Complete Genome Sequences of 38 Gordonia sp. Bacteriophages

ABSTRACT

We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity.

GENOME ANNOUNCEMENT

The bacteriophage population is vast, dynamic, and old, with an estimated population of 10³¹ virions and 10²³ productive infections/s on a global scale (1). The genomic diversity of the population is poorly understood, with fewer than 3,000 complete genome sequences in GenBank. In general, phages isolated on phylogenetically unrelated hosts share little or no sequence similarity, but considerable insights can be gleaned by comparative genomics of phages isolated on a common host, as illustrated for enterobacteriophages and mycobacteriophages (2, 3). The Howard Hughes Medical Institute (HHMI) Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program provides an undergraduate course-based research experience that contributes to our understanding of phage diversity and evolution through bacteriophage discovery and genomics, using Actinobacteria, including mycobacteria and Gordonia sp. strains, as isolation hosts.

Gordonia phages were isolated by enrichment or direct plating of filtered soil samples using Gordonia terrae 3612 or Gordonia neofelifaecis NRRL 59395 as a host (Table 1). Thirty-eight individual phages were isolated, and electron microscopy shows that all have siphoviridal morphotypes. Plaque-purified phages were amplified, and their double-stranded DNA (dsDNA) was extracted and sequenced using an Illumina MiSeq, as described previously (4). The 140-base reads were assembled using Newbler and Consed, with average coverages between 447- and 3,241-fold. Sequence ambiguities and genome termini were resolved either by sequencing directly from genomic templates or from PCR products. Genomes were annotated using DNA Master (http://cobamide2.bio.pitt.edu), coding sequences were predicted using GeneMark (5) and Glimmer (6), and tRNAs were predicted using Aragorn (7) and tRNAscan-SE (8). Functional assignments were made using BLASTP (9) and HHpred (10, 11) against the publically available databases GenBank, the Protein Data Bank, and Pfam.

TABLE 1 .

Gordonia phage genometrics

Phage name	GenBank accession no.	Genome size (bp)	G+C content (%)	No. of tRNAs	No. of CDSsa	End typeb	Host strain
Bachitac	KU998247	93,843	61.9	8	182	CGCGACGCTC	G. terrae 3612
Bantamd	KX557272	92,580	64.7	2	168	CGCAGCACTC	G. terrae 3612
BatStarre	KX557273	53,432	66.6	0	83	CGGCTGGGGA	G. terrae 3612
Blueberryc	KU998236	54,990	67	0	86	TGGCCGGTGA	G. terrae 3612
BritBratc	KU998233	55,524	65	0	98	CGTATGGCAT	G. terrae 3612
CaptainKirk2e	KX557274	47,898	67.4	0	79	TCGCCGGTGA	G. terrae 3612
CarolAnne	KX557275	54,167	66.9	0	80	TGGCCGGTGA	G. terrae 3612
ClubLc	KU998246	92,618	61.9	9	179	CGCGACGCTC	G. terrae 3612
Cozzc	KU998239	46,600	60	0	68	CGGTAGGCTT	G. terrae 3612
Cucurbitaf	KX557276	93,686	62	9	178	CGCGACGCTC	G. terrae 3612
Demosthenesc	KU998242	74,073	59.3	0	95	Dir. Term. Repeat	G. terrae 3612
Eyree	KX557277	44,929	67.5	0	74	CCCTGCGCTGA	G. terrae 3612
Ghobese	KX557278	45,285	65.2	0	59	TGCCCGAGGTA	G. terrae 3612
Hedwige	KX557279	44,536	67.2	0	70	TCCCGCGGTA	G. terrae 3612
Howec	KU252585	53,182	65.6	0	79	TGCCAAGGGGA	G. terrae 3612
JSwagd	KX557280	52,726	61.9	3	101	CGGGTGGTTA	G. terrae 3612
Jumbod	KX557281	78,302	54.5	0	102	Dir. Term. Repeat	G. terrae 3612
Kampec	KU998254	80,649	47	2g	115	Dir. Term. Repeat	G. terrae 3612
KatherineGc	KU998251	52,689	61.9	3	99	CGGGTGGTTA	G. terrae 3612
Kvothec	KU998243	75,462	59.5	0	99	Dir. Term. Repeat	G. terrae 3612
Nyceiraee	KX557282	41,857	67.5	0	61	CGCGGGGGA	G. terrae 3612
OneUpc	KU998245	93,577	61.5	9	163	CGCGACGCTC	G. terrae 3612
Orchidc	KU998253	80,650	47	2g	114	Dir. Term. Repeat	G. terrae 3612
PatrickStarc	KU998252	80,729	47	2g	115	Dir. Term. Repeat	G. terrae 3612
Remusc	KX557283	52,738	62	3	98	CGGGTGGTTA	G. terrae 3612
Rosalindc	KU998250	52,684	61.9	3	99	CGGGTGGTTA	G. terrae 3612
Smoothiec	KU998244	93,139	61.9	8	179	CGCGACGCTC	G. terrae 3612
Soupsc	KU998249	52,924	61.9	3	98	CGGGTGGTTA	G. terrae 3612
Splinterc	KU998238	45,858	66.1	0	80	TCCGGGCCGGTA	G. terrae 3612
Strosahld	KX557284	52,738	62	3	98	CGGGTGGTTA	G. terrae 3612
Terrapine	KX557285	66,611	59.6	0	97	Circ. Permuted	G. terrae 3612
Twister6e	KX557286	57,804	67.7	0	93	Circ. Permuted	G. terrae 3612
Utzc	KU998248	49,768	67.7	0	71	TCGCCGGTGA	G. terrae 3612
Vendettac	KU998237	45,858	66.1	0	81	TCCGGGCCGGTA	G. terrae 3612
Wizardc	KU998234	58,308	67.9	0	89	Circ. Permuted	G. terrae 3612
Zirinkae	KX557287	52,077	66.7	0	79	CGGCTGGGGA	G. terrae 3612
Jeaniec	KU998256	17,118	68.6	0	25	AGCCCCCGGT	G. neofelifaecis
McGonagallc	KU998255	17,119	68.6	0	25	AGCCCCCGGT	G. neofelifaecis

