An Attenuated CRISPR-Cas System In Enterococcus faecalis Permits DNA Acquisition

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dc.contributor.ORCID0000-0002-7343-9271 (Palmer, KL)
dc.contributor.authorHullahalli, Karthik
dc.contributor.authorRodrigues, Marinelle
dc.contributor.authorUyen Thy Nguyen
dc.contributor.authorPalmer, Kelli L.
dc.contributor.utdAuthorHullahalli, Karthik
dc.contributor.utdAuthorRodrigues, Marinelle
dc.contributor.utdAuthorUyen Thy Nguyen
dc.contributor.utdAuthorPalmer, Kelli L.
dc.date.accessioned2019-06-28T18:13:35Z
dc.date.available2019-06-28T18:13:35Z
dc.date.created2018-05-01
dc.description.abstractAntibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Fnterococcus faecolis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPRCas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in F. faecolis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly. DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR- assisted genome-editing scheme. Our results suggest that E. faecatis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense. IMPORTANCE CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in E. faecafis with negligible off target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR- induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.
dc.description.departmentSchool of Natural Sciences and Mathematics
dc.description.sponsorshipNIH grant no. R01 AI116610
dc.identifier.bibliographicCitationHullahalli, Karthik, Marinelle Rodrigues, , and Kelli Palmer. 2018. "An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition." mBio 9(3): e00414-18, doi:10.1128/mBio.00414-18
dc.identifier.issn2150-7511
dc.identifier.issue3
dc.identifier.urihttps://hdl.handle.net/10735.1/6625
dc.identifier.volume9
dc.language.isoen
dc.publisherAmer Soc Microbiology
dc.relation.urihttp://dx.doi.org/10.1128/mBio.00414-18
dc.rightsCC BY 4.0 (Attribution)
dc.rights©2018 The Authors
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.source.journalmBio
dc.subjectCRISPR-Cas Systems
dc.subjectEscherichia coli
dc.subjectStreptococcus thermophilus
dc.subjectImmune System
dc.subjectGenome
dc.subjectRNA
dc.subjectBacteriophages
dc.subjectProkaryotic Cells
dc.subjectProteins
dc.subjectMicrobiology
dc.subjectEnterococcus faecalis
dc.subjectGene Editing
dc.subjectGene Transfer, Horizontal
dc.titleAn Attenuated CRISPR-Cas System In Enterococcus faecalis Permits DNA Acquisition
dc.type.genrearticle

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