The complete genome sequence of EC1-UPM, a novel N4-like bacteriophage that infects Escherichia coli O78:K80

Genomic features of bacteriophage EC1-UPM

The genome of bacteriophage EC1-UPM consists of 70,912 bp (GC content of 42.9%). A total of 80 protein coding genes were predicted with an average length of 816 bp (92.5% coding density). Annotation of the majority of predicted CDS showed confident hits against proteins of the N4-like bacteriophages, indicating that bacteriophage EC1-UPM may be a member of the N4-like bacteriophages (Additional file 1: Table S1). N4-like bacteriophages are bacteriophages of the Podoviridae family. Members of this group are lytic against their hosts. The host specificity of N4-like bacteriophages is rather diverse, ranging from enterobacteria such as E. coli and Pseudomonas aeruginosa to marine bacteria such as Sulfitobacter sp. EE-36 and Silicibacter pomeroyi DSS-3 [6–9]. Bacteriophage N4 which infects E. coli K-12 is currently the most studied strain for this group of bacteriophages [10]. In addition to having its complete genome sequenced, the identity and locations of several of its structural proteins have been established through comparisons of three-dimensional, cryo-electron microscopic structures of wild-type N4 and its mutants [11]. Based on our analysis, the annotated proteins of bacteriophage EC1-UPM can be categorized into the following functional groups: bacteriophage structure and packaging (portal protein, major coat protein, tail protein, tailspike protein, structural protein, capsid decorating protein), DNA replication/modification (DNA helicase, DNA polymerase, endonuclease, terminase), signal transduction and regulatory function (ssDNA-binding protein, RNA polymerase), nucleotide metabolism (thymidilate synthase, dCTP deaminase) and host lysis (holin, N-acetylmuramidase). The presence of lysis gene but not lysogeny-related gene indicates that bacteriophage EC1-UPM is a lytic bacteriophage. The largest gene encodes for the virion polymerase and is 10,839 bp, which is approximately 15% of the whole genome length (Figure 1). The presence of bacteriophage-encoded RNA polymerase of such size is a signature of N4 bacteriophage. It was demonstrated in N4 bacteriophage that this particularly large RNA polymerase was packaged into its capsid and ejected into the host cell thus eliminating the need to rely on host RNA polymerase for the transcription of its early genes [11]. No tRNA was identified in the genome of bacteriophage EC1-UPM, thus indicating that upon entry into the host, it is completely reliant on the host tRNA makeup for its protein synthesis.

Codon usage of bacteriophage EC1-UPM

Comparison of the codon usage in E. coli O78:K80 and bacteriophage EC1-UPM revealed a rather similar codon frequency (Figure 2). Compatibility of phage codon-usage with that of its host will confer an evolutionary advantage as this will aid in the energy-demanding and laborious mechanism of protein synthesis. Three codons however were overrepresented in bacteriophage EC1-UPM, namely, codons ACT, GTA and GCT that correspond to the amino acids threonine, valine and alanine, respectively. Interestingly, bacteriophage EC1-UPM does not adapt to the codon usage dissimilarity as evidence by the absence of tRNA gene in its genome. In the evolution of dsDNA phages, the presence of tRNAs particularly those that are abundant in the phage and less common in the host allows the phage to outcompete its potential competitors in the host by translating its proteins more effectively, speeding up the reproduction rate and reducing its latency time [12]. In T4 phages, deletion of the tRNA genes was shown to decrease burst sizes and protein synthesis rates, demonstrating the growth advantage of carrying tRNA genes in phages [13]. The complete absence of tRNA in bacteriophage EC1-UPM may imply that its ability to infect E. coli O78:K80 could have been a recent acquisition.