^aCDSs, coding sequences.

^bEnd types are 3′-single-stranded overhangs, unless otherwise noted as Dir. Term. Repeat (direct terminal repeat) or Circ. Permuted (circularly permuted).

^cPhage Hunters Integrating Research and Education (PHIRE) program, University of Pittsburgh.

^dScience Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES), University of Wisconsin-River Falls.

^eSEA-PHAGES, University of Pittsburgh.

^fSEA-PHAGES, Calvin College.

^gThis total includes one transfer-messenger RNA (tmRNA).

The 38 newly isolated Gordonia phages exhibit considerable diversity (Table 1). The smallest genomes, Jeanie and McGonagall, at ~17,000 bp, have the highest G+C content (68%) and are each predicted to contain only 25 genes, including those encoding structural proteins, integrase and immunity repressor, endolysin, and a DnaQ-like subunit of DNA polymerase III. Three phages (PatrickStar, Kampe, and Orchid) have G+C contents (47%) that are strikingly lower than that of their host (67.77%), and lower than the G+C% of any mycobacteriophage; these phages may be relatively recent arrivals to the Gordonia neighborhood (12) (Table 1). These phages, together with Kvothe, Jumbo, and Demosthenes, have genomes with direct terminal repeats, a feature not observed in any mycobacteriophages. Many of the Gordonia phage genomes have defined ends with 3′ single-stranded extensions (Table 1), and only three (Terapin, Twister6, and Wizard) are circularly permuted.

Most of the Gordonia phages form turbid plaques, and 27 of the 38 encode either tyrosine or serine integrases; another six phages encode putative ParAB partitioning systems. Temperate lifestyles thus appear to be common for these phages. Some of the phages have all or part of a second integrase gene, and although these are mostly predicted to be nonfunctional, they perhaps reflect relatively recent genomic rearrangements. Finally, we note that six phages, KatherineG, Rosalind, Strosahl, Remus, Soups, and JSwag, are sufficiently similar to some mycobacteriophages to warrant grouping within Cluster A (13).

Accession number(s).

Nucleotide sequence accession numbers are shown in Table 1.