# High-throughput conjugation reveals strain specific recombination patterns enabling precise trait mapping in Escherichia coli

## TL;DR

A high-throughput conjugation method in Escherichia coli reveals variable DNA fragment sizes during recombination, enabling precise trait mapping and challenging previous assumptions about conjugation.

## Contribution

A novel high-throughput conjugation method was developed to study recombination patterns and enable precise trait mapping in E. coli.

## Key findings

- Recombined DNA fragment sizes in E. coli range from less than 10 kilobases to over a megabase.
- Short recombined fragments enable precise identification of selected loci with gene-level resolution.
- Strain-specific recombination patterns suggest genetic control of the process.

## Abstract

Genetic exchange is a cornerstone of evolutionary biology and genomics, driving adaptation and enabling the identification of genetic determinants underlying phenotypic traits. In Escherichia coli, horizontal gene transfer via conjugation and transduction not only promotes diversification and adaptation but has also been instrumental in mapping genetic traits. However, the dynamics and variability of bacterial recombination remain poorly understood, particularly concerning the patterns of recombined DNA fragments. To elucidate these patterns and simultaneously develop a tool for trait mapping, we designed a high-throughput conjugation method to generate recombinant libraries. Recombination profiles were inferred through whole-genome sequencing of individual clones and populations after selection of a marker from the donor strain in the recipient. This analysis revealed an extraordinary range of recombined fragment sizes, spanning less than ten kilobases to over a megabase—a pattern that varied across the three tested strains. Mathematical modelling indicated that this diversity in recombined fragment size enables precise identification of selected loci following genetic crosses. Consistently, population sequencing pinpointed a selected marker at kilobase-scale accuracy, offering a robust tool for identifying subtle genetic determinants that could include point mutations in core genes. These findings challenge the conventional view that conjugation always transfers large fragments, suggesting that even short recombined segments, traditionally attributed to transduction, may originate from conjugation.

Escherichia coli demonstrates a remarkable ability to adapt to diverse environments. This adaptability is in part connected to its ability to recombine incoming DNA in its chromosome, as illustrated by the vast gene content diversity observed in the species. However, the precise dynamics and patterns of bacterial recombination remain poorly understood. To address this knowledge gap, we developed a high-throughput method to observe genetic transfer in the laboratory based on conjugation. Surprisingly, we found that the size of transferred DNA fragments varied from less than 10 kilobases up to a megabase. These results challenge the long-held belief that conjugation primarily transfers only large DNA segments. This diversity in fragment sizes has important consequences. First as a genetic tool, it enables us to pinpoint the location of specific traits in the genome with gene resolution. Second and more importantly, short fragment recombination allows a specific selected loci to be transferred from one strain to another with minimum collateral material allowing an almost surgical precision in genome evolution. Finally, using three different stains as recipient, we observe a strain specific pattern of recombination suggesting a genetic control of that process that will require further investigation.