Comparative genomics of bacteriophage EC1-UPM and closely related N4-like bacteriophages

Initial whole genome BLAST of bacteriophage EC1-UPM against the NCBI database showed that bacteriophage EC1-UPM is closely related to three N4-like viruses namely KBNP21, vB_EcoP_G7C and IME11 (data not shown). Bacteriophages KBNP21, vB_EcoP_G7C and IME11 were isolated from chicken farm, horse feces and hospital sewage, respectively [6, 14, 15]. Bacteriophage IME11 could infect 13 of 31 enteropathogenic clinical E. coli strains [6]. Bacteriophage vB_EcoP_G7C could infect E. coli strain 4 while bacteriophage KBNP21 could infect E. coli strains KBP21 and KBP135 [14, 15]. However, the lack of O and K antigen data of the tested E. coli strains severely impeded host specificity determination based on surface antigen. Using Blast Ring Image generator (BRIG), it was shown that the majority of genomes are conserved across the four bacteriophages (Figure 3), except in the genomic region encompassing the genes coding for tail fiber and tail spike proteins. Tail fiber and tail spike proteins are often associated with the binding and degradation of host cell surface antigen [14]. Given the differences in the isolation source of each E. coli-infecting N4-like bacteriophage, it is likely that their E. coli hosts are of different strains that lack similarity in surface antigen profile. The gene coding for tail spike protein in bacteriophage EC1-UPM is conserved only in bacteriophage KBNP21 which was similarly isolated from avian source. Although, InterProscan annotation of the tail spike protein did not show any significant protein domain (Additional file 1: Table S1), augmentation of the protein annotation with HHpred indicates three major domains associated with carbohydrate binding and degradation between residues 120 to 190, 230 to 500 and 550 to 700 (Figure 4). Tail spike has been shown to be a vital component particularly in the infection of bacteria possessing both O and K antigens [14]. Some K antigens could prevent the direct recognition of receptor on the O antigen by the phage tail due to the large K-antigen size (up to 4000 A). The tail spike protein functions to overcome such limitation by creating a tunnel in the cell capsule and exposing the receptor to the tail protein [15, 16].

Novel region in the C-terminus of tail fiber protein

Based on BRIG visualization, the genomic region corresponding to the C-terminus of bacteriophage EC1-UPM tail fiber protein was not shared by any member of the closely related N4-like bacteriophages (Figure 3). Similar to tail spike protein, the tail fiber of bacteriophage has also been implicated in host specificity. The C-terminus region of tail fiber protein has been reported to alter the host range of a bacteriophage [17]. Divergences in the C-terminus may therefore represent the phage adaptation towards the huge variation of O and K antigens in E. coli[18]. Expanding the BLAST search towards the whole NR database did not reveal any significant hit in the C-terminus region. Based on HHpred annotation, the C-terminus of tail fiber protein showed significant degree of structural homology with various polysaccharide degrading enzymes possessing the right-handed β-helix structure (Figure 4 and Additional file 2: Table S2). Notably, residues 402 to 731 of the tail fiber displayed significant similarity to a β-helix structure-containing exo-poly-α-D-galacturonosidase of Thermotoga maritima, a thermophilic bacteria (E-value = 4.44 × 10^-44) (Figure 4 and Additional file 2: Table S2). Exo-poly-α-D-galacturonosidase is involved in the hydrolysis of pectic from the non-reducing to release digalacturonate. Initially identified in pectate lyase C, the right-handed β-helix is commonly found in the catalytic domains of various proteins that process oligo- and polysaccharides [19]. Given the structural homology of the tail fiber C-terminus to various polysaccharide degrading enzymes (Additional file 2: Table S2), it is plausible that the tail fiber may be associated with binding and/or degradation of the polysaccharide present on the bacterial antigens. The presence of hydrolytic phage tail fiber proteins has been shown to allow bacteriophage ΦK1-5 to attach and degrade both K1 and K5 capsular polysaccharides [20]. Further enzymatic study is necessary to validate the function of bacteriophage EC1-UPM tail fiber protein and its substrate range. This novel tail fiber protein consisted of 928 amino acid residues with molecular mass and pI of 99.3 kDa and 4.85, respectively.

Host lysis

It is widely accepted that lysis of the host is indispensable for the release and dispersion of progenies in all bacteriophages, except in filamentous bacteriophages [21, 22]. In bacteriophage EC1-UPM, genes involved in the holin-endolysin system are located adjacent to each other. The N-acetylmuramidase (NCBI accession number: AGC31576) which may be responsible for the endolysis activity contains a domain implicated in glycosyl hydrolase (PF05838) from residues 22 to 113. The glycosyl hydrolase family functions via the hydrolysis of β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of bacteria. The presence of a peptidoglycan binding domain (PF09374) from residue 116 to 195 after the glycosyl hydrolase domain may further enhance the activity of N-acetylmuramidase by localizing the protein to its target region. Membrane topology visualization of bacteriophage EC1-UPM holin protein indicates that it contains two transmembrane domains with both N-and C-terminus (data not shown) located within the cytoplasmic region, thus classifying it as a class II holin [21, 22]. The genes coding for the holin-endolysin system are well-conserved in bacteriophages KBNP21, vB_EcoP_G7C, IME11 and EC1-UPM (Figure 3), indicating a common mechanism for host lysis.

