Complete Genome Sequence of Microbacterium Bacteriophage Erla

We characterized the complete genome sequence of Siphoviridae bacteriophage Erla, an obligatory lytic subcluster EA1 bacteriophage infecting Microbacterium foliorum NRRL B-24224, with a capsid width of 65 nm and a tail length of 112 nm. The 41.5-kb genome, encompassing 62 predicted protein-coding genes, is highly similar (99.52% identity) to that of bacteriophage Calix.

T he discovery of antibiotic agents in 1928 by Alexander Fleming has completely changed the way clinicians treat bacterial infections. However, with new therapeutic challenges resulting from antibiotic resistance, scientists have now turned to bacteriophages as promising clinical alternatives to antibiotics (1). Aiding this endeavor is the vast, though largely uncharacterized, diversity of bacteriophages across the globe (current estimates range in the order of 10 31 [2]).
Here, we report the complete genome sequence of bacteriophage Erla. Erla was obtained from a soil sample collected at 16°C from a garden in Ottawa, Ontario, Canada (45.428778 N, 75.677246 W). Following the standard procedures outlined in the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Sciences (SEA-PHAGES) Discovery Guide (https://seaphagesphagediscoveryguide .helpdocsonline.com/home), the sample was mixed in an equal volume of peptoneyeast-calcium agar (PYCa) medium and shaken (250 rpm) at 30°C for 2 h. Next, the sample was filtered (pore size, 0.22 mm), mixed with 500 ml host cells (Microbacterium foliorum NRRL B-24224), and grown for 72 h with shaking (220 rpm). A 1-ml sample was filtered (pore size, 0.22 mm), and 100-ml samples of 10-fold serial dilutions were mixed with 300 ml host cells (Microbacterium foliorum NRRL B-24224) and 3.5 ml PYCa top agar. This mixture was incubated at 30°C for 2 to 3 days until plaques formed. Erla was purified by repeated rounds of plaque picking into phage buffer, serial dilution, and plaque assays of 100 ml of diluted phage, as described above. Amplification was performed by flooding "webbed" plates with phage buffer overnight at 4°C. Erla forms medium-sized plaques with a faint bullseye in the middle (Fig. 1a) and exhibits a Siphoviridae morphology with an icosahedral capsid (diameter, 65 nm) enclosing the double-stranded DNA, attached to a flexible, noncontractile tail (length, 112 nm; Fig. 1b).
In order to sequence Erla's genome, DNA was extracted from the phage lysate using a phenol-chloroform assay, followed by ethanol precipitation. Next, a sequencing library was prepared using the NEB Ultra II FS kit and sequenced on an Illumina MiSeq instrument (150bp single-end reads) to .3,300Â coverage (976,546 reads). Following Russell (3), the raw reads were assembled using Newbler v.2.9 (4), resulting in a single linear contig 41,538 bp in size with a GC content of 63.4%, similar to the 68.7% GC content of its host, Microbacterium foliorum NRRL B-24224. The assembly was checked for completeness, accuracy, and genome termini using Consed v.29.0 (5). As neither read start buildups nor substantial variations in coverage were observed, Erla's genome is most likely circularly permuted.
Data availability. Whole-genome sequencing data are available through NCBI's Sequence Read Archive (BioProject accession number PRJNA488469; run accession number SRR13108336). The annotated genome assembly has been deposited in GenBank under accession number MW291026.

ACKNOWLEDGMENTS
Erla was sequenced as part of the Howard Hughes Medical Institute (HHMI) SEA-PHAGES undergraduate research program. Computations were partially performed at Arizona State University's High Performance Computing facility. We are grateful to Suhail Ghafoor for IT support, Billy Biederman, Graham Hatfull, Deborah Jacobs-Sera, Welkin Pope, Daniel Russell, and Vic Sivanathan for library preparation, sequencing, and assembly, as well as providing faculty training for the SEA-PHAGES program and guidance with our genome annotation.
This study was supported by Arizona State University's School of Life Sciences.