Producing Υ- Poly(glutamic acid)
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Abstract
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Bacillus licheniformis 9945a releases a natural γ-poly(glutamic acid)(γ-PGA) into fermentation broth and shows a mucoid phenotype on the solid agar medium. Transformation of mucoid cells of Bacillus species has not been simple and straightforward. The transpositional activity of Tn10 in B. licheniformis also has not been known either. Thus, a spontaneous non-mucoid derivative of the B. licheniformis was obtained first. Shuttle vector pHV1248 containing mini-Tn10 was introduced into the non-mucoid derivative by the method of protoplast transformation. The resulting transformant was reverted to the wild mucoid phenotype, and then mutated randomly with the mini-transposon by heat induction. Auxotrophs requiring arginine, lysine, or tryptophan were isolated by replica plating method. Southern blotting and DNA-DNA hybridzation analysis showed that these auxotrophs were generated by mini-Tn10 insertion into the chromosomal DNA. This method of transformation and mutation using pHV1248 would be useful for the generation of diverse mutants of B. licheniformis 9945a.
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