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Applied and Environmental Microbiology, July 2004, p. 4136-4143, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4136-4143.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of the Riboflavin Synthase Gene (ribC) as a Model for Development of an Essential Gene Disruption and Complementation System for Haemophilus influenzae

Amna Saeed-Kothe,1,{dagger} Wei Yang,2 and Scott D. Mills2*

Department of Biology, Northeastern University, Boston, Massachusetts 02115,1 Molecular Sciences, AstraZeneca R&D Boston, Waltham, Massachusetts 024512

Received 21 January 2004/ Accepted 5 April 2004

We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20. We constructed two "suicide" complementation vectors (pASK5 and pASK6) containing 5' and 3' regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of H. influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of ribC for survival of H. influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain with ribC deleted.

* Corresponding author. Mailing address: AstraZeneca R&D Boston, 35 Gatehouse Dr., Waltham, MA 02451. Phone: (781) 839-4619. Fax: (781) 839-4570. E-mail: scott.mills{at}astrazeneca.com.

{dagger} Present address: Biogen, Inc., Cambridge, MA 02142.

Applied and Environmental Microbiology, July 2004, p. 4136-4143, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4136-4143.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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