The authors declare that there are no conflicts of interest present.
Bacteriophage Myram was isolated from sandy soil in South Portland, ME on
(A) Plaques formed by phage Myram on
Actinobacteriophage are being developed for use in the treatment of multidrug-resistant bacterial infections (Hatfull 2020; Hibstu 2022).
Myram was isolated from sandy soil at the top of a cliff overlooking Willard Beach in South Portland, Maine (Global Positioning System 43.645555 N, 70.227498 E) using standard procedures (Zorawik et al., 2024). Five grams of soil were suspended in PYCa (peptone, yeast extract, and calcium chloride) liquid media and the suspension inoculated with
Phage DNA was extracted from a lysate using the Promega Wizard DNA clean-up kit. The sequencing library was prepared using the NEB Ultra II Library Kit, then sequenced on the Illumina NextSeq 1000 sequencer with v3 reagents. This yielded 166M 100-base single-end reads, providing 9,347-fold coverage. Raw reads were trimmed and filtered by cutadapt 4.7 using the -nextseq-trim 30 option (Martin 2011), and skewer 0.2.2 using options -q 20 -Q 30 -n -l 50 (Jiang et al., 2014 ) and genomic termini and completeness was evaluated with Consed V29 (Gordon and Green, 2013) using default parameters. The complete Myram genome length is 17,362-bp with a 68.5% GC content and 3' single-stranded ends (5' CCCGCCCCA).
Myram's genome sequence was automatically annotated in DNA Master v5.23.6, build 2705 (Pope and Jacobs-Sera, 2017). GeneMark v2.5p (Besemer and Borodovsky, 2005) and Glimmer v3.02b (Delcher et al., 2007) were used to assess coding potential and identify protein-coding genes. BLASTp v2.16.0 using the Actinobacteriophage and the NCBI non-redundant protein sequences [nr] databases (Altschul et al., 1990), HHPred v2.08 using the PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains [CD] databases (Söding et al., 2005), Phamerator v597, Actino_Draft(Cresawn et al., 2011) using the Actinodraft database, and Starterator v3.02(Pacey, 2016) were used to refine start selection and ascertain predicted gene functions. Aragorn v1.2.41.c. (Laslett and Canback, 2004) and tRNAscanSE v2.0.12 (Lowe and Eddy, 1997) were utilized to detect possible tRNAs. DeepTMHMM v1.2.33.c. (Hallgren et al., 2022) and SOSUI v1.11 (Hirokawa et al., 1998) were used for transmembrane domain detection. All software was operated on default settings. This analysis identified 25 putative protein-coding genes, of which 18 could be assigned a putative function.
Myram was assigned to cluster EE, based on gene content similarity (GCS) of at least 35% to phages in the same cluster (Pope et al.,2017; Russell and Hatfull, 2017). As with previously characterized cluster EE phages, Myram encodes structural proteins in the left third of the genome, a putative lysis cassette on the right arm containing an endolysin and two transmembrane proteins, and a predicted programmed translational frame shift encoding the tail assembly chaperones. All genes are transcribed unidirectionally, with the exception of three consecutive genes at the right end of the genome that encode for two DNA-binding proteins and an Lsr2-like DNA bridging protein. There are no identifiable integrase or immunity repressor functions suggesting that Myram is unlikely to establish lysogeny.
The Myram sequence and annotation are available at GenBank with Accession No.
We’d like to thank Kayla O’Rourke for discovering and assisting in the isolation of phage Myram. A special thanks to Vic Sivanathan and Howard Hughes Medical Institute (HHMI) for support in our research as well as Dr. Graham Hatfull and the SEA-PHAGES program. We are also grateful to Dan Russell, Becky Garlena, and Deborah Jacobs-Sera for sequencing and guidance through the annotation process. We thank the University of Maryland Baltimore County (UMBC) and Tagide deCarvalho at the Keith R. Porter Imaging Facility for electron microscopy of phage Myram.