A cryptic plasmid, pBL1 of 4.3 kb, has been found in lysine-producing strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for I, I, II, III and I. It had four sites for I, seven for III, eight for I and a very large number for I, but no sites were found for I, RI or HI. The estimated copy number was 30. Three different pBL1-pBR322 hybrids named pUL1, pUL10 and pUL20 were constructed. Transposon Tn5 was inserted by transposition into either the pBR322 or the pBL1 components of plasmid pUL1, pUL10 and pUL20. A shuttle vector able to replicate in and was constructed by cloning pBL1 into the plasmid pIJ860, a bifunctional vector carrying the and genes. A polyethylene glycol-assisted transformation system for protoplasts was developed. Transformation frequencies of 10 transformants (μg DNA) were obtained. The resistance gene from Tn5 was expressed very efficiently in (up to 200 μg ml). A smaller plasmid, pUL62, was constructed in which the (thiostrepton resistance) gene of pUL61 was deleted.


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