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. Thes e vectors are based on the P15A origin of replication (ori) and incorp orate sacB from Bacillus subtilis, which is inducible by sucrose and i s lethal when expressed in Gram-negative bacteria. The vectors also ha ve a convenient antibiotic-resistance market (gentamicin resistance) a nd the lacZalpha system which allows blue/white selection of cloned fr agments. Three different multiple cloning sites, allowing several dist inct 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 acc ess to most of the restriction sites within the cloned fragment for th e purpose of insertion of various cassettes and interposons. The vecto rs carry the mob region from the broad-host-range plasmid RP4 and are thus mobilisable by conjugation into a wide range of Gram-negative bac teria; since they will not replicate in bacteria other than enterobact eria, they function as 'suicide' vectors. Variants of the vectors carr ying the phage lambda cos site were also constructed. We have used the se vectors to carry out gene replacement experiments in the fixN regio n of Rhizobium leguminosarum and have demonstrated that they are extre mely useful in eliminating long and tedious screening procedures.