The evolutionary fate of duplicate tRNA genes in the bacterium Pseudomonas fluorescens SBW25

The transfer RNA (tRNA) content of cells affects the efficiency of protein synthesis. To study how organisms can adapt to novel translational demands, our laboratory uses two engineered strains of Pseudomonas fluorescens SBW25, each lacking one or more tRNA genes. Previously, we have shown that these slow-growing strains rapidly recover fitness in serial transfer evolution experiments by duplicating large chromosome segments (up to 1 Mb), each containing a small (~100 bp) compensatory tRNA gene. However, while adaptive, these large duplications are mechanistically unstable and are unlikely to persist over longer evolutionary time scales. This project investigates the evolutionary fate of the duplications and the new tRNA gene copies that they contain. It restarted and extended the evolution experiment to 100 transfers (~750 generations) and characterised the evolving lines.
Except for one SNP promoter mutant observed in one line, all the identified mutations compensating the tRNA gene(s) loss were duplication mutations. Within each evolving population, various duplication fragments rapidly arise and compete. Over time, progressively smaller – and mechanistically more stable – duplication fragments arise and dominate in all lines. The smallest is a duplication fragment of only 236 bp, encompassing the compensatory tRNA gene and promoter. Population sequencing revealed the duplication mutants with small duplication fragments were increasing in relative abundance in the population. This suggested they will dominate in the population and may outcompete other less fit mutations.
Together, the results provide a detailed, real-time example of a bacterial tRNA gene set evolving in response to translational challenges. This study investigated the enormously growing interest in how recombination-related mutations shape living organisms in evolution and the evolutionary success of bacteria via changing their gene copy numbers.


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