Abstract
Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance when overexpressed supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no effects on stress tolerance, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems’ homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.
Highlights
Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent
In order to analyse the benefits and costs of multitude TA systems, the 13 TA loci were deleted from P. putida PaW85 chromosome in the following order: graTA (PP_1586-1585), res-xre (PP_2433-2434), higBA (PP_1199-1198), hicAB-1 (PP_14801479), relE2-higA2 (PP_5435-PP_0274), mqsRA (PP_4205-4204), mazEF (PP_0770-0771), PP_1716-1717, brnTA (PP_4530-4529), PP_4151-4152, relBE (PP_1268-1267), yefM-yoeB (PP_2940-2939) and relB2-parE (PP_24992500) (Table 1)
The idea of chromosomal TA systems acting as stress response elements and contributing to bacterial stress tolerance has been prevalent in TA-related studies
Summary
Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Pseudomonas putida, a model soil bacterium known for its metabolic versatility and remarkable environmental adaptability[35], is predicted to contain up to 15 chromosomal TA operons[36] Four of these TA loci – graTA, mqsRA, res-xre and mazEF – are verified to comprise functional TA proteins. GraT has ability to affect stress tolerance both positively and negatively, yet deletion of the graTA operon has no effect on P. putida fitness[28]. P. putida mqsRA locus codes for a quite mild toxin, given that the removal of the antitoxin gene mqsA does not significantly affect cell growth[41]. The P. putida MazEF system has been analysed only in mechanistic sense in a study demonstrating that the purified MazF acts as an mRNase which cleaves UAC sequence[43]
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