Article Figures & Data
- FIG 1
Genome organization of lytic phages φVPE25 and φVFW. Whole-genome alignments were performed using MAFTT version 1.3 (61). Open reading frames for φVPE25 and φVFW were determined using RAST version 2.0, and the resulting data were imported into Geneious 6.0.6. Modular gene organization based on predicted function is color coded. Vertical lines indicate regions with a high degree of nucleotide heterogeneity between φVPE25 and φVFW. Transmission electron microscopy revealed that φVPE25 and φVFW are noncontractile tailed siphophages.
- FIG 2
φVPE25 and φVFW DNA is modified at cytosine residues. (A) Structure of methylation and glycosylation modifications that occur at cytosine residues in DNA. Cytosine can be methylated in the form of a single methyl group (5mC) or hydroxyl-methylated (5hmC). 5hmC can be converted to a glucose-linked cytosine (5ghmC) by glucosyltransferase. (B) DNA sequencing analysis of sodium bisulfite-treated E. faecalis OG1RF genomic DNA or φVPE25 and φVFW genomic DNA. Unmodified cytosine in E. faecalis genomic DNA is converted to uracil after bisulfite treatment and when sequenced appears as thymidine. φVPE25 and φVFW genomic DNA resists bisulfite conversion, confirming cytosine modification. Dots indicate that these nucleotides match those in the consensus sequence. (C) Restriction endonuclease digestion of genomic DNA from φVPE25, φVFW, and E. faecalis V583. Incomplete digestion by PvuRts1I may be due to a minimum number of glycosylation sites or to inefficient DNA cleavage.
- FIG 3
EF0858 encodes PIPEF, promotes phage infection, and harbors a hypervariable region. (A) Cross streak and plaque assays using φVPE25 show the susceptibility or resistance profiles of E. faecalis V583 and the isogenic PIPEF deletion strain BDU50. Introduction of the pLZPIP plasmid, which contains the entire open reading frame of PIPEF restores phage infectivity of E. faecalis BDU50. pLZ12 is the empty vector. (B) Topological cartoon of E. faecalis V583 PIPEF generated using TOPCONS (62). The cartoon depicts PIPEF as an integral membrane protein that spans the membrane six times. The ~160-amino-acid variable region of PIPEF is represented as the black box with a black netting pattern in the large extracellular domain. (C) Pairwise amino acid sequence alignments of 19 PIPEF homologs were performed using Geneious 6.0.6. The N and C termini are indicated.
- FIG 4
A variable region in PIPEF determines phage tropism in E. faecalis. (A) Schematic of PIPEF from E. faecalis V583. A variable region covering ~160 amino acids is located in the center of the PIPEF coding sequence. (B) Both φVPE25 cross streak and plaque assays show that truncations in the PIPEF variable region abolish phage infectivity regardless of location. The deletions (ΔA, ΔB, and ΔC) correspond to the colored amino acids highlighted in the magnified area of the variable region shown in panel A. (C) Susceptibility profiles of 19 E. faecalis strains for phages φVPE25 and φVFW. (D) Clustering of PIPEF variable region amino acid alignments from 19 strains of E. faecalis. Strains cluster according to their susceptibility patterns as determined in panel C. These strains are indicated by color coding as follows: strains sensitive to killing by both phages (black), strains sensitive to only φVPE25 (blue), and strains sensitive only to φVFW (red). Strains can be further grouped into five specific clades based on PIPEF variable region amino acid identity. (E) Representative clade-specific mutation frequencies for phages φVPE25 and φVFW. NT, not tested (due to natural resistance to the phage of interest).
- FIG 5
PIPEF swapping alters phage tropism. (A) Using the E. faecalis strain E1Sol, which is naturally resistant to infection by φVPE25, both cross streak and plaque assays showed that E1Sol can acquire φVPE25 susceptibility by expressing the E. faecalis V583 PIPEF gene (pLZPIP). Single-copy replacement of the E. faecalis E1Sol PIPEF homolog with V583 PIPEF in the E1Sol chromosome also confers φVPE25 sensitivity (PIPV583). A chimera of E. faecalis E1Sol PIPEF and the variable region of V583 PIPEF show that the PIPEF variable region determines phage tropism (pLZEV). (B and C) Plaquing efficiency of φVPE25 (B) and φVFW (C) on E. faecalis V583, E1Sol, and PIPV583 transgenic E1Sol strains. The value that was significantly different (P < 0.01) by Student’s t test from the value for E. faecalis V583 is indicated by an asterisk. (D) Cross streak assay showing that expression of E. faecalis V583 PIPEF from plasmid pPBPIP can confer φVPE25 sensitivity on E. faecium strains 1,141,733 and Com12, but not strain Com15.
- FIG 6
PIPEF promotes DNA entry, but not initial phage adsorption. (A to C) Initial phage adsorption to E. faecalis cells was measured by determining the percentage of phages remaining in the supernatant after the addition of various E. faecalis strains. NB, no bacteria added. (D to F) Southern blotting was performed using an φVPE25 whole-genome probe on DNA isolated from whole cells infected with φVPE25. (D) E. faecalis V583 compared to the isogenic PIPEF mutant strain BDU50. (E) φVPE25 replication can be restored in strain BDU50 when PIPEF is provided in trans. (F) The variable region of strain V583 is sufficient to allow φVPE25 DNA entry into E. faecalis, because φVPE25 DNA can replicate in strain E1Sol only if the strain expresses a PIPEF chimera carrying the V583 PIPEF variable region (pLZEV) in the large extracellular facing domain. pLZ12 is the empty vector control.
