We present here the complete genomes of eight phages that infect Paenibacillus larvae, the causative agent of American foulbrood in honeybees. Phage PBL1c was originally isolated in 1984 from a P. larvae lysogen, while the remaining phages were isolated in 2014 from bee debris, honeycomb, and lysogens from three states in the USA

Berg, Jordan A.

Breakwell, Donald P.

Dhalai, Aziza

Dingman, Douglas W.

Fajardo, Chris P.

Graves, Kiel

Grose, Julianne H.

Hill, Hunter L.

Hilton, Jared A.

Hope, Sandra

Imahara, Cameron

Knabe, Bradley K.

Mangohig, James

Merrill, Bryan D.

Monk, Josh

Mun, Heejin

Payne, Ashley M.

Salisbury, Alicia

Stamereilers, Casey

Tsourkas, Philippos K.

Velez, Kathie

Walker, Jamison K.

Ward, Andy T.

English

University of Nevada, Las Vegas Repository

Complete Genome Sequences of Paenibacillus larvae PhagesBN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, andTadhanaJamison K. Walker,a Bryan D. Merrill,a Jordan A. Berg,a Aziza Dhalai,b Douglas W. Dingman,c Chris P. Fajardo,a Kiel Graves,aHunter L. Hill,a Jared A. Hilton,a Cameron Imahara,b Bradley K. Knabe,a James Mangohig,b Josh Monk,b Heejin Mun,bAshley M. Payne,a Alicia Salisbury,b Casey Stamereilers,b Kathie Velez,b Andy T. Ward,a Donald P. Breakwell,aJulianne H. Grose,a Sandra Hope,a Philippos K. TsourkasbaDepartment of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah, USAbSchool of Life Sciences, University of Nevada, Las Vegas, Nevada, USAcDepartment of Entomology, Connecticut Agricultural Experiment Station, New Haven, Connecticut, USAABSTRACT We present here the complete genomes of eight phages that infectPaenibacillus larvae, the causative agent of American foulbrood in honeybees. PhagePBL1c was originally isolated in 1984 from a P. larvae lysogen, while the remainingphages were isolated in 2014 from bee debris, honeycomb, and lysogens from threestates in the USA.The Gram-positive bacterium Paenibacillus larvae is the causative agent of Americanfoulbrood, currently the most destructive bacterial disease affecting the honeybee,Apis mellifera (1). With the rise of antibiotic-resistant strains of P. larvae (2), there isgrowing interest in phages that infect this pathogen. The ﬁrst P. larvae phages wereisolated in the 1950s (3), and the ﬁrst complete P. larvae genome was published in 2013(4). There are currently 18 complete P. larvae phage genomes in the literature (4–7).Here, we present eight complete P. larvae phage genomes obtained from samplesacross the United States. The phages’ GenBank accession numbers, isolation sources,geographical provenance, and assembly results are shown in Table 1.Phage PBL1c was isolated from a lysogen in 1984 by Dingman et al. (8) but was notsequenced until 2018 at Brigham Young University (BYU). The remaining seven phageswere isolated over the period 2014 to 2016 from samples from the USA states of Utah,Idaho, and Wisconsin (Table 1) as part of the Phage Hunters course at BYU.The phages were isolated from bee debris, honeycomb, and lysogens and ampliﬁedin P. larvae ﬁeld isolates. Phage genomic DNA was isolated from high-titer lysates usingNorgen phage DNA isolation kits (Norgen Biotek, Thorold, ON, Canada). Phage ge-nomes were sequenced in the BYU DNA Sequencing Center using the Illumina HiSeq2500 platform (Illumina, Hayward, CA, USA) and were assembled using Geneious 8software (Biomatters Inc., Newark, NJ, USA).All nine phages are members of the family Siphoviridae with linear double-strandedDNA genomes. The DNA packaging strategy