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

Abstract

Antibiotic-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 Enterococcus faecalis, 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. CRISPR-Cas 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 E. faecalis 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. faecalis 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.

The CRISPR-Cas systems were selectively inactivated during evolution of Bacillus cereus group for adaptation to diverse environments

“…However, the factors that determined such inactivation pathways of CRISPR-Cas systems in B. cereus group, and the detailed mechanisms and significance involved remain unclear and need further studies. Other inactivation models of CRISPR-Cas systems include loss of nuclease components or repression of the expression of nucleases [14,15] and attenuating the function by diverse anti-CRISPR proteins [53]. In addition, loss of CRISPR-Cas elements through recombination has been reported [54].…”

“…For example, the CRISPR loci in Staphylococci counteracts multiple routes of HGT to limit the spread of antibiotic resistance genes [4]. Similarly, in Streptococcus pneumoniae the CRISPR-Cas prevents the acquisition of virulence related genes [11], and in Enterococcus faecalis it was reported that the potential for DNA acquisition could be maximized by attenuating its CRISPR-Cas system [14,15]. Previously, a bioinformatic study showed that there is no detectable influence of CRISPR-Cas systems on the HGT rate over evolutionary timescales among species [16].…”

“…The existing studies mostly were conducted with individual strains, but these analyses cannot truly reflect the relationship between CRISPR-Cas systems and HGT within species at the population scale. In addition, the CRISPR-Cas systems have been proposed as important factors contributing for the evolution of virulence in several pathogens [11,[14][15][16]18], but the impact of such systems on bacterial evolution at population level is also poorly understood. The increasing availability of sequenced genomes will facilitate the study and understanding of the role interplayed among CRISPR-Cas systems and HGT, and its impact on bacterial evolution within species.…”

Ethanolamine Utilization and Bacterial Microcompartment Formation Are Subject to Carbon Catabolite Repression

“…The first approach was performed as described by Kristich et al (37) after conjugation of strain EFKK27 with the ΔccpA mutant and EFKK35 with SD289, resulting in strains EFKK29 and EFKK36, respectively. After conjugation of strain EFKK33 with OG117, the second approach, which is based on a CRISPR-Cas system, was performed as described by Hullahalli et al (38), resulting in strain EFKK34.…”

Mobile Genetic Elements in Vancomycin-Resistant Enterococcus faecium Population

“…Conversely, commensal E. faecalis contain type II CRISPR-Cas systems, but multi-drug resistant (MDR) strains not carry complete CRISPR systems. Thus, MDR E. faecalis are prone for acquiring antibiotic resistance genes [31].…”