Arch Virol DOI 10.1007/s00705-015-2654-1 ANNOTATED SEQUENCE RECORD Genomic characterization of Ralstonia solanacearum phage /RS138 of the family Siphoviridae Bich Van Truong Thi1,4 • Nguyen Huan Pham Khanh1 • Ryuta Namikawa1 Kaito Miki1 • Akihiro Kondo2 • Phuong Thao Dang Thi3 • Kaeko Kamei1 • Received: 17 July 2015 / Accepted: 19 October 2015 Ó Springer-Verlag Wien 2015 Abstract /RS138, a bacteriophage of the family Siphoviridae that lyses Ralstonia solanacearum, was isolated The genomic DNA of /RS138 was 41,941 bp long with a GC content of 65.1 % and contained 56 putative open reading frames The /RS138 genome could be divided into three regions based on similarities to other genomes: (1) a region containing genes encoding a putative transcriptional regulator and an integrase, similar to the prophage genes in Ralstonia solanacearum K60-1; (2) a region encoding proteins related to structural modules and virion morphogenesis, similar to genes in the Pseudomonas phages of the family Siphoviridae; and (3) a region highly similar to the genomes of other Ralstonia solanacearum strains Ralstonia solanacearum causes wilt disease in more than 200 plant species, thereby exerting a significant financial impact on the agricultural industry In the present study, a Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2654-1) contains supplementary material, which is available to authorized users & Kaeko Kamei kame@kit.ac.jp Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan Hiyoshi Corporation, 908 Kitanosho, Omihachiman, Shiga 523-8555, Japan Department of Molecular and Environmental Biotechnology, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam Department of Molecular Biotechnology, Biotechnology Research and Development Institute, Can Tho University, Cantho City, Vietnam new phage /RS138, which lyses R solanacearum, was isolated from the soil of a tomato field in 2010 (Japan, 35.01655 N latitude, 135.563849 E longitude) using a double agar overlay plaque assay [1] /RS138 lysed R solanacearum MAFF 730138 and 106603 (race 1, biovar 3), 730139 (race 1, biovar 4), and 211272 (race 4, biovar 4), but this phage did not lyse MAFF 211556, 106611 (race 1, biovar 4), 211270 (race 1, biovar N2), or 211558 (race 3, biovar N2) All bacteria used in this study were obtained from the National Institute of Agrobiological Sciences Genebank, Japan Using R solanacearum MAFF 730138 cells as a host, the growth parameters of /RS138 were determined to be as follows: eclipse phase period, *90 min; latent period, *120 min; rise period, *120 min; average burst size, 120–130 plaque-forming units per infected cell Imaging of /RS138 by electron microscopy revealed that its virion structure consisted of a flexible tail *220 nm in length and *10 nm in width and an icosahedral head *60 nm in diameter (Supplementary Fig 1) This morphology is similar to that of k-like phages of the family Siphoviridae /RS138 had a longer tail than other well-characterized members of the Siphoviridae, such as Pseudomonas aeruginosa phage MP22, with a 150-nm tail [2], and P aeruginosa phage D3, with a 113-nm tail [3] At 220 nm, the length of the /RS138 tail is similar to that of the Pseudomonas Mutype bacteriophage, RcapMu [4] The phage genome was extracted using a QIAGEN Lambda Mini Kit; it was then digested with SalI and randomly cloned into the pGEX6-P1 vector (GE Healthcare) To obtain partial DNA sequence information, sequencing of random clones was performed at the Biotechnology Center of Akita Prefectural University (Japan) using BigDye Terminator Sequencing Next, the full genome 123 B Van Truong Thi et al 60 K 40 K 20 K R solanacearum K60 (A) 5K 10 K 15 K 20 K 25 K 30 K 35 K 40 K RS138 30 K 20 K 10 K Pseudomonas phage JBD88a (B) 5K 15 K 20 K 25 K 30 K 35 K 40 K 3M 2M 1M R solanacearum CMR15 10 K RS138 (C) 5K 10 K 15 K 20 K 25 K 30 K 35 K 40 K RS138 Fig Matrix plot of the /RS138 genomic DNA sequence with chromosomal DNAs of R solanacearum K60-1 (A), Pseudomonas phage JBD88a (B), and R solanacearum CMR15 (C) Matrix plots were