Complete Genome Sequences of Mycobacteriophages OKaNui and DroogsArmy

Mycobacteriophages OKaNui and DroogsArmy were isolated from soil using the bacterial host Mycobacterium smegmatis mc2155, which belongs to the phylum Actinobacteria. OKaNui was discovered in east Mississippi and DroogsArmy in west Alabama in the United States. The genomes of OKaNui and DroogsArmy were 51,424 bp and 53,254 bp long, respectively.

A mong biological agents, bacteriophages (phages) are the most populous and ubiquitous in the environment (1). The phages named OKaNui and DroogsArmy were isolated from the bacterial host Mycobacterium smegmatis mc 2 155. Most Mycobacteria species are saprophytic and reside in the soil or water or on plants (2). Although M. smegmatis is a nonpathogenic bacterium, an understanding of phage infection in this strain may contribute to advances in phage therapy for other Mycobacteria species. M. smegmatis may be used as a delivery system for phages intended for infection of M. avium and M. tuberculosis (3). Phages isolated from M. smegmatis have be used in the experimental treatment of closely related hosts, including M. ulcerans (4). OKaNui was discovered in Meridian, Mississippi, in moist soil in a shaded area, and DroogsArmy was found in Lisman, Alabama, in a dark-colored, moist soil ( Table 1).
The soil samples were inoculated with the host bacterium and incubated at 37°C with shaking in 7H9 liquid medium (5). A filtrate was then plated on a lawn of M. smegmatis mc 2 155 along with a negative control (5). The formation of plaques following incubation at 37°C were indicative of phage presence. Isolates were purified using serial dilutions and a filtered high-titer lysate (HTL) collected from webbed plates (5). Phage genomic DNA was extracted using the Wizard DNA cleanup system with modified protocols (Promega, Madison, WI) (5). DNA libraries were built and pooled for sequencing using the NEBNext Ultra II FS kit with dual-indexed barcoding (New England BioLabs, Ipswich, MA). Sequencing was performed using the Illumina MiSeq platform at the Pittsburgh Bacteriophage Institute. The genome lengths, GϩC content, and coverage depths are listed in Table 1. OKaNui yielded ϳ316,000 single-end 150-base reads, and DroogsArmy yielded ϳ410,000 reads. Both genomes displayed defined ends with 10-bp overhangs (CGGCCGGTAA). Assembly was performed using Newbler 2.9 with default settings (6). A single contig for each genome was produced and used to determine the genome ends; the beginning of each genome was chosen based on similar genomes. These were then checked for completeness and accuracy using Consed 2.0 (7).
When plated with the host, OKaNui produced relatively large plaques with a halo, and DroogsArmy produced small plaques. Both OKaNui and DroogsArmy were identified as lysogenic phages because they produced turbid plaques, and the genomes contained genes for lysogeny (14). Transmission electron microscopy and genome analysis indicated that the phages share the Siphoviridae morphotype (5). Based on nucleotide similarity and synteny across the genome, OKaNui has been placed in the A4 subcluster of mycobacteriophages and DroogsArmy in A7 (15).
Data availability. The complete genome sequences of OKaNui and DroogsArmy are available from GenBank under the accession numbers MT490373.1 and MT553337.1, respectively. The raw Illumina reads for OKaNui and DroogsArmy are available on NCBI's Sequence Read Archive under accession numbers SRX8622883 and SRX8622882, respectively.

ACKNOWLEDGMENTS
This material is based upon work supported by the National Science Foundation under grant number 1911457. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. This project was supported by the Howard Hughes Medical Institute Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science program (www.seaphages.org) and the University of West Alabama 2018 -2020 Phage Hunters.
We thank Daniel A. Russell and Rebecca A. Garlena for conducting genome sequencing, quality control, and assembly at the University of Pittsburgh. We thank Kimberly Lackey with the Optical Analysis Facility at the University of Alabama for assisting with transmission electron microscopy.