Complete Genome Sequence of Escherichia coli Myophage Mangalitsa

Enteropathogenic Escherichia coli is a prevalent Gram-negative bacterium that can lead to fatal complications from infection in humans. Here, we present the isolation and complete annotation of the 52,329-bp genome of enteropathogenic E. coli ATCC 23545 myophage Mangalitsa. Predicted terminal repeats and temperature sensitivity for plaque formation place Mangalitsa with similar unclassified myoviruses.

gens that are prevalent in both communal and clinical settings (1). They have been identified as one of the leading causes of persistent diarrhea, which is the second largest contributor to childhood mortality, accounting for 1.3 million deaths per year (2,3). Here, we present the complete genome sequence of enteropathogenic E. coli myophage Mangalitsa.
Bacteriophage Mangalitsa was isolated from a chloroform-sterilized and enriched swine fecal sample collected in College Station, TX, based on its ability to grow on the enteropathogenic E. coli strain ATCC 23545. While the host was typically grown aerobically at 37°C in Luria broth (BD) and standard soft agar overlay methods were used (4), Mangalitsa only produced plaques at 30°C or 22°C. Phage genomic DNA was isolated using the shotgun library prep modification of the Promega Wizard DNA clean-up system (5). A genomic library prepared with the TruSeq Nano low-throughput kit was sequenced by an Illumina MiSeq platform with paired-end 250-bp reads. A total of 480,501 reads were in the phage index. Quality control was performed with FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/). Sequence reads were then trimmed using the FASTX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). The genome was assembled with 1,333-fold coverage using SPAdes v3.5.0 and closed by PCR (forward primer, 5=-AGTGCACGGTAT TCTTCGTTAG-3=; reverse primer, 5=-CTAACGCATCGAATCTCTTCTCA-3=) and Sanger sequencing (6). Structural annotations were made with GLIMMER v3.0 and MetaGene-Annotator v1.0, and ARAGORN v2.36 did not reveal any tRNAs (7)(8)(9). Protein-coding gene function was predicted with InterProScan v5.33-72 and BLAST v2.2.31 (10,11). The BLAST analysis queried the NCBI nonredundant and UniProtKB Swiss-Prot and TrEMBL databases with a 0.001 maximum expectation value cutoff (12). Rho-independent termination sites were analyzed using TransTermHP v2.09 (13). Whole-genome similarity was calculated by the progressiveMauve v2.4.0 algorithm (14). The annotation tools used are in the Galaxy and Web Apollo instances hosted by the Center for Phage Technology (https://cpt.tamu.edu/galaxy-pub). The morphology of the phage sample was determined by negative staining with 2% uranyl acetate and visualized with transmission electron microscopy at the Texas A&M University Microscopy and Imaging Center (15).
Data availability. The genome sequence and associated data for phage Mangalitsa were deposited under GenBank accession no. MN045229, BioProject no. PRJNA222858, SRA no. SRR8869233, and BioSample no. SAMN11360419.

ACKNOWLEDGMENTS
This work was supported by funding from the National Science Foundation (awards EF-0949351 and DBI-1565146) and the Bill and Melinda Gates Foundation (project OPP1139800). Additional support came from the Center for Phage Technology (CPT), an Initial University Multidisciplinary Research Initiative supported by Texas A&M University and Texas AgriLife, and from the Department of Biochemistry and Biophysics of Texas A&M University.
We are grateful for the advice and support of the CPT staff. This announcement was prepared in partial fulfillment of the requirements for BICH464 Bacteriophage Genomics, an undergraduate course at Texas A&M University.