Spontaneous resistance to phage NPV1 confers increased daptomycin susceptibility in 1 Enterococcus faecalis OG1RF

13 Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the


Introduction
Enterococcus faecalis is a Gram-positive bacterium that inhabits the human gastrointestinal tract and is associated with nosocomial infections (1).Infections caused by E. faecalis can be difficult to treat because of the high frequency of resistance to multiple antibiotics among E. faecalis clinical isolates (2).The antibiotic daptomycin can be used to treat certain infections caused by multidrug-resistant enterococci.Daptomycin is a lipopeptide antibiotic that interacts with the enterococcal cell surface and disrupts membrane structure and function (3).
Bacteriophages (phages) are bacterial viruses and natural predators of bacteria.It is reasonable to expect that phages can be employed to treat bacterial infections.However, phages have not been extensively studied in the Western world in the context of therapeutic application until recently due to the availability of antibiotics (4).In recent years, interest in using phages to treat bacterial infections (phage therapy) has reemerged because of the emergence of multidrugresistant bacteria.For E. faecalis, promising studies include the use of phage to eliminate biofilm, a major barrier to antibiotic treatment, and to increase survival rates in mouse models of enterococcal infection (5,6).
One advantage of phage therapy is limited damage to the native microbiome because of the specificity of the phage to its host (7).The first step to a successful phage infection is the attachment of the phage particle to the proper receptor present on the surface of the host cell.
Phage receptors have been extensively studied in certain phage families, including the T series phages, Mu, and λ for Gram-negative bacteria (8)(9)(10)(11).Some phage receptors have been characterized in Gram-positive bacteria, including receptors for ϕSPP-1 of Bacillus subtilis (12) and the phage c2 group of Lactococcus lactis (13,14).YueB, the ϕSPP-1 receptor, and PIP, the phage c2 receptor, are orthologs and are required for irreversible phage adsorption (12).Enterococcal phage receptors have not been well-characterized.Previously, we and collaborators identified PIP as a receptor and potential DNA channel for the E. faecalis phages ϕVPE25 and ϕVFW (15).Bacteria can evolve phage resistance.Mechanisms of phage resistance include modification or loss of the phage receptor (16).However, as phage receptors generally serve physiological functions in the cell, the modification or loss of a receptor could come at a cost for the bacterial host.Indeed, spontaneous phage-resistant mutants have altered antibiotic sensitivity in P. aeruginosa (17).Phages utilizing receptors that have roles in antibiotic resistance could be advantageous for re-sensitizing resistant bacteria to antibiotics.
Considering the increasingly limited treatment options for E. faecalis infections and the revival of interest in using phage therapy to treat bacterial infections, it is crucial that we know the receptor(s) of enterococcal phages since effective phage cocktails use phages targeting multiple different receptors (18).Moreover, the roles of these receptors in enterococcal physiology should be elucidated.In this report, we used a combination of genomic and genetic approaches to identify a receptor for the tailed, virulent phage ϕNPV1 (19,20), which infects E. faecalis OG1RF by a PIP-independent mechanism.

