Abstract
Pectobacterium carotovorum subsp. carotovorum, a member of the Enterobacteriaceae family, is an important plant-pathogenic bacterium causing significant economic losses worldwide. P. carotovorum subsp. carotovorum bacteriophage My1 was isolated from a soil sample. Its genome was completely sequenced and analyzed for the development of an effective biological control agent. Sequence and morphological analyses revealed that phage My1 is a T5-like bacteriophage and belongs to the family Siphoviridae. To date, there is no report of a Pectobacterium-targeting siphovirus genome sequence. Here, we announce the complete genome sequence of phage My1 and report the results of our analysis.
GENOME ANNOUNCEMENT
The plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum (formerly known as Erwinia carotovora subsp. carotovora) produces numerous plant cell wall-degrading enzymes to cause soft rot diseases in diverse plants (5, 6, 13, 19) and, consequently, causes severe economic losses. Bacteriophages have been considered novel natural biocontrol agents for bacterial plant diseases (8, 14). Several phages infecting Erwinia carotovora subsp. carotovora (14, 18) or other Erwinia spp. have been isolated (4, 11, 12), and a few of these species have been sequenced. Although we have announced the complete sequence of P. carotovorum subsp. carotovorum Podoviridae phage PP1 (10), a genome sequence of a Pectobacterium-targeting phage belonging to the Siphoviridae family has not been reported to date. In this study, we isolated the P. carotovorum subsp. carotovorum-targeting Siphoviridae named My1 and completely sequenced its genome.
Genomic DNA extraction of bacteriophage My1 was carried out using SDS-proteinase K and phenol-chloroform extraction methods (15, 21). Purified DNA was sequenced using a Genome Sequencer FLX (GS-FLX) Titanium by Macrogen Inc. (Seoul, Republic of Korea), with 52-fold coverage. Quality filtered reads were assembled using a 454 Newbler 2.3 assembler. The open reading frames (ORFs) were predicted using the Glimmer v3.02 (3), GeneMarkS (2), and FgenesB (Softberry, Inc., Mount Kisco, NY) and then confirmed by the RBSfinder (17). Transfer RNAs were predicted using the tRNAscan-SE program (16). Functional analysis of ORFs was conducted using the BLASTP (1) and InterProScan (22) programs.
In silico complete genome sequence analysis of bacteriophage My1 revealed that it consists of 122,024 bp in length, with an average GC content of 40.61%, 149 ORFs, and 20 tRNA genes. The predicted ORFs were highly homologous to those of T5 (20) or T5-like phages (7, 9) in the Siphoviridae family. Interestingly, this genome contains redundant 12,854-bp terminal repeats at both ends.
Of the 149 predicted ORFs, 51 (34.23%) have been annotated and categorized into five functional groups as follows: phage structure and packaging (portal protein, prohead protease, tail protein, terminase large subunit), DNA replication/modification (DNA helicase, DNA primase, DNA polymerase, endonuclease, NAD-dependent DNA ligase, RNase H), signal transduction and regulatory functions (PhoH-like protein, serine/threonine protein phosphatase, single-stranded DNA [ssDNA]-binding transcriptional regulator), nucleotide metabolism (thymidylate synthase, dihydrofolate reductase, thioredoxin), and host lysis (endolysin, holin, cell wall hydrolase). However, lysogeny-related genes were not detected. Two lysis proteins and no detection of lysogeny-related genes suggest that this phage could be a useful biocontrol agent. YadA domain-containing protein showed high similarity to the L-shaped tail fiber protein among T5 phage genes but low coverage, suggesting that it probably contributes to host-phage-specific interaction. The others were annotated as hypothetical proteins, probably due to insufficient information about Pectobacterium phage genes.
This first report of the complete genome sequence of Pectobacterium carotovorum subsp. carotovorum bacteriophage My1 will extend our knowledge about the interaction between the Pectobacterium host and its phages and provide a novel potential as a biocontrol agent to prevent plant diseases caused by Pectobacterium.
