The complete genome, structural proteome, comparative genomics and phylogenetic analysis of a broad host lytic bacteriophage ϕD3 infecting pectinolytic Dickeya spp.
© Czajkowski et al. 2015
Received: 1 May 2015
Accepted: 17 September 2015
Published: 24 September 2015
Plant necrotrophic Dickeya spp. are among the top ten most devastating bacterial plant pathogens able to infect a number of different plant species worldwide including economically important crops. Little is known of the lytic bacteriophages infecting Dickeya spp. A broad host lytic bacteriophage ϕD3 belonging to the family Myoviridae and order Caudovirales has been isolated in our previous study. This report provides detailed information of its annotated genome, structural proteome and phylogenetic relationships with known lytic bacteriophages infecting species of the Enterobacteriaceae family.
Pectinolytic Dickeya spp. can cause disease on a number of arable and ornamental crops worldwide including potato, tomato, carrot, onion, pineapple, maize, rice, hyacinth, chrysanthemum and calla lily resulting into severe economic losses . Dickeya spp. are recognized to be among the top ten most important bacterial pathogens in agriculture . To date there is no effective control of Dickeya spp. in agriculture due to the lack of practical measures and strategies .
Lytic bacteriophages have been proposed as potential biological control agents against various pathogenic bacterial species including plant pathogens . Their potential to control plant bacterial diseases has been evaluated among others against Erwinia amylovora , Xanthomonas pruni, Ralstonia solanacearum and also were experimentally tested against Pectobacterium spp. and Dickeya spp. in different crop systems . In the case of Pectobacterium spp. and Dickeya spp. lytic bacteriophages, only limited attempts have been made so far to isolate and characterize these bacteriophages in detail [5, 6] and to provide information on their genomes and structural proteomes .
At present, only two Dickeya spp. lytic bacteriophages: LimeStone1 and ϕD5 were characterized in detail, viz. their complete genomes are available in the Genbank (accessions: NC019925 and KJ716335, respectively) and information on other features (e. g. structural proteomes and host range, multiplicity of infection and adsorption to bacterial hosts) is also available [6, 7].
Genome sequencing information
Genome project history
A number of recent studies have shown that bacteriophages play a substantial role in global ecosystems and have a direct bearing on the ecology and evolution of their hosts. The ϕD3 genome is the third (after LimeStone1 and ϕD5) complete genome of lytic bacteriophage virulent to plant pathogenic Dickeya spp. available to the scientific community. Genome sequencing and analysis provide a better possibility to deduce phage infections in host cells and phage interaction with a variable environment. This genome project was deposited in NCBI Genbank as Bioproject PRJNA242299 under the title: “Bacteriophages of Pectobacterium spp. and Dickeya spp. Genome sequencing”. A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation
D. solani IPO2222 (type strain for D. solani ), grown on tryptone soya agar (Oxoid) and/or in tryptone soya broth (Oxoid), was used in all experiments as a ϕD3 host. Bacteriophage ϕD3 was isolated as described previously  from Dickeya spp.-free garden soil which may indicate that the phage can infect also different soil-borne bacteria as additional hosts. Purification and concentration of phage particles followed the previous protocols and included: DNase I and RNase A treatments, CsCl gradient ultracentrifugation and dialysis to remove CsCl from phage concentrated samples . Purified phage particles were resuspended in 500 μl of 5 mM MgSO4 or in 1/4 Ringer’s buffer (Merck) and stored at 4 °C in the dark. The ϕD3 genomic DNA was purified using CTAB method as described in .
Genome sequencing and assembly
The genome was sequenced using the Illumina next generation technology at Baseclear, The Netherlands, following the manufacturer’s instructions (Illumina). The sequencing library yielded ca. 270 Mb clean data reads after sets of rigorous filtrations against bacterial host genomic DNA ( D. solani strain IPO2222, Genbank accession: AONU00000000). De novo assembly of the ϕD3 genome from the resulting raw reads was performed using CLC Genomic Workbench 7.5 (CLC bio) as described earlier  which provided >1500 x coverage of the genome.
