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 infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
Thomson
V J
VJ
Molecular Genetics Program, Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York 12208, USA.
Bhattacharjee
M K
MK
Fine
D H
DH
Derbyshire
K M
KM
Figurski
D H
DH
D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D002374:Catalase / Q000235:genetics*; D004251:DNA Transposable Elements; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010940:Plant Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D004251:DNA Transposable Elements; C081231:KATA protein, Arabidopsis; D010940:Plant Proteins; D002374:Catalase