Complete Genome Sequences of Two Bacteroides uniformis Bacteriophages

ABSTRACT

Here, we describe the isolation and genomic annotation of two novel siphovirus species of bacteriophages that infect Bacteroides uniformis: Bacteroides phage EMB1 and Bacteroides phage EMB2. EMB1 has a 34,204-bp genome with 48 coding sequences, and EMB2 has a 34,008-bp genome with 47 coding sequences.

ANNOUNCEMENT

Bacteroides species are among the most abundant bacteria in the gut microbiome and have been linked to human health and disease (1). Bacteriophages (phages) are a crucial factor in shaping the structure and function of the human gut microbiome (2) and may be key components of future clinical interventions (3, 4). In this work, we sought to isolate and characterize phages for Bacteroides uniformis, which may have beneficial metabolic effects (5–7), as tools to study host-phage interactions in the human gut. Thus far, very few phages that infect B. uniformis have been isolated (8, 9).

Bacteroides phages EMB1 and EMB2 were isolated from filtered (0.22-μm pore size) primary effluent wastewater (collected 16 June 2021 from King County Wastewater Treatment Division’s West Point Treatment Plant, Seattle, WA). Host B. uniformis (strain ATCC 8492) cells were grown anaerobically at 37°C in a nutrient-rich bacterial growth medium (10). Phage enrichment and isolation were performed in growth medium supplemented with 100 μM taurocholic acid (catalog number T4009; Sigma-Aldrich), 100 μM glycocholic acid (catalog number G7132; Sigma-Aldrich), and 0.5% (wt/vol) mixed bile salts (catalog number 48305; Sigma-Aldrich). EMB1 and EMB2 were propagated on B. uniformis using liquid cultures in growth medium and the soft agar overlay method (11).

Genomic DNA was extracted using a phage DNA isolation kit (catalog number 46800; Norgen Biotek Corp.). Sequencing libraries were prepared using the Illumina DNA prep kit and IDT 10-bp unique dual indexes (UDI) and sequenced by the Microbial Genome Sequencing Center (MiGS, Pittsburgh, PA) on the Illumina NextSeq 2000 using 2 × 151-nt paired-end sequencing. Demultiplexing, quality control, and adapter trimming were performed by MiGS using bcl-convert version 3.9.3 (12). Read quality was assessed with FastQC version 0.11.9 (13), and quality filtering performed using BBMap version 38.92 (14). Fifty thousand paired forward and reverse reads (15) were randomly selected using Seqtq version 1.3-GCC-8.3.0 (16) and used for de novo assembly into contigs using MEGAHIT version 1.2.9 (17). Small contigs (213 to 2,412 bp) were determined to be residual bacterial genome sequencing, whereas phage genomes assembled into single contigs greater than 30,000 bp in size, had high coverage, and did not align to the B. uniformis genome. Quality trimmed reads were then mapped back onto each phage genome using BWA-MEM version 0.7.17-GCC-10.2.0 (18). Average genome coverage was determined using SAMtools Depth version 1.11-GCC-10.2.0 (19). Protein coding sequences (CDS) and tRNA genes were predicted and preliminarily annotated using Prokka version 1.14.5 (20). Putative functions were determined using BLASTp version 2.9.0 on the NCBI nonredundant protein sequence database (21) using a maximum expectation value of 0.001 (22). PhageTerm was used to predict the phage termini and packaging mechanism (23). The closest relatives to EMB1 and EMB2 were determined using nucleotide BLAST search (21) on the nucleotide collection (nr/nt) standard database. Intergenomic similarities of EMB1 and EMB2 to closest relatives and to each other were calculated using VIRIDIC Web (24). PhageTerm, Quast, and “GenBank Format to Five Column Format” were accessed through the Center for Phage Technology’s Galaxy and Web Apollo (https://cpt.tamu.edu/galaxy-pub) (25). Genome assembly results and accession numbers are summarized in Table 1.

TABLE 1.

Phage genome assembly results and accession numbers

Phage	No. of:		Genome coverage (×)	GC content (%)	No. of:		Genome length (bp)	Packing mechanism	Termini	GenBank accession no.	SRA accession no.
	Sequencing reads	Filtered reads			CDS	tRNA genes
EMB1	5,451,260	5,251,968	7,832	45.54	48	0	34,204	Unknown	Circularly permuted	ON721384	SRR19527454
EMB2	6,353,794	6,134,744	7,878	45.80	47	0	34,008	Headful (pac)	Circularly permuted	ON721385	SRR19527453

EMB1 and EMB2 plaques are clear. Both phages have icosahedral heads (Fig. 1), and their head and tail sizes are consistent with Siphoviridae morphology (27). EMB1 is most closely related to phage ctND05 (GenBank accession number BK016558.1), with a nucleotide similarity of 86.6%, falling below the 95% average nucleotide identity (ANI) species cutoff (28); thus, EMB1 is a novel phage isolate. EMB2 is most closely related to phage ctND05 (GenBank accession number BK016558.1), with a nucleotide similarity of 87.9%. EMB1 and EMB2 have a nucleotide similarity of 90.8% to one another; therefore, EMB2 is a novel siphovirus as well.

FIG 1.

Transmission electron microscopy images of EMB1 (A) and EMB2 (B). Phage particles were fixed in 1/2 strength Karnovsky’s fixative overnight at 4°C and captured on Formvar/carbon-coated glow-discharged grids. Grids were negatively stained with 1% uranyl acetate and imaged on a ThermoFisher Talos L120c transmission electron microscope at an accelerating voltage of 120 kV. Six phage particles per isolate were measured using ImageJ (26) to determine approximate head and tail size.

Data availability.

The GenBank accession numbers are ON721384 for EMB1 and ON721385 for EMB2. The SRA accession numbers are SRR19527454 for EMB1 and SRR19527453 for EMB2. Metadata are deposited under BioProject number PRJNA844182. BioSample accession numbers are SAMN28795846 for EMB1 and SAMN28795847 for EMB2.