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. Bacterial chromosomes contain multiple copies of toxin-antitoxin (TA) gene pairs which code for a toxic protein and an antagonist of the toxin. Chromosomal TA systems most probably originate from plasmids 1-3 , where they contribute to plasmid maintenance during host replication 4. The mechanism behind the plasmid stabilization by TA modules is the unequal stability of the toxin and antitoxin, with the latter being usually less stable than the toxin 5,6. Loss of plasmid and termination of antitoxin production therefore results in release of toxin from the antitoxin-mediated control which ultimately leads to toxin-caused cell death or growth arrest and plasmid-free bacteria are outcompeted from the population 7,8. While the role of TA systems in plasmid addiction is highly recognized, the biological importance of chromosomal TA loci has remained enigmatic despite years of extensive research. Several functions have been attributed to the chromosomal TA systems including stabilization of genomic mobile elements 9,10 , defence against bacteriophages 11,12 as well as other invading mobile DNA 13 and modulation of bacterial stress tolerance 14. However, there are also a lot of contradictions and discrepancy between different studies. For example, chromosomal TA loci were considered to be the major players in formation of metabolically dormant and antibiotic tolerant persister cells but recently this concept was rebutted 15-17. Also, despite multiple studies show that TA toxins can affect the stress tolerance 18-22 , these studies mostly involve artificial overexpression of the toxin which will likely never occur from the single-copy chromosomal TA locus. It is also important to note...

The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly understood. Here, we used whole-cell proteomics to study the cellular effects of the Pseudomonas putida toxin GraT that is known to inhibit growth and ribosome maturation in a cold-dependent manner when the graA antitoxin gene is deleted from the genome. Proteomic analysis of P. putida wild-type and ΔgraA strains at 30 °C and 25 °C, where the growth is differently affected by GraT, revealed two major responses to GraT at both temperatures. First, ribosome biogenesis factors, including the RNA helicase DeaD and RNase III, are upregulated in ΔgraA. This likely serves to alleviate the ribosome biogenesis defect of the ΔgraA strain. Secondly, proteome data indicated that GraT induces downregulation of central carbon metabolism, as suggested by the decreased levels of TCA cycle enzymes isocitrate dehydrogenase Idh, α-ketoglutarate dehydrogenase subunit SucA, and succinate-CoA ligase subunit SucD. Metabolomic analysis revealed remarkable GraT-dependent accumulation of oxaloacetate at 25 °C and a reduced amount of malate, another TCA intermediate. The accumulation of oxaloacetate is likely due to decreased flux through the TCA cycle but also indicates inhibition of anabolic pathways in GraT-affected bacteria. Thus, proteomic and metabolomic analysis of the ΔgraA strain revealed that GraT-mediated stress triggers several responses that reprogram the cell physiology to alleviate the GraT-caused damage.

14Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, 15 despite extensive studies in the last decade, their biological importance remains ambivalent. The ability 16 of TA-encoded toxins to affect stress tolerance supports the hypothesis of TA systems being associated 17 with stress adaptation. However, the deletion of TA genes has usually no fitness consequences, 18 supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of 19 chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer 20 fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, 21 persistence and biofilm formation. Our results instead show that TA loci are costly and decrease the 22 competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain 23 conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for 24 toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or 25 moderately potent proteins. Analysis of P. putida TA systems' homologs among fully sequenced 26 Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems 27 spread among bacteria by horizontal gene transfer.28

Chaperone proteins are crucial for proper protein folding and quality control, especially when cells encounter stress caused by non-optimal temperatures. DnaK is one of such essential chaperones in bacteria. Although DnaK has been well characterized, the function of its intrinsically disordered C-terminus has remained enigmatic as the deletion of this region has been shown to either enhance or reduce its protein folding ability. We have shown previously that DnaK interacts with toxin GraT of the GraTA toxin-antitoxin system in Pseudomonas putida. Interestingly, the C-terminal truncation of DnaK was shown to alleviate GraT-caused growth defects. Here, we aim to clarify the importance of DnaK in GraT activity. We show that DnaK increases GraT toxicity, and particularly important is the negatively charged motif in the DnaK C-terminus. Given that GraT has an intrinsically disordered N-terminus, the assistance of DnaK is probably needed for re-modelling the toxin structure. We also demonstrate that the DnaK C-terminal negatively charged motif contributes to the competitive fitness of P. putida at both high and optimal growth temperatures. Thus, our data suggest that the disordered C-terminal end of DnaK enhances the chaperone functionality.