- FIG 7
In vivo phage predation selects for E. faecalis PIPEF mutants. Germfree C57BL6/J mice were orally inoculated with E. faecalis V583 followed by an oral treatment of φVPE25. (A) Fecal burden of E. faecalis from mice with and without phage treatment over a 9-day period. Each symbol represents the value for an individual mouse, and the short black bar represents the mean of the group of mice. The means that are significantly different (P < 0.001) by Student’s t test are indicated by a bar and asterisk. (B) φVPE25 particle numbers from gnotobiotic mouse feces as determined by a plaque assay. (C) Percentage of φVPE25-resistant E. faecalis clones isolated from gnotobiotic mice following phage treatment. The percentage of φVPE25 resistance was calculated by determining the number of phage-resistant isolates by cross streaking and then dividing the number of resistant isolates by the total number of isolates acquired at each time point and multiplying by 100. Symbols: ♦, treated with φVPE25; ●, not treated with phage.
Supplemental Material
Figure S1
Comparative analysis of the φVPE25 and φVFW genomes. (A) Comparative whole-genome alignments of φVPE25 and φVFW and seven other siphophages that infect E. faecalis were performed using Mauve 2.3.1 (A. E. Darling, B. Mau, and N. T. Perna, PLoS One 5:e11147, 2010, http://dx.doi.org/10.1371/journal.pone.0011147). Lines indicate genomic regions that have connectivity based on nucleotide sequence similarity. (B) A BLASTn analysis was performed on φVPE25 and φVFW to identify genome relatedness to phages outside of known enterococcal siphophages. φVPE25 was set as the reference sequence for circular alignment using Brig 0.95 (N. F. Alikhan, N. K. Petty, N. L. Ben Zakour, and S. A. Beatson, BMC Genomics 12:402, 2011, http://dx.doi.org/10.1186/1471-2164-12-402). Download Figure S1, PDF file, 1.4 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S2
Phage-resistant E. faecalis isolates do not harbor integrated prophages. Genomic DNA isolated from E. faecalis cells that developed spontaneous resistance to φVPE25 and φVFW infection (see Table S2 in the supplemental material) lacks detectable phage DNA as determined by Southern blotting. The NS-mix lane contains a pool of E. faecalis V583 phage-resistant isolates recovered from scraping the soft agar of a semiconfluent φVPE25 lysis plate that was serially passaged in BHI three times prior to extraction of total genomic DNA for Southern blot analysis. The V583 lane contains genomic DNA from phage-sensitive wild-type E. faecalis V583. Purified φVPE25 and φVFW DNAs are included as controls. Download Figure S2, PDF file, 0.8 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S1
Enterococcal bacteriophage genome organization and features. Table S1, PDF file, 0.2 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S2
Spontaneous mutations in EF0858 (PIPEF) result in phage resistance. Table S2, PDF file, 0.04 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S3
φVPE25 infection of transgenic E. faecium 1,141,733. (A) Growth kinetics of E. faecium 1,141,733 carrying the E. faecalis V583 PIPEF expression plasmid pPBPIP in the presence (●) and absence (♦) of φVPE25. The arrow indicates the time of φVPE25 addition to the culture. (B) φVPE25 particle numbers from infected E. faecalis V583 cells carrying pPBPIP immediately after phage addition (Input) or 2 h after phage infection (2 hours post). (C) Quantitative real-time PCR of the φVPE25 transcripts orf_106 (lysin), orf_117 (major tail protein), and orf_123 (major capsid protein) isolated from E. faecium 1,141,733 or E. faecium 1,141,733 carrying plasmid pPBPIP. E. faecalis V583 and the PIPEF mutant strain BDU50 are included as controls. Transcript abundances are plotted on a logarithmic scale. (D) Viable phage particles recovered from wild-type and PIPEF transgenic E. faecium 1,141,733 after cell disruption using lysozyme and sonication. E. faecalis E1Sol was included as a control strain that is resistant to φVPE25 infection. ND, none detected. Download Figure S3, PDF file, 0.1 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S3
Sewage PIP read mapping to clade-specific PIPEF variable region. Table S3, PDF file, 0.04 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S4
Sequence variation among the PIPEF variable regions of E. faecalis sewage isolates. (A) Schematic of the variable region of PIPEF (amino acids 342 to 494). The amino acids where variation was detected by direct PCR from raw sewage (EBOX) or from pooled enterococcal isolates grown on selective agar (P1) are indicated in red and green, respectively. For both EBOX and P1 samples, the majority of the amino acid content of the PIPEF variable region matched E. faecalis V583 (52.00% of contigs for P1 and 41.61% for EBOX). The top four or five representative contigs containing variant amino acid composition compared to the E. faecalis V583 PIPEF variable region sequence as a reference are indicated. (B) Alignment of the E. faecalis V583 (clade 4) and E1Sol (clade 5) PIPEF variable regions. Download Figure S4, PDF file, 1.4 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S4
Mutations conferring φVPE25 resistance in E. faecalis from gnotobiotic mouse feces. Table S4, PDF file, 0.04 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Text S1
Supplemental Materials and Methods. Download Text S1, PDF file, 0.1 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S5
Bacterial strains, phages, plasmids, and primers used in this study. Table S5, PDF file, 0.1 MB.
Copyright © 2016 Duerkop et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Additional Files
Supplementary Data
Supplementary Data
- Text s1, PDF - Text s1, PDF
- Figure sf1, PDF - Figure sf1, PDF
- Figure sf2, PDF - Figure sf2, PDF
- Figure sf3, PDF - Figure sf3, PDF
- Figure sf4, PDF - Figure sf4, PDF
- Table st1, PDF - Table st1, PDF
- Table st2, PDF - Table st2, PDF
- Table st3, PDF - Table st3, PDF
- Table st4, PDF - Table st4, PDF
- Table st5, PDF - Table st5, PDF
