Enterobacteria Phage SV76 Host Range and Genomic Characterization

Abstract

Background: Increasing the quantity and detail of bacteriophage genomic data is critical to broadening our understanding of how bacteriophages operate to allow us to harness their unique properties for biotechnology advancements. In this study we present the complete sequence of phage SV76's assembled and annotated genome (Accession No. OM339528). SV76 has previously been classified as a T4-like bacteriophage belonging to the Tequatrovirus genus within the Myoviridae family of contractile tailed bacteriophages.

Materials and Methods: Whole genome sequencing, assembly, and annotation were performed on SV76. Double-agar spot assays were utilized to determine SV76's host range against a panel of 72 Escherichia coli isolates meant to represent the diversity of E. coli, as well as a series of knockouts designed to identify required receptor binding proteins. The genome and host range were compared with the closely related phage, T2.

Results: Spot assays revealed that SV76 could plaque on 10 of the 72 strains (13.9%) and nine of the nine E. coli K12 single gene knockout of known phage receptors (100%). SV76 did not plate on a ΔfadL E. coli suggesting a requirement as a receptor binding protein.

Conclusions: SV76 is closely related to T2 with similar host ranges within ECOR. This study presents novel host range and genomic data on SV76 phage, providing a foundation for future studies to further characterize SV76 to understand more about SV76 and other T4-like phages that can be applied to create novel biotechnologies.

Introduction

Enterobacteria phage SV76 (NCBI:txid69613) was isolated by Dr. Grimont of the Institut Pasteur in Paris, France, and was first published by Ackermann and DuBrow in 1987.^1,2 SV76 is classified as member of the Tequatrovirus genus within the Myoviridae family of contractile tailed bacteriophages.³ The proposed changes in taxonomy would consider these phages in the family of Straboviriade, subfamily Tevenvirinae, and bionomial species Tequatrovirus SV76. This genus consists of many Enterobacteria phages as well as some Shigella, Aeromonas, and Cyanophages.³

SV76 is considered a T-even phage belonging to the T4-like superfamily, a group consisting of some commonly studied phages including T2, T4, and T6. T4-like phages all share similar elongated icosahedral capsid morphology, 160–260 kb double-stranded DNA genomes, and contractile tails terminated by a complex base plate with six long tail fibers.^4–7 Phages in this superfamily mainly infect Escherichia coli and closely related Enterobacteria species.

Limited research has been done on the genomic and structural characterization of SV76. The tail fiber adhesion (gp38) and single-stranded DNA-binding protein (gp32) of SV76 have been described and are available in NCBI's protein database.⁸ There have been a few studies involving SV76's host range. In one study by Trojet et al., E. coli K12 strains with single gene deletions of known phage receptors to identify that infection by SV76 require host receptors OmpF, FhuA, and full-length core oligosaccharide of the lipopolysaccharide to all be present.⁹

The data also indicate that although SV76 only varies by two residues from T2 phage in the hyper variable regions of the long tail fibers that are a major determinant of host range, T2 requires a single host receptor for infection that is completely different, FadL.^9,10 In another study, SV76's ability to infect Yersinia pseudotuberculosis was utilized to modify the host range of a T4 phage by swapping regions of its long tail fiber with SV76.^10,11

In this study, we evaluate the host range of SV76 against a panel of 72 E. coli isolates intended to represent the diversity of E. coli, the ECOR Reference Library, as well as an additional nine E. coli K12 strains with single gene deletions in known phage receptors.^12,13 We also present the complete sequence of SV76's genome, which we further annotated using web-based bioinformatics platforms, Galaxy and Apollo. The SV76 host range and adhesion sequence were compared with T2 phage, due to high genomic sequence similarity.

SV76's genome (Accession No. OM339528) was submitted to NCBI as a BioSample (SAMN25208553) in BioProject PRJNA579348, and the raw whole genome sequence reads were made publicly available as an SRA (#17713127). This study produced SV76's annotated genome as well as an initial host range analysis, which can be utilized in future studies to uncover more about SV76 and other phages in the T4-like superfamily.

Materials and Methods

Bacteria, phage, and reagents

Bacterial cultures were grown in Luria–Bertani (LB) broth (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and shaking at 90 rpm for 18 h. The ECOR Reference Library was obtained from the Ochman Lab¹² at the University of Texas (EC990774–EC990845). The E. coli K12 strains with single gene knockouts in known phage receptors JW0146-2 (ΔfhuA), JW0401-1 (Δtsx), JW0940-6 (ΔompA), JW2203-1 (ΔompC), JW3605-1 (ΔwaaP), JW3606-1 (ΔwaaG), JW3619-1 (ΔyicC), JW2341-1 (ΔfadL) were received from the Coli Genetic Stock Center at Yale University.¹³ SV76 was generously donated by H.M. Krisch from Centre National de la Recherche Scientifique, Toulouse, France.

