The First Complete Genome Resource of a Ralstonia solanacearum Phage UAM5 from Colombia
2022; American Phytopathological Society; Volume: 35; Issue: 6 Linguagem: Inglês
10.1094/mpmi-01-22-0033-a
ISSN1943-7706
AutoresCristian D. Grisales-Vargas, Camilo Andrés Ramírez-Cuartas, Juan E. Pérez‐Jaramillo,
Tópico(s)Bacteriophages and microbial interactions
ResumoHomeMolecular Plant-Microbe Interactions®Vol. 35, No. 6The First Complete Genome Resource of a Ralstonia solanacearum Phage UAM5 from Colombia PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseThe First Complete Genome Resource of a Ralstonia solanacearum Phage UAM5 from ColombiaCristian D. Grisales-Vargas, Camilo A. Ramírez-Cuartas, and Juan E. Pérez-JaramilloCristian D. Grisales-Vargashttps://orcid.org/0000-0002-4037-2971Unidad de Bioprospección y Estudio de Microbiomas, Programa de Estudio y Control de Enfermedades Tropicales–PECET, Facultad de Medicina, Universidad de Antioquia, Medellín, 1226, ColombiaInstituto de Biología, Universidad de Antioquia, Medellín, 050010, Colombia, Camilo A. Ramírez-CuartasInstituto de Biología, Universidad de Antioquia, Medellín, 050010, ColombiaGrupo de Bacteriología Agrícola y Ambiental–BA&A, Universidad de Antioquia, Medellín, 050010, Colombia, and Juan E. Pérez-Jaramillo†Corresponding author: J. E. Pérez-Jaramillo; E-mail Address: [email protected]Unidad de Bioprospección y Estudio de Microbiomas, Programa de Estudio y Control de Enfermedades Tropicales–PECET, Facultad de Medicina, Universidad de Antioquia, Medellín, 1226, ColombiaInstituto de Biología, Universidad de Antioquia, Medellín, 050010, ColombiaAffiliationsAuthors and Affiliations Cristian D. Grisales-Vargas1 2 Camilo A. Ramírez-Cuartas2 3 Juan E. Pérez-Jaramillo1 2 † 1Unidad de Bioprospección y Estudio de Microbiomas, Programa de Estudio y Control de Enfermedades Tropicales–PECET, Facultad de Medicina, Universidad de Antioquia, Medellín, 1226, Colombia 2Instituto de Biología, Universidad de Antioquia, Medellín, 050010, Colombia 3Grupo de Bacteriología Agrícola y Ambiental–BA&A, Universidad de Antioquia, Medellín, 050010, Colombia Published Online:8 Apr 2022https://doi.org/10.1094/MPMI-01-22-0033-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementRalstonia solanacearum is a major threat to agroecosystems because of its wide host range and broad geographical distribution. Therefore, several strategies to control this plant pathogen based on pesticides, biocontrol agents, and integrated pest management have been developed. For instance, the use of bacteriophages (viruses that infect bacteria) has recently shown promising results to control different R. solanacearum strains. Here, we provide the first complete and annotated genome sequence of a R. solanacearum phage (UAM5) from Colombia. The UAM5 phage had a 59,830-bp linear double-stranded genome with 65% of GC content. It contained 72 coding sequences (CDS) and one transfer RNA (tRNA). These CDS were associated with endonucleases, transcriptional regulators, and a recombinase. This phage genome resource will be of great value for future R. solanacearum phage therapy studies and phage genomics.Ralstonia solanacearum (Smith 1896) is one of the top plant pathogens worldwide (Mansfield et al. 2012). It is a soilborne, gram-negative β-proteobacteria, which is considered a major threat to agroecosystems because of its wide host range and broad geographical distribution (Kannan et al. 2015; Mansfield et al. 2012; Strange and Scott 2005). Consequently, several strategies to control this plant pathogen based on pesticides, biocontrol agents, and integrated pest management have been developed (Yuliar et al. 2015). For instance, the use of bacteriophages (phages) (viruses that infect bacteria) has shown promising results to control R. solanacearum strains (Elhalag et al. 2018; Ramírez et al. 2020; Wang et al. 2019). R. solanacearum phages have been reported from different families (Podoviridae, Myoviridae, Siphoviridae, and others) and with different phage lifestyles (Trotereau et al. 2021). Because of their fast propagation, high specificity, and inhibitory activity against R. solanacearum, there is an increasing effort to genomically characterize these phages in order to promote their use as biocontrol agents and to gain knowledge into their virulence mechanisms, phage lifestyles, and phage-host interactions (Biosca et al. 2021; Philipson et al. 2018; Trotereau et al. 2021; Yamada 2012). Thus, we have sequenced a previously described R. solanacearum phage, M5 (hereafter UAM5), which is currently being studied as a biocontrol agent in "Moko disease" of plantain and banana crops in Colombia (Ramírez et al. 2020).The UAM5 phage was isolated from plantain rhizosphere soils with previous reports of R. solanacearum 'Moko' strains in Colombia (Ramírez et al. 2020). It infected 64 of 65 Moko strains tested in vitro and inhibited the disease progression up to 80% in a cocktail preparation with another phage isolated by our research group (UAM8) under greenhouse conditions (Ramírez et al. 2020). We