5916 Nucleic Acids Research, 2006, Vol. 34, No. 20 plasmid, Sok-RNA binds to the mature form of hok mRNA and represses translation by preventing ribosome entry (15–17). In addition, formation of the hok mRNA::Sok- RNA duplex leads to rapid RNase III-mediated degradation (scavenging) of the mature and translatable form of hok mRNA (18). In cases where the plasmid is lost, the Sok-RNA pool is depleted through rapid decay and this frees hok mRNA for translation and toxin production kills the cell (19). Hok toxin causes dramatic changes in cell morphology, resulting in characteristic ‘ghost cells’, where the cell poles appear dense relative to the cell centre (9,10). Therefore, the hok/sok system provides plasmid stability through post-segregational killing of plasmid free progeny. This mechanism of controlled expres- sion of Hok toxin confers increased plasmid maintenance, and this may provide a net beneﬁt to cells by maintain- ing plasmid encoded virulence or stress resistance traits. A simpliﬁed model of the hok/sok system is shown in Figure 1. Additional details of the hok/sok system and other TA systems were reviewed by Gerdes and co- workers (20). We aimed to test the sense/antisense pairing model for hok mRNA::Sok-RNA interactions in cells and also test possibili- ties to induce Hok-mediated killing using an antisense agent. Short synthetic peptide nucleic acid (PNA) oligomers complementary to Sok sequences were designed to act as competitive inhibitors of sense/antisense interactions. Anti Sok PNAs added to growing cells caused growth arrest and were bactericidal. Also, PNA-treated cells showed the ‘ghost’ cell morphology and RNA maturation changes characteristic of Hok toxin-mediated cell killing. Finally, using database searches, we revealed a large number of hok/sok and another family of antisense RNA-regulated toxin genes, ldr, in enteric bacterial genomes. MATERIALS AND METHODS Bacteria, plasmids and PNAs Escherichia coli K-12 strain CSH50 was used as host and transformed with two plasmids with and without hok/ sok genes (pPR95 and pOU82, respectively) (Table 1). Anti Sok and control PNAs were synthesized and high- performance liquid chromatography puriﬁed by Oswel Ltd (UK) (21) (Table 1). All PNAs were synthesized with an attached peptide (KFFKFFKFFK) to enhance cell uptake (22). Bacterial growth and minimal inhibitory concentration (MIC) measurement Bacteria were grown in Mueller Hinton (MH) broth containing ampicillin (30 mg/ml) under aeration to mid-log phase of growth. Inoculums from mid-log phase cultures were used to initiate cultures at 105 c.f.u./ml in the wells of ultra low attachment 96-well microplate (Costar 3474, Corn- ing incorporated, NY) and incubated with PNA at 37 C for 20 h. The lowest PNA concentrations that prevented measur- able cell growth after 20 h were scored as the MIC. C.f.u. were determined by serial dilutions of cell culture with PBS followed by plating on LB plates. Figure 1. Schematic model of the hok/sok TA system and the Sok-RNA inhibition strategy used in this study. (A) Full-length hok mRNA folds into a compact form in which the 50 and 30 ends of the molecule pair. The ends of the molecule make an exact match, thus generating a highly folded and ‘blunt-ended’ RNA structure. The sequestering of the mRNA 30 end reduces the rate of 30-processing by polynucleotide phosphorylase and ribonuclease II (15). However, the 30 exoenzymes removes the terminal 39 nt at the 30 end of hok mRNA at a low rate. The arrow-head points to the bottom of the stem– loop structure, which acts as a ‘road-block’ for the 30 end trimming. (B) Via its 50 end single-stranded tail, Sok-RNA (shown in red) recognises a single- stranded stem–loop present only in the truncated, refolded hok mRNA. This is because the 30 trimming of full-length hok mRNA releases the very 50 end of the mRNA and this release triggers a major refolding of the mRNA 50 end that results in the formation of the antisense RNA binding stem–loop structure (26,27). The refolded isoform of the mRNA is metabolically very stable and binds Sok-RNA avidly, but can also be bound by ribosomes and therefore be translated (26). In the presence of excess Sok-RNA rapid binding of the antisense RNA prevents ribosome binding to hok mRNA and thus prevents its translation (12,26). Eventually, the hok mRNA::Sok-RNA duplex is formed and then rapidly cleaved (scavenged) by RNase III (18). Therefore, the truncated form of hok mRNA does not accumulate in Sok-RNA containing cells. The RNase III cleavage of the hok mRNA::Sok-RNA duplex is not required for inhibition of hok mRNA translation (41). (C) Sok-RNA inhibition strategy. PNAs complementary to the single-stranded 50 end of Sok-RNA (shown in blue) prevent Sok-RNA binding to hok mRNA and thereby induce hok translation, synthesis of the lethal Hok protein and cell killing. Microscopy Cells morphology was analysed by phase-contrast microscopy (Zeiss) at 1000· total magniﬁcation. Prior to microscopy, cultures were treated with 8 mM PNA or 150 mg/ml rifampicin for 3 h and then collected by centrifugation. Digi- tal images were captured using a CCD camera and Openlab imaging software, version 3.1.5. (Improvision Ltd). Northern analysis of mRNA maturation Total RNA was extracted from mid-log growth phase cultures using the acid-phenol method as described (23). RNA was fractionated in a 6% polyacrylamide/urea/TBE gel and electrotransferred onto a GeneScreen Plus nylon membrane (Perkin Elmer, USA). Transcripts containing hok sequences