Project description:Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5'-phosphate gDNA, 14-30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4-12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5'-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10-16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology.
Project description:The genus Lactobacillus contains over 100 different species that were traditionally considered to be uniformly non-motile. However, at least twelve motile species are known to exist in the L. salivarius clade of this genus. Of these, Lactobacillus rumnis is the only motile species that is also autochthonous to the mammalian gastrointestinal tract. The genomes of two L. ruminis strains, ATCC25644 (human isolate, non-motile) and ATCC27782 (bovine isolate, motile) were sequenced and annotated to identify the genes responsible for flagellum biogenesis and chemotaxis in this species. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644 during the motile growth phase.