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Applied and Environmental Microbiology, November 2005, p. 7542-7547, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7542-7547.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

Yue Chen,1* Bruce A. McClane,2 Derek J. Fisher,2 Julian I. Rood,3 and Phalguni Gupta1

Department of Infectious Diseases and Microbiology, Graduate School of Public Health,1 Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania,2 Department of Microbiology, Monash University, Victoria, Australia3

Received 4 January 2005/ Accepted 9 June 2005

In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

* Corresponding author. Mailing address: Department of Infectious Diseases and Microbiology, University of Pittsburgh, 430 Parran Hall, GSPH, 130 DeSoto Street, Pittsburgh, PA 15261. Phone: (412) 624-5398. Fax: (412) 624-4953. E-mail: cheny{at}pitt.edu.

Applied and Environmental Microbiology, November 2005, p. 7542-7547, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7542-7547.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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