Results
Deletion of epaR alters susceptibility to ϕNPV1.We isolated a OG1RF∆PIP (15) strain with spontaneous resistance to ϕNPV1 (Figure 1).We refer to this strain as OG1RF-C.The genome sequence of OG1RF-C was determined.We identified non-synonymous substitutions in epaR, bgsB, iolA2, and OG1RF_10252 (Table 2).epaR is one of the 18 conserved genes of the epa gene cluster (epaA-epaR), which codes for synthesis of the enterococcal polysaccharide antigen (Epa) (20).epaR encodes a putative glycosyltransferase with 5 predicted transmembrane domains, and its role in Epa biosynthesis has not been investigated.The .CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made available under The copyright holder for this preprint (which was not this version posted April 16, 2018.; https://doi.org/10.1101/301929doi: bioRxiv preprint product of bgsB is a putative cytoplasmic protein catalyzing the transfer of glucose from UDPglucose to diacylglycerol (DAG) to form monoglucosyl-DAG.An additional glucose is added to glucosyl-DAG by bgsA, forming diglucosyl-DAG.From diglucosyl-DAG, the polymerization of glycerol-phosphate can occur, resulting in lipoteichoic acid (LTA) (21).iolA2 is predicted to encode a methylmalonate-semialdehyde dehydrogenase, which catalyzes the breakdown of malonic semialdehyde to acetyl-CoA and CO 2 (22).OG1RF_10252 is predicted to encode a Acyl-ACP_TE domain (pfam01643; e-value 7.510e -114 ) which catalyzes the termination of fatty acyl group extension by hydrolyzing an acyl group on the fatty acid.
To begin to elucidate the roles of these genes in ϕNPV1 susceptibility, we constructed in-frame deletions of epaR and bgsB, generating strains OG1RF∆epaR and OG1RF∆bgsB, respectively, and a double deletion strain, OG1RF∆epaR∆bgsB.Phage susceptibility of each of these mutants was assayed.Deletion of epaR alone was sufficient to confer phage resistance (Figure 1).In contrast, deletion of bgsB alone did not alter phage susceptibility.These results indicate that variation in epaR is the major factor conferring resistance to ϕNPV1 in OG1RF-C.Since we observed that deletion of epaR in OG1RF conferred phage resistance to the same extent as that observed for OG1RF-C, we did not investigate the effects of iolA2 and OG1RF_10252 on phage resistance in this study.
The OG1RF-C epaR allele confers ϕNPV1 resistance.To determine whether epaR mutation is the major contributor to ϕNPV1 resistance in OG1RF-C, we generated strain OG1RF∆epaR_Ec, an OG1RF∆epaR strain complemented in cis with the epaR allele from OG1RF-C.We also generated strain OG1RF∆epaR_Ew, an OG1RF∆epaR strain with a reconstituted wild-type epaR.Complementation with the epaR allele of OG1RF-C conferred phage resistance to OG1RF∆epaR (Figure 2).The wild-type epaR allele restored phage susceptibility to OG1RF∆epaR (Figure 2).Because the mutated epaR allele from OG1RF-C .CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made available under The copyright holder for this preprint (which was not this version posted April 16, 2018.; https://doi.org/10.1101/301929doi: bioRxiv preprint confers a phage resistance phenotype, as did deletion of epaR, we infer that the epaR mutation in OG1RF-C confers loss of function. epaR is required for phage adsorption.We were next interested in how epaR inactivation protects OG1RF from ϕNPV1 infection.After 15 minutes incubation with ϕNPV1, ~95% of the phage adsorbed to wild-type OG1RF (Figure 3).In contrast, under the same conditions, ~1-2% of the phage adsorbed to OG1RF-C or OG1RF∆epaR.Consistent with the observation that bgsB deletion alone confers no significant phage resistance, ~90% of the phage adsorbed to OG1RF∆bgsB within the same experimental settings.These data indicate that epaR is required for ϕNPV1 adsorption.
