INSERTIONS WITHIN IAP GENE OF LISTERIA-MONOCYTOGENES GENERATED BY PLAMID-PLIV ARE NOT LETHAL

To carry out efficient insertional mutagenesis in Listeria monocytogen es 1040S and to facilitate the characterisation of disrupted genes, th ree 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 applie d to most genes, even those that are essential. The method is unique a nd particularly effective by the use of a temperature-sensitive pE 194 replicon to facilitate the insertion of the gene. After transformatio n of the plasmid into L. monocytogenes it is possible to select for in tegration of the plasmid into the chromosome at 42-degrees-C. High ins ertion frequency and convenience for looking for specific mutations wi th known sites of insertion make them the plasmid derivatives of choic e for insertional mutagenesis in any bacteria that support replication of pE 194 TS. Three insertional mutants of L. monocytogenes are descr ibed. Two insertions were shown to be within iap gene, one in hly gene . The supernatants and the pellets from the iap mutants had no detecta ble haemolytic titer when assayed without the reducing agent.