Insertions within iap gene of Listeria monocytogenes generated by plasmid pLIV are not lethal

To carry out efficient insertional mutagenesis in Listeria monocytogenes 1040S and to facilitate the characterisation of disrupted genes, three novel derivatives of plasmid pACYC 184 were constructed, p-LIV 1, pLIV-2 and pLIV-3. The technique is simple and rapid and can be applied to most genes, even those that are essential. The method is unique and particularly effective by the use of a temperature-sensitive pE 194 replicon to facilitate the insertion of the gene. After transformation of the plasmid into L. monocytogenes it is possible to select for integration of the plasmid into the chromosome at 42 degrees C. High insertion frequency and convenience for looking for specific mutations with known sites of insertion make them the plasmid derivatives of choice for insertional mutagenesis in any bacteria that support replication of pE 194 TS. Three insertional mutants of L. monocytogenes are described. Two insertions were shown to be within iap gene, one in hly gene. The supernatants and the pellets from the iap mutants had no detectable haemolytic titer when assayed without the reducing agent.