Nucleotide sequence accession number.
The complete genome sequence of P. carotovorum subsp. carotovorum bacteriophage My1 has been deposited in GenBank under the accession number JX195166.
ACKNOWLEDGMENT
This work was supported by the Rural Development Administration (RDA) fund (grant PJ008615).
REFERENCES
- 1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410 [DOI] [PubMed] [Google Scholar]
- 2. Besemer J, Lomsadze A, Borodovsky M. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29:2607–2618 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Gill JJ, Svircev AM, Smith R, Castle AJ. 2003. Bacteriophages of Erwinia amylovora. Appl. Environ. Microbiol. 69:2133–2138 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Gnanamanickam SS. 2006. Plant-associated bacteria. Springer, Dordrecht, The Netherlands [Google Scholar]
- 6. Hauben L, et al. 1998. Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst. Appl. Microbiol. 21:384–397 [DOI] [PubMed] [Google Scholar]
- 7. Hong J, et al. 2008. Identification of host receptor and receptor-binding module of a newly sequenced T5-like phage EPS7. FEMS Microbiol. Lett. 289:202–209 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Jones JB, et al. 2007. Bacteriophages for plant disease control. Annu. Rev. Phytopathol. 45:245–262 [DOI] [PubMed] [Google Scholar]
- 9. Kim M, Ryu S. 2011. Characterization of a T5-like coliphage, SPC35, and differential development of resistance to SPC35 in Salmonella enterica serovar Typhimurium and Escherichia coli. Appl. Environ. Microbiol. 77:2042–2050 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Lee JH, et al. 2012. Complete genome sequence of phytopathogenic Pectobacterium carotovorum subsp. carotovorum bacteriophage PP1. J. Virol. 86:8899–8900 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Lehman SM, Kropinski AM, Castle AJ, Svircev AM. 2009. Complete genome of the broad-host-range Erwinia amylovora phage ΦEa21-4 and its relationship to Salmonella phage Felix O1. Appl. Environ. Microbiol. 75:2139–2147 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Müller I, Kube M, Reinhardt R, Jelkmann W, Geider K. 2011. Complete genome sequences of three Erwinia amylovora phages isolated in North America and a bacteriophage induced from an Erwinia tasmaniensis strain. J. Bacteriol. 193:795–796 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Pérombelon MCM. 2002. Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathol. 51:1–12 [Google Scholar]
- 14. Ravensdale M, Blom T, Gracia-Garza J, Svircev A, Smith R. 2007. Bacteriophages and the control of Erwinia carotovora subsp. carotovora. Can. J. Plant Pathol. 29:121–130 [Google Scholar]
- 15. Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Google Scholar]
- 16. 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:W686–W689 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Suzek BE, Ermolaeva MD, Schreiber M, Salzberg SL. 2001. A probabilistic method for identifying start codons in bacterial genomes. Bioinformatics 17:1123–1130 [DOI] [PubMed] [Google Scholar]
- 18. Toth I, Perombelon M, Salmond G. 1993. Bacteriophage ΦKP mediated generalized transduction in Erwinia carotovora subspecies carotovora. J. Gen. Microbiol. 139:2705–2709 [Google Scholar]
- 19. Toth IK, Bell KS, Holeva MC, Birch PR. 2003. Soft rot erwiniae: from genes to genomes. Mol. Plant Pathol. 4:17–30 [DOI] [PubMed] [Google Scholar]
- 20. Wang J, et al. 2005. Complete genome sequence of bacteriophage T5. Virology 332:45–65 [DOI] [PubMed] [Google Scholar]
- 21. Wilcox SA, Toder R, Foster JW. 1996. Rapid isolation of recombinant lambda phage DNA for use in fluorescence in situ hybridization. Chromosome Res. 4:397–398 [DOI] [PubMed] [Google Scholar]
- 22. Zdobnov EM, Apweiler R. 2001. InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848 [DOI] [PubMed] [Google Scholar]