VERSATILE SUICIDE VECTORS WHICH ALLOW DIRECT SELECTION FOR GENE REPLACEMENT IN GRAM-NEGATIVE BACTERIA

A set of vector plasmids which greatly facilitate gene replacement and reverse genetics in many Gram-negative bacteria was constructed. These vectors are based on the P15A origin of replication (ori) and incorporate sacB from Bacillus subtilis, which is inducible by sucrose and is lethal when expressed in Gram-negative bacteria. The vectors also have a convenient antibiotic-resistance market (gentamicin resistance) and the lacZalpha system which allows blue/white selection of cloned fragments. Three different multiple cloning sites, allowing several distinct cloning and gene replacement strategies, are available in the 5' end of lacZ on different vectors. One of these cloning sites, which we synthesised, contains only a NotI-SmaI-NotI sequence; this allows access to most of the restriction sites within the cloned fragment for the purpose of insertion of various cassettes and interposons. The vectors carry the mob region from the broad-host-range plasmid RP4 and are thus mobilisable by conjugation into a wide range of Gram-negative bacteria; since they will not replicate in bacteria other than enterobacteria, they function as 'suicide' vectors. Variants of the vectors carrying the phage lambda cos site were also constructed. We have used these vectors to carry out gene replacement experiments in the fixN region of Rhizobium leguminosarum and have demonstrated that they are extremely useful in eliminating long and tedious screening procedures.