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

<i>Borrelia,</i> a highly prevalent tick-borne pathogen, has a genome with a linear chromosome and numerous linear and circular plasmids. There are three groups of <i>Borrelia</i>: Lyme borreliosis, relapsing fever, and Echidna-reptile. In Europe, <i>Borrelia afzelii</i> and <i>Borrelia garinii</i> 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 <i>Borrelia</i> species available in the REBASE database. Additionally, it investigates the effect of the RMS on the transformation efficiency of low-passage <i>B. afzelii</i> and <i>B. garinii</i> isolates. Upon comparing the RMS of 18 <i>Borrelia</i> 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 <i>Borrelia</i> recognises pre-methylated vectors as native DNA. The knowledge gained in this study contributes to the understanding of <i>Borrelia</i> 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 <i>Borrelia.</i>