Inactivation of epaR leads to a loss of a polymer.Next, we sought to determine whether the Epa polymer was intact in mutants defective for ϕNPV1 adsorption.The Epa polymer has been extracted and visualized by different groups using different methods (20,23).We based our method on that from Teng, et al. (20).We found that solubilizing the precipitation with 50% acetic acid improved visualization of the polymers.Gel electrophoresis analysis of carbohydrate extracts found that OG1RF with either an epaR deletion or the epaR allele from OG1RF-C exhibited loss of a band (P1) that is present in wild-type OG1RF, OG1RF∆bgsB, and the reconstituted epaR strain, OG1RF∆epaR_Ew (Figure 4).We conclude that P1 represents an epaR-dependent polymer.Since NPV1 cannot bind to epaR mutants, we hypothesized that the P1 polymer is the phage receptor.However, we when pre-incubated NPV1 with crude carbohydrate extract prior to infection of host cells, we did not observe a decrease in PFU for any extract (Figure S1), suggesting that the polymers in the crude extract are not sufficient for phage adsorption.We also observed an increase in the intensity of the P2 band in OG1RF∆bgsB and OG1RF∆bgsB∆epaR compared to wild-type OG1RF, suggesting that the product P2 is increased in these two deletion strains.Since deletion of bgsB in E. faecalis results in accumulation of LTA (21), product P2 may represent LTA.
NPV1-resistant mutants have increased susceptibility to daptomycin.Dale et al. reported increased daptomycin susceptibility in an OG1RF derivative with a deletion in epaO (23).
Moreover, we identified a bgsB mutation in a laboratory-evolved E. faecium isolate with decreased daptomycin susceptibility (24).Because of these results, we investigated the daptomycin MIC of our OG1RF mutants (Figure 5).We found that OG1RF-C, OG1RFΔepaR, and OG1RFΔepaR complemented with the OG1RF-C epaR allele were each significantly more susceptible to daptomycin than OG1RF.Interestingly, deletion of bgsB also conferred increased daptomycin susceptibility (Figure 5).This was complemented by expression of the wild-type bgsB allele in cis, but not by expression of the OG1RF-C bgsB allele in cis (Figure 5).Finally, daptomycin susceptibility was substantially altered in the OG1RF∆epaR∆bgsB mutant, with 3 of 6 experimental trials resulting in an MIC below the level of detection of the E-test strip (<0.016 µg/mL; a value of 0.008 µg/mL was used for these data points in statistical analysis).Without complete data regarding the MIC of OG1RF∆epaR∆bgsB, we did not quantitatively determine whether there is a synergistic relationship between bgsB and epaR regarding daptomycin susceptibility.
epaR protects OG1RF from osmotic stress.The epa gene cluster was up-regulated when E. faecalis V583 was grown with 6.5% sodium chloride supplementation, indicating that the Epa polymer has a role in osmotic stress response (25).As such, we investigated the effect of sodium chloride on our epaR mutants.We tested our mutants for their tolerance for sodium chloride stress using BHI agar supplemented with sodium chloride at concentrations of 0%, 2.5%, 5%, and 7.5%.Overnight cultures in stationary phase were serially diluted and spotted on these agars.We observed fewer CFU for OG1RF-C, OG1RF∆epaR, and OG1RF ∆epaR∆bgsB compared to the wild-type at 7.5% sodium chloride after 72 h incubation (Figure 6).When sodium chloride concentrations of 5% or lower were used, no effect on growth was observed.
Interestingly, when cells from an overnight culture were spotted, OG1RF∆bgsB showed no difference in CFU compared to the wild-type (Figure 6).However, when cells were cultured in fresh BHI broth 1 h prior to spotting, a CFU difference was observed for OG1RF∆bgsB with 7.5% sodium chloride (Figure S2).These data suggest that both epaR and bgsB play roles in sodium chloride-induced osmotic stress response.
EpaR constitutes a major mutational pathway for ϕNPV1 resistance.We isolated 9 spontaneous ϕNPV1-resistant mutants of OG1RF and sequenced the epaR region of these mutants.All 9 mutants have non-synonymous substitutions in epaR (Table 3).Since we know that mutations in epaR affect daptomycin susceptibility, we also determined the daptomycin MIC of these ϕNPV1-resistant strains and found that all were more significantly more susceptible to daptomycin than the wild-type (Figure S3).
Most of the EpaR sequence (amino acid positions 35-458 of 484 total) is a predicted sugar transferase domain (TIGR03025; E-value: 2.45e -137 ).This domain consists of a conserved Cterminal region responsible for the sugar transferase activity (pfam02397) and a variable Nterminal region with predicted flippase activity.All epaR mutations in spontaneously ΦNPV1resistant OG1RF strains occur in the C-terminal region of the sugar transferase domain (Table 3).We identified other proteins containing the same predicted sugar transferase domain as EpaR, and we determined that the altered amino acid positions in our mutants are conserved across 6 of these proteins (Figure S4).

