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Abstract
A cryptic plasmid, pBL1 of 4·3 kb, has been found in lysine-producing Brevibacterium lactofermentum strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for BalI, BclI, HaeII, HindIII and HpaI. It had four sites for AvaI, seven for HaeIII, eight for MboI and a very large number for AluI, but no sites were found for PstI, EcoRI or BamHI. 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 Escherichia coli, Streptomyces lividans and B. lactofermentum was constructed by cloning pBL1 into the plasmid pIJ860, a bifunctional E. coli-S. lividans vector carrying the tsr, bla and kan genes. A polyethylene glycol-assisted transformation system for B. lactofermentum protoplasts was developed. Transformation frequencies of 102 transformants (µg DNA)−1 were obtained. The kan resistance gene from Tn5 was expressed very efficiently in B. lactofermentum (up to 200 µg ml−1). A smaller plasmid, pUL62, was constructed in which the tsr (thiostrepton resistance) gene of pUL61 was deleted.
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