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Infection and Immunity, December 2004, p. 7147-7154, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7147-7154.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

BBE02 Disruption Mutants of Borrelia burgdorferi B31 Have a Highly Transformable, Infectious Phenotype

Hiroki Kawabata,1* Steven J. Norris,2 and Haruo Watanabe1

Department of Bacteriology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan,1 Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, Texas2

Received 7 April 2004/ Returned for modification 11 May 2004/ Accepted 30 June 2004

We constructed highly transformable and infectious Borrelia burgdorferi B31 by inactivating BBE02, a putative restriction-modification gene on the linear plasmid lp25. The low-passage-number B31 clones 5A4 (containing all plasmids) and 5A18 (lp28-4– lp56–) were used for this study, and BBE02 was disrupted by homologous recombination. The transformation efficiency with the shuttle vector pBSV2C03::gnt{Delta}kan was increased from <1 to ~10 colonies per µg of DNA for 5A4 and 5A4 BBE02::Kanr and from 14 to approximately 600 colonies per µg of DNA for 5A18 and 5A18 BBE02::Kanr. lp25, which is required for infectivity in mice, was retained in BBE02 mutants transformed with pBSV2C03::gnt{Delta}kan, but lp25 was not detected in transformants of the parental clones 5A4 and 5A18. BBE02 disruptants and pBSV2C03::gnt{Delta}kan transformants of these clones remained infectious in C3H/HeN mice, and the 50% infective doses of the BBE02 disruptants were <102 organisms per mouse. The inactivation of BBE02 thus eliminates a transformation barrier for infectious B. burgdorferi B31 and will provide a valuable tool for studying the virulence factors of Lyme disease.

* Corresponding author. Mailing address: Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1163. E-mail: kbata{at}nih.go.jp.

Editor: J. T. Barbieri

Infection and Immunity, December 2004, p. 7147-7154, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7147-7154.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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