Isolation and Annotation of Cluster EE Bacteriophage Loca

Aurod Ounsinegad, Dasire Brawley, Kendall Johnson, Megan Ashcraft, Grace Clements, Austin Densmore, Ashley Fannin, Alexis Gastin, Sydney Keyon, Marisol Luciano, Cole Moore, Virginia Munoz, Aryana Pernarelli, Maci Pitner, Esmae Velsen, Kara Wiggam, Marlee Goppert, Dustin Edwards

Bacteriophage Loca was isolated from a shopping cart handle swab in Stephenville, Texas, and was part of a collection of 11 bacteriophages isolated in Erath County in 2021. The swab was suspended in peptone-yeast extract-calcium liquid media and supernatant was filtered through a 0.22 µm filter before incubation with host Microbacterium foliorum NRRL-24224 SEA. Following two rounds of 10-fold serial dilutions and plaque assays with a soft agar overlay, Loca formed small, defined lytic plaques less than 1 cm in diameter. Negative-staining transmission electron microscopy revealed Siphoviridae morphology with an approximate tail length of 102 nm and capsid diameter of 43 nm. Bacteriophage DNA was extracted with a modified zinc chloride precipitation method and sequenced to 73-fold genome coverage by the Pittsburgh Bacteriophage Institute using Illumina next-generation sequencing. Bacteriophage Loca has a double-stranded DNA genome that is 17,475 base pairs in length with a 9-base 3’ sticky overhang (CCCGCCCCA) and 68.7% G+C content. Loca is the eighth-largest member of cluster EE, and is most closely related to Microbacteriophages Leafy, Quaker, Livingwater, and Hulk, with nucleotide sequence identity >96%. Whole-genome sequence analysis using PECAAN, PhagesDB, NCBI BLASTn and BLASTp, HHPRED, and tRNAscan-SE software determined 25 protein-coding genes, including DNA-binding and bridging proteins, one HNH endonuclease, and no predicted tRNAs. This whole-genome sequence contributes to our understanding of Microbacteriophage genetics.