The raw sequence reads from Whole Genome Sequencing of all ECOR strains can be found as BioSamples SAMN13109282 to SAMN13116764 within NCBI BioProject PRJNA579348. Propagation strain, E. coli DH5 Alpha (ATCC 68233), was purchased from ATCC (ATCC, Manassas, VA, USA). SV76 was a gift given to our group as part of a collaborative effort from the Krisch Research Group in Toulouse, France, and was deposited in our strain collection as NRG-P0013. SV76 was propagated using standard procedure as described by Bonilla and Barr.¹⁴

Phage lysate was purified by centrifuging at 3260 × g for 30 min to pellet large cell debris followed by supernatant filtration through 0.22-μm pore size filter PDVF vacuum filter systems from Corning (Corning, NY, USA). Phages were concentrated through centrifugation (JA17 rotor, 39,800 × g, 3 h, 4°C) and resuspension in 500 μL of SM buffer. Phage titer was determined through double-agar spot assay.¹⁵

Host range assay

SV76 and T2's ability to plaque on 72 ECOR isolates and nine E. coli K-12 strains with single gene knockouts in known phage receptors was assessed through double-agar spot assay.¹⁵ Molten 0.8% LB top agar was inoculated with 500 μL of overnight bacterial culture, mixed, and then poured onto an already solidified 1.4% LB agar square plate. SV76 phage at a starting titer of 2 × 10¹⁰ pfu/mL and T2 phage at a starting titer of 6 × 10¹⁰ pfu/mL were serially diluted in SM buffer, whereas the top agar was allowed to solidify at room temperature. SV76 and T2 dilutions negative one through negative eight were spotted (10 μL) onto each bacteria-seeded plate. Phage spots were allowed to fully dry at room temperature before incubating plates overnight at 37°C.

An isolate was considered a host if lysis gradually progressed through the dilution series to achieve individual plaques on the higher dilutions. An isolate was not considered a host if there was no lysis or only lysis on the lower dilutions but no gradual progression to single plaques. The lysis seen in the later situation could be a result of abortive infection or virion induced lysis-from-without, both of which are a result of incomplete phage replication cycle that do not produce phage progeny.^16,17

Genome assembly and annotation

SV76 genome was isolated using the “extended protocol” from Norgen Biotek's Phage Genome Isolation kit (Norgen Biotek, ON, Canada). The genomes were submitted for whole genome sequencing as a service from the Goodman Lab (Cornell University College of Veterinary Medicine Animal Health Diagnostic Center, Ithaca, NY, USA). Analysis was performed using the Illumina MiSeq NGS platform with a Nextera XT DNA Library Kit. Data collection and quality control analysis were performed in BaseSpace Sequence Hub (Illumina, San Diego, CA, USA) and all returned reads were evaluated to ensure that >85% of bases called had a quality score of 30 or higher.

Raw sequence reads were assembled and analyzed using the Geneious Prime 2021.2.2 software with both forward and reverse sequences being imported as paired-end reads (inward pointing). The “BBDuk” plugin tool was used to trim and filter paired reads using an error probability limit of 0.05.¹⁸ Reads were then normalized using default settings from the “BBNorm” plugin (target coverage level = 40; minimum depth = 6).

Trimmed and normalized SV76 phage reads were assembled using Geneious Prime's internal de novo assembly algorithm. Because SV76 is a T-even bacteriophage, the genomic repeat left behind as an erroneous sequencing artifact from de novo assembly was identified and deleted after SV76's rIIa gene was made the first CDS of its genome. To identify related phages, SV76's genome was evaluated using NCBI's BLASTn database with the settings: standard database, RefSeq Genome Database, organism—viruses (taxid:10239), and optimized for somewhat similar sequences (blastn).¹⁹

For SV76 genome annotation, a FASTA file of the assembly was exported from Geneious Prime and uploaded to the Galaxy Project,²⁰ using server workspace (https://cpt.tamu.edu/galaxy-pub) provided by the Center for Phage Technology (CPT). The CPT's “PAP Structural Workflow v2021.02” in Galaxy was run to predict the location of possible tRNAs, tmRNAs, open reading frames, CDSs, Shine–Dalgarno sequences, and Rho-independent terminators in the SV76 genome. The location of identified genetic elements was viewed and defined or “called” using the genome browser software, Apollo.^21,22 The structurally defined SV76 genome was run through CPT's “PAP Functional Workflow v2021.01” in Galaxy.

