A mutational hotspot that determines highly repeatable evolution can be built and broken by silent genetic changes

Abstract

Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work, we reveal that a near-deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observe this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determine that this evolutionary disparity is owed to just 6 synonymous variations within the ntrB locus, which we demonstrate by swapping the sites and observing that we are able to both break (>95% to 0%) and build (0% to 80%) a deterministic mutational hotspot. Our work reveals a key role for silent genetic variation in determining adaptive outcomes.

A Near-Deterministic Mutational Hotspot in Pseudomonas fluorescens Is Constructed by Multiple Interacting Genomic Features

“…The frequency of the SNP dropped from 95.2% in AR2 to 50% in AR2 miniTn7[ ntrBC -Lag], yielding a significantly different mutation spectrum overall ( P = 0.0015, Chi-square test). Furthermore, local nucleotide context continued to play a major role in mutation frequency at this site, as the introduction of six synonymous changes around position 289 in the novel genomic context (closer to OriC) generated a similar effect to that observed previously ( Horton et al 2021 ), with A289C frequency falling from 50% to 0% ( fig. 2 B ).…”

“…Contrary to the expectation that mutations simply provide a random supply of genetic diversity for selection to act upon, differences in mutation rates across genomic position ( Monroe et al 2022 ) and mutation types ( Cano et al 2022 ) can introduce bias into the mutational spectra that has been shown to shape adaptive trajectories. In some circumstances, such biases can be strong enough that a particular position mutates at a rate far higher than expected by chance, generating what is referred to as a mutational hotspot ( Zhang et al 2018 ; Horton et al 2021 ). Mutation bias is of key interest to those wishing to make forecasts of adaptive evolution, as mutational hotspots can facilitate highly repeatable, and by extension reliably predictable, adaptive outcomes.…”

“…In previous work, we found that a mutational hotspot drove repeatable outcomes in immotile variants of Pseudomonas fluorescens SBW25 (denoted AR2; see Alsohim et al 2014 ; Taylor et al 2015 ). During the re-evolution of flagellar motility, >95% of replicate lines realized an identical de novo single nucleotide polymorphism (SNP), A289C in the ntrB gene ( Horton et al 2021 ). Just six synonymous SNPs defined whether strong mutation bias occurred at position 289 within the ntrB locus, and these changes were theorized to impact the formation of a single stranded DNA secondary structure ( Horton et al 2021 ).…”

“…During the re-evolution of flagellar motility, >95% of replicate lines realized an identical de novo single nucleotide polymorphism (SNP), A289C in the ntrB gene ( Horton et al 2021 ). Just six synonymous SNPs defined whether strong mutation bias occurred at position 289 within the ntrB locus, and these changes were theorized to impact the formation of a single stranded DNA secondary structure ( Horton et al 2021 ). Elucidating biased hotspots of similar potency in other bacterial genomes would inform predictive evolutionary models and bring us closer to forecasting adaptive trajectories.…”

Localized pmrB hypermutation drives the evolution of colistin heteroresistance

“…First, the molecular mechanism of pmrB hypermutability is unclear. Hypermutability in bacterial genomes is classically driven by repeated sequence motifs that are prone to acquiring insertions and deletions ( Moxon et al., 2006 ), but repeats are conspicuously absent from pmrB , suggesting that alternative mechanisms drive the hypermutability of this gene ( Horton et al., 2021 ). Second, our study did not provide insight into phenotypic mechanisms of adaptation to colistin.…”