Complete Genome Sequence of Rahel, a C1 ClusterMycobacteriophage

Fernando E. Nieto-Fernandez,a Christos Noutsos,a Jillian Nissen,a Yara Abdelsalam,a Jessica Ackloo,a Navpreet Banger,a

Hason Chan,a Tarana Chittineedi,a Isabel Duplessy,a Mark Dyce,a Daesha Garrison,a Jaime Gonzalez,a Sandra John,a Imanjot Kahlon,a

Tania Kumar,a Adwoa Lewis,a Karthik Madhira,a Rivka Mullokandova,a Nellie Pirzadeh,a Iman Raja,a Kevin Ram,a Ravi Ramdhari,a

Rita Reddy,a Briana S. Saed,a Phalan Smith,a Steeve Sproul,a Jane Thomas,a Avia Yossefi,a Jorge Moralesb

aDepartment of Biological Sciences, SUNY Old Westbury, Old Westbury, New York, USAbThe City College of New York, New York, New York, USA

ABSTRACT Rahel is a lytic Myoviridae bacteriophage that infects Mycobacterium smeg-matis mc2155. It has 1,555,955bp and 64.7% G1C content. Rahel has a circularly per-muted genome with 270 genes, 53 of them of known function, 33 tRNAs, and 1 trans-fer-messenger RNA (tmRNA). Only five genes are coded on the reverse strand.

Rahel was isolated as part of the Science Education Alliance-Phage Hunters AdvancingGenomics and Evolutionary Science (SEA-PHAGES) at SUNY Old Westbury. Rahel was

isolated in Hicksville, Nassau County, New York (lat 40.7679, long 273.5405). It is a C1 sub-cluster lytic phage that infects Mycobacterium smegmatis mc2155. Clusters are groups ofphages with sequence similarity over 50% of their genome (1). It was isolated by enrichmentand two cycles of purification and amplification in 7H9 top agar at 37°C (2). For transmissionelectron microscopy (TEM), phages were collected from a high-titer lysate by high-speedcentrifugation, mounted on carbon-stabilized, Formvar-coated copper TEM grids stainedwith uranyl acetate, and imaged with a JEOL JEM-2100 TEM (2). Rahel is a Myoviridae of 178nm total length with an isometric head and tail of 89nm (Fig. 1). DNA was extracted from ahigh-titer lysate using a Promega DNA Wizard kit with a modified protocol (2). GenomicDNA libraries were generated using an Ultra II FS kit (New England Biolabs [NEB], Ipswich,MA) with dual-indexed barcoding. Pooled libraries were run on an Illumina MiSeq system atthe University of Pittsburgh, which yielded at least 547,000 single-end 150-base reads. Readcoverage depth was 471� without Sanger finishing reactions. The reads were assembledusing Newbler and Consed (3). A full genome sequence was present, as the contigs on bothends overlapped and the genome circularized. Rahel was annotated using DNA Master(http://cobamide2.bio.pitt.edu/computer.htm) and the gene prediction tools GLIMMER v3.0(4), GeneMark v2.5 (5), and Starterator v1.1 to determine gene start sites. tRNA and transfer-messenger RNA (tmRNA) predictions were made using ARAGORN v1.2.38 (6) and tRNAscan-SE v3.0 (7). Functional assignments were made using BLAST v2.9 (8), HHpred (9), andPhamerator (10). Default settings were used for all programs.

Rahel has 155,955 bp, 64.7% G1C content, 270 open reading frames (ORFs), 33 tRNAs,1 tmRNA, 53 genes of known function, and 182 hypothetical proteins (67.6%); only fiveORFs are coded on the reverse strand. Rahel has a circularly permuted genome, as all ofthe reads assembled into a large contig and there was no large buildup of reads or cover-age variation at the ends (2). When linearized, the coding region of gene 270 is disrupted.Structural genes include genes 99 (the major capsid protein) and 100 (the capsid decora-tion protein), genes 126 to 144 (the tail structural proteins, including the tail assemblychaperone), and genes 129 and 130. Rahel has a tail sheath protein gene, 126, and twobaseplate wedge protein genes, 138 and 139, but a tail tube protein was not identified.Genes involved in nucleotide metabolism and DNA replication are found on the right

Citation Nieto-Fernandez FE, Noutsos C,Nissen J, Abdelsalam Y, Ackloo J, Banger N,Chan H, Chittineedi T, Duplessy I, Dyce M,Garrison D, Gonzalez J, John S, Kahlon I, KumarT, Lewis A, Madhira K, Mullokandova R,Pirzadeh N, Raja I, Ram K, Ramdhari R, Reddy R,Saed BS, Smith P, Sproul S, Thomas J, Yossefi A,Morales J. 2020. Complete genome sequenceof Rahel, a C1 cluster mycobacteriophage.Microbiol Resour Announc 9:e01071-20.https://doi.org/10.1128/MRA.01071-20.

Editor John J. Dennehy, Queens College

Copyright © 2020 Nieto-Fernandez et al. Thisis an open-access article distributed under theterms of the Creative Commons Attribution 4.0International license.

Address correspondence to Fernando E.Nieto-Fernandez, nietof@oldwestbury.edu.

Received 3 October 2020Accepted 21 October 2020Published 5 November 2020

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end of the genome from gene 147 on to the end. In Rahel, the lysis system is encodedby genes 252 and 254, lysin A and B, respectively, followed by a terminase, gene 255. Wedid not find a holin gene as part the lysis system.

Data availability. The complete sequence for Rahel’s genome can be found inGenBank under accession number MK359348 and SRA accession number SRX9162484.

ACKNOWLEDGMENTSWe thank the HHMI SEA-PHAGES program for the opportunity to be part of this project.

We are grateful to the School of Arts and Sciences at SUNY Old Westbury and theDepartment of Biology for supporting this work. We also thank the University of PittsburghSEA-PHAGES team members involved in sequencing, assembly, and quality control (QC)annotation.

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FIG 1 TEM of Rahel mounted on Formvar-coated copper grids stained with uranyl acetate and imagedwith a JEOL JEM-2100 microscope. Rahel’s virions are 178 nm in total length, with an isometric head andtail of 89nm (�50,000 magnification).

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