generated via BLASTN analysis sequence was analyzed using primers designed based on the partial DNA sequence determined in this study The full genome sequence was determined bi-directionally using the primer-walking method The experiment was repeated more than two times for both strands The complete genome comprised 41,941 bp of dsDNA with a GC content of 65.1 % The genome sequence had no similarity to that of other dsDNA-tailed phages that infect R solanacearum, including /RSA1 and /RSL1of the family Myoviridae [5, 6] and /RSB1 of the family Podoviridae [7] The results of matrix plot (Fig 1) and similarity search 123 using the BLAST program (Table 1) revealed that the / RS138 genome can be divided into three regions based on similarity with other genes The region from the 50 end to approximately 11.5 kbp of the /RS138 genome possessed a sequence similar to the chromosomal DNA of R solanacearum K60-1 The corresponding region in the R solanacearum K60-1 genome is located in a unique region bearing no similarity to genome sequences of other R solanacearum strains (Supplementary Fig 2) [8] The similarities in the genomic sequence of /RS138 and R solanacearum K60-1, which were isolated in the United States, may shed light on the origin of genes in this region The region from approximately 12 kbp to 25 kbp of the / RS138 genome exhibited high similarity to Pseudomonas phages of the family Siphoviridae with Mu-type characteristics, such as JBD88a, JBD5 [9], and MP22 [2] This region also included a sequence similar to the genome of Burkholderia vietnamiensis The right-end region from around 25 kbp showed high similarity to regions of other R solanacearum strains such as CMR15 and GMI1000 These similarities might suggest that /RS138 originated as a Pseudomonas phage of the family Siphoviridae with Mutype characteristics The /RS138 genome was analyzed using the online program ORF Finder (NCBI) to identify putative ORFs A total of 56 putative ORFs were found in the /RS138 genome; these are summarized in Supplementary Table Many ORFs were similar to those in the Mu and Mu-like prophages ORFs 3–9 include putative genes for a transcriptional regulator, an integrase, and a transposase ORFs 25–46 encode proteins related to structural modules and virion morphogenesis ORF28 encodes a putative lysis protein ORF46 was identified as the putative tail tape measure protein that determines tail length and, expectedly, was the largest protein (1565 aa) The larger size of this protein compared to those of other phages is consistent with /RS138 having a longer tail and is similar to that of Burkholderia phage AH2 (accession no AEY69560.1; length, 1578 aa; tail, approximately 220 nm) These results agreed well with the morphology of /RS138, with a longer tail of *220 nm Some other R solanacearum phages, such as RSA1 [5] and RSL1 [6], and several Siphoviridae phages, such as clP1 and D3 [10, 11], possess tRNA genes that can increase the overall translation rate by transcribing tRNA that recognizes rare codons However, tRNAscan analysis did not identify such a tRNA gene in the /RS138 genome [12, 13] This is the first report of the whole genome sequence of the R solanacearum phage /RS138 belonging to the family Siphoviridae Nucleotide sequence accession number The complete genome sequence of bacteriophage /RS138 is available in Ralstonia solanacearum phage of the family Siphoviridae Table Similarities to the /RS138 genome identified by BLASTN search Region of /RS138 genome Description Query cover (%) Max identity (%) Evalue* Accession no 1–11.5 kbp R solanacearum K60-1*, whole genome shotgun sequence 31 74 0.0 CAGT01000060.1 12–25 kbp Burkholderia pseudomallei MSHR2543 chromosome I, complete sequence 13 71 0.0 CP009478.1 Burkholderia sp 2002721687 chromosome I, complete sequence 21 71 0.0 CP009549.1 Burkholderia vietnamiensis G4 chromosome 1, complete sequence 12 70 0.0 CP000614.1 Burkholderia pseudomallei MSHR491 chromosome I, complete sequence 13 70 0.0 CP009485.1 30.6–41.6 kbp Pseudomonas phage