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.
Agrobacterium tumefaciens strains bearing a tumor-inducing (Ti) plasmid are the etiological agents of crown gall disease. Most genes required for pathogenicity are located on the plasmids (
17,
33). Ti plasmids are kept stable at a low copy number equivalent to that of the chromosomal DNA in the bacterial cells (
32) due to the
repABC locus (
16,
30,
34). The stability of Ti plasmids differs among strains (
11).Many genes for keeping plasmids stable have been reported in eubacteria, and these are divided into three categories based on their mechanism: multimer resolution systems, active partitioning systems, and toxin-antitoxin (TA) systems (
15). Multimer resolution systems increase the number of plasmid molecules by resolving a multimer plasmid into monomers, resulting in a higher probability of plasmid distribution to daughter cells during cell division even when plasmid distribution occurs randomly (
29). Active partitioning systems deliver plasmid copies to each progeny cell at cell division (
21). In the
repABC locus, the RepA and RepB proteins and
parS site(s) ensure stable plasmid inheritance by the active partitioning system (
2). TA systems contribute to plasmid maintenance in cell populations by initiating growth inhibition or death of plasmid-free cells and are widely distributed among eubacterial and archaeal plasmids as well as their chromosomes (
9). Generally, the TA module consists of two genes which encode toxin and antitoxin. The antitoxin neutralizes the action of a cognate toxin by interaction with the toxin or its target molecules. When a plasmid harboring the TA module is lost from a host cell, the antitoxin molecules decrease to an ineffective level because the antitoxin is degraded quickly or diluted by cell division (
15). Thereafter, the toxin exerts its toxicity and inhibits the host cell growth. RNA antitoxins can suppress toxin expression by binding to the toxin mRNA as an antisense RNA or repress toxicity effects by an unknown mechanism (
6,
4). In pTi-SAKURA, the Ti plasmid in the
A. tumefaciens strain MAFF301001, it was shown that the
tiorf24 and
tiorf25 module increased plasmid stability by the TA mechanism (
40; also S. Yamamoto, unpublished data).Differences in Ti plasmid stability are critical for plasmid engineering and evolution (
33). However, little is known about the stability factors of Ti plasmids other than the
repABC locus. In our previous study (
40),
tiorf24 and
tiorf25 were shown to increase the segregational stability and incompatibility of Ti plasmids and reduce the efficiency of transconjugants by the introduction of incompatible plasmids into host cells. The two genes are located 2.5-kbp downstream of
repABC (
8). Generally, incompatibility has been defined as a situation where two plasmids contain a related replication and/or partitioning system and are unable to exist in a cell simultaneously without external selection (
1).
A. tumefaciens strain C58, which contains a Ti plasmid pTiC58, allows entry of an incompatible
repABC plasmid into the cell 60-fold less efficiently than a derivative of C58. The derivative of C58 harbors a small
repABC vector instead of pTiC58 (
40). This suggests the presence of incompatibility-enhancing genes on pTiC58.In this study, we located the responsible genes in pTiC58 and found that the novel genes
ietA and
ietS enhance the incompatibility and stability of the plasmid by the TA mechanism.
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