Discussion
Due to the high frequency of antibiotic resistance in E. faecalis, alternatives to antibiotics, such as phage therapy, are of increasing interest in the United States.In this study, we investigated mechanisms for spontaneous phage resistance in E. faecalis.We have reported here that epaR is indispensable for ϕNPV1 adsorption to E. faecalis OG1RF, and that inactivating mutations in epaR constitute a major pathway for ϕNPV1 resistance in this strain background.We also found that inactivating mutations in epaR and bgsB result in increased susceptibilities to daptomycin and sodium chloride stress.Our results show that resistance to ϕNPV1 comes at a cost.
Adsorption to the host is the first step to a productive phage infection.For a tailed phage particle to successfully adsorb to the host, the tail apparatus on the phage must recognize the corresponding receptor(s) on the host cell surface.When challenged with a high phage titer in a resource-limited environment, receptor mutations in host cells are favored over use of intracellular defense mechanisms (26).This preference for receptor mutations is an especially important consideration in the design of phage cocktails, as using phage that recognize the same receptors could result in decreased efficacy of the treatment (18).Receptors for enterococcal phages have not been well studied.We and collaborators recently identified PIP as a receptor for phage ϕVPE25 and ϕVFW, but PIP is not the sole player in host cell recognition, as ϕVFW and ϕVPE25 can still adsorb to a PIP deletion strain (15).PIP may act as a DNA channel as is implicated in studies of L. lactis (14).
The epa gene cluster is involved in the synthesis of a cell wall rhamnose polysaccharide referred to as the enterococcal polysaccharide antigen (Epa).There is precedence for cell wall rhamnose polysaccharides as phage receptors.The structure of the rhamnose polysaccharide dictates phage host range in L. lactis and S. mutans (27,28).In E. faecalis, the epa gene cluster consists of 18 core genes (epaA to epaR) and a set of strain-variable genes that occur downstream (20,29).Unfortunately, the Epa structure has not been determined (30), which is a critical gap in knowledge about the enterococcal cell surface.
The connection between the epa gene cluster and ϕNPV1 resistance was first investigated by Teng et al, who assessed ΦNPV1 susceptibilities of E. faecalis OG1RF mutants with disruptions in epaA, epaB, epaE, epaM, and epaN (20).No ϕNPV1 plaques were obtained for epaB, epaE, epaM, and epaN mutants, and plaque production was reduced by 50% in the epaA mutant as compared to the wild-type.However, when the wild-type strain and the epaA and epaB mutants were assessed for ϕNPV1 adsorption, no differences were noted.Teng et al also examined the polysaccharide content of their mutants and found that production of the 'P1' product was absent in the epaB, epaM, epaN and epaE mutants, but a new polysaccharide product referred to as 'PS12' was synthesized.For the epaA mutant, both P1 and P12 were produced.The results from Teng et al. suggest that a complete Epa product is required for productive ϕNPV1 infection.Our results support this conclusion as ϕNPV1 does not absorb to our epaR mutants, nor do epaR mutants synthesize the P1 (or P12) product.However, the P1 product may not be the only requirement for ϕNPV1 adsorption because no significant decrease in PFU was observed when ϕNPV1 was pre-incubated with crude polysaccharide extracts from OG1RF strains with either wild-type or mutant epaR (Figure S1).Alternatively, the availability of P1 to the phage may differ in whole cells versus crude extracts.
Daptomycin is a lipopeptide antibiotic that is used to treat certain Gram-positive bacterial infections (31).The mechanism of action for daptomycin in B. subtilis begins with daptomycin binding to the cell membrane, and ultimately leads to the displacement of membrane-associated proteins essential for cell wall and phospholipid biosynthesis (32).In our study, we found that inactivating mutations in epaR and in bgsB lead to increased daptomycin susceptibility in E. faecalis.Deletion of bgsB results in loss of glycolipids in the membrane, a longer chain length in the LTA, and increased charge density of the membrane (21).A higher charge density might contribute to daptomycin susceptibility through charge-charge interaction with the calciumbound daptomycin, but this is speculative.Dale et al. reported that deletion of epaO results in increased daptomycin susceptibility and loss of the Epa polymer, suggesting that the complete Epa polymer is important for daptomycin resistance (23).The loss of the Epa polymer also results in defects in cell wall architecture (20,33).More research on the Epa polymer is required to mechanistically assess its contribution to antibiotic susceptibility in enterococci.
In summary, in this study we characterized a mechanism for spontaneous ϕNPV1 resistance in E. faecalis OG1RF.The major advance of this study is that we determined that phage resistance comes at a cost to E. faecalis OG1RF; specifically, increased daptomycin susceptibility.E. faecalis OG1RF is a natively daptomycin-sensitive strain that became more sensitive as a result of phage resistance.We did not assess whether ϕNPV1 treatment could reverse resistance in daptomycin-resistant clinical isolates.This is an exciting area for future research.

