The poison–antidote stability system of the broad‐host‐range Thiobacillus ferrooxidans plasmid pTF‐FC2

Abstract

SummaryIn plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison-antidote plasmid stability system. The three genes were named pasA, pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which 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 occurs in the ParD system of R1. In spite of this, the presence of the Pas poison-antidote system resulted in a relatively modest threefold stabilization 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 to have become integrated within the pTF-FC2 replicon module.

The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins

“…The sequence identity\homology between PasA and ParD were 27\50 % for the N-terminal domain (Arg$-Trp%') and 26\47 % for the whole sequence, respectively. These values differ slightly from the ones published previously [25] due to minor deviations in alignment. Identities and homologies between ParD and members of the ribbon-helix-helix family were calculated and showed surprisingly low sequence identities but high homologies (Figure 4).…”

“…Homology has been reported for ccdA and pemI [40] as well as for mazE (chpAI ) [41], and within the large family of relBE systems [14]. Amongst all the characterized antidote proteins, the closest related to ParD is PasA from the Gram-negative acidophilic bacterium Thiobacillus ferrooxidans [25]. The complete amino acid sequence of PasA (74 residues) can be aligned with that of ParD and, consequently, the ribbon-helix-helix motif is proposed for the N-terminal domain (Figure 4).…”

“…A multiple sequence alignment of ParD, other known members of the ribbon-helix-helix family of proteins and the antidote PasA [25] was generated using CLUSTAL W (version 1.8, June 1999) [26] with the following parameters : weight matrix, blosum ; gap-opening penalty, 15 ; gap-extension penalty, 0.05 ; hydrophilic gaps and residue-specific gap penalties, on ; hydrophilic residues, GPSDQERK. In addition, secondary-structure predictions and database threading were performed using the programs PSIPred [27] and GenTHREADER [28].…”

Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

“…They were firstly found on plasmids and considered prevalently to be associated with stable plasmid inheritance at cell division [9][10][11]. The well-known plasmid-encoded TA loci contained CcdA/ CcdB of F factors [12,13], Kis/Kid of R1 [14], Phd/Doc of P1 [15], ParD/ParE of RK2 [16], pas locus of pTF-CF2 [17], ω-ε-ξ operon of pSM19035 [18], stb locus of pMY-SH6000 [19] and relBE locus of P307 [20].…”

Evolution of compatible replicons of the related IncQ-like plasmids, pTC-F14 and pTF-FC2

“…However, as the RepB primase is essential for IncQ-type plasmid replication, and expression of the repB gene is from the mobB promoter, pTC-F14 is likely to have retained control over its own replication. The role of the pas in the replication of pTC-F14 and pTC-FC2 is unknown and puzzling, as the entire pas of both pTF-FC2 and pTC-F14 (Smith & Rawlings 1997;Deane & Rawlings, 2004) could be deleted without a detectable effect on plasmid replication or copy number. This pasA cross-regulation is the subject of a recent study (Deane & Rawlings, 2004).…”