Abstract
Fire blight, a plant disease of economic importance caused by Erwinia amylovora, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three E. amylovora-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an Erwinia tasmaniensis strain that is antagonistic for E. amylovora. The American phages resemble two already-described viral genomes, whereas the E. tasmaniensis phage displays a singular genomic sequence in BLAST searches.
Several Erwinia amylovora phages from North America have been partially characterized before by restriction digests and proposed for control of fire blight (5). We determined the genomes of φEa1h, φEa100, and φEa104 by shotgun sequencing on ABI3730XL capillary sequencers (3) and by primer walking. The shotgun sequences were assembled with PHRAP and edited with Consed, GAP4, and Clonemanager 5. Open reading frames (ORFs) were predicted with GLIMMER3. Partial sequences were aligned with the sequence of phage φEra103 (GenBank/EMBL/DDB accession number EF160123) for φEa1h and φEa100 and the sequence of phage φEa21-4 (accession number EU710883) for φEa104, and gaps were filled by sequencing PCR fragments.
The genomes of φEa1h and φEa100, members of the Podoviridae, have almost identical sizes of 45,522 bp and 45,554 bp, respectively, and average GC contents of 49.7%. They differed in six base pairs and a 32-bp repeat for φEa100.
Annotation revealed 50 major ORFs and a putative function for 27 genes, including genes coding for an extracellular polysaccharide (EPS) depolymerase and HNH endonucleases and genes associated with DNA replication, but no tRNA genes.
The highly related genomes of φEa1h/φEa100 and φEra103 diverged in 10 genes and a degenerated terminal repeat of 277 bp. Another peculiarity is a split gene for DNA polymerase and the associated 5′-3′ exonuclease.
The phages φEa1h and φEa100 may use direct repeats in their genomes for replication as concatemers, similar to E. coli phage T7 (2). A 54-mer is repeated twice and is present in both genomes.
Phage φEa104 belongs to the Myoviridae and has a genome length of 84,565 bp with 43.9% GC content. One hundred eighteen ORFs were identified with similarity to ORFs in φEa21-4. Morphogenesis-related proteins were predicted for 10 genes, and an endolysin and a holin gene were identified. φEa104 carries genes involved in nucleotide metabolism but no EPS depolymerase. A cluster of 24 tRNAs was detected for φEa104, similar to phage φEa21-4 (4), with 23 genes coding for standard tRNAs without pseudogenes. For φEa104, a circularly permutated linear genome is assumed.
Comparison of φEa104 with φEa21-4 revealed 98% identity for their genomes of 84,565 and 84,576 bp, respectively. Regions with high levels of mismatch but >97% protein identity were noted for genes encoding RIIA, a hypothetical protein, and DNA polymerase. BLAST search revealed low similarities to Salmonella phage Felix O1 (AF320576) (6), Escherichia coli O157:H7 phage wV8 (EU877232), and Staphylococcus phage SA1 (GU169904). The genome structure of φEa104 is completely different from that of φEa1h and φEa100.
Erwinia tasmaniensis strain Et1/99, antagonistic to E. amylovora (1), carries prophage information (3). From the Australian strain Et88, the novel phage φEt88 was induced with mitomycin C. It belongs to the Myoviridae and has a genome size of 47.3 kb. After shotgun sequencing and assembly, 68 ORFs were predicted. Eight genes encode morphogenesis-related proteins, seven have functions in nucleotide metabolism, and five are related to lysogenicity. Additionally, a lysis protein gene, an associated holin gene, and a single tRNA gene were found.
BLASTX search revealed only low similarity to other viral proteins, including a large terminase subunit, a hypothetical protein, an integrase and a DNA helicase, indicating a singular genomic sequence for φEt88.
Nucleotide sequence accession numbers.
The sequences of the E. amylovora and E. tasmaniensis phages have been submitted to the EMBL database under accession no. FQ482084 (φEa1h), FQ482086 (φEa100), FQ482083 (φEa104), and FQ482085 (φEt88).
Acknowledgments
We thank the Julius Kuehn Institute and the Max Planck Society for financial support of the sequencing projects.
Footnotes
Published ahead of print on 19 November 2010.
REFERENCES
- 1.Jakovljevic, V., S. Jock, Z. Du, and K. Geider. 2008. Hypersensitive response and acyl-homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae. Microb. Biotechnol. 1:416-424. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Kornberg, A. 1980. DNA replication. W. H. Freeman and Co, San Francisco, CA.
- 3.Kube, M., et al. 2008. The genome of Erwinia tasmaniensis strain Et1/99, a non-pathogenic bacterium in the genus Erwinia. Environ. Microbiol. 10:2211-2222. [DOI] [PubMed] [Google Scholar]
- 4.Lehman, S. M., A. M. Kropinski, A. J. Castle, and A. M. Svircev. 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]
- 5.Schnabel, E. L., and A. L. Jones. 2001. Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora. Appl. Environ. Microbiol. 67:59-64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Whichard, J. M., et al. 2010. Complete genomic sequence of bacteriophage Felix O1. Viruses 2:710-730. [DOI] [PMC free article] [PubMed] [Google Scholar]