Rapid mapping of cloned DNA fragments on the Salmonella chromosome.

Genetic analysis in bacteria often involves mapping novel mutations. Traditionally, mapping has been performed using time-consuming methods such as conjugation and transductional analysis. However, an alternative strategy is to use physical mapping using restriction enzymes, which cleave bacterial chromosomal DNA at relatively few sites, generating DNA fragments that can be resolved by pulsed-field gel electrophoresis (PFGE) (6). For example, the Salmonella typhimurium LT2 chromosome can be cut into 11, 23 and 7 fragments using the restriction endonucleases BlnI, XbaI and CeuI, respectively (3,5,11). We and others have used transposons, such as TnphoA (7) and polymerase chain reaction (PCR) with degenerate primers, to identify novel regions of S. typhimurium DNA, for example those required for virulence. As a step towards the full characterization of novel loci, the influence of individual genes within the loci and any polar effects should be fully assessed. To achieve this, it is usually necessary to map the region and construct a series of defined mutations. Here we report a simple technique, particularly applicable to Salmonella, that allows the simultaneous construction of defined mutations and rapid physical mapping of the novel loci within the genome. S. typhimurium C5 TnphoA::440 is a novel, unmapped TnphoA insertion mutant of S. typhimurium C5. Salmonella strains harboring TnphoA::440 are attenuated for virulence in mice (7). The salmonella genomic sequences bordering the TnphoA::440 transposon insertion site of TnphoA::440 were