Complete Genome Sequence of the Gordonia Bacteriophage BiggityBass

ABSTRACT Here, we characterized the complete genome of the Siphoviridae BiggityBass, a lytic subcluster DR bacteriophage infecting Gordonia terrae CAG3. Its 63.2-kb genome contains 84 protein-coding genes, of which 40 could be assigned a putative function. BiggityBass is related most closely to AnClar and Yago84 with 90.61% and 90.52% nucleotide identity, respectively.

B acteriophages are one of the most abundant organisms on our planet (1), and yet, their diversity remains poorly characterized. As part of the Howard Hughes Medical Institute Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (HHMI SEA-PHAGES) course-based undergraduate research experience, we characterized the complete genome sequence of BiggityBass, an obligatory lytic Gordonia terrae CAG3 bacteriophage.
BiggityBass was obtained by direct plating of a filtered soil sample collected in Manhattan, Kansas, at the top of a hill on the Konza Prairie Nature Trail (39.108056 N, 96.596944 W), purifying a well-isolated plaque, and amplifying the plaque in Gordonia terrae CAG3 (which was propagated in peptone-yeast extract-calcium [PYCa] broth at 30°C for 48 h with aeration in an orbital incubator at 250 rpm), following the procedures outlined in the SEA-PHAGES Discovery Guide (https://seaphagesphagediscoveryguide.helpdocsonline.com/home).
Copper electron microscopy grids were prepared using uranium acetate staining, which demonstrated that BiggityBass exhibits a Siphoviridae morphology, characterized by a long, noncontractile tail and an icosahedral capsid that contains the double stranded DNA (dsDNA) (Fig. 1). Genomic DNA was extracted using the Promega Wizard DNA clean-up kit (washing columns twice with 80% propanol, without RNase or DNase pretreatment), and a library was prepared using the NEBNext Ultra II FS kit and sequenced on an Illumina MiSeq instrument to more than 900-fold coverage. Next, 411,782 high-quality, 150-bp single-end raw sequencing reads were de novo assembled using Newbler v.2.9 (2), yielding a complete genome of length 63,202 bp with circularly permuted ends. The genome exhibits a GC content of 69.4%, which is similar to that of its host Gordonia terrae (67.8%). The assembly was checked for completeness, accuracy, and genome termini using Consed v.29.0 (3). All software was executed using default settings.
Data availability. The whole-genome sequencing data are available through NCBI Sequence Read Archive (BioProject accession number PRJNA488469; run number SRR20167194). The annotated genome assembly is available through NCBI GenBank under accession number ON260813.

ACKNOWLEDGMENTS
This work was supported by a National Science Foundation CAREER grant to S.P.P. (DEB-2045343), Howard Hughes Medical Institute SEA-PHAGES program, and Arizona State University School of Life Sciences.
Bacteriophage isolation was performed at Kansas State University, Manhattan, KS; library preparation, sequencing, and de novo assembly were performed at the University of Pittsburgh, Pittsburgh, PA; and genome annotations and comparative analyses were performed at Arizona State University, Tempe, AZ.