High-resolution genomic analysis reveals abundant mosaic outcomes of bacterial natural transformation independent of MutS-mediated mismatch repair.

The nature and breadth of horizontal gene transfer outcomes specific to natural transformation remain elusive. We present a genome-scale analysis of location-specific information associated with single-round transformation events in Bacillus subtilis. Using distributed selectable markers to remove location bias, we found transformant genomes often contained multiple discontinuous segments of donor sequence in close proximity. These highly mosaic sites span multiple length scales, with an abundance of shorter segments. We found that the small segments scale with the length of the nearest stretch of perfect homology, and these segments defy minimal, efficient homologous recombination rules. Sites of transformation and their associated intervening recipient sequences were not distinguished by overall percent identity, GC content, or median gene expression. Mismatch repair activity by MutS also failed to explain the breadth and frequency of mosaic patches. High-resolution mapping of donor and recipient alleles across sites of transfer demonstrates that natural transformation can contribute a breadth of allelic diversity, especially within short, clustered patches of genetic exchange. These observations point to a need to further investigate the complex mechanisms that drive distinct outcomes of natural transformation.IMPORTANCESeveral works have suggested the potential for discontinuity for donor DNA in transforming DNA. This work developed robust bioinformatic and genomic approaches to assess the full breadth of exchange between divergent genomes during natural transformation. The results demonstrate that simplistic sequence and expression-based associations are not sufficient to explain highly variable transformation outcomes. Similarly, transformant genomes are frequently incongruent with previously defined rules for homology-mediated recombination. MutS-mediated mismatch repair, a frequently proposed contributor to mosaic recombination, is also insufficient to explain discontinuity. Therefore, widespread molecular mechanisms intrinsic to recombination have the potential to generate significant genetic diversity during transformation, ranging from the scale of individual alleles to full operons. These results further reinforce the role of natural transformation in shaping genetic diversity within bacterial populations.