Materials and Methods

An Attenuated CRISPR-Cas System inEnterococcus faecalisPermits DNA Acquisition

MATERIALS AND METHODSBacterial strains, growth conditions, and routine molecular biology procedures. Enterococcus faecalis was routinely cultured at 37°C in brain heart infusion (BHI) broth without agitation. Escherichia coli was routinely cultured at 37°C in lysogeny broth with agitation at 220 rpm. Routine PCR was performed with Taq DNA polymerase, and PCR for cloning purposes was performed with Q5 DNA polymerase (New England Biolabs). T4 polynucleotide kinase (New England Biolabs) was used for routine phosphorylation. PCR products were purified with the PureLink PCR purification kit (Invitrogen). Plasmids were purified using the GeneJet plasmid purification kit (Fisher). Primers were synthesized by Sigma-Aldrich. Routine DNA sequencing was performed at the Massachusetts General Hospital DNA Core Facility. E. coli EC1000 was used for routine plasmid propagation (52). E. faecalis and E. coli competent cells were prepared as described previously (25). Genomic DNA was extracted using the Mo Bio microbial DNA isolation kit (Qiagen). Antibiotics were used in the following concentrations: chloramphenicol, 15 µg/ml; streptomycin, 500 µg/ml; spectinomycin, 500 µg/ml; vancomycin (Van), 10 µg/ml; erythromycin (Erm), 50 µg/ml; rifampin, 50 µg/ml; fusidic acid, 25 µg/ml; tetracycline, 10 µg/ml; gentamicin (Gent), 300 µg/ml. A full list of primers can be found in Table S2 in the supplemental material.TABLE S2 Primers used in this study. Download TABLE S2, DOCX file, 0.02 MB.Copyright © 2018 Hullahalli et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Strain and plasmid construction. A schematic of the plasmid construction used in this study is shown in Fig. S5. All strains and plasmids used in this study are shown in Table S3. CRISPR-edited strains are shown in Table 1. All CRISPR-editing plasmids can be derived in a single step from pGR-ermB (accession number {"type":"entrez-nucleotide","attrs":{"text":"MF948287","term_id":"1297896060","term_text":"MF948287"}}MF948287). The derivation of pGR-ermB is described below.FIG S5 Plasmid construction scheme. The general plasmid workflow is shown (components not to scale). CRISPR repeats are depicted by thin, light-blue rectangles; the colored rectangles adjacent to the repeats represent various spacers. All CRISPR editing plasmids can be derived from pGR-ermB as either one-step or two-step assemblies. Generic primer schematic for generating CRISPR editing deletion plasmids from a single step is shown as arrows indicating 5′-to-3′ directionality. The primer pairs used in each reaction are colored identically (i.e., the two red arrows represent the primers that are used in the same reaction to amplify one fragment). Homologous overhangs for subsequent Gibson assembly are shown. Thirty-base-pair overhangs were used in all cloning procedures. Download FIG S5, TIF file, 0.6 MB.Copyright © 2018 Hullahalli et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.TABLE S3 Strains and plasmids used in this study. Download TABLE S3, DOCX file, 0.02 MB.Copyright © 2018 Hullahalli et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.To generate chromosome-targeting constructs, pCR2-ermB was linearized to remove 160 bp upstream from the ermB spacer and simultaneously introduce the promoter of bacA from pPD1, which is constitutive (PbacA) (25, 35). This procedure also removed the upstream repeat. The linear product was phosphorylated and self-ligated to generate an intermediate plasmid referred to as pSR-ermB. This plasmid was once again linearized around cat, and a fragment containing cat and pheS* from pLT06 was blunt end ligated (53). The original cat was deleted to simplify the cloning procedure. The final plasmid was designated pGR-ermB and was fully sequenced (accession number {"type":"entrez-nucleotide","attrs":{"text":"MF948287","term_id":"1297896060","term_text":"MF948287"}}MF948287).To modify the spacer, pGR-ermB was linearized at PbacA and the downstream repeat; primers contained the entirety of the spacer sequence to be inserted. The exception was pGR-IS256, which was generated without ligation by taking advantage of the ability of E. coli EC1000 to recombine linear DNA (i.e., linear DNA was recombined in vivo). All pGR derivatives were sequence verified to ensure spacer integrity prior to introduction into strain C173 for conjugation. Homologous recombination templates were introduced using the NEB HiFi DNA assembly master mixture (New England Biolabs). For simplicity, the spacer was included as overhangs during Gibson assembly, and therefore, a plasmid containing two fragments for homologous recombination and the appropriate spacer could be generated in a single step. The same linearization-phosphorylation-ligation procedure was used to modify the plasmid to insert PbacA upstream from cas9. Knock-in protocols were performed essentially as previously described (54). A streamlined protocol for CRISPR-assisted genome editing in E. faecalis using our system is outlined in Fig. S6, and the primer schematic for generating CRISPR editing plasmids is shown in Fig. S5.FIG S6 CRISPR-Cas genome-editing protocol for E. faecalis. A workflow for achieving CRISPR-assisted genome editing in E. faecalis is shown. (1) Overnight cultures of donors (D) and recipients (R) are grown. Donors must possess the appropriate CRISPR editing