Finding novel factors affecting the mutation frequency: a case study of tRNA modification enzymes TruA and RluA
Date
2022-11-02
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Bakterid suudavad elada kõikjal, kuid karmide ja muutlike keskkonnatingimustega kohanemiseks on aga vaja geneetilist varieeruvust. Bakterites on selle põhiliseks allikaks mutatsioonid. Evolutsiooni mõistmiseks on vaja selgitada molekulaarseid mehhanisme, mis mõjutavad mutatsioonide tekkesagedust. Käesolevas töös kirjeldasin ja analüüsisin uut testsüsteemi, mis võimaldab tuvastada mutatsioonisagedust mõjutavaid faktoreid bakteriperekonnas Pseudomonas. Kirjeldatud testsüsteemi abil õnnestus mullabakteris Pseudomonas putida tuvastada nii varem kirjeldatud kui ka uusi mutatsioonisagedust mõjutavaid geene. Üllatavaim leid oli tRNA modifikatsiooniensüümide TruA ja RluA mõju mutatsioonisagedusele. tRNAd on väikesed molekulid, mis valgusünteesil kannavad valkude ehituskive ribosoomi. Selleks, et paremini oma funktsiooni täita, on paljud nukleotiidid tRNAdes modifitseeritud. Modifikatsioonidel võib olla palju ülesandeid, näiteks aitavad modifikatsioonid tRNAdel saavutada õiget struktuuri või suurendavad translatsiooni täpsust. TruA ja RluA modifitseerivad U nukleotiidi pseudouridiiniks, tehes seda erineval poolel tRNA antikoodonist. Näitasime, et TruA ja RluA tehtud modifikatsioonide puudumisel suureneb P. putidas mutatsioonisagedus. Mõistmaks paremini nende ensüümide olulisust, analüüsisime translatsiooni täpsust, stressi taluvust, proteoomi ja üldist elulemust P. putida TruA ja RluA defektsetes tüvedes. Lisaks sellele selgitasime võrdlevalt TruA ja RluA rolle ka Pseudomonas aeruginosa ja Escherichia coli rakkudes. Saadud tulemustest on näha, kuidas konserveerunud funktsiooniga valgud võivad põhjustada erinevates bakterites erinevaid fenotüüpe. Samuti ilmestab käesolev töö, kuivõrd mitmekesised ja ootamatud tegurid võivad mõjutada DNAs mutatsioonide teket.
Bacteria can live everywhere. To cope with harsh and everchanging environmental conditions there is a constant need for genetic versatility. Mutations are the main source of genetic versatility in bacteria. To understand the evolution and adaptive abilities of bacteria, it is vital to investigate the processes affecting the mutation frequency. We have created, verified, and applied a new test system for detecting factors affecting the mutation frequency in bacteria from the genus Pseudomonas. By exploiting the new assay, we identified several genes affecting the mutation frequency in the soil bacterium Pseudomonas putida, many of which were not previously associated with the mutation frequency. Most surprising finding was that the tRNA modification enzymes TruA and RluA affect mutagenesis. tRNAs are small adaptor molecules that carry the building blocks of enzymes to the ribosome and thus are essential for translation. To improve their performance, tRNAs are extensively modified. Among other roles, modifications help tRNA’s to achieve the correct structure and improve the translation fidelity. TruA and RluA modify U nucleotide into pseudouridines in the close vicinity of tRNA anticodon. We demonstrated that the lack of TruA- and RluA-catalyzed modifications remarkably increases the mutation frequency in P. putida. To further analyze the importance of the modification enzymes TruA and RluA, we measured the stress tolerance, translation fidelity, protein expression and general fitness of P. putida strains lacking TruA or RluA. For comparison we analyzed the phenotypes caused by TruA and RluA deficiency in Pseudomonas aeruginosa and Escherichia coli cells. Our research demonstrates how an enzyme with conserved function can cause diverse phenotypes in different bacteria. Also, the thesis illustrates how the complex world of DNA mutations can be affected by many nonobvious factors.
Bacteria can live everywhere. To cope with harsh and everchanging environmental conditions there is a constant need for genetic versatility. Mutations are the main source of genetic versatility in bacteria. To understand the evolution and adaptive abilities of bacteria, it is vital to investigate the processes affecting the mutation frequency. We have created, verified, and applied a new test system for detecting factors affecting the mutation frequency in bacteria from the genus Pseudomonas. By exploiting the new assay, we identified several genes affecting the mutation frequency in the soil bacterium Pseudomonas putida, many of which were not previously associated with the mutation frequency. Most surprising finding was that the tRNA modification enzymes TruA and RluA affect mutagenesis. tRNAs are small adaptor molecules that carry the building blocks of enzymes to the ribosome and thus are essential for translation. To improve their performance, tRNAs are extensively modified. Among other roles, modifications help tRNA’s to achieve the correct structure and improve the translation fidelity. TruA and RluA modify U nucleotide into pseudouridines in the close vicinity of tRNA anticodon. We demonstrated that the lack of TruA- and RluA-catalyzed modifications remarkably increases the mutation frequency in P. putida. To further analyze the importance of the modification enzymes TruA and RluA, we measured the stress tolerance, translation fidelity, protein expression and general fitness of P. putida strains lacking TruA or RluA. For comparison we analyzed the phenotypes caused by TruA and RluA deficiency in Pseudomonas aeruginosa and Escherichia coli cells. Our research demonstrates how an enzyme with conserved function can cause diverse phenotypes in different bacteria. Also, the thesis illustrates how the complex world of DNA mutations can be affected by many nonobvious factors.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
bacteria, mutations, enzymes, transport RNA, mutagenicity tests, modifications, genes