Student Theses and Dissertations


Victor Chen

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Laboratory


Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient genetic methods. Current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. Additional genetic infrastructure, including robust inducible promoters and sitespecific integration vectors have yet to be developed in E. faecium. Furthermore, a programmable gene silencing tool useful to selectively perturb essential genes in bacteria remains undeveloped for E. faecium. To address these limitations in gene editing of E. faecium, Chapter 2 reports the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Here, I utilize a recombineering system using a latent bacterial prophage RecT that is compatible with E. faecium and other related species. By combining recombineering with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 counterselection, I was able to produce both scar-less mutants via point mutations as well as controllable deletions of various sizes. Additionally, I report my system to have gene editing activity with PCR generated dsDNA templates. Overall, I show the versatility of my RecT-mediated recombineering method to produce substitution, deletion, and insertion mutants in E. faecium to enable facile genetic-based studies. Notably, the expression of RecT in combination with CRISPR-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less singlestranded DNA recombineering to generate specific gene editing mutants in E. faecium. Moreover, I demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic selectable markers to generate gene deletion mutants. As further proof-of-principle, in Chapter 3, I use CRISPR-Cas9 or dsDNA mediated recombineering to perform further knock out studies to investigate E. faecium microbiology or E. faecium host-microbe interactions. Using CRISPR-Cas9 mediated recombineering, I knocked out both genomically encoded sortase A genes in E. faecium for downstream functional characterization. Additionally, I used CRISPR-Cas9 mediated recombineering was to knock out E. faecium tyrosine decarboxylase to genetically validate E. faecium’s interaction with the orphan human G-protein-coupled receptor, GPRC5A. Furthermore, using dsDNA recombineering, I generated HA-tagged and knockouts of E. faecium bile acid salt hydrolase to genetically confirm E. faecium’s interactions with bile acids. The general RecT-mediated recombineering methods described here should significantly enhance reverse genetic studies of E. faecium and other closely related species for functional and mechanistic studies. To make it easier to study perturbations of essential genes in E. faecium, I describe my efforts to generate genetic infrastructure to enable the application of CRISPR interference programmed gene silencing in Chapter 4. Here, I demonstrate the generation of a compatible integration vector de novo for large gene insertions for E. faecium. Leveraging my integration system, I create a stably expressing and genome integrated gfp gene in E. faecium Com15 strain. I then demonstrate efficient targeting by Streptococcus pyogenes or Streptococcus thermophilis dead Cas9 (dCas9) of gfp produces ample gene silencing in E. faecium Com15. In Chapter 5, I present foundational experiments and the roadmap to generating functional CRISPRi in vancomycin-resistant E. faecium (VREfm). By utilizing my integration system, I was able to create genome integrated inducible dCas9 controlled by IPTG or agmatine inducible systems in VREfm. Furthermore, I generate and characterize a compact, highly transformable single-guide RNA (sgRNA) delivery vector for CRISPRi assays. In developing CRISPRi for VREfm, I discover a putative anti-CRISPR protein that has CRISPR inhibitory effects in E. faecium Com15. Overall, these experiments provide a detailed roadmap for implementing a CRISPRi system into VREfm. Overall, the projects described here should accelerate the genetic methodological landscape for E. faecium.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Sunday, May 18, 2025

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