TOXIN-ANTITOXIN MODULES IN SYMBIOTIC NITROGEN-FIXING SOIL BACTERIA

One of the major limiting nutrients for plant growth is utilizable nitrogen in the environment. Acquisition and assimilation of nitrogen is therefore second in importance only to photosynthesis. Soil bacteria belonging to Rhizobiaceae are able to form a symbiotic relationship with leguminous plants, and in new plant organs, the root nodules, reduce the most abundant nitrogen source, the atmospheric nitrogen to ammonia. Host plants utilize the fixed nitrogen and in turn, provide carbon source and energy for nitrogen fixing bacteroids.
We investigate the presence and role of toxin-antitoxin (TA) systems in rhizobia under conditions of free-living state and during symbiosis with host plants. TA modules may participate in the adjustment of bacterial metabolism to varying environmental conditions. Moreover, the drastic physiological changes during the transition from free-living to symbiotic state may also require the active contribution of TA modules to metabolic regulation.

Toxin-antitoxin (TA) modules consisting of two partially overlapping genes are ubiquitous among bacteria and archaea. TA systems encode proteins that form a complex acting as repressors for the TA operons. The mechanism of action of TA modules is based on the different stabilities of toxin and antitoxin proteins. Signals triggered by various stress factors lead to a decrease in the amount of labile antitoxin, thus the free stable toxin exerts its effect on various cellular targets. The physiological function of chromosomally located toxins is still controversial. Certain toxins, when activated by stress conditions induce significant loss of viability leading to programmed cell death. Other observations demonstrated that ectopic expression of toxins resulted in bacteriostasis rather than bactericidal effect. TA loci were also shown to participate in bacterial persistence and biofilm formation.

The proposed role of TA loci as general stress managers adjusting the metabolic rates under varying environmental stimuli may be of special importance during the adaptation of soil bacteria to oligotrophic conditions. In addition, symbiotic nitrogen fixing soil bacteria, which develop an intimate interaction with leguminous plants also have the ability to adapt and function within the plant host cells during symbiosis.

Our aim is to investigate the presence and role of TA systems in Sinorhizobium meliloti and Bradyrhizobium japonicum, the microsymbionts of two agriculturally important crops, alfalfa and soybean, respectively. We have shown that the ntrPR operon of Sinorhizobium meliloti represents a vapBC-type TA system, which is the most abundant group of the seven typical TA gene families. Insertion of transposon Tn5 into the ntrR gene resulted in increased transcription of both nodulation genes (responsible for the production of bacterial nodulation signals the Nod factors) and nitrogen fixation genes determining the enzyme nitrogenase. Moreover, the dicarboxylate transport system providing carbon source for bacteroids in nodules during symbiosis was also expressed at an increased level. As a result, alfalfa plants inoculated by this mutant strain had increased nitrogen content and biomass production as compared to those of the plants inoculated by the wild-type strain.

TA modules are surprisingly abundant in bacterial genomes. They are present in the chromosomes of almost all prokaryotes, often in very high numbers. Interestingly, obligate intracellular organisms have no functional TA loci, suggesting that these organisms which multiply under constant environmental conditions do not require TA modules as stress response mechanisms. Sinorhizobium meliloti also carries multiple copies of TA modules representing different TA families. These loci are distributed on the chromosome as well as on the megaplasmids of Sinorhizobium meliloti. Some of them show high sequence homology to the ntrPR operon; however, our preliminary data suggest that these additional copies are involved in the control of metabolic functions other than the regulation of nodulation and nitrogen fixation gene expression. Our aim is to determine the importance and role of TA copies, their possible cooperation, and their target sites in the cell. We investigate the molecular mechanism of the toxin molecules and the effect of TA systems on the stress tolerance of bacteria under free-living and symbiotic conditions. Understanding their function may help to improve the efficiency of the symbiotic nitrogen fixation interaction with host plants as was already demonstrated for the ntrPR operon in Sinorhizobium meliloti.