11 hours ago
10
IJMS, Vol. 25, Pages 11343: Optimising Transformation Efficiency in Borrelia: Unravelling the Role of the Restriction-Modification System of Borrelia afzelii and Borrelia garinii
International Journal of Molecular Sciences doi: 10.3390/ijms252111343
Authors: Margarida Ruivo Noémi Zsuzsa Kovács Anna-Margarita Schötta Theresa Stelzer Laura Hermann Verena Mündler Andreas Bergthaler Michael Reiter Michiel Wijnveld
Borrelia spp. are transmitted to humans by the bite of an infected tick. In Europe, Borrelia afzelii and Borrelia garinii are the main causative agents of Lyme borreliosis, one of the most prevalent tick-borne diseases in the northern hemisphere. In bacteria such as Borrelia spp., a restriction-modification system (RMS) protects against the harmful introduction of foreign DNA. The RMS comprises two activities: methyltransferase and endonuclease. This study is aimed to characterize the RMS of B. afzelii and B. garinii. First, we identified potential RMS genes. The predicted genes were cloned into a methylase-deficient Escherichia coli strain and digested with methylation-sensitive restriction enzymes to verify methyltransferase activity. Additionally, the RMS proteins were purified to evaluate endonuclease activity. Subsequently, methylated and unmethylated plasmids were used to investigate the effect of methylation on endonuclease activity and transformation efficiency. We identified four possible RMS genes in B. afzelii and four RMS genes in B. garinii. We analyzed the presence of these genes in patient isolates and observed a high degree of heterogeneity. The restriction pattern of DNA methylated by each of the four recombinantly expressed genes provided strong evidence that all encode adenine-specific methyltransferases. After 24 h of incubation with purified RMS proteins, we observed complete digestion of unmethylated plasmid DNA, demonstrating endonuclease activity. Finally, we proved that methylation protects against endonuclease activity and increases transformation efficiency.
