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Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can 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 of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium Re