Orthologs identification and multilocus phylogenetic analysis of N4-like bacteriophages

The recent steady increase and availability of the complete genomes of various N4-like bacteriophages in the GenBank provides an opportunity to identify the orthologs that are well-conserved in all N4-like bacteriophages. Initial orthologs identification in bacteriophage EC1-UPM and its closely related strains vB_EcoP_G7C, IME11 and KBNP21 revealed that at 70% identity and 70% aligned cutoffs, a total of 44 proteins were conserved across the bacteriophages (Table 1). The average identities of the orthologs shared in bacteriophages vB_EcoP_G7C, IME11 and KBNP21 as compared to that of EC1-UPM were 91%, 93% and 95%, respectively. No orthologs can be identified when the analysis was expanded to include the more distant N4-like bacteriophages (Table 2) using the similar setting. Nevertheless, by lowering the identity cutoff to 40%, seven orthologous clusters were identified namely DNA polymerase I, N4 gp42-like protein, DNA primase, N4 gp44-like protein, portal protein, terminase and N4 gp69-like protein. The functional assignment of proteins in the orthologous clusters is in agreement with the notion that viruses share homologous “hallmark genes” encoding for proteins involved in genome replication and virion structure formation [23]. The phylogenetic tree analysis utilizing the trimmed and concatenated alignment of proteins in these orthologous clusters showed that the evolutionary relationship of the N4-like bacteriophage is very much correlated with their host specificity. Comparison of several phylogenetic trees constructed using individual orthologous clusters with known function (DNA polymerase, terminase, portal protein and DNA primase) showed that phylogenetic tree analysis based solely on sequence alignment of DNA polymerase resulted in a slightly different tree topology with a lower bootstrap support as compared to the multi-locus phylogenetic tree (Figure 5). This demonstrates that although DNA polymerase is a good indicator of evolutionary relationship in general, caution should be taken when resolving closely related N4-like bacteriophages. In contrast, phylogenetic tree construction based on DNA primase is highly comparable with the multi-locus phylogeny approach in terms of tree topology and bootstrap support value.

Protein ID	Product Description	% Identity			InterProscan ID
		E	I	K
AGC31510	hypothetical protein	87	95	97
AGC31513	RNA polymerase RNAP1 subunit A	96	94	98
AGC31517	hypothetical protein	94	93	96
AGC31519	putative phage protein	74	90	84
AGC31522	hypothetical protein	82	99	99
AGC31523	hypothetical protein	91	88	91
AGC31525	RNA polymerase subunit B	95	96	99
AGC31526	RNA polymerase subunit A	88	96	97	IPR002092
AGC31527	capsid decorating protein	79	91	93	IPR003599; IPR007110; IPR013783
AGC31528	hypothetical protein	98	97	97
AGC31531	hypothetical protein	95	97	95
AGC31533	hypothetical protein	95	95	95	IPR027417
AGC31534	hypothetical protein	95	97	99	IPR018698; IPR025154
AGC31537	putative membrane immunity protein	86	98	94	IPR016410
AGC31539	hypothetical protein	88	94	95
AGC31540	thymidilate synthase	84	90	95	IPR003669
AGC31543	hypothetical protein	76	80	82
AGC31545	rIIA-like protein	86	91	94	IPR003594
AGC31546	rIIB-like protein	88	89	87
AGC31547	hypothetical protein	90	96	95
AGC31549	DNA helicase	98	99	99	IPR027417; IPR027785
AGC31550	hypothetical protein	97	99	98
AGC31551	DNA polymerase	98	98	97	IPR001098; IPR002298; IPR012337
AGC31552	hypothetical protein	92	91	99
AGC31555	hypothetical protein	97	95	98
AGC31557	DNS protein	97	96	100	IPR014820
AGC31558	hypothetical protein	99	99	99
AGC31559	ssDNA-binding protein	95	97	97
AGC31560	hypothetical protein	99	99	98
AGC31561	hypothetical protein	76	92	94
AGC31567	putative structural protein	88	85	85
AGC31568	hypothetical protein	92	93	94
AGC31569	putative tail protein	94	82	96
AGC31570	hypothetical protein	96	97	99
AGC31571	major coat protein	97	97	100
AGC31572	hypothetical protein	95	93	96
AGC31573	hypothetical protein	99	96	100
AGC31574	portal protein	97	97	99
AGC31576	N-acetylmuramidase	95	97	98	IPR008565; IPR018537
AGC31502	terminase subunit A	98	98	100	IPR004921
AGC31503	hypothetical protein	98	98	100
AGC31504	hypothetical protein	80	91	85
AGC31505	hypothetical protein	81	76	77
AGC31506	hypothetical protein	87	74	80

