Figure 1.

Functional interrogation of toxic genes. (A) Endonuclease activity of a gap-residing gene. ORF #2459 in Synechococcus elongatus PCC 7942 was translated in vitro, and the translated product was incubated for 2 h with 1 μg of phage lambda genomic DNA (48.5 kb long). The band pattern on the agarose gel indicates digestion of the lambda DNA. Negative control lanes: lambda DNA incubated with in vitro translation reaction only (+IVT) or not incubated (−IVT). (B) shosA/shosT is a bi-gene operon that undergoes extensive horizontal transfer. The genomic context surrounding the gene pair in seven genomes is shown to be different for every genome, indicative of horizontal gene transfer. Genes are depicted as block arrows. (Blue and red filled arrows) The predicted antitoxin (shosA) and toxin (shosT), respectively. Genes marked by an asterisk are transposase and integrase genes, which are hallmarks of horizontal gene transfer. (C) ShosA/ShosT is a toxin–antitoxin system. The predicted toxin and antitoxin were cotransformed into Escherichia coli BL21(DE3)pLysS on compatible plasmids: the toxin cloned under the control of an IPTG-responsive promoter, and the antitoxin under the control of an arabinose-responsive promoter. Induction of toxin expression (100 μM IPTG) and induction of antitoxin (0.3% arabinose) were performed. Cells do not grow when toxin is induced unless antitoxin is coinduced. Cells without any inducer also do not grow, most probably due to toxin leakage from the IPTG-inducible promoter. (D) ShosT has a bacteriostatic effect. The bacterial OD₆₀₀ was measured over time when only antitoxin was induced (dark blue); only toxin was induced (red); and when antitoxin expression was induced 2.5 h after toxin induction (gray). Each experiment was performed in six repetitions (biological triplicate and technical duplicate).