The ϕD3 genome was mapped and annotated using available bacteriophage genomic sequences deposited in GenBank. Structural and functional annotations for the ϕD3 genome were obtained from the Annotation Service Automatic Pipeline (Institute for Genome Science, School of Medicine, University of Maryland, USA) and confirmed using RAST set to auto settings. Additional analysis of the gene predictions and annotations was supplemented using Manatee accessed via the website of IGS, University of Maryland, USA. The lifestyle of ϕD3 (temperate [lysogenic] or lytic) was predicted using PHACTS . To find potential genes acquired by ϕD3 coding for toxins and allergens, the genome sequence was analyzed bioinformatic analysis using Virulence Finder 1.2 and VirulentPred.
Classification and general features of Dickeya spp. bacteriophage ϕD3
Domain: Viruses, dsDNA viruses, no RNA viruses
pH range; Optimum
Obligate intracellular parasite of Dickeya spp.
Lytic virus of Dickeya spp.
Poland / Kujawsko-Pomorskie (Kuyavian-Pomeranian Province)
February 18, 2013
One paired-end library
CLC Genomics Workbench, version 7.0.3
Gene calling method
RAST version 4.0, IGS Annotation Service (Manatee)
GenBank Date of Release
16.07.2016 (earlier upon publication)
Source Material Identifier
Biological effects in soil and plant environments
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Comparisons with other genomes of Dickeya spp. bacteriophages and bacteriophage T4
Interestingly, the majority of the genes found in ϕD3 do not have homologs in T4 (one of the best described and characterized Myoviridae bacteriophages) and only two genes are present in both phages viz. (i) phage recombination protein and (ii) phage endoribonuclease translational repressor of early genes.
Bacteriophage capsid assembly protein (gp20) was used for phylogenetic analysis as previously described [16, 17]. Nucleotide sequences of gp20 proteins of LimeStone1 (Genbank accession: NC019925), bacteriophage ϕD5 (KJ716335), Shigella phage phiSboM-AG3 (NC013693), Salmonella phage SKML-39 (NC019910), Klebsiella phage 0507-KN2-1 (NC022343), Salmonella phage vB SalM SJ3 (NC024122), Escherichia coli phage PhaxI (NC0194521), E. coli phage vB_EcoM_CBA120 (NC016570), Salmonella phage SFP10( NC016073), Salmonella phage PhiSH19 (NC019530), Salmonella phage Maynard (NC022768), Salmonella phage Marshall (NC022772), E. coli phage ECML-4 (NC025446), Salmonella phage vB SalM SJ2 (NC023856), Salmonella phage Vi01 (NC015296) were obtained from GenBank. ClustalX was used to align nucleotide sequences and to manually correct aligned sequences prior to further analyses. Phylogeny studies were performed with the use of the Phylip program  and Molecular Evolutionary Genetic Analysis (MEGA6) software . Dendrograms were created using the Maximum likelihood method followed by calculating the p-distance matrix for aligned gp20 nucleotide sequences (length of gp20 nucleotide sequences: 600 bp, nucleotide substitution model: K80 Kimura) with the bootstrap support fixed to 1000 re-samplings. To root the tree, a gp20 nucleotide sequence from Enterobacteriaceae bacteriophage T4 derived from its complete genome (NC000866) was used.
As expected, ϕD3 showed the highest similarity to the other described Dickeya spp. bacteriophages (LimeStone1 and ϕD5). On the basis of the gp20 phylogenetic analysis, ϕD3 was also closely related to Shigella phage phiSboM-AG3 and Salmonella phage SKML-39. The largest phylogenetic distance was observed between ϕD3 and Enterobacteriaceae phage T4 (Fig. 3b).
As far we know, the ϕD3 is the third bacteriophage able to infect (and kill) several species of Dickeya that has been genetically characterized in depth and is also the second Dickeya spp. lytic bacteriophage isolated in Poland. We expect that the availability of an additional Dickeya spp. specific bacteriophage would improve our understanding of bacteriophage – bacteria interactions and gives an insight on conservation and evolution of Dickeya spp. lytic bacteriophages as well as improve our knowledge on Dickeya spp. ecological fitness in complex (soil, rhizosphere and phyllosphere) environments.
