Examining the Effect of Microbial Community on CRISPR-Cas Activity in Enterococcus Faecalis

Enterococcus faecalis is an opportunistic pathogen involved in causing various hospital-acquired infections. Their propensity to develop antibiotic resistance by horizontal gene transfer via conjugative mobile genetic elements makes the treatment of E. faecalis infection increasingly difficult. Pheromone-responsive plasmids are involved in the transfer of mobile genetic elements in E. faecalis. CRISPR-Cas acts as a barrier against foreign genome transfer in bacteria. E. faecalis CRISPR-Cas activity varies in vivo (murine intestinal model) and in vitro. The functional differences may be due to various biotic and abiotic factors such as nutrient availability, community diversity, donor-to-recipient ratios, and cas9 regulatory differences. A previous study has shown that the efficacy of CRISPR-Cas varies in the presence of a microbial community. Here we study the effect of Escherichia coli, also a resident of the mammalian gut, on CRISPR-Cas defense in vitro using E. faecalis containing pAM714 plasmid. Through our work, we are able to demonstrate that CRISPR-Cas efficacy is not altered by the presence of E. coli during conjugation between donor OG1SSp (pAM714) and recipients T11RF and T11RFΔcas9. Our goal is to provide insights into CRISPR-Cas function of Gram-positive bacteria in the microbial community.