JBD88a*, complete genome 12 68 0.0 JX434033.1 Pseudomonas phage JBD5*, complete genome 10 68 0.0 JX434030.1 Pseudomonas Phage MP22*, complete genome 12 68 0.0 DQ873690.1 Pseudomonas phage F_HA0480sp/Pa1651*, complete genome 12 68 0.0 JN808773.1 Pseudomonas aeruginosa DNA, complete genome, strain: NCGM257 15 67 0.0 AP014651.1 Pseudomonas phage MP48*, complete genome 12 67 0.0 KF475786.1 Pseudomonas phage PA1/KOR/2010*, complete genome 10 67 0.0 HM624080.1 Pseudomonas phage MP29*, complete genome 10 67 0.0 EU272036.1 Pseudomonas phage JD024*, complete genome 11 67 0.0 KJ477077.1 Pseudomonas aeruginosa strain Carb01 63, complete genome 14 67 0.0 CP011317.1 R solanacearum CMR15 chromosome*, complete genome R solanacearum GMI1000 chromosome*, complete sequence 13 80 0.0 FP885895.1 11 85 7e-145 AL646052.1 R solanacearum strain YC45, complete genome CP011997.1 11 69 3e-147 R syzygii R24, genomic contig 00002-1628 70 2e-142 FR854087.1 R solanacearum str PSI07 chromosome, complete genome 69 2e-142 FP885906.2 11 69 7e-136 CP004012.1 R solanacearum FQY_4, complete genome * The probably of obtaining a match by chance, determined by BLASTN analysis the DNA Data Bank of Japan (DDBJ) (http://www.ddbj nig.ac.jp/) under accession number AB916497 Acknowledgments We thank Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Center for providing the soil from the tomato field References Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay Methods Mol Biol 501:69–76 Heo YJ, Chung IY, Choi KB, Lau GW, Cho YH (2007) Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22 Microbiology 153(Pt 9):2885–2895 Gilakjan ZA, Kropinski AM (1999) Cloning and analysis of the capsid morphogenesis genes of Pseudomonas aeruginosa bacteriophage D3: another example of protein chain mail? J Bacteriol 181(23):7221–7227 Fogg PC, Hynes AP, Digby E, Lang AS, Beatty JT (2011) Characterization of a newly discovered Mu-like bacteriophage, RcapMu, in Rhodobacter capsulatus strain SB1003 Virology 421(2):211–221 Fujiwara A, Kawasaki T, Usami S, Fujie M, Yamada T (2008) Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000 J Bacteriol 190(1):143–156 Yamada T, Satoh S, Ishikawa H, Fujiwara A, Kawasaki T, Fujie M, Ogata H (2010) A jumbo phage infecting the phytopathogen Ralstonia solanacearum defines a new lineage of the Myoviridae family Virology 398(1):135–147 Kawasaki T, Shimizu M, Satsuma H, Fujiwara A, Fujie M, Usami S, Yamada T (2009) Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage J Bacteriol 191(1):422–427 123 B Van Truong Thi et al Remenant B, Babujee L, Lajus A, Medigue C, Prior P, Allen C (2012) Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum J Bacteriol 194(10):2742–2743 doi:10.1128/JB.00249-12 Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR (2013) Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system Nature 493(7432):429–432 10 Kelly D, O’Sullivan O, Mills S, McAuliffe O, Ross RP, Neve H, Coffey A (2012) Genome sequence of the phage clP1, which infects the beer spoilage bacterium Pediococcus damnosus Gene 504(1):53–63 123 11 Kropinski AM (2000) Sequence of the genome of the temperate, serotype-converting, Pseudomonas aeruginosa bacteriophage D3 J Bacteriol 182(21):6066–6074 12 Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence Nucleic Acids Res 25(5):955–964 13 Schattner P, Brooks AN, Lowe TM (2005) The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs Nucleic Acids Res 33(Web Server issue):W686–W689 ... the /RS138 genome [12, 13] This is the first report of the whole genome sequence of the R solanacearum phage /RS138 belonging to the family Siphoviridae Nucleotide sequence accession number The. .. genome sequence of bacteriophage /RS138 is available in Ralstonia solanacearum phage of the family Siphoviridae Table Similarities to the /RS138 genome identified by BLASTN search Region of /RS138... /RSL 1of the family Myoviridae [5, 6] and /RSB1 of the family Podoviridae [7] The results of matrix plot (Fig 1) and similarity search 123 using the BLAST program (Table 1) revealed that the /