Material and Methods
Bacterial strains, media, and bacteriophages.A complete list of bacterial strains and bacteriophage used in this study can be found in Table 1.E. faecalis strains were cultured in brain heart infusion (BHI; Difco) at 37°C without agitation.E. coli strains were cultured in LB broth at 37°C with shaking at 225 rpm unless otherwise stated.Plates of the appropriate media were made by adding 1.5% agar to the broth prior to autoclaving.For MIC testing, Muller Hinton media supplemented with 1.5% agar (MHA) was used.Phages were stored in phage buffer as previously described (34).Chloramphenicol (Cm) was used at a concentration of 15 µg/mL when required for selection.5-bromo 4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was used at 120 µg/mL and 40 µg/ml for E. faecalis and E. coli, respectively.The upper soft agar for phage assays was M17 medium supplemented with 0.75% agar, while the lower layer was BHI supplemented with 1.5% agar.
Routine molecular techniques and DNA sequencing.Routine PCR reactions were performed using Taq polymerase (NEB) per the manufacturer's instructions.Phusion polymerase (Fisher) was used for cloning procedures per the manufacturer's instructions.
Plasmid was purified using the GeneJET Plasmid miniprep kit (Fisher).Genomic DNA was isolated using the Ultraclean microbial DNA isolation kit (MoBio).Restriction enzymes, Klenow fragment, T4 polynucleotide kinase (PNK), T4 DNA ligase, and calf intestinal phosphatase (CIP) from NEB were used as instructed by the manufacturer.DNA sequencing was performed at the Massachusetts General Hospital DNA sequencing facility.A complete list of primers used in this study can be found in Table S1.
Phage spot assay.0.5 mL of an exponentially growing culture was added to 3 mL soft agar and poured onto BHI agar. 10 µL of the of phage mixture to be titered was spotted onto the soft agar.Plaques were counted after 16 h incubation at 37°C, unless otherwise stated.
Phage propagation and storage.Phage stocks were prepared by mixing 450 µL of an overnight culture of E. faecalis OG1RF with ϕNPV1 at an M.O.I of 10 -2 .The mixture was incubated at 37°C for 15 min and subsequently added to 3 mL M17 soft agar maintained at 55°C.The soft agar was then poured onto BHI agar and incubated at 37°C for 18 h.5 mL of phage buffer was added to the confluently lysed plate and incubated for 20 min at 37°C with shaking at 75 rpm.The lysate was then collected and centrifuged at 16.6 x g for 1 min to remove cellular debris.The supernatant was filtered with a Whatman 0.2 µm filter to obtain the phage stock.The phage stock was stored at 4°C in the dark.Phage titer was determined using phage spot assays.
Generation of OG1RF deletion mutants.Gene deletion was carried out via the markerless deletion procedure described by Thurlow et al. (35) with some modifications.Briefly, two 1.0 kb regions flanking epaR were amplified with primers 1-4 from Table S1.The two amplified products were ligated with an overlap PCR extension through a 21 bp complementary region underlined in Table S1.The approximately 2.0 kb product was purified and digested with BamHI and EcoRI.The digested product was ligated to plasmid pLT06 through restriction sites added on the primers (highlighted in red in Table S1).The ligation product was then purified and electroporated and propagated in E. coli EC1000.OG1RF was made electrocompetent using the glycine method (36) (3% glycine) and transformed with 1 µg of the plasmid.OG1RF transformants were screened for successful transformation and subsequently inoculated in BHI supplemented with Cm at 30°C.The culture was diluted 1:100 in BHI and incubated at 30°C for 2 h followed by 42°C for 4 h.Dilutions of the culture were plated on BHI agar supplemented with Cm and X-gal, and large blue colonies were screened for plasmid integration using primers 5 and 19.The positive colonies were then restruck, incubated at 42°C, and screened once again for plasmid integration.Positive clones were cultured in BHI broth at 30°C for 18 h.To counterselect against clones harboring the plasmid, dilutions of the culture were made on MM9YEG agar, and the deletion of epaR was determined by colony PCR with primers 5 and 6 after 36 h incubation at 37°C.Clones positive for the deletion were then restruck on BHI agar and screened again using the same primers.Positive clones were verified for plasmid loss by streaking on BHI agar supplemented with Cm.The epaR region was sequenced to confirm the deletion.Deletion of bgsB was obtained in a similar fashion.
Complementation.Complementation of epaR in an OG1RF∆epaR background was obtained using a similar strategy to deletion.The insert containing the epaR gene and flanking 500 bp upstream and downstream regions was amplified from either OG1RF or OG1RF-C using primers 7 and 8. pLT06 was digested with SphI and blunt-ended with Klenow fragment; the blunt-end product was then treated with CIP.The insert was phosphorylated with T4 PNK and blunt-end ligated to pLT06.The plasmid was purified and transformed into EC1000.Clones with the correct insert size were screened, and their plasmids isolated.Subsequent steps for transformation of OG1RF, integration of the plasmid and counterselection on MM9YEG were as described above for the deletion process.Positive clones for the complementation were confirmed with primers 5 and 6 after counter-selection on MM9YEG.The complemented epaR allele was verified through Sanger sequencing.
Assessment of phage resistance and sodium chloride stress tolerance.For assessment of phage resistance, 500 µL of an 8×10 9 PFU phage stock was added to 3 mL M17 soft agar.The mixture was then poured onto BHI agar.Bacterial culture dilutions were spotted on the soft agar and incubated at 37°C for 18 h.For assessment of osmotic stress tolerance, BHI plates were supplemented with NaCl (0%, 2.5%, 5%, and 7.5%).Overnight cultures of bacteria are serially diluted and spotted on NaCl-supplemented plates.Plates were imaged after 72 h incubation.
For some experiments, overnight cultures were subcultured into fresh BHI broth for 1 h prior to serial dilution and spotting.
Phage adsorption assay.An overnight bacterial culture was diluted 1:5 in fresh BHI broth.The culture was then equilibrated at 37°C for 20 min in a water bath.ϕNPV1 was added at an M.O.I of 10 -3 .After 15 min, a 1 mL aliquot was centrifuged at 16.6 x g for 1 min at room temperature.500 µL of the supernatant was collected, and its titer is determined with the phage spot assay.A medium with only phage added (no bacteria) was used as control.Percent adsorption was determined as follow: Isolation of ϕNPV1 resistant mutants.For isolation of a ϕNPV-1 resistant strain from an OG1RF∆PIP background, ϕNPV-1 was used to infect OG1RFΔPIP in a soft agar overlay.The confluently lysed plates were incubated until presumptive phage-resistant colonies were observed.These colonies were cultured in BHI broth and used as hosts for ϕNPV1 infection to confirm phage resistance.A confirmed ϕNPV-1 resistant strain, referred to in our study as OG1RF-C, was stocked and used for genome sequencing.
For isolation of ϕNPV-1 resistant strains from an OG1RF background, 500 µL of an overnight culture of OG1RF was infected with ϕNPV1 at an M.O.I of 10 -1 in a soft agar overlay.10 colonies that arose on the confluently lysed plate were struck on BHI plates and incubated at 37°C for 18 h.Single colonies from each of the plates were tested for phage resistance by cross-streaking against ϕNPV1.9 colonies that showed little to no lysis were stocked and used for daptomycin susceptibility testing and epaR sequencing.

