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Applied and Environmental Microbiology, May 2004, p. 3158-3162, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3158-3162.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

Isaac K. O. Cann,{dagger} Kensey R. Amaya,{dagger} Maurice W. Southworth, and Francine B. Perler*

New England Biolabs, Inc., Beverly, Massachusetts 01915

Received 3 September 2003/ Accepted 21 January 2004

A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.

* Corresponding author. Mailing address: New England Biolabs, Inc., 32 Tozer Rd., Beverly, MA 01915. Phone: (978) 927-5054, ext. 326. Fax: (978) 921-1350. E-mail: perler{at}neb.com.

{dagger} Present address: Laboratory of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

Applied and Environmental Microbiology, May 2004, p. 3158-3162, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3158-3162.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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