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Journal of Bacteriology, December 1999, p. 7298-7307, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans

Valeri J. Thomson,^1, Mrinal K. Bhattacharjee,² Daniel H. Fine,³ Keith M. Derbyshire,^1,* and David H. Figurski²

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 12208¹; Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032²; and Department of Oral Pathology and Biology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103³

Received 14 July 1999/Accepted 24 September 1999

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, IS903kan, 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. IS903kan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl--D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903kan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903kan 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 IS903kan 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 IS903kan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903kan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

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^* Corresponding author. Mailing address: Wadsworth Center, David Axelrod Institute, 120 New Scotland Ave., Albany, NY 12208. Phone: (518) 473-6079. Fax: (518) 486-7971. E-mail: keith.derbyshire{at}wadsworth.org.

Present address: Department of Biology, Bard College, Annandale-on-Hudson, NY 12504-5000.

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Journal of Bacteriology, December 1999, p. 7298-7307, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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