was identiﬁed as “cohesive ends with 3=overhangs,” as explained in references 9 and 10. The overhangs were identiﬁed bysequence similarity with previously published phages (3–7). The overhangs are “CGACT-GCCC” for phages BN12, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana, and“CGACGGACC” for phage Dragolir. The genomes were rearranged by setting the ﬁrstbase of the genome to be the base immediately after the 3= overhang.Genome length is in the 37 kb to 42 kb range, and the GC content was in the 41to 44% range, consistent with 3= cohesive ends for P. larvae phages (11). Preliminaryanalysis shows that phages Pagassa and Tadhana are closely related to each other, withReceived 3 January 2018 Accepted 8 May2018 Published 14 June 2018CitationWalker JK, Merrill BD, Berg JA, DhalaiA, Dingman DW, Fajardo CP, Graves K, Hill HL,Hilton JA, Imahara C, Knabe BK, Mangohig J,Monk J, Mun H, Payne AM, Salisbury A,Stamereilers C, Velez K, Ward AT, Breakwell DP,Grose JH, Hope S, Tsourkas PK. 2018. Completegenome sequences of Paenibacillus larvaephages BN12, Dragolir, Kiel007, Leyra, Likha,Pagassa, PBL1c, and Tadhana. GenomeAnnounc 6:e01602-17. https://doi.org/10.1128/genomeA.01602-17.Copyright © 2018 Walker et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to Philippos K.Tsourkas, philippos.tsourkas@unlv.edu.VIRUSEScrossmVolume 6 Issue 24 e01602-17 genomea.asm.org 1 on July 13, 2018 by guesthttp://mra.asm.orgDownloaded from the other phages slightly more distant; phage Dragolir was shown to be an outlier. Alleight phages encode a large terminase, a major tail protein, two tail assembly proteins,a tail tape measure protein, and an N-acetylmuramoyl-L-alanine amidase, amongothers. The tail assembly proteins appear to have a programmed translational frame-shift similar to the G and G-T genes of phage lambda (12, 13), located in the 3= regionof gp12 (the upstream tail assembly protein). We tentatively identiﬁed the heptanucle-otide slippery sequence as “AAAAAAG” in phages BN12, Kiel007, Likha, Leyra, Pagassa,PBL1c, and Tadhana, and possibly “AAAAAAC” in phage Dragolir. Future studies willinvestigate this and other features of P. larvae phage genomes and also provide adetailed comparative genomic analysis of these and other P. larvae phages.Accession number(s). The genome sequences of the P. larvae phages reported herehave been deposited in GenBank under the accession numbers listed in Table 1.ACKNOWLEDGMENTSA.D., A.S., C.S., and P.K.T. are supported by the National Institute of General MedicalSciences GM103440 (NV INBRE). S.H. acknowledges Edwin Wilcox at the BYU DNASequencing Center.REFERENCES1. de Graaf DC, Alippi AM, Antúnez K, Aronstein KA, Budge G, De Koker D,De Smet L, Dingman DW, Evans JD, Foster LJ, Fünfhaus A, Garcia-Gonzalez E, Gregore A, Human H, Murray KD, Nguyen BK, Poppinga L,Spivak M, van Engelsdorp D, Wilkins S, Genersch E. 2013. Standardmethods for American foulbrood research. J Apic Res 52:1–28. https://doi.org/10.3896/IBRA.1.52.1.11.2. Miyagi T, Peng CYS, Chuang RY, Mussen EC, Spivak MS, Doi RH. 2000.Veriﬁcation of oxytetracycline-resistant American foulbrood pathogenPaenibacillus larvae in the United States. J Invertebr Pathol 75:95–96.https://doi.org/10.1006/jipa.1999.4888.3. Gochnauer TA. 1955. The isolation of a bacteriophage (bacterial virus)from Bacillus larvae. Bee World 36:101–103. https://doi.org/10.1080/0005772X.1955.11094880.4. Oliveira A, Melo LDR, Kropinski AM, Azeredo J. 2013. Complete genomesequence of the broad-host-range Paenibacillus