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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.

T he bacteriophage population is vast, dynamic, and old, with an estimated population of 10 31 virions and 10 23 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.
The 38 newly isolated Gordonia phages exhibit considerable diversity ( Table 1). The smallest genomes, Jeanie and McGonagall, at~17,000 bp, have the highest GϩC 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 GϩC contents (47%) that are strikingly lower than that of their host (67.77%), and lower than the GϩC% 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.