The fitness effects of spontaneous mutations nearly unseen by selection in a bacterium with multiple chromosomes

Mutation accumulation (MA) experiments employ the strategy of minimizing the population size of evolving lineages to greatly reduce effects of selection on newly arising mutations. Thus, most mutations fix within MA lines independently of their fitness effects. This approach, more recently combined with genome sequencing, has detailed the rates, spectra, and biases of different mutational processes. However, a quantitative understanding of the fitness effects of mutations virtually unseen by selection has remained an untapped opportunity. Here, we analyzed the fitness of 43 sequenced MA lines of the multi-chromosome bacterium Burkholderia cenocepacia that had each undergone 5554 generations of MA and accumulated an average of 6.73 spontaneous mutations. Most lineages exhibited either neutral or deleterious fitness in three different environments in comparison with their common ancestor. The only mutational class that was significantly overrepresented in lineages with reduced fitness was the loss of the plasmid, though nonsense mutations, missense mutations, and coding insertion-deletion mutations were also overrepresented in MA lineages whose fitness had significantly declined. Although the overall distribution of fitness effects was similar between the three environments, the magnitude and even the sign of the fitness of a number of lineages changed with the environment, demonstrating that the fitness of some genotypes was environmentally dependent. These results present an unprecedented picture of the fitness effects of spontaneous mutations in a bacterium with multiple chromosomes and provide greater quantitative support of the theory that the vast majority of spontaneous mutations are neutral or deleterious.