Physiological effects of the Pseudomonas putida toxin GraT
Kuupäev
2018-09-13
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Looduses peavad bakterid olema võimelised toime tulema keskkonnatingimuste muutumisega üpris laiades piirides. Selles on bakteritele abiks paljud raku elutegevust ja kasvu kontrollivad süsteemid. Eriti omapärasteks bakteri kasvu regulaatoriteks arvatakse olevat toksiin-antitoksiin (TA) süsteemid. Enamasti koosnevad need kahest väikesest valgust: toksiin on võimeline inhibeerima bakteri elutähtsaid protsesse, kuid tavatingimustel takistab seda temaga seondunud antitoksiin. Selleks, et mõista konkreetse TA süsteemi olulisust ja mõju bakterile, on vaja kirjeldada nii toksiini otsest sihtmärki rakus kui ka bakteri füsioloogilist vastust toksiini toimele.
Selle töö eesmärgiks oli tuvastada mullabakteri Pseudomonas putida ühe TA toksiini, GraT, sihtmärk rakus ning kirjeldada toksiini mõju raku elutegevusele. GraT on omapärane toksiin, kuna selle efekt on temperatuuritundlik ning süveneb külmas. Töö tuvastas, et GraT lagundab transleeritavaid mRNA-sid. GraT struktuuri analüüs viitab, et toksiinidele ebatüüpiliselt on valgu esimene viiendik paindlik ja ilma kindla struktuurita. Samuti tuvastati, et GraT toksilisust võimendab teiste valkude voltumist abistav šaperonvalk DnaK, mis võib olla seotud GraT paindliku regiooni voltumisega.
GraT füsioloogilisi efekte jälgiti mudelsüsteemis, kus toksiini toodetakse looduslikust genoomsest lookusest, millest puudub antitoksiin. Võrreldes tihti rakendatavate toksiini kunstliku ületootmise süsteemidega, on kasutatud mudel paremaks lähenduseks toksiini loomulikule aktivatsioonile. GraT mõjul täheldati kahetist efekti bakteri stressitaluvusele: mõningate stressiallikate puhul see suureneb, kuid teiste puhul väheneb. Lisaks häirib GraT ribosoomide biogeneesi, mida pole sarnaste toksiinide kohta varem kirjeldatud. GraT mõju all kasvavate rakkude valgulise koostise analüüs näitas, et bakter üritab GraT toksilisust leevendada, suurendades ribosoomide biosünteesi abivalkude hulka ja vähendades süsinikumetabolismi intensiivsust. Kuigi GraT roll P. putida elus ei ole endiselt selge, kirjeldati GraT puhul mitmeid TA toksiinidele ebaharilikke omadusi, mis täiendavad meie arusaamu nende ebatavaliste regulaatorite mitmekesisusest.
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
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
Kirjeldus
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Märksõnad
bacterial toxins, antitoxins, physiological effect