THE POISON-ANTIDOTE STABILITY SYSTEM OF THE BROAD-HOST-RANGE THIOBACILLUS-FERROOXIDANS PLASMID PTF-FC2

In plasmid pTF-FC2, three small open reading frames (ORFs) are situate d between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by t ests for plasmid stability and host cell growth indicated that the ORF s encoded a poison-antidote plasmid stability system. The three genes were named pasA, pasB and pasC (plasmid addiction system), in which Pa sA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruptio n of the pasA gene resulted in two different spontaneous deletions, wh ich inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter , induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occ urs in the ParD system of R1. In spite of this, the presence of the Pa s poison-antidote system resulted in a relatively modest threefold sta bilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison-antidote plasmid stability module, which appears t o have become integrated within the pTF-FC2 replicon module.