Direct selection of IS903 transposon insertions by use of a broad-host-range vector: Isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequen t transposition events. We have developed an IS903-based transposon mutagen esis system for diverse gram-negative bacteria that is not limited by trans fer efficiency, The transposon, IS903 phi kan, carries a cryptic kart gene, which can be expressed only after successful transposition. This allows th e stable introduction of the transposon delivery vector into the host. Gene ration of insertion mutants is then limited only by the frequency of transp osition. IS903 phi kan was placed on an IncQ plasmid vector with the transp osase gene located outside the transposon and expressed from isopropyl-beta -D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase indu ction, IS903 phi kan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903 phi kan to isolate th ree catalase-deficient mutants of the periodontal pathogen Actinobacillus a ctinomycetemcomitans from a library of random insertions. The mutants displ ay increased sensitivity to hydrogen peroxide, and all have IS903 phi kan i nsertions within an open reading frame whose predicted product is closely r elated to other bacterial catalases. Nucleotide sequence analysis of the ca talase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the co re DNA uptake signal sequence for natural transformation of Haemophilus inf luenzae. Our results demonstrate the utility of the IS903 phi kan mutagenes is system for the study of A. actinomycetemcomitans. Because IS903 phi kan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.