On 15 November at 10:15 Andres Ainelo will defend his doctoral thesis „Physiological effects of the Pseudomonas putida toxin GraT”.
Senior Research Fellow in Genetics Rita Hõrak, Institute of molecular and cell biology, University of Tartu
Professor of Molecular Biology Jaanus Remme, Institute of molecular and cell biology, University of Tartu
Associate Professor Ditlev Egeskov Brodersen, PhD, Molecular biology and genetics, Aarhus University
Free-living bacteria must cope with very different environmental conditions. This is enabled by many systems that control their growth and metabolism. Toxin-antitoxin (TA) systems are thought to be a peculiar example of such regulators. These usually consist of two small proteins: the toxin blocks vital cellular processes but is normally inhibited by antitoxin binding. To understand the importance of a TA system in bacterial life, it is necessary to pinpoint the toxin’s molecular target and to also describe the physiological response to the toxicity.
This thesis aimed to identify the target of GraT, a TA toxin in the soil bacterium Pseudomonas putida, and to study the toxin’s effect on bacterial physiology. GraT is an intriguing toxin, as its effect is more pronounced at lower growth temperatures. This work identified that GraT degrades translated mRNAs. Structural analysis of GraT suggests that, atypically for TA toxins, the first one fifth of the protein is disordered. Additionally, it was seen that the chaperone DnaK enhances GraT toxicity, which may be linked to folding the GraT disordered region.
The physiological effects of GraT were studied in a model where the toxin is produced from its native locus that lacks the antitoxin gene. Compared to the common systems of artificial toxin overexpression, the applied model is a better estimation of natural toxin activation. GraT was found to have a dual effect on P. putida stress tolerance: the bacterium is more sensitive to some stressors while more resilient against others. Additionally, GraT inhibits ribosome biogenesis, which has not been described before for similar toxins. Analysing the protein composition of cells growing under GraT stress showed that bacteria work to alleviate GraT toxicity by upregulating ribosome assembly factors and downregulating carbon metabolism. Even though the role of GraT in P. putida biology is still uncertain, the thesis described several GraT features unusual for TA toxins and thus enriched our knowledge of TA systems’ diversity.