Mutation – whilst stochastic – is frequently biased toward certain loci. When combined with selection this results in highly repeatable and predictable evolutionary outcomes. Immotile variants of the bacterium Pseudomonas fluorescens (SBW25) possess a ‘mutational hotspot’ that facilitates repeated occurrences of an identical de novo single nucleotide polymorphism when re-evolving motility, where ≥95% independent lines fix the mutation ntrB A289C. Identifying hotspots of similar potency in other genes and genomic backgrounds would prove valuable for predictive evolutionary models, but to do so we must understand the genomic features that enable such a hotspot to form. Here we reveal that genomic location, local nucleotide sequence, gene strandedness and presence of mismatch repair proteins operate in combination to facilitate the formation of this mutational hotspot. Our study therefore provides a framework for utilising genomic features to predict and identify hotspot positions capable of enforcing near-deterministic evolution.
The survival of a population during environmental shifts depends on whether the rate of phenotypic adaptation keeps up with the rate of changing conditions. A common way to achieve this is via change to gene regulatory network (GRN) connections conferring novel interactions on transcription factors. To understand the success of rapidly adapting organisms, we therefore need to determine the rules that create and constrain opportunities for GRN innovation. Here, using an experimental microbial model system we construct a maladapted GRN, through deletion of a master transcription factor, and challenge evolution to fix this network. We identify three key properties - high activation, high expression, and pre-existing low-level affinity for novel target genes - that facilitate transcription factor innovation via gain of functional promiscuity. Ease of acquiring these properties is constrained by pre-existing GRN architecture, which was overcome in our experimental system by both targeted and global network alterations. This work reveals the key properties that determine transcription factor evolvability, and as such, the evolution of GRNs.
Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.
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