The /RS138 genome could be divi-ded into three regions based on similarities to other gen-omes: 1 a region containing genes encoding a putative transcriptional regulator and an integrase
Trang 1A N N O T A T E D S E Q U E N C E R E C O R D
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
iso-lated 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
divi-ded into three regions based on similarities to other
gen-omes: (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
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 Mu-type bacteriophage, RcapMu [4]
The phage genome was extracted using a QIAGEN Lambda Mini Kit; it was then digested with SalI and ran-domly 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 Big-Dye Terminator Sequencing Next, the full genome
material, which is available to authorized users.
& Kaeko Kamei
kame@kit.ac.jp
Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
Shiga 523-8555, Japan
University of Science, Vietnam National University,
Ho Chi Minh City, Vietnam
Research and Development Institute, Can Tho University,
Cantho City, Vietnam
Arch Virol
DOI 10.1007/s00705-015-2654-1
Trang 2sequence 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
com-plete 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
sola-nacearum, 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
using the BLAST program (Table1) 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 sola-nacearum 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 charac-teristics, 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 Mu-type 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 1 Many ORFs were similar to those in the Mu and Mu-like prophages ORFs 3–9 include putative genes for a tran-scriptional 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 rec-ognizes 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
RS138
RS138
RS138
(A)
(B)
(C)
chromosomal DNAs of R solanacearum K60-1 (A), Pseudomonas
phage JBD88a (B), and R solanacearum CMR15 (C) Matrix plots
were generated via BLASTN analysis
B Van Truong Thi et al.
Trang 3the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.
nig.ac.jp/) under accession number AB916497
For-estry, and Fisheries Technology Center for providing the soil from the
tomato field.
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Region of /RS138
genome
(%)
Max identity (%)
E-value*
Accession no.
sequence
complete sequence
Burkholderia sp 2002721687 chromosome I, complete sequence
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NCGM257
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genome
R solanacearum GMI1000 chromosome*, complete sequence
R solanacearum str PSI07 chromosome, complete genome
* The probably of obtaining a match by chance, determined by BLASTN analysis
Ralstonia solanacearum phage of the family Siphoviridae
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