Polysaccharide analysis.
Polysaccharide extraction was performed as described by Teng et al. with some modifications (20).200 mL of an overnight culture was centrifuged and resuspended in 750 µL 50 mM Tris buffer, pH 7.5.Mutanolysin (0.25 U/µL) and lysozyme (5 mg/mL) were added to the suspension.The suspension was incubated at 37°C for 2 h.Subsequently, 10 mM MgSO 4 , 2.5 mM CaCl 2 , 0.15 mg/mL DNase I, and 0.15 mg/mL RNase A were added.After an additional 2 h incubation at 37°C, the suspension was centrifuged, and the cellar debris discarded.Proteinase K (100 µg/mL) was added to the clear supernatant, and the mixture was incubated for 16 h.Afterwards, the supernatant was extracted twice with chloroform-phenol-isoamyl alcohol (Sigma Aldrich) and once with chloroform.Ethanol was added to a final concentration of 80% to precipitate the polysaccharide.The precipitate was collected by centrifugation and air-dried.The pellet was resuspended in 50% acetic acid (v/v) in deionized water, and the insoluble material was removed by centrifugation.30 µL was loaded onto an 1% agarose gel and electrophoresed for 30 min at 130 V.The gel was soaked in staining solution containing Stains-All (Alfa Aesar) and left overnight with gentle rocking.The staining solution was 25% isopropanol, 10% formamide, 65% water, and 0.005% Stains-All.
After 18 h, the gel was destained under light for 40 min prior to visualization.
Daptomycin MIC.Daptomycin MIC was assessed using Etest strips (BioMérieux).3-5 colonies of similar sizes were resuspended in 500 µL BHI and evenly distributed over a MHA plate using a sterile cotton swab.A daptomycin Etest strip was placed onto the plate, and the plate was incubated for 18 h at 37°C.MIC was determined by recording the number closest to the zone of inhibition.The MIC reported for each strain is the average of at least three trials.For trials in which the daptomycin MIC was below the detection limit of the strip (<0.016 µg/mL), the MIC was reported as 0.008 µg/mL for the purposes of statistical analysis.Data were analyzed using the two-tailed unpaired Student's t test.
Whole genome sequencing and analysis of OG1RF-C.OG1RF-C genomic DNA was isolated from overnight culture using the Ultraclean Microbial DNA Isolation kit (Mo Bio) per the manufacturer's instruction.The gDNA was sequenced using MiSeq with 2 x 150 bp chemistry at MR DNA (Shallowater, Texas).After sequencing, the reads were mapped to the complete OG1RF reference (NC_017316.1)using CLC Genomics Workbench (Qiagen).Putative mutations were detected using the basic variant detector in CLC Genomic Workbench.Variants occurring at ≥50% frequency in the read assembly and resulting in non-synonymous substitutions were confirmed with Sanger sequencing.BlastP and NCBI Conserved Domains were used to analyze conserved domains in proteins.Amino acid alignment was performed with CLUSTALW (37).Transmembrane helices were predicted with TMHMM version 2.0 (38).
Accession numbers.The Illumina reads for resequencing of OG1RF-C have been deposited in the Sequence Read Archive under accession number PRJNA450206.

