Riding the wave of genomics to investigate aquatic coliphage diversity and activity

Abstract

Bacteriophages infecting Escherichia coli (coliphages) have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that is present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterized coliphages from three coastal locations in the United Kingdom and Poland. Comparative genomics and phylogenetic analysis of phage isolates facilitated the identification of putative new species within the genera Rb69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, genomic and proteomic analysis combined with host range analysis allowed the identification of a putative tail fibre that is likely responsible for the observed differences in host range of phages vB_Eco_mar003J3 and vB_Eco_mar004NP2.

Figure 1

Phylogenetic analysis of phages within the genus Rb69virus. The tree is based on the nucleotide sequence of nine concatenated genes [nrdC (GeneID:1494209), rnlA (GeneID:1494352), ndd (GeneID:1494384), regA (GeneID:1494173), g52 (GeneID:1494381), g14 (GeneID:1494292), td (GeneID:1494357), g053 (GeneID:1494168) and g30.3 (GeneID:1494331)] using a GTR+F+ASC+R2 model of evolution, with 1000 bootstrap replicates using IQTREE (Nguyen et al., 2015). Current phage species as defined by the ICTV are marked with an *. Bootstrap values above 70% are marked with a filled circle, with the size proportional to the bootstrap value. The ANI value between phages is represented as a heatmap, with only values >97% coloured. The phages included in the tree are RB69 (acc:AY303349), Shf125875 (acc: KM407600), phiC120 (acc:KY703222), vB_EcoM_PhAPEC2 (acc:KF562341), SHSML‐52‐1 (acc:KX130865), ST0 (acc:MF044457), HX01 (acc:JX536493), vB_EcoM_JS09 (acc:KF582788), E. coli O157 typing phage 3 (acc:KP869101), E. coli O157 typing phage 6 (acc:KP869104), APCEc01 (acc:KR422352), and vB_Eco_mar005P1 (acc:LR027390). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Phylogenetic analysis of phages within the genus T5virus. The tree is based on the nucleotide sequence of two concatenated genes (locus tags: MAR004NP2_00031 and MAR004NP2_00005) using a GTR+F+ASC+R2 model of evolution, with 1000 bootstrap replicates using IQTREE (Nguyen et al., 2015). Current phage species as defined by the ICTV are marked with an *. Bootstrap values above 70% are marked with a filled circle, with the size proportional to the bootstrap value. The ANI value between phages is represented as a heatmap, with only values >97% coloured. The phages included in the tree are T5 (acc:AY543070), T5 strain ATCC 11303‐B5 (acc:AY587007), T5 strain st0 deletion mutant (acc:AY692264), EPS7 (acc:CP000917), phiR201 (acc:HE956708), SPC35 (acc:HQ406778), bV_EcoS_AKFV33 (acc:HQ665011), AvB_EcoS_FFH1 (acc:KJ190157), Stitch (acc:KM236244), DT57C (acc:KM979354), DT571/2 (acc:KM979355), Shivani (acc:KP143763), APCEc03 (acc:KR422353), 100268_sal2 (acc:KU927497), 118970_sal2 (acc:KX017521), SP01 (acc:KY114934), phiLLS (acc:KY677846), BSP22A (acc:KY787212), SSP1 (acc:KY963424), slur09 (acc:LN887948), SH9 partial (acc:MF001363), OSYSP (acc:MF402939), chee24 (acc:MF431730), pork27 (acc:MF431731), pork29 (acc:MF431732), saus47N (acc:MF431733), saus111K (acc:MF431734), poul124 (acc:MF431735), chee130_1 (acc:MF431736), saus132 (acc:MF431737), poul149 (acc:MF431738), chee158 (acc:MF431739), cott162 (acc:MF431740), saus176N (acc:MF431741), LVR16A partial (acc:MF681663), SP3 partial (acc:MG387042), vB_SenS_PHB06 (acc:MH102285), vB_Eco_mar003J3 (acc:LR027389) and vB_Eco_mar004NP2 (acc:LR027384). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Phylogenetic analysis of phages within the subfamily Tunavirinae. The tree is based on the nucleotide sequence of four concatenated genes (locus tags, MAR001J1_00001, MAR001J1_00004, MAR001J1_00010, and MAR001J1_00077) using a GTR+F+ASC+G4 model of evolution, with 1000 bootstrap replicates using IQTREE (Nguyen et al., 2015). Current phage genera as defined by the ICTV are marked with the first coloured strip chart. Bootstrap values above 70% are marked with a filled circle, with the size proportional to the bootstrap value. The ANI value between phages is represented as a heatmap, with only values >97% coloured. The phages included in the tree are Rtp (acc:NC_007603), vB_Eco_ACG‐M12 (acc:NC_019404), phiEB49 (acc:NC_023743), e4/1c (acc:NC_024210), JK06 (acc:NC_007291), vB_EcoS_Rogue1 (acc:NC_019718), phiJLA23 (acc:KC333879), C119 (acc:KT825490), bV_EcoS_AHP24 (acc:KF771236), vB_EcoS_AHS24 (acc:NC_024784), vB_EcoS_AKS96 (acc:NC_024789), vB_EcoS_AHP42 (acc:NC_024793), PKP126 (acc:NC_031053), F20 (acc:JN672684), KLPN1 (acc:KR262148), 1513 (acc:KP658157), Sushi (acc:KT001920), MezzoGao (acc:MF612072), GML‐KpCol1 (acc:MG552615), KP36 (acc:NC_029099), Shfl1 (acc:NC_015456), ADB‐2 (acc:NC_019725), pSf‐2 (acc:NC_026010), T1 (acc:NC_005833), JMPW2 (acc:KU194205), JMPW1 (acc:KU194206), Esp2949‐1 (acc:NC_019509), Stevie (acc:NC_027350), TLS (acc:NC_009540), SP126 (acc:KC139513), YSP2 (acc:MG241338), pSf‐1 (acc:KC710998), vB_Eco_swan01 (acc:LT841304), SECphi27 (acc:LT961732) and vB_Eco_mar002J2 (acc:LR027385). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Genomic comparison of phages within the genus T5virus. All phages were compared to phage T5 (accession: AY692264) with BRIG (Alikhan et al., 2011) using blastn settings of minimum e‐value 0.001 and minimum length of 100. Each phage is represented by a single ring of different colour. Nucleotide identity of 80%–100% is shaded in colour for each ring, with darker shading representing higher identity. An identity between 50%–80% is shaded in light grey. The outer two rings contain the genes from T5 (accession: AY692264) and annotation. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Morphology of phage isolates. Phages vB_Eco_swan01, vB_Eco_mar005P1, vB_Eco_mar002J2, vB_Eco_mar003J3, vB_Eco_mar004NP2 were stained with 2% (w/v) uranyl acetate and imaged in a JEOL JEM‐1400 TEM with an accelerating voltage of 100 kV.