This functional workflow takes the genetic elements that were defined during the structural workflow evaluations and searches those sequences within known and trusted databases such as BLAST and SwissProt. These evaluations provide evidence tracks used to define the predicted functions of the previously identified genetic elements. Final annotation calls were made in Apollo utilizing the resulting hits and evidence tracks from the functional workflow.

SV76's annotated genome was exported back into Galaxy as a merged GFF3/FASTA file so it could be split into a raw FASTA file and a GFF3 file. The FASTA file was downloaded and the GFF3 file was further modified using the “GFF3 to GenBank conversion” tool the “GenBank to 5 Column Table” tool was used on the GFF3 file, which converted the file into a five-column table that could be submitted through the BankIt portal (BankIt submission #2542745). This submission was done from within our group's previously made BioProject (PRJNA579348), and all raw WGS data were submitted to NCBI as an SRA (#17713127) in the same location. All NCBI submission details are found in Table 1.

Table 1.

Information from NCBI Submission of SV76's Annotated Genome

BankIt submission	#2542745
Phage name	Bacteriophage SV76 Complete Genome
BioSample	SAMN25208553
Raw WGS data (SRA)	SRA#17713127
BioProject	PRJNA579348
Accession no.	OM339528

WGS, whole genome sequencing.

Results and Discussion

SV76 genome characteristics

The complete sequence of SV76's annotated genome (Accession No. OM339528) is part of NCBI BioProject PRJNA579348. De novo assembly of SV76's genome used 99.1% of all available sequence reads and resulted in a depth of sequencing coverage of 400 × . The consensus sequence is 163,826 bp in length, with a GC content of 35.3%. After assembly, SV76's rIIA (located within the well-conserved lysis inhibition operon, r^11,23) was made the first CDS in its genome: a common practice when sequencing and annotating the genomes of newly isolated T-even phages. SV76 is classified as a “T4-like” phage and has high similarity to T4 phage (97.66% identity), but BLASTn evaluations indicated SV76's genome is most closely related to Enterobacteria phage T2 (99.99% identity).

In accordance with previous reports on the sequence of SV76's gp38,⁹ we also found that it only differs from T2 phage's gp38 (UniProtKB-A0A6J3ZU77) by two amino acid residues. Figure 1 shows a hydrophobic alanine and uncharged glycine in T2 were replaced with a polar threonine and negatively charged aspartic acid in SV76. The substitution of amino acids with different properties in the hypervariable region of the long tail fiber has potential to alter interactions the long tail fiber has with bacteria receptors, which was later evaluated through host range analysis. SV76's genome is also closely related (>91% identity) to a handful of other phages including 49 Escherichia, 12 Shigella, 10 Enterobacteria, 3 Citrobacter, 3 Yersinia, 2 Salmonella, and 1 Serratia.

FIG. 1.

T2 and SV76 adhesion comparison. Alignment of T2 and SV76 Gp38 adhesion protein sequences. Dashes in the SV76 sequence indicate the same amino acid in that position as T2.

A list of the top 100 genomes with the most nucleotide similarity to SV76 is given in Supplementary Table S1. As described in Supplementary Table S2, the final predicted annotations from Galaxy and Apollo revealed that SV76 has 9 tRNAs and 287 coding regions. Finally, the GenBank flat file that contains SV76's complete genome and all locations from various genetic elements is given in Supplementary Tables S3a and b. Although the whole genome sequence of SV76 has not previously been reported, several gene sequences have been.^1–3,9 We confirmed the reported sequence matched our findings.

SV76 host range against ECOR isolates

The spot assay results displayed in Table 2 show that SV76 could plaque on 10 of the 72 ECOR strains (13.9%) and all 9 of the E. coli K12 single gene knockout strains (100%). T2 phage had very high genomic sequence similarity to SV76 (99.99% identity), so we performed host range analysis of T2 with the same bacteria strains to see whether the genomic similarity translated to host range phenotypic similarity. T2 had the same plaquing host range with the ECOR strains as SV76 but could only plaque on eight of the nine E. coli K12 single gene knockout strains (88.9%). T2 phage's inability to plaque on ΔfadL was consistent with a previous study by Trojet et al., however, our results for SV76 differed significantly with their results, indicating it could only plaque on four out of eight knockout strains tested.⁹

Table 2.