This work was financially supported by the National Science Centre, Poland (Narodowe Centrum Nauki, Polska) via a postdoctoral research grant FUGA1 (DEC-2012/04/S/NZ9/00018) to Robert Czajkowski. The authors are grateful to Michel C. M. Perombelon (ex. SCRI, Dundee, Scotland) for helpful discussion and his editorial work on the manuscript.
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- Toth IK, van der Wolf JM, Saddler G, Lojkowska E, Hélias V, Pirhonen M, et al. Dickeya species: an emerging problem for potato production in Europe. Plant Pathol. 2011;60:385–99.View ArticleGoogle Scholar
- Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald M, et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol. 2012;13:614–29.View ArticlePubMedGoogle Scholar
- Czajkowski R, Pérombelon MCM, van Veen JA, van der Wolf JM. Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review. Plant Pathol. 2011;60:999–1013.View ArticleGoogle Scholar
- Jones JB, Jackson LE, Balogh B, Obradovic A, Iriarte FB, Momol MT. Bacteriophages for plant disease control. Annu Rev Phytopathol. 2008;45:245–62.View ArticleGoogle Scholar
- Czajkowski R, Ozymko Z, Lojkowska E. Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 (‘D. solani’). Plant Pathol. 2014;63:758–72.View ArticleGoogle Scholar
- Adriaenssens EM, Van Vaerenbergh J, Vandenheuvel D, Dunon V, Ceyssens PJ, De Proft M, et al. T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by ‘Dickeya solani’. PLoS ONE. 2012;7(3), e33227.PubMed CentralView ArticlePubMedGoogle Scholar
- Czajkowski R, Ozymko Z, Zwirowski S, Lojkowska E. Complete genome sequence of a broad-host-range lytic Dickeya spp. bacteriophage ϕD5. Arch Virol. 2014;159:3153–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Boulanger P. Purification of bacteriophages and SDS-PAGE analysis of phage structural Proteins from ghost particles. In: Bacteriophages. Methods in Molecular Biology™: Humana Press. Edited by Clokie MJ, Kropinski A, 2009: 227-38.
- Kurowski MA, Bujnicki JM. GeneSilico protein structure prediction meta-server. Nucl Acids Res. 2003;31:3305–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Kawabata T, Arisaka F, Nishikawa K. Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches. Gene. 2000;259:223–33.View ArticlePubMedGoogle Scholar
- Frampton RA, Taylor C, Holguín Moreno AV, Visnovsky SB, Petty NK, Pitman AR, et al. Identification of bacteriophages for biocontrol of the kiwifruit canker phytopathogen Pseudomonas syringae pv. actinidiae. Appl Environ Microbiol. 2014;80:2216–28.PubMed CentralView ArticlePubMedGoogle Scholar
- Sykilinda NN, Bondar AA, Gorshkova AS, Kurochkina LP, Kulikov EE, Shneider MM, et al. Complete genome sequence of the novel giant Pseudomonas phage PaBG. Genome Announc. 2014;2.
- McNair K, Bailey BA, Edwards RA. PHACTS, a computational approach to classifying the lifestyle of phages. Bioinformatics. 2012;28(5):614–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Darling AE, Mau B, Perna NT. ProgressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS ONE. 2010;5(6), e11147.PubMed CentralView ArticlePubMedGoogle Scholar
- Blom J, Albaum S, Doppmeier D, Puhler A, Vorholter F-J, Zakrzewski M, et al. EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics. 2009;10(1):154.PubMed CentralView ArticlePubMedGoogle Scholar
- Brewer TE, Stroupe ME, Jones KM. The genome, proteome and phylogenetic analysis of Sinorhizobium meliloti phage ϕM12, the founder of a new group of T4-superfamily phages. Virology. 2014;450–451:84–97.View ArticlePubMedGoogle Scholar
- Comeau AM, Krisch HM. The Capsid of the T4 phage superfamily: the evolution, diversity, and structure of some of the most prevalent proteins in the biosphere. Mol Biol Evol. 2008;25:1321–32.View ArticlePubMedGoogle Scholar
- Felsenstein J. PHYLIP (phylogeny inference package), version 3.5 c. Joseph Felsenstein.; 1993.
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25:25–9.PubMed CentralView ArticlePubMedGoogle Scholar