Novel Toxin-Antitoxin System Composed of Serine Protease and AAA-ATPase Homologues Determines the High Level of Stability and Incompatibility of the Tumor-Inducing Plasmid pTiC58

Abstract

Stability of plant tumor-inducing (Ti) plasmids differs among strains. A high level of stability prevents basic and applied studies including the development of useful strains. The nopaline type Ti plasmid pTiC58 significantly reduces the transconjugant efficiency for incoming incompatible plasmids relative to the other type, such as octopine-type plasmids. In this study we identified a region that increases the incompatibility and stability of the plasmid. This region was located on a 4.3-kbp segment about 38 kbp downstream of the replication locus, repABC. We named two open reading frames in the segment, ietA and ietS, both of which were essential for the high level of incompatibility and stability. Plasmid stabilization by ietAS was accomplished by a toxin-antitoxin (TA) mechanism, where IetS is the toxin and IetA is the antitoxin. A database search revealed that putative IetA and IetS proteins are highly similar to AAA-ATPases and subtilisin-like serine proteases, respectively. Amino acid substitution experiments in each of the highly conserved characteristic residues, in both putative enzymes, suggested that the protease activity is essential and that ATP binding activity is important for the operation of the TA system. The ietAS-containing repABC plasmids expelled Ti plasmids even in strains which were tolerant to conventional Ti-curing treatments.

A cooperative virulence plasmid imposes a high fitness cost under conditions that induce pathogenesis

“…Stable vertical transmission of these plasmids depends upon an efficient partitioning system involving the RepA and RepB proteins, which physically links replication of the plasmid to the process of cell division [35]. In addition, recent work has identified a toxin-antidote system on pTiC58 that further enhances the stability of this Ti plasmid [36]. Cells that lose the plasmid after segregation are often killed owing to poor stability of the antidote and unconstrained toxin activity [37].…”

The ParE2–PaaA2 toxin–antitoxin complex fromEscherichia coliO157 forms a heterodocecamer in solution and in the crystal

“…This toxin-neutralizing segment binds to or even complements the fold of the toxin and inhibits its biochemical activity (Kamada et al, 2003;Kamada & Hanaoka, 2005;Garcia-Pino et al, 2008;Li et al, 2009;De Jonge et al, 2009;Brown et al, 2009). This activity depends on the family of toxin considered and may include poisoning DNA gyrase (Bernard & Couturier, 1992;Jiang et al, 2002), cleaving RNA in a ribosome-dependent or independent fashion (Zhang et al, 2003;Zhang & Inouye, 2011), direct ribosome inhibition (Liu et al, 2008), chemical modification of ribosomes (Vesper et al, 2011), modifying initiator tRNA or a number of other activities that directly alter the basic physiology of the cell (Yamamoto et al, 2009;Tan et al, 2010;Mutschler et al, 2011).…”

An efficient stress-free strategy to displace stable bacterial plasmids

“…By contrast, the pCURE approach is highly effectively and does not appear to involve loss of viability of the bacteria due to the displacement at any stage. The recent report of coupling the new Ti plasmid PSK system to the previously described replication-based displacement vector (9,45) appears to corroborate this as an effective approach.…”

“…Therefore, to improve a plasmid displacement strategy based on incompatibility, we proposed that the curing vector should be supplemented with the plasmid's PSK system's antitoxins or anti-sense RNA genes. This point does not seem to have been generally recognized in the design of plasmid displacement strategies to date, although while this manuscript was being prepared, a paper that implicitly covers this point with respect to the Agrobacterium tumefaciens Ti plasmid was published (9). …”