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Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

ABSTRACT: Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phage vB_CskP_GAP227 and Dev-CD-23823. Based on phylogenetic analysis, PP2 and its homologous bacteriophages form a new group within the subfamily Autographivirinae in the family Podoviridae, suggesting the need to establish a new genus. No lysogenic-cycle-related genes or bacterial toxins were identified.

SUBMITTER: Lim JA

PROVIDER: S-EPMC5506502 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

Lim Jeong-A JA Heu Sunggi S Park Jinwoo J Roh Eunjung E

Archives of virology 20170413 8

Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phag ...[more]

PMID: 28409265

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Project description:Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P < 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence. IMPORTANCE Pectobacterium carotovorum subsp. carotovorum is a phytopathogen that causes soft rot disease in various crops by producing plant cell wall-degrading enzymes (PCWDEs). Although antibiotics and copper-based pesticides have been extensively applied to inhibit P. carotovorum subsp. carotovorum, the emergence of antibiotic-resistant bacteria and demand for harmless antimicrobial products have emphasized the necessity of finding alternative therapeutic strategies. To address this problem, we developed a phage cocktail consisting of three P. carotovorum subsp. carotovorum-specific phages that recognize colanic acids and flagella of P. carotovorum subsp. carotovorum. The phage cocktail treatments significantly decreased P. carotovorum subsp. carotovorum populations, as well as soft rot symptoms in napa cabbage. Simultaneously, they resulted in virulence attenuation in phage-resistant P. carotovorum subsp. carotovorum, which was represented by decreased PCWDE production and decreased flagellum-mediated swimming motility. These results suggested that preparations of phage cocktails targeting multiple receptors would be an effective approach to biocontrol of P. carotovorum subsp. carotovorum in crops.

Project description:Pectobacterium carotovorum is an economically important phytopathogen and has been identified as the major causative agent of bacterial soft rot in carrots. Control of this phytopathogen is vital to minimizing carrot harvest losses. As fully efficient control measures to successfully avoid the disease are unavailable, the phage-mediated biocontrol of the pathogen has recently gained scientific attention. In this study, we present a comprehensive characterization of the P. carotovorum phage vB_PcaM_P7_Pc (abbreviated as P7_Pc) that was isolated from infected carrot samples with characteristic soft rot symptoms, which were obtained from storage facilities at market places in Gampaha District, Sri Lanka. P7_Pc is a myovirus, and it exhibits growth characteristics of an exclusively lytic life cycle. It showed visible lysis against four of the tested P. carotovorum strains and one Pectobacterium aroidearum strain. This phage also showed a longer latent period (125 min) than other related phages; however, this did not affect its high phage titter (>1010 PFU/mL). The final assembled genome of P7_Pc is 147,299 bp in length with a G+C content of 50.34%. Of the 298 predicted open reading frames (ORFs) of the genome of P7_Pc, putative functions were assigned to 53 ORFs. Seven tRNA-coding genes were predicted in the genome, while the genome lacked any major genes coding for lysogeny-related products, confirming its virulent nature. The P7_Pc genome shares 96.12% and 95.74% average nucleotide identities with Cronobacter phages CR8 and PBES02, respectively. Phylogenetic and phylogenomic analyses of the genome revealed that P7_Pc clusters well within the clade with the members representing the genus Certrevirus. Currently, there are only 4 characterized Pectobacterium phages (P. atrosepticum phages phiTE and CB7 and Pectobacterium phages DU_PP_I and DU_PP_IV) that are classified under the genus, making the phage P7_Pc the first reported member of the genus isolated using the host bacterium P. carotovorum. The results of this study provide a detailed characterization of the phage P7_Pc, enabling its careful classification into the genus Certrevirus. The knowledge gathered on the phage based on the shared biology of the genus will further aid in the future selection of phage P7_Pc as a biocontrol agent. IMPORTANCE Bacterial soft rot disease, caused by Pectobacterium spp., can lead to significant losses in carrot yields. As current control measures involving the use of chemicals or antibiotics are not recommended in many countries, bacteriophage-mediated biocontrol strategies are being explored for the successful control of these phytopathogens. The successful implementation of such biocontrol strategies relies heavily upon the proper understanding of the growth characteristics and genomic properties of the phage. Further, the selection of taxonomically different phages for the formulation of phage cocktails in biocontrol applications is critical to combat potential bacterial resistance development. This study was conducted to carefully characterize and resolve the phylogenetic placement of the P. carotovorum phage vB_PcaM_P7_Pc by using its biological and genomic properties. Phage P7_Pc has a myovirus morphotype with an exclusively lytic life cycle, and the absence of genes related to lysogeny, toxin production, and antibiotic resistance in its genome confirmed its suitability to be used in environmental applications. Furthermore, P7_Pc is classified under the genus Certrevirus, making it the first reported phage of the genus of the host species, P. carotovorum.

Dataset Information

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

Publications

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

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