plasmid, and recipients must possess a sufficiently expressed cas9. (2) Overnight cultures are diluted in BHI without antibiotics, regrown for 1.5 h, and (3) subsequently plated at a donor-to-recipient ratio of 1:9 on BHI. (4) The conjugation reaction mixture is incubated overnight and then scraped and plated on appropriate selective medium to obtain transconjugants (TCs), which are then (5) restruck on medium containing chloramphenicol. (6) Colonies are then inoculated into BHI broth, cultured until turbid, and (7) plated on MM9YEG plus p-Cl-Phe to counterselect for the plasmid. Edited clones are subsequently confirmed to be chloramphenicol sensitive. Media are color coded. BHI, BHI plus chloramphenicol, and MM9YEG plus p-Cl-Phe are shown in red, brown, and green, respectively. The bacteria present at each step of the process are also indicated. The appropriate number of colonies to screen from the initial transconjugant selection is dependent on each experiment, but we find that proceeding with 6 unique transconjugants is sufficient to recover at least two edited clones. Download FIG S6, TIF file, 0.8 MB.Copyright © 2018 Hullahalli et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.For CRISPR-assisted editing, the appropriate plasmid was first transformed into E. faecalis C173 or CK111SSp(pCF10-101). Conjugation into the desired recipient strain was then performed, and transconjugants were selected on agar medium containing chloramphenicol and appropriate antibiotics for recipient strain selection. Transconjugant colonies were restruck for isolation on agar medium containing chloramphenicol, and single colonies were inoculated into 1 to 5 ml of BHI broth lacking antibiotics and incubated at 37°C until turbid. Cultures were then struck on MM9YEG (36) containing para-chloro-phenylalanine (p-Cl-Phe) to counterselect against the plasmid backbone. By this point, the recipient strain will have received the CRISPR editing plasmid, recombined with the editing template, and then lost the backbone plasmid. In total, this procedure can take as little as 2 days once transconjugants are obtained. We observed that an additional passage in MM9YEG–p-Cl-Phe was helpful for eliminating residual chloramphenicol resistance, since the counterselection is imperfect. This extra passage was utilized whenever frequencies needed to be determined and there was no marker to phenotypically screen for, since preliminary experiments occasionally yielded some chloramphenicol-resistant clones which interfered with an accurate assessment of successful editing rates. Once presumptive CRISPR-edited mutants were obtained, colony PCR to confirm the desired edit was performed in all cases except for deletion of pstB; the larger amplicon required that genomic DNA be extracted. Genotypes of representative clones were verified through Sanger sequencing (for deletion of pstB, pstSCAB, and vanB) or whole-genome sequencing (for deletion of EF3217).Conjugation assays. Conjugation assays were performed essentially as described previously (25). C173 was used as the donor in all experiments, except for experiments using CRISPR-mediated editing to delete vanB. For deletion of vanB, the erythromycin-sensitive strain CK111SSp(pCF10-101) was used as the donor, since transconjugant selection during this experiment required erythromycin instead of vancomycin and C173 is erythromycin resistant.Transcriptomics analysis. To assess the transcriptional response to CRISPR self-targeting, transconjugants of V649(pGR-tetM) (control) and V649(pGR-IS256) (test) selected on vancomycin and chloramphenicol were incubated on agar medium for 2 days. Cells were scraped from plates, resuspended in RNA-Bee (Tel-Test, Inc.), and lysed by bead beating in lysis matrix B (MP Biomedicals). After RNA-Bee extraction, the aqueous layer was subjected to ethanol precipitation. The RNA was treated with DNase (Roche) and concentrated using the GeneJet RNA cleanup and concentration kit (Fisher). For assessment of the transcriptional response to levofloxacin (LVX)-induced stress, cells were treated essentially as previously described (25). Briefly, overnight cultures of V649 were diluted in fresh medium and grown to an optical density at 600 nm (OD600) of 0.3, at which point cultures were split. Some cells were harvested for control transcriptomic analysis, and LVX was added to the remaining cells at a concentration of 1 µg/ml. After 2 h of incubation with LVX, the remaining cells were harvested. RNA was isolated and treated with DNase as described above. Three biological replicates were performed under both experimental conditions.RNA-Seq analysis was performed at MR DNA (Molecular Research LP). The concentration of total RNA was determined using the Qubit RNA assay kit (Life Technologies, Inc.). Baseline-Zero DNase (Epicentre) was used to remove DNA contamination, and the RNA was purified using RNA Clean and Concentrator-5 columns (Zymo Research). Subsequently, rRNA was removed by using the Ribo-Zero gold rRNA removal (epidemiology) kit (Illumina) and purified with RNA Clean and Concentrator-5 columns (Zymo Research). rRNA-depleted samples were subjected to library preparation using the TruSeq RNA LT sample preparation kit (Illumina) according to the manufacturer’s instructions. The libraries were pooled and paired-end sequenced for 300 cycles using the HiSeq 2500 system (Illumina).RNA-sequencing data were analyzed using CLC