GnT motifs: short nucleotide tracts of >=8bp that can increase T:A->G:C mutation rates >1000-fold in bacteria

Nucleotides across a genome do not mutate at equal frequencies. Instead, specific nucleotide positions can exhibit much higher mutation rates than the genomic average due to their immediate nucleotide neighbours. These mutational hotspots can play a prominent role in adaptive evolution, yet we lack knowledge of which short nucleotide tracts drive hotspots. In this work, we employ a combinatorial approach of experimental evolution with Pseudomonas fluorescens and bioinformatic analysis of various Salmonella species to characterise a short nucleotide motif ([≥]8bp) that drives T:A[->]G:C mutation rates >1000-fold higher than the average T[->]G rate in bacteria. First, we experimentally show that homopolymeric tracts ([≥]3) of G with a 3 T frequently mutate so that the 3 T is replaced with a G, resulting in an extension of the guanine tract, i.e., GGGGT [->] GGGGG. We then demonstrate that the potency of this T:A[->]G:C hotspot is dependent on the nucleotides immediately flanking the GnT motif. We find that the dinucleotide pair immediately 5 to a G4 tract and the nucleotide immediately 3 to the T strongly affect the T:A[->]G:C mutation rate, which ranges from [~]5-fold higher than the typical rate to >1000-fold higher depending on the flanking elements. Therefore the T:A[->]G:C hotspot motif is a product of several modular nucleotide components (1-4bp in length) which each exert a significant effect on the mutation rate of the GnT motif. This work advances our ability to accurately identify the position and quantify the mutagenicity of hotspot motifs predicated on short tracts of nucleotides.