Abstract
Erwinia amylovora is the causative agent of fire blight, a serious disease of some Rosaceae plants. The newly isolated bacteriophage PhiEaH2 is able to lyse E. amylovora in the laboratory and has reduced the occurrence of fire blight cases in field experiments. This study presents the sequenced complete genome and analysis of phage PhiEaH2.
GENOME ANNOUNCEMENT
Erwinia amylovora, a member of Enterobacteriaceae, is the causative agent of fire blight, a serious disease of some Rosaceae plants (8, 9). One alternative treatment to control fire blight could be the application of bacteriophages (2, 6, 7).
We isolated E. amylovora phage PhiEaH2 from a soil sample in Hungary. This phage demonstrated strong lytic effect against E. amylovora in the laboratory and reduced the occurrence of fire blight cases in a field experiment when no artificial infection was applied (D. Dömötör, G. Schneider, G. Rákhely, B.G. Polyák, and T. Kovács, submitted for publication). These observations indicate that this phage might be able to be used as a biocontrol agent against this plant-pathogenic bacterium.
The genomic DNA of phage PhiEaH2 was extracted and purified by using a Roche High Pure viral nucleic acid kit (Roche Diagnostics GmbH, Germany) according to the protocol supplied. A shotgun library was created and sequenced using two platforms (Roche GS Junior, ABI 3500XL genetic analyzer) with 20-fold coverage of the phage genome. Open reading frame (ORF) prediction was done by using Genemark, Baysys, and Rast. Sequence annotations were performed by Baysys and Rast. The genomic sequence of PhiEaH2 phage is 243,050 bp in length with a G+C content of 51.28 mol%. No phages against E. amylovora with such a large genome have been sequenced before (1, 4, 5). The genome showed 262 ORFs, and 205 ORFs were annotated as encoding hypothetical proteins; most of them had the highest similarity to the Salmonella phage SPN3US (accession no. JN641803) (3). Additionally, 57 ORFs were annotated as functional genes. Thirty-five ORFs were predicted to encode proteins involved in the structure and assembly of virions, and 15 ORFS were found to encode proteins related to nucleic acid metabolism and modification and DNA replication (thymidylate synthase, thymidylate kinase, DNA adenine methylase, endodeoxyribonuclease, RNase H, dihydrofolate reductase, a transcriptional regulator, DNA-dependent RNA polymerase beta subunits, helicases, and an SMC domain-containing protein). One ORF encodes a protein containing an HD domain, two ORFs encode endolysins, and another two are for acetyltransferases. The product of one ORF is involved in amylovoran biosynthesis, and two proteins contain radical SAM superfamily domains.
In conclusion, we analyzed the complete genomic sequence of the newly isolated E. amylovora phage PhiEaH2. PhiEaH2 is a good candidate for use as biocontrol agent against this plant-pathogenic bacterium. However, sequencing its genome revealed the presence of the amsF gene, which codes for a protein that is essential in amylovoran biosynthesis. This complex polysaccharide is necessary for E. amylovora to evoke the pathogenic process in the host plant. The presence of this gene in PhiEaH2 must be taken into consideration if practical use of this phage strain is intended.
Nucleotide sequence accession number.
The complete genome sequence of E. amylovora phage PhiEaH2 has been submitted to GenBank and assigned accession number JX316028.
ACKNOWLEDGMENTS
This work was funded by the European Union and by the Hungarian Government; projects GVOP-3.3.3-05/2.-2006-01-0045/3.0, GOP-1.1.1-07/1-2008-0038, GOP-1.3.2.-09-201-0023. The Hungarian National Technology Program (projects FAGCNTER and MFCDiagn) also supported this work.
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