Published online 25 October 2006 Nucleic Acids Research, 2006, Vol. 34, No. 20 5915–5922 doi:10.1093/nar/gkl750 Competitive inhibition of natural antisense Sok-RNA interactions activates Hok-mediated cell killing in Escherichia coli Omid R. Faridani, Abbas Nikravesh, Deo Prakash Pandey1, Kenn Gerdes2,3 and Liam Good* Department of Cell and Molecular Biology, Karolinska Institutet, Berzelius va¨ g 35, 171 77, Stockholm, Sweden, 1De´ partement de Biologie Cellulaire Universite´ de Gene` ve, Sciences III 30 Quai Ernest-Ansermet 1211 Gene` ve 4, Switzerland, 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark and 3Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, NE2 4HH, UK Received August 11, 2006; Revised and Accepted September 19, 2006 ABSTRACT Short regulatory RNAs are widespread in bacteria, and many function through antisense recognition of mRNA. Among the best studied antisense transcripts are RNA antitoxins that repress toxin mRNA translation. The hok/sok locus of plasmid R1 from Escherichia coli is an established model for RNA antitoxin action. Base-pairing between hok mRNA and Sok-antisense-RNA increases plasmid maintenance through post-segregational-killing of plasmid-free progeny cells. To test the model and the idea that sequestration of Sok-RNA activity could provide a novel antimicrobial strategy, we designed anti Sok peptide nucleic acid (PNA) oligomers that, according to the model, would act as competitive inhibitors of hok mRNA::Sok-RNA interactions. In hok/sok-carrying cells, anti Sok PNAs were more bactericidal than rifampicin. Also, anti Sok PNAs induced ghost cell morphology and an accumulation of mature hok mRNA, consistent with cell killing through synthesis of Hok protein. The results sup- port the sense/antisense model for hok mRNA repression by Sok-RNA and demonstrate that anti- sense agents can be used to out-compete RNA::RNA interactions in bacteria. Finally, BLAST analyses of 200 prokaryotic genomes revealed that many enteric bacteria have multiple hok/sok homologous and analogous RNA-regulated toxin–antitoxin loci. Therefore, it is possible to activate suicide in bacteria by targeting antitoxins. INTRODUCTION Non-coding regulatory RNAs are widely expressed in many genomes (1,2). A large number of non-coding RNAs are complementary to active open reading frames, yet there is only limited evidence for direct sense/antisense interactions. Antisense transcripts are encoded both in-cis and in-trans and are believed to modulate RNA processing, decay and translation through direct pairing with complementary target sequences (3). Bacterial genomes and plasmids contain a number of annotated as well as predicted sense and antisense genes. Despite predictions of widespread sense/antisense pairing in several species (4–7), there have been few attempts to experimentally probe these structures and test the effects of disrupted interactions (8). A paradigm for sense/antisense RNA pairing is the hok/sok toxin–antitoxin (TA) plasmid stabilization locus of the R1 plasmid in E. coli (9). The hok/sok locus codes for three genes: hok (host killing) encodes a highly toxic trans- membrane protein that irreversibly damages the cell mem- brane (10). The mok (modulation of killing) reading frame overlaps with hok sequences and is required for hok expres- sion and translation. Finally, the sok (suppression of killing) gene encodes a small antisense RNA in-cis that blocks trans- lation of the mok reading frame and thus inhibits expression of hok mRNA (11). Pairing between Sok and hok transcripts is supported by in vitro and phylogenetic studies (12–14). Sok-RNA is very unstable (half-life in the order of 30 s) but driven by a strong promoter. In contrast, the full-length hok transcript is heavily structured, stable (half-life in the order of 30 min) and inaccessible to either ribosome initiation or Sok-RNA binding. Slow 30 end processing of hok mRNA results in mature transcripts that are accessible for translation and Sok-RNA binding. In the presence of a hok/sok-carrying *To whom correspondence should be addressed. Tel: +46 8 5248 6385; Fax: +46 8 32 39 50; Email: liam.good@ki.se 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.