Isolation and Annotation of Cluster EB Bacteriophage IndyLu

Ashley Suris, Raylon Huckaby, Jaime Merrill, Selina Alvarado, Tommy Butler, Carlos Canales, Matthew Castro, Julia Gaston, Marlee Goppert, Jesse Laposky, Jessica Lee, Elizabeth Mullins, Damla Ustundag, Josue Zunigue, Faith Cox, Dustin Edwards

With the growing concern surrounding antibiotic resistance, there is increased interest in bacteriophage-based therapies as an alternative treatment strategy. Bacteriophage IndyLu was directly isolated from a soil sample taken from a dry area near a horse barn in Stephenville, Texas, and incubated in host Microbacterium foliorum NRRL-24224 SEA. Following two rounds of serial dilutions and plaque assays with a soft agar overlay, IndyLu formed small, defined lytic plaques less than 1cm in diameter. Negative-staining transmission electron microscopy revealed Siphoviridae morphology with an approximate tail length of 140 nm and capsid diameter of 60 nm. Phage DNA was extracted with a modified zinc chloride precipitation method and sequenced to 1156-fold genome coverage by the Pittsburgh Bacteriophage Institute using Illumina Next Generation Sequencing. A double-stranded DNA genome of 41,958 base-pairs with a 10 base 3’ sticky overhang (ACTCCCGACA) was determined, making IndyLu the sixth largest member of cluster EB, with an average G+C content of 66.2% for the cluster, and most closely related to Microbacterium phages Didgeridoo (96% coverage) and Lahqtemish (95%). Whole-genome sequence analysis using PECAAN, PhagesDB, NCBI BLASTn and BLASTp, HHPRED, and TmHmm revealed 72 protein-coding genes transcribed rightwards (94.5% of genome) and leftwards (5.6% of genome). Putative genes include structural proteins, a HNH endonuclease, Holliday junction resolvase, and Cas4 family exonuclease have already been identified.