5920 Nucleic Acids Research, 2006, Vol. 34, No. 20 Table 2. Numbers of hok/sok, ldr and protein-regulated TA loci in enteric bacteria1 hok genes ldr genes protein-regulated TA loci2 Organism E.coli K-12 E.coli CFT073 E.coli 0157 H7 E.coli 0157 H7 EDL933 S.flexneri 2a 301 S.flexneri 2a 2457T Salmonella typhi S.typhi Ty2 Salmonella enterica paratypi S.typhimurium LT2 Photorhabdus luminescens E.carotivora atroseptica Vibrio vulnificus CMCP6 chrII Total number of genes 5 5 13 13 10 9 1 0 0 0 3 1 1 61 4 3 3 4 5 3 2 2 2 2 0 0 0 30 5 5 8 8 3 2 6 6 4 8 50 14 1 120 1V.vulnificus is closely related to enterobacteria but not classified as such. 2Seven gene-families of protein-regulated TA loci are known: relBE, higBA, parDE, mazEF, phd/doc, vapBC and ccdAB. competition for Sok-RNA in cells requires an excess of PNA relative to hok mRNA. Such an excess is likely to exist as hok genes are driven by weak promoters and PNA appears to accumulate in cells and is not removed by drug efﬂux pumps (30). Enterobacteria contain a large numbers of hok/sok and ldr toxin loci that are regulated by antisense RNAs Given the efﬁcient killing of E.coli via activation of hok described above, we found it interesting to describe in detail the phylogenetic distribution of antisense RNA-regulated toxin-encoding genes. E.coli K-12 encodes ﬁve hok/sok loci (32) and four ldr (long direct repeats) loci. ldr loci are analo- gous (but not homologous) to hok/sok loci and encode a small toxin gene of 30 codons, a small cis-encoded antisense RNA that represses translation of the toxin gene. Similar to hok/sok genes, the toxin-encoding mRNA is stable and the antisense RNA is unstable (33). We searched 218 fully sequenced genomes exhaustively for the presence of hok and ldr genes using an approach described previously (34). An overview of the results is shown in Table 2. Our analysis shows that ldr loci are found only in a narrow spectrum of enteric bacteria, including E.coli and Salmonella and Shigella ﬂexneri species, whereas hok/sok loci are found in a broader spectrum of enterobacteria, in one Vibrio species, and in one of four Salmonella species examined. In contrast, protein- regulated TA genes (e.g. relBE, mazEF etc.) have a much broader phylogeny (34). We conclude that ldr and hok/sok loci are conﬁned to enteric and closely related bacteria. The genomic localizations of hok/sok and ldr loci on two E.coli strains and 10 additional enteric pathogen species are shown in Figure 6 and Supplementary Figure S1. For comparison, the locations of protein-regulated TA loci were also included. The ldr loci tend to cluster, often on directly opposite chromosomal locations (i.e. in the four E.coli spe- cies and in one S.ﬂexneri species). In contrast, hok/sok and protein-regulated TA loci were almost randomly scattered on the chromosomes, except in the case of Photorhabdus hokA ldrD mazEF-1 mazEF-2 E.coli K-12 4.6 MB hokC (gef) relBE-2 hokE hok-13 ldr-3 ldrA ldrB ldrC hokB hokD mazEF-1 relBE-1 hok-12 relBE-3/yoeB mazEF-2 hok-1 E.coli 0157 H7 5.5 MB ccdAB-1 relBE-1 parDE-1 hok-2 hok-3 parDE-2 hok-4 hok-5 hok-6 ldr-1 ldr-2 hok-7 hok-8 hok-9 higBA-1hok-11 hok-10 parDE-3 Figure 6. Localization of hok/sok (red) and the hok/sok-like ldr loci (green) on two E.coli chromosomes. Protein-regulated TA loci (blue) were included for comparison (34). The genes were found using tblastn (Supplementary Table S1) and annotated with VectorNTI. luminiscens, which has three clustered hok/sok loci and a bewilderingly large number of protein-regulated TA loci (50), many of which are clustered at the terminus (terC) region. In E.coli K-12, protein-regulated TA loci function as stress-response elements that regulate cellular metabolism in starvation conditions or as genetic stabilization elements that increase the genetic stability of the chromosome segments on which they reside (35). The cellular functions of the chromosome-encoded hok/sok and ldr loci are not yet known but their presence in large numbers is consistent with a role as genetic stabilization elements (20). DISCUSSION Three conclusions can be drawn from the results of these experiments. First, the data support the sense/antisense model for hok mRNA::Sok-RNA interactions. Second, anti Sok PNAs are bactericidal to E.coli containing a hok/ sok plasmid. Third, the results demonstrate that antisense agents can be used as competitive inhibitors to study RNA::RNA interactions in bacteria. Furthermore we extend the previous knowledge of RNA TA systems distribution in Enterobacteria. The hok/sok TA system is a paradigm for sense/antisense RNA interactions (Figure 1). Here we show that anti Sok PNAs selectively kill hok/sok-carrying E.coli cells. Also, anti Sok PNAs induced ‘ghost’ cell morphology and accumu- lation of the mature, 30 end truncated hok mRNA. All of these effects are characteristic of Hok toxin-mediated cell killing. Together, the results support the model of sense/antisense RNA interaction in the hok/sok TA plasmid stability system in cells. The presence of hok and other toxin-encoding genes in many free-living enteric bacteria (Figure 6, Supplementary Figure S1 and Table S1) provides possibilities to induce bacterial suicide. The basic idea is to target the antitoxin inside cells to release expression or activity of the endoge- nous toxin. The cell-killing activity of Hok toxin is well established, and puriﬁed Hok toxin is bactericidal when electroporated into E.coli (36). Therefore, hok induction should kill E.coli. The challenge is to develop molecules that can enter E.coli and release hok mRNA for translation.