# Comparative analysis of Borrelia’s Defence mechanisms and their impact on genetic manipulation of low-passage isolates of Borrelia afzelii and Borrelia garinii

## TL;DR

This study shows that modifying DNA with in vitro methylation improves genetic transformation in Borrelia, helping researchers better study these tick-borne pathogens.

## Contribution

The study demonstrates that in vitro methylation of vectors enhances transformation efficiency in low-passage Borrelia isolates.

## Key findings

- Borrelia species differ in their restriction-modification system (RMS) gene numbers and locations.
- In vitro methylated vectors significantly increase transformation efficiency in low-passage Borrelia strains.
- The RMS recognizes pre-methylated vectors as native DNA, supporting the hypothesis of methylation-based defense.

## Abstract

•The Borrelia restriction-modification system (RMS) differs by species group.•The RMS affects the transformation efficiency of Borrelia.•Increase in transformation efficiency observed with an in vitro methylated vector.•Transformation efficiency increased in low-passage Borrelia strains.•In vitro methylation can be a facilitating tool for Borrelia genetic manipulation.

The Borrelia restriction-modification system (RMS) differs by species group.

The RMS affects the transformation efficiency of Borrelia.

Increase in transformation efficiency observed with an in vitro methylated vector.

Transformation efficiency increased in low-passage Borrelia strains.

In vitro methylation can be a facilitating tool for Borrelia genetic manipulation.

Borrelia, a highly prevalent tick-borne pathogen, has a genome with a linear chromosome and numerous linear and circular plasmids. There are three groups of Borrelia: Lyme borreliosis, relapsing fever, and Echidna-reptile. In Europe, Borrelia afzelii and Borrelia garinii are the main causative agents of Lyme borreliosis.

The primary defence mechanism of bacteria against bacteriophages and other invading DNA elements is the restriction-modification system (RMS), which discriminates between native and foreign DNA based on their distinct methylation patterns.

This present study compares the RMS of all the Borrelia species available in the REBASE database. Additionally, it investigates the effect of the RMS on the transformation efficiency of low-passage B. afzelii and B. garinii isolates.

Upon comparing the RMS of 18 Borrelia species, differences in the number, location and characteristics of genes were observed between groups. Given that Lyme borreliosis species exhibit higher genomic plasticity, we hypothesise that they possess a greater number of RMS genes to ensure functionality of the RMS even if some plasmids are lost.

In this study, we demonstrate a large increase in transformation efficiency of low-passage strains by using an in vitro methylated shuttle vector, confirming our hypothesis that the RMS of Borrelia recognises pre-methylated vectors as native DNA.

The knowledge gained in this study contributes to the understanding of Borrelia defence mechanisms and provides possible explanations for the relatively low transformation efficiency observed in previous studies. Consequently, in vitro methylation can serve as a valuable tool for facilitating studies involving genetic manipulation of Borrelia.

Image, graphical abstract