We isolated a replication-thermosensitive mutant of the broad-host-range replicon pVWVOl. The mutant pVE6002 is fuly thermosensitive above 35°C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon TnlO derivative in BaciUus subtilis, we observed transposition frequencies of about 1%.

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Summary -The construction of recombinant strains of lactic bacteria has become an important objective for many researchers. New strains may resolve sorne of the pre-existing problems in industrial fermentation, and may offer new approaches to meeting medical and pharmaceutical needs. Such an approach is limited by technical problems of manipulation of the lactic bacteria. We have therefore focused on the methodology for the isolation of recombinant strains. Here, we describe the development of a broad host range thermosensitive (Ts) vector, pG+host, which allows efficient integration of DNA by single and double crossing over into the chromosome of a variety of Gram-posItive bacteria. pG+host has been adapted with an origin of transfer which allows it to be mobilized in trans by a conjugative helper plasmld derived from broad host range plasmid p1P501. We describe a method, using pG+host, which permits the construction of food grade recombinant strains, le, the integration of foreign DNA (or other modifications) without leaving a selectable marker behind. Using this method, 1-40 % of a bacterial population underwent replacement recombination in the absence of selection for the replacing DNA. With selection, 50-98 % of chromosomal replacements were selected. Possible desirable features of recombinant lactic strains are discussed.