Phage	Isolation source	Host	Accession number	Reference
EC-UPM	Chicken fecal material	E. coli	KC206276	This study
KBNP21	Chicken farm	E. coli	JX415535	[24]
vB_EcoP_G7C	Horse fecal material	E. coli	HQ259105	[25]
IME11	Hospital sewage	E. coli	JX880034	[6]
N4	Sewer	E. coli	EF056008	[11]
EamP-S6	Fruit production environment	Erwinia amylovora	NC_019514	[17]
DSS3P2	Seawater sample	Silicibacter pomenyi	Nc_012697	[7]
EE36P1	Seawater sample	Sulfitobacter sp.	NC_012696	[7]
LIT1	Pond	Pseudomonas aeruginosa	NC_013692	[9]
LUZ7	Hospital waste sample	Pseudomonas aeruginosa	NC_013691	[9]

Genome sequencing and annotation of bacteriophage EC1-UPM

Methods for propagation, purification and genomic DNA extraction of bacteriophage EC1-UPM followed those described by Lau et al. [4]. The purified DNA was sequenced on the Illumina Hiseq 2000 (100-bp paired-end reads). The reads were assembled de novo using Velvet v1.1.07. Subsequent scaffolding and gap filling were performed to obtain a complete genome of bacteriophage EC1-UPM. Protein coding sequences were predicted using combined gene model from GeneMarkS and Glimmer3 [26]. The predicted proteins were annotated by BLAST against the NCBI non-redundant database. Additional annotation was subsequently performed using Blast2Go and InterProscan [27, 28]. Possible presence of tRNA in the genome was determined using tRNAscan-SE v1.23.

Genome visualization of bacteriophage EC1-UPM and comparison with other closely related bacteriophage genomes

The annotated complete genome of bacteriophage EC1-UPM was visualized using CLC Genomics Workbench 6.0 (CLC Bio, Denmark). To identify the closely related bacteriophage, BLASTN was performed on the complete genome of bacteriophage EC1-UPM against the NCBI non-redundant database. Blast Ring Image Generator [29] was used to provide the genomic comparison of bacteriophage EC1-UPM against its closely related bacteriophage (40% identity cut-off).

In-silicoanalysis of bacteriophage EC1-UPM tailspike and tail fiber proteins

Additional annotation of the tail fiber protein was performed using the HHpred interactive server [30]. Additionally, the computation of various physical and chemical parameters of the tail fiber protein was performed using ProtParam [31].

Classification of bacteriophage EC1-UPM holin

Protein sequence of the putative holin was subjected to a transmembrane helices prediction using the TMHMM Server v. 2.0. The text output from the prediction was subsequently piped into to TMRPres2D [32] to create uniform, two-dimensional high analysis graphical images of the putative transmembrane protein.

Evolutionary relationship of the N4-like bacteriophages

The currently available genomes of N4-like bacteriophage were retrieved from NCBI in Genbank format. PanOCT [33] was used to identify the genes that are conserved across the N4-like bacteriophages (40% or 70% identity cut-off, at least 70% protein length overlap). Each orthologs cluster was aligned using MUSCLE [34]. Then, the individual alignment was trimmed with TrimAl using the “strictplus” parameter which is optimized for NeighborJoining tree construction [35] and concatenated for phylogenetic analysis using MEGA. A phylogenetic tree was constructed using the NeighborJoining method with 1000 bootstrap repetitions.

Nucleotide sequence accession number

The Genbank accession number for the complete genome sequence of bacteriophage EC1-UPM is KC206276.