Host Range Analysis of SV76 and T2

Strain	SV76 host	T2 host	Strain	SV76 host	T2 host	Strain	SV76 host	T2 host
ECOR 1	−	−	ECOR 28	+	+	ECOR 55	−	−
ECOR 2	−	−	ECOR 29	−	−	ECOR 56	−	−
ECOR 3	−	−	ECOR 30	−	−	ECOR 57	−	−
ECOR 4	+	+	ECOR 31	−	−	ECOR 58	−	−
ECOR 5	+	+	ECOR 32	−	−	ECOR 59	−	−
ECOR 6	−	−	ECOR 33	−	−	ECOR 60	−	−
ECOR 7	−	−	ECOR 34	−	−	ECOR 61	−	−
ECOR 8	−	−	ECOR 35	−	−	ECOR 62	−	−
ECOR 9	+	+	ECOR 36	−	−	ECOR 63	−	−
ECOR 10	−	−	ECOR 37	−	−	ECOR 64	−	−
ECOR 11	−	−	ECOR 38	−	−	ECOR 65	−	−
ECOR 12	−	−	ECOR 39	−	−	ECOR 66	−	−
ECOR 13	+	+	ECOR 40	−	−	ECOR 67	−	−
ECOR 14	−	−	ECOR 41	−	−	ECOR 68	−	−
ECOR 15	−	−	ECOR 42	−	−	ECOR 69	−	−
ECOR 16	+	+	ECOR 43	−	−	ECOR 70	−	−
ECOR 17	−	−	ECOR 44	−	−	ECOR 71	−	−
ECOR 18	−	−	ECOR 45	−	−	ECOR 72	−	−
ECOR 19	−	−	ECOR 46	−	−	ΔompA	+	+
ECOR 20	−	−	ECOR 47	+	+	ΔompF	+	+
ECOR 21	−	−	ECOR 48	+	+	ΔompC	+	+
ECOR 22	−	−	ECOR 49	−	−	Δtsx	+	+
ECOR 23	−	−	ECOR 50	−	−	ΔfhuA	+	+
ECOR 24	−	−	ECOR 51	−	−	ΔfadL	+	−
ECOR 25	−	−	ECOR 52	−	−	ΔwaaG	+	+
ECOR 26	+	+	ECOR 53	−	−	ΔwaaP	+	+
ECOR 27	+	+	ECOR 54	−	−	ΔyicC	+	+

Double-agar spot assays were performed using serially diluted phage aliquots. A “+” indicates a host and a “−” indicates not a host. A strain was considered a host if lysis gradually progressed throughout the dilution series to achieve individual plaques on the higher dilutions. A strain was not considered a host if there was no lysis or only lysis on lower dilutions but no gradual progression to single plaques.

We both used serial dilution spot assays to evaluate host range with the only differing factors in the methods being type of top agar medium used (LB vs. Htop) and volume of dilutions plated (10 μL vs. 3 μL). Medium components can affect expression of E. coli outer membrane proteins that could be a reason for the variation, but a definitive source of the varying host range results is still unknown.^24,25

Conclusion

In this study, Enterobacteria phage SV76's complete genome was assembled and annotated, in addition to initial host range analysis with 72 ECOR isolates. BLASTn evaluations revealed that SV76's genomic sequence is most like bacteriophages T2 (99.99% identity) and T4 (97.66% identity), but is also related to a handful of other Enterobacteria phages. SV76's genome was submitted to NCBI through the BankIt portal and was given the accession number OM339528. Host range analyses of SV76 against the ECOR Reference Library through double-agar spot assays revealed SV76 can plaque on 10 of the 72 strains (13.9%) and 9 out of 9 E. coli K12 single gene knockout strains (100%).

SV76 had the same ECOR host range as T2, but SV76 could plaque on ΔfadL while T2 could not. Future studies to further characterize SV76 are still needed, however, the study presented here provides a solid foundation of data to support additional genomic and host range analyses. The genomic and host range similarity between SV76 and T2 in this study suggests these phages are variants with one differentiation being the T2 requirement of FadL (long-chain fatty acid transport protein) in the host E. coli.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study is supported, in part, by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) R01EB027895. This study was also supported by USDA NIFFA predoctoral fellowships 2020-09959 and 2018-07728.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3