The authors declare no conflict of interest.
Bacteriophage Spink was isolated using host bacterium
Bacteriophage Spink was isolated in 2017 from soil in Arkadelphia, Arkansas (GPS: 34.1258° N, 93.0532° W), using standard protocols for enriched isolation as described (Poxleitner et al., n.d.). Soil samples were washed in peptone-yeast extract-calcium (PYCa) liquid medium for two hours at 30°C with shaking to suspend phage particles. The suspension was then filtered through a 0.22 µm filter. The filtrate was then inoculated with
The host range of phage Spink was investigated by performing spot titers of the phage on the following Gordonia species:
Genomic DNA was extracted from the phage lysate using the Promega Wizard DNA cleanup kit. Sequencing of Spink DNA was performed on an Oxford Nanopore Technologies MinION platform using a FLO-MIN106 flow cell. The library was prepared using the SQK-LSK109 ligation sequencing kit according to the manufacturer's specifications. Sequencing produced 2,750 reads and 16.39 Mb of data over five hours. Basecalling of the raw FAST5 files was performed using Guppy v6.5.7 (Wick et al., 2019) with default parameters for FLO-MIN106 and SQK-LSK109. The reads were then filtered using Filtlong v2.0 (Wick, 2017) with the following parameters: mean quality weight of 30, minimum read length of 2,000 bp, and target bases of 5,000,000. This resulted in 1,537 filtered reads totaling 13,221,164 bases.
The filtered reads were then assembled to a genome length of 49,479 bp (~267× coverage) using Flye v2.8.3 (Kolmogorov et al., 2019) with the following parameters: --nano-raw, 100 kb estimated genome size, 1,000 bp minimum overlap, four threads, and haplotype retention enabled. The assembly was followed by polishing via Medaka v2.0.1 (Oxford Nanopore Technologies, 2022). The genome's GC content of 67.4% is similar to its isolation host
The Spink genome was auto-annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5 (Besemer et al., 2005). It was then refined through manual annotation with DNA Master v5.23.6 (Lawrence, 2007), PECAAN v20241104 (Rinehart et al., 2024), BLAST (Camacho et al., 2009), DeepTMHMM v1.0.24 (Hallgren et al., 2022), and HHpred (Söding et al., 2005). Databases accessed by these programs include the following: BLAST used the Actinobacteriophage database (Russell and Hatfull, 2017) and the NCBI non-redundant database; HHpred used PDB_mmCIF70 and Pfam v36; and the NCBI Conserved Domain Database v3.20. Aragorn v1.2.41 (Laslett, 2004) detected no tRNA genes. All software was used with default parameters.
Throughout annotation, functions were assigned to 39 of 78 annotated genes in the 49,479 bp genome. The majority of genes are transcribed rightward, with ten leftward-transcribed genes located from genes 33 to 44. As with all cluster CV phages, Spink encodes an immunity repressor and a tyrosine integrase, suggesting Spink is able to establish lysogeny. Spink also encodes a RelE-like toxin and RelB-like antitoxin adjacent to its tyrosine integrase gene. Spink was found to encode six minor tail proteins along with three membrane proteins of unknown function containing two or more membrane regions.
Sequence data of phage Spink are available under SRA accession
We would like to thank Dr. Han Tan for genome assembly and Dr. Elizabeth Padilla-Crespo for sequencing. We also thank Kayla Brown, Katlin Jacobs, and Jordan Ford for isolating the phage and Jeffrey A Kamykowski at Digital Microscopy Core for visualization.