Supplemental Figures
Figure S1: Percent PFU recovery from pre-incubation of phiNPV1 with polysaccharide extract.Polysaccharide extract was prepared as described above except for the final solvent being phage buffer rather than 50% acetic acid.phiNPV1 was added at a PFU of 10 5 and the mixture was incubated for 15 minutes at 37°C.The PFU was evaluated with phage spot assay.
The percent PFU recovery was calculated using a phage buffer only as the base value.The experiment was performed in duplicate.MIC data points below the detection limit of 0.016 µg/mL were taken to be 0.008 µg/mL for statistical treatment.***, p <0.001.BHI agar BHI agar with phiNPV1 BHI agar BHI agar with phiNPV1

Figure 1 .
Figure 1.Phage susceptibility of E. faecalis OG1RF and derivatives.Overnight cultures were diluted in PBS and spotted on BHI plates with or without 10 9 PFU/mL phiNPV1.Images

Figure 2 .
Figure 2. Phage susceptibility of complemented strains of OG1RF∆epaR.Overnight cultures were diluted in PBS and spotted on BHI plates with or without 10 9 PFU/mL phiNPV1.

Figure 4 .
Figure 4. Carbohydrate extract analysis.Carbohydrate was extracted from 200 mL overnight

Figure S2 :
Figure S2: Effects of osmotic pressure on epaR and bgsB deletion strains of OG1RF.500 µL of overnight cultures of indicated strains were centrifuged.The pellets were then

Figure 1 .
Figure 1.Phage susceptibility of E. faecalis OG1RF and derivatives.Overnight cultures were diluted in PBS and spotted on BHI plates with or without 10 9 PFU/mL phiNPV1.Images were taken after 18 h incubation at 37°C.The image shown is representative of three independent trials.

Figure 2 .
Figure 2. Phage susceptibility of complemented strains of OG1RF∆epaR.Overnight cultures were diluted in PBS and spotted on BHI plates with or without 10 9 PFU/mL phiNPV1.Images were taken after 18 h incubation at 37°C.The image shown is representative of three independent trials.

Figure 6 .
Figure 6.Susceptibility to sodium chloride-induced osmotic stress.Overnight cultures were diluted in PBS and spotted on BHI plates with or without sodium chloride.Images were taken after 72 h incubation.The image shown is representative of three independent trials.

Table 1 .
Strains and plasmid used.