TRANSPOSON MUTAGENESIS IN ACTINOBACILLUS-PLEUROPNEUMONIAE WITH A TN10DERIVATIVE

A transposon mutagenesis procedure functional in the gram-negative swi ne pathogen Actinobacillus pleuropneumoniae was developed for the firs t time. The technique involved the use of a suicide conjugative plasmi d, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactop yranoside (IPTG)-inducible transposase located outside the mobile elem ent (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:65 57-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal tr ansfer functions provided by the chromosome of the donor strain. When IPFG was present in the mating medium, A. pleuropneumoniae CM5 transpo son mutants were obtained at a frequency of 10(-5), while no mutants w ere detected in the absence of IPTG. Since the frequency of conjugal t ransfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the exp ression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon inserti ons occurred at random, as determined by Southern blotting of chromoso mal DNA of randomly selected mutants and by the ability to generate mu tants defective for the selected phenotypes. Almost all the mutants an alyzed resulted from a single insertion of the Tn10 element. About 1.2 % of the mutants resulted from the cointegration of pLOF/Km into the A . pleuropneumoniae chromosome. The applicability of this transposon mu tagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis sys tem in genetic studies of A. pleuropneumoniae is discussed.