Genomics Workbench. rRNA and tRNA reads were first removed, and the unmapped reads were mapped to the V649 reference genome. Transcripts-per-million (TPM) values were used to quantitate expression. False discovery rate (FDR)-adjusted P values were used to assess significance. Genes were filtered by first removing those for which both CRISPR self-targeting and LVX treatment yielded FDR-adjusted P values of >0.05. Subsequently, genes for which both LVX treatment and CRISPR self-targeting had fold changes of <2 were removed. The remaining list consisted of genes that were significantly up- or downregulated by either LVX treatment or CRISPR self-targeting. Raw reads for RNA sequencing and whole-genome sequencing have been deposited in the Sequence Read Archive under accession number PRJNA420898.RT-qPCR to verify increased cas9 expression was performed as previously described (25). RNA was harvested from cultures of strains V649 and V117 at an OD600 of 0.3.Phage resistance assay. Approximately 105 to 106 PFU/ml of ΦNPV-1 was added to 5 ml of M17 soft agar (Fisher) plus chloramphenicol and overlaid on BHI agar plus chloramphenicol (41). Overnight cultures of strains OG1RF and OG117 containing pGR-tetM or pGR-NPV1 were spotted on the soft agar containing ΦNPV1. pGR-NPV1 targets a predicted phage lysin gene. A simultaneous control lacking soft agar and phage was included to enumerate total bacterial CFU. Using identical amounts of ΦNPV-1 in each experiment was essential for consistent results.Detection of circular Phage01 DNA. Cultures were treated identically to those prepared for RNA sequencing. Cells were pelleted, and genomic DNA was extracted using the Mo Bio microbial DNA isolation kit (Qiagen) according to the manufacturer’s instructions. qPCR was performed using the AzuraQuant green fast qPCR mixture LoRox (Azura) according to the manufacturer’s instructions. Similar to a previously reported approach for circular-phage detection (39), circular Phage01 DNA was detected using primers qpp1c For and qpp1c Rev, which amplify across the junction of the circularized phage.Phage lysis assay. Cultures were induced with LVX as described in a previous section. Induced cultures were pelleted, and the supernatant was filtered using 0.2-µm polyethersulfone filters. Similarly, transconjugant colonies of V649(pGR-tetM) and V649(pGR-IS256) were scraped from agar plates using 2 ml phosphate-buffered saline (PBS) (identical to the protocol used for transcriptomics analysis) and pelleted, and the supernatant filtered. Filtrates were spotted on soft agar containing lawns of E. faecalis ATCC 29212, which is susceptible to infection by V583 prophages (55). To prepare the lawns, overnight cultures of ATCC 29212 were diluted in fresh medium and cultured to an OD600 of 0.4. Amounts of 10 μl of culture were added to 2-ml amounts of melted soft agar (BHI broth, 0.2% agarose, 10 mM MgSO4), and the mixtures were poured onto 100-mm-diameter standard BHI agar plates (1.5% agar). We observed that varying the amount of bacteria added and the thickness of the soft agar affected the visibility of phage plaques; the protocol we present here yielded the clearest zones of lysis.Genome sequencing. Whole-genome sequencing was performed at MR DNA (Molecular Research LP). Briefly, libraries were prepared with the Nextera DNA sample preparation kit (Illumina) using 50 ng of total genomic DNA. Libraries were pooled and paired-end sequenced for 300 cycles using the Illumina HiSeq system. Reads were mapped to the V117 genome in CLC Genomics Workbench. Mapping graphs were generated to identify deleted (zero-coverage) regions, and basic variant detection was performed on read mappings to identify smaller single-nucleotide polymorphisms (SNPs), deletions, and insertions using the default parameters. Raw reads for RNA sequencing and whole-genome sequencing have been deposited in the Sequence Read Archive under accession number PRJNA420898.Statistics. P values for conjugation frequencies and CFU measurements were calculated using a one-tailed Student’s t test from log10-transformed values. P values for qPCR data were calculated using a one tailed Student’s t test. Geometric mean values and geometric standard deviations are shown for all data except those presented in Table 1 (CRISPR editing experiments).Accession number(s). The pGR-ermB sequence has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"MF948287","term_id":"1297896060","term_text":"MF948287"}}MF948287. Raw reads for RNA sequencing and whole-genome sequencing have been deposited in the Sequence Read Archive under accession number PRJNA420898.

Article TitleAn Attenuated CRISPR-Cas System inEnterococcus faecalisPermits 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 pathogenEnterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensalE. faecalisstrains, the genomes of multidrug-resistant (MDR)E. faecalisclinical isolates are enriched for mobile genetic elements (MGEs) and lackclusteredregularlyinterspacedshortpalindromicrepeats (CRISPR) andCRISPR-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 inE. faecalisis 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 thatE. faecaliscan 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 lowcas9expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest thatE. faecalishas 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.


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