A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems

Abstract

With the advance of high-throughput sequencing, the molecular basis of coevolutionary interaction between viruses and host microorganisms is predominantly elucidated by in silico genomic analyses, which revealed potential communication of genetic materials related to microbial immune systems such as the restriction-modification (R-M) system. However, the sequence-dependent information is often insufficient to output a conclusive argument without biochemical characterizations, particularly for homologs of rare genes considered less accurately annotated. We proposed a 1-day and one-pot workflow covering in vitro protein synthesis and enzymatic assays to confirm the exact function of putative R-M genes only with manual pipetting operations of microliter-scale liquids. The proof-of-demonstration experiments mainly focused on a series of putative R-M enzymes from our recently found deep-sea temperate bacteriophage and its host bacterium. Two new restriction endonucleases and two new methyltransferases with respective unambiguous substrate specificities, superior catalytic performance, or unique sequence preferences were quickly identified. A frequent discrepancy between sequence similarity search and single-molecule methylation-sensitive sequencing toward the prediction of recognition motifs can get settled with the established direct biochemical characterization. The proposed approach under the cell-free one-pot concept allows for preliminary characterizations of diverse categories (e.g., Types I, II, and III) of putative R-M systems at most laboratories with minimum equipment and time costs. IMPORTANCE The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.