Razzouk, R.; Hadjadj, L.; Militello, M.; Beye, M.; Pinault, L.; Rolain, J. M.; Bittar, F.

The Candidate Phyla Radiation (CPR) consists of microorganisms with highly reduced genomes and limited metabolic capabilities, including the inability to synthesize nucleotides and amino acids. Previous studies revealed an unexpected abundance of antimicrobial resistance-related genes in CPR genomes, with methyltransferases (MTs) accounting for up to 47.34% of detected resistance determinants. Here, we investigated the distribution, diversity, and potential functions of MTs across 12,552 CPR genomes, focusing on their roles in antimicrobial resistance and restriction-modification (R-M) systems. Sequence similarity searches were performed using BLASTp, followed by domain validation with the NCBI Conserved Domain Database (CDD). Putative restriction endonucleases (REs) associated with these systems were also identified and analyzed. We detected 20,232 resistance-associated MTs, 7,391 hsdM-like MTs, and 4,737 REs, revealing a high prevalence of putatively functional MTs relative to REs across CPR genomes. Functional CPR MTs and REs displayed extensive sequence divergence from known bacterial homologs and exhibited greater domain diversity and functional complexity than their bacterial counterparts. Selected high-confidence MT candidates were heterologously expressed in Escherichia coli BL21 and characterized using BIOLOG phenotypic assays and antimicrobial susceptibility testing. Transformed strains showed enhanced utilization of methyl pyruvate as a carbon source, whereas no significant antimicrobial resistance phenotype was detected. Collectively, these findings suggest that CPR-encoded MTs are primarily associated with R-M systems, supporting defense against foreign DNA and facilitating nucleotide scavenging rather than direct antimicrobial resistance. This study provides new insights into the adaptive strategies of CPR bacteria and the multifunctional roles of MTs in metabolically streamlined microorganisms.