Investigating the restriction-modification barrier to DNA delivery in human gut probiotic bacteria for streamlined genetic tool development

Abstract

Human gut bacteria have the potential to serve as next-generation therapeutics to many disease conditions, although poor genetic tractability limits mechanistic understanding and engineering capabilities in most strains. A major barrier to genetic tool development is bacterial defense systems such as the highly abundant restriction-modification systems that restrict DNA transformed into the cell. Although these systems are well-known barriers to DNA delivery across bacteria, their prevalence and diversity within probiotic human gut species has not been investigated. In this review, we detail the importance of DNA delivery in establishing genetic tools in bacteria and illustrate the role of restriction-modification systems in preventing DNA delivery. We then perform a computational analysis of available sequencing data to compile restriction-modification abundance and complexity in many human gut species with large probiotic potential. Through this analysis, we elucidate the large number of restriction-modification systems present in many species and highlight the diversity of restriction-modification systems in closely related strains. Among the four main types of restriction-modification systems, we pinpoint the main type(s) present in different species. To highlight strains with high potential to inhibit DNA delivery, we determine the percentage of strains from each species with multiple restriction-modification systems present. Finally, we cover established methods of bypassing the restriction-modification barrier to DNA delivery. Overall, this generalizable analysis should help others extend DNA delivery to more relevant strains across human gut bacteria to establish next generation living therapeutics from a wider range of probiotic strains.

Keywords: DNA delivery; Live biotherapeutic products; Methyltransferase; Non-model; Probiotics; Restriction enzyme; Restriction-modification.