RecT recombinase expression enables efficient gene editing inEnterococcus

AbstractEnterococcus faeciumis a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains ofE. faeciumcan confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection ofE. faeciumfunctions and mechanisms has been restricted by inefficient gene editing methods. To address these limitations, here we report the expression ofE. faeciumRecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains ofE. faeciumand otherEnterococcusspecies such asE. duransandE. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less single-stranded DNA recombineering to generate specific gene editing mutants inE. faecium. Moreover, we demonstrate thatE. faeciumRecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic selectable markers to generate gene deletion mutants. As further proof-of-principle, we use CRISPR-Cas9 mediated recombineering to knock out both sortase A genes inE. faeciumfor downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies ofE. faeciumand other closely related species for functional and mechanistic studies.ImportanceEnterococcus faeciumis widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensalEnterococcusstrains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy ofEnterococcusspecies and strains highlights the need for in-depth studies to discover and characterize the genetic components underlining its diverse activities. However, current genetic engineering methods inE. faeciumstill require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid intoE. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in genomic DNA ofE. faecium. These improved recombineering methods should facilitate functional and mechanistic studies ofEnterococcus.