larvae phage phi-IBB_Pl23. Genome Announc 1(5):e00438-13. https://doi.org/10.1128/genomeA.00438-13.5. Carson S, Bruff E, DeFoor W, Dums J, Groth A, Hatﬁeld T, Iyer A, Joshi K,McAdams S, Miles D, Miller D, Oufkir A, Raynor B, Riley S, Roland S, RozierH, Talley S, Miller ES. 2015. Genome sequences of six Paenibacillus larvaeSiphoviridae phages. Genome Announc 3(3):e00101-15. https://doi.org/10.1128/genomeA.00101-15.6. Beims H, Wittmann J, Bunk B, Spröer C, Rohde C, Günther G, Rohde M,von der Ohe W, Steinert M. 2015. Paenibacillus larvae-directed bacterio-phage HB10c2 and its application in American foulbrood-affected honeybee larvae. Appl Environ Microbiol 81:5411–5419. https://doi.org/10.1128/AEM.00804-15.7. Tsourkas P, Yost D, Krohn A, Leblanc L, Zhang A, Stamereilers C, Amy PS.2015. Complete genome sequences of nine phages capable of infect-ing Paenibacillus larvae, the causative agent of American foulbrooddisease of honeybees. Genome Announc 3(5):e01120-15. https://doi.org/10.1128/genomeA.01120-15.8. Dingman DW, Bakhiet N, Field CC, Stahly DP. 1984. Isolation of twobacteriophages from Bacillus larvae, PBL1 and PBL0.5, and partial char-acterization of PBL1. J Gen Virol 65:1101–1105. https://doi.org/10.1099/0022-1317-65-6-1101.9. Casjens SR, Gilcrease EB. 2009. Determining DNA packaging strategy byanalysis of the termini of the chromosomes in tailed-bacteriophagevirions, p. 91–111. In Clokie MRJ, Kropinski AM (ed), Bacteriophages:methods and protocols, volume 2: molecular and applied aspects.Humana Press, New York, NY. https://doi.org/10.1007/978-1-60327-565-1_7.10. Merrill BD, Ward AT, Grose JH, Hope S. 2016. Software-based analysis ofbacteriophage genomes, physical ends, and packaging strategies. BMCGenomics 17:679. https://doi.org/10.1186/s12864-016-3018-2.11. Stamereilers C, LeBlanc L, Yost D, Amy PS, Tsourkas PK. 2016. Compar-ative genomics of 9 novel Paenibacillus larvae bacteriophages. Bacterio-phage 6:e1220349. https://doi.org/10.1080/21597081.2016.1220349.12. Xu J, Hendrix RW, Duda RL. 2004. Conserved translational frameshift indsDNA bacteriophage tail assembly genes. Molecular Cell 16:11–21.https://doi.org/10.1016/j.molcel.2004.09.006.13. Xu J, Hendrix RW, Duda RL. 2013. A balanced ratio of proteins from geneG and frameshift-extended gene GT is required for phage lambda tailassembly. J Mol Biol 425:3476–3487. https://doi.org/10.1016/j.jmb.2013.07.002.TABLE 1 P. larvae phages, GenBank accession numbers, and genome assembly resultsPhage name GenBank accession no. Isolation source Location Genome length (bp) GC content (%)BN12 MG727695 Bee debris Cedar City, Utah, USA 39,485 42.6Dragolir MG727697 Bee debris Wisconsin, USA 41,131 44Kiel007 MG727696 Bee debris Salt Lake City, Utah, USA 37,985 41.8Leyra MG727701 Bee debris Idaho, USA 42,276 41.4Likha MG727702 Honeycomb American Fork, Utah, USA 39,778 41.3Pagassa MG727699 P. larvae lysogen Provo, Utah, USA 40,035 42PBL1c MG727698 P. larvae lysogen Iowa City, Iowa, USA 40,611 41.2Tadhana MG727700 P. larvae lysogen Provo, Utah, USA 37,880 42.1Walker et al.Volume 6 Issue 24 e01602-17 genomea.asm.org 2 on July 13, 2018 by guesthttp://mra.asm.orgDownloaded from 

Complete Genome Sequences of Paenibacillus Larvae Phages BN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana

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Complete Genome Sequences of Paenibacillus Larvae Phages BN12, Dragolir, Kiel007, Leyra, Likha, Pagassa, PBL1c, and Tadhana

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