Bacteriophages are important key biological agents that regulate bacterial populations (Hatfull GF, 2015). The isolation and characterization of bacteriophages can advance our understanding of microbial population dynamics and the development of therapeutics for controlling bacterial growth (Hatfull GF, 2022). Here, we describe 24 bacteriophages isolated from soil samples collected in the Flint Hills region of Kansas (GPS coordinates provided in Table 1) using various actinobacteria as hosts. In total, 16 phages were isolated on Gordonia terrae CAG3, 6 phages on Mycobacterium smegmatis mc ² 155, and 1 phage on Microbacterium foliorum NRRL B-24224.

All phages were isolated by washing the soil samples in liquid medium and filtering the wash using a 0.2 µm filter. The medium used to grow G. terrae CAG3 and M. foliorum NRRL B-24224 was PYCa, and the medium used to grow M. smegmatis mc ² 155 was 7H9. The filtrate was then plated in top agar with host bacteria (direct isolation) or first inoculated with host bacteria and incubated with shaking at 30°C for 1 – 3 days before being refiltered and plated in top agar with host bacteria (enriched isolation) (Table 1). Plates were incubated for 1 – 3 days at 30˚C to form plaques, which were then plaque-purified through 2 – 3 rounds of additional plating. Liquid lysates were then prepared for each purified phage, which were used to image virions by negative-stain (1% uranyl acetate) transmission electron microscopy. All phages displayed a siphovirus morphology, with the exception of CherryTomatoes, which possesses a myovirus morphology. Phage DNA was extracted from the lysate using the Wizard DNA prep kit from Promega, and prepared for sequencing using the NEB FS Ultra II kit before being sequenced using an Illumina MiSeq (v3 reagents) to generate 150 base reads. Raw reads were then assembled using Newbler v2.9 and checked for completeness and genome ends using Consed v29 (Gordon et al., 1998). Sequencing parameters and genome characteristics are presented in Table 1.

All genomes were annotated using DNAMaster (http://cobamide2.bio.pitt.edu/, v5.0.2) and PECAAN (https://discover.kbrinsgd.org , v20240320), Glimmer v3.02 (Delcher et al. 2007, v3-3.02b), GeneMark v2.5 (Besemer and Borodovsky 2005), and Phamerator v578 (Cresawn et al. 2011, web version). tRNAs were identified using ARAGORN v1.2.41 (Laslett and Canback 2004) and tRNAscan-SE (Lowe and Eddy 1997). Functional assignment was performed using BLAST (Altschul et al. 1990.), searching against the Actinobacteriophage and NCBI non-redundant databases, and HHPRED (Soding et al. 2005), searching against the PDB_mmCIF70, Pfam- v.36, NCBI Conserved Domains databases. Phages were assigned to clusters based on gene content similarity of >35% to phages in the Actinobacteriophage database, phagesDB (https://phageDB.org) (Russell and Hatfull 2017; Pope et al. 2017). All software were used with default settings. Genome content is described in Table 1.

Each genome has a set of genes related to phage structure and assembly, including major capsid, minor tail, tape measure, terminase, and portal protein. These genes were located in the right arm of the genome, except for phage CherryTomatoes, where they are located in the middle of the genome. Among the phages characterized in this study, terminase is indicated as either a single gene or two genes (small and large subunits).