The function and origin of multi-copy tRNA genes in Pseudomonas fluorescens

Transfer RNAs play a central role in protein synthesis. Organisms contain sets of genes that code for different tRNA isotypes, and these sets differ among species and strains. An interesting question is how and why these differences come to exist. This study is focused on answering this question by applying the Pseudomonas fluorescens as a model system. First, in the introduction chapter, a review of the functions and life cycle of a tRNA molecule, and the genetic organization of genes encoding tRNAs in all domains of life, is provided. Next, the tRNA gene sets characteristics in several strains of Pseudomonas fluorescens, with particular focus on three strains with differing degrees of evolutionary divergence (SBW25, A506 and Pf0-1), are discussed for further analyses. By comparing the tRNA gene sets of these strains, the tRNAs present in all three genomes (core tRNAs) and those present in only a subset (accessory tRNAs) are identified. In the next section, the fitness effects of removing accessory tRNAs from individual genomes are investigated. Unexpectedly, the results indicate no significant deleterious effects of removing accessory tRNAs from A506 and Pf0-1. Considering the general tolerance of A506 and Pf0-1 to the deletion of one copy of multi-copy tRNA genes, I conclude that there is a degree of redundancy in tRNA gene sets. This redundancy provides a certain level of robustness to tRNA mutations. I next hypothesized that there is a threshold for this redundancy. By deleting more than one copy of the redundant tRNA-GluUUC and tRNA-GlyGCC genes from the SBW25 background, the threshold was passed, and phenotyping showed a significant fitness defect. This strain was then used as a founder for five independent lineages of an evolution experiment along with five independent wild type control lineages. 1% of each population was passaged daily into fresh medium, for 21 days. The fitness defect was recovered after ~210 generations. To unravel the genetic bases of this fast recovery, the genomic DNA of a single isolate from day 20 of each lineage was subjected to whole genome sequencing. Based on the sequencing analyses, genomic rearrangements, which resulted in duplications as large as 1Mb, are the main molecular mechanisms behind the rapid adaptation. Interestingly, this duplicated region includes a copy of the tRNA-GlyGCC gene in every case. Thus, the duplications have increased the tRNA-GlyGCC gene dosage, likely resulting in a compensatory increase in mature tRNA-GlyGCC. This study has provided a real time example of gene duplication as a mechanism in the evolution of tRNA gene sets.



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