Abstract
Pectobacterium carotovorum subsp. carotovorum is a phytopathogen causing soft rot disease on diverse plant species. To control this plant pathogen, P. carotovorum subsp. carotovorum-targeting bacteriophage PP1 was isolated and its genome was completely sequenced to develop a novel biocontrol agent. Interestingly, the 44,400-bp genome sequence does not encode any gene involved in the formation of lysogen, suggesting that this phage may be very useful as a biocontrol agent because it does not make lysogen after host infection. This is the first report on the complete genome sequence of the P. carotovorum subsp. carotovorum-targeting bacteriophage, and it will enhance our understanding of the interaction between phytopathogens and their targeting bacteriophages.
GENOME ANNOUNCEMENT
Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) is a phytopathogenic Gram-negative bacterium causing soft rot on various plant species (5, 7, 10). This pathogen causes serious loss of produce quality during its growth, transit, and even storage. Recently, a novel bacteriocin, carocin D, was isolated from P. carotovorum subsp. carotovorum strain Pcc21 to develop a biocontrol agent to control this phytopathogen (9). Furthermore, to increase this antibacterial activity, an additional biocontrol agent such as bacteriophage treatment is needed. This approach could be one of the best biocontrol agents to maximize the growth inhibition of this pathogen in plants. Here, we isolated P. carotovorum subsp. carotovorum-targeting bacteriophage PP1 and fully sequenced its genome to understand its inhibition mechanism against this pathogen.
The genome of phage PP1 was isolated using the alkaline lysis and phenol extraction method (11). Physically shared DNA was cloned to T vector, and libraries were completely sequenced using ABI3730 (GGBio, South Korea). The open reading frames (ORFs) were bioinformatically predicted using Glimmer3 (4), GeneMarkS (2), and FgenesB (Softberry, Inc., Mount Kisco, NY) and confirmed by RBS finder (J. Craig Venter Institute, Rockville, MD). The functions of ORFs were predicted by BLASTP (1) and InterProScan with protein motif databases (12). The complete genome sequence and annotation information were edited and handled using Artemis14 (3).
The genome of P. carotovorum subsp. carotovorum-targeting phage PP1 consisted of a length of 44,400 bp with a GC content of 49.74%, encoding 48 ORFs with no tRNA. Although six phage genomes are available in the GenBank database infecting Erwinia amylovora, E. tasmaniensis, and E. pyrifoliae to date (6, 8), there is no report on the complete genome sequence of the P. carotovorum subsp. carotovorum-infecting phage. Therefore, more than 50% of the annotated ORFs (26 of 48) encode hypothetical proteins due to insufficient genome annotation information on this phage. The genes encoded in this genome are categorized into five groups: DNA replication/manipulation (DNA-directed RNA polymerase, DNA primase, DNA polymerase, HNH endonuclease, 5′-3′ exonuclease, and DNA ligase), phage structure (head-tail connector, scaffolding protein, major capsid protein, tail tubular proteins, and internal virion proteins), phage packaging (large and small subunits of terminase), host lysis (lysozyme domain protein and holin), and host specificity (tail fiber protein). Interestingly, this genome has only one gene encoding tail fiber protein for host specificity, suggesting that this gene may be important to recognize and to infect the host. After infection of this host, the genes encoding lysozyme and holin may play important roles in the host lysis. However, this genome does not encode proteins involved in lysogen formation, suggesting that this phage probably does not make lysogen after phage infection. The absence of genes involved in lysogen formation may be very useful to develop an effective biocontrol agent with this phage. This is the first report on the complete genome sequence of the P. carotovorum-targeting bacteriophage, and it should enhance our understanding of bacterial pathogenesis to plants and its control between P. carotovorum subsp. carotovorum and its infecting phages.
Nucleotide sequence accession number.
The complete genome sequence of P. carotovorum subsp. carotovorum-targeting bacteriophage PP1 is available under GenBank accession number JQ837901.
ACKNOWLEDGMENTS
This work was supported by Rural Development Administration (RDA) grant PJ008283. H. Shin was the recipient of a graduate fellowship provided by the Ministry of Education, Science and Technology through the Brain Korea 21 Project.
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