propagated UAM5 phage according to Ramírez et al. (2020). Briefly, we inoculated a 20-ml culture of R. solanacearum strain UA1591 (1 × 108 CFU/ml) and 0.312 ml of UAM5 phage (4 × 109 PFU/ml) into 140 ml of 10% tryptic soy broth medium (Himedia) supplemented with sucrose 0.1% (Bio Basic) and 1 M 0.1% CaCl2. After culturing for 24 h, we removed the cells by centrifugation at 10,000 × g for 10 min at 4°C. The supernatant was passed through a 0.2-μm membrane filter (Advantec). Subsequently, we extracted the DNA from two samples of this phage solution, one with a DNA isolation kit (Norgen Biotek), following the manufacturer's instructions, and the other with the phenol:chloroform extraction method. We also evaluated phage DNA integrity and concentration through an agarose gel (1.0%), Nanodrop Spectrophotometer 1000 (Thermo Scientific), and Qubit 4 fluorometer (Invitrogen). Phage genomic DNA library preparation and sequencing were performed by Admera Health. Phage genomic libraries were prepared with the Kapa Hyper library prep kit (min cycle), and genomic sequencing was done on an Illumina HiSeq X platform (2 × 150 bp).We analyzed the quality of the raw reads through FastQC v.0.11.9. Next, we filtered out low-quality bases (Phred value < 30) and adapters with Trimmomatic v.0.39 (Bolger et al. 2014). Then, we removed human hg19 and R. solanacearum UA1591 (GCF_003860705.1) sequences from these filtered reads with Bowtie2 v.2.3.5.1 (Langmead and Salzberg 2012). We used these decontaminated reads for de novo assembly with SPAdes v.3.15.3 (Bankevich et al. 2012), with default settings. We used BLASTn at the NCBI website on the draft assembly in order to find the highest coverage contigs with the highest similarity to other phages. Later, we extracted the sequences from the decontaminated reads that belonged to the previous phage contigs with BBDuk from the BBMap v.38.87 package. We used these phage sequences to assemble the UAM5 phage genome with SPAdes. Also, we used Bandage v.0.8.1 (Wick et al. 2015) and BLASTn to visualize UAM5′s closed genome and look for the most similar phages, respectively. Additionally, we assessed the quality of the UAM5 genome through CheckV v.0.8.1 (Nayfach et al. 2021). Afterward, we identified the UAM5 phage termini and packaging mechanism, and rearranged the genome accordingly through PhageTerm v.1.0.11 (Garneau et al. 2017). Further, we predicted the potential CDS of the UAM5 genome through RAST pipeline v.2.0 (Aziz et al. 2008) with default settings. Additionally, we manually annotated these CDS with BLASTp, HMMER v.3.3 (Zimmermann et al. 2018), and the ACLAME database v.0.4 (Leplae et al. 2004). We also predicted the tRNAs and head-neck-tail modules of the UAM5 genome through tRNAscan-SE v.2.0.9 (Chan et al. 2021) and VIRFam (Lopes et al. 2014), respectively. Finally, we visualized the annotated UAM5 phage genome with the Linear Genome Plot tool (Zulkower and Rosser 2020) through the CPT Phage Galaxy v.1.0 (Ramsey et al. 2020).The phage UAM5 had a 59,830-bp linear double-stranded genome with 65% GC, 72 CDS, and 1 tRNA (Table 1). We only found 37.5% of the CDS with putative functions, which were associated with structural proteins, endonucleases, transcriptional regulators, and a prophage-associated recombinational DNA repair protein (RecT) (Fig. 1). Phage termini prediction showed that UAM5 phage had a permuted genome with a headful (pac) packaging mechanism, fixed termini, and redundant ends. Moreover, it had the highest similarity to R. pseudosolanacearum phage Gerry from the Podoviridae family (Table 1) (Trotereau et al. 2021). Also, the head-neck-tail module of the UAM5 genome was similar to Podoviridae members (data not shown). These findings were in concordance with previous assumptions of this phage belonging to the Podoviridae family because of the phage morphology shown through transmission electron microscopy (Ramírez et al. 2020). To our knowledge, we have provided the first complete genome sequence of a R. solanacearum phage from Colombia. This new genomic resource will be of great value for future studies of R. solanacearum phage therapy and phage genomics.Table 1. Summary information of Ralstonia solanacearum phage UAM5 genomeGenome size (bp)Average raw readsCoverageGC (%)Transfer RNAsCoding sequences (percentage with putative functions)Best match (GenBank accession number)aCoverage (%)aIdentity (%)a59,8301,017,633252×65172 (37.5)Ralstonia phage Gerry (NC_054959.1)6987,58aBLASTn query results.Table 1. Summary information of Ralstonia solanacearum phage UAM5 genomeView as image HTML Fig. 1. Genome map of Ralstonia solanacearum phage UAM5. Arrows indicate coding sequences (CDS) or transfer RNAs (tRNAs) and the direction of the transcription. The color of each row indicates the functional category: CDS with putative function (green arrow), hypothetical proteins (orange arrow), and tRNAs (red arrow). Scale units are base pairs.Download as PowerPointData AvailabilityThe genome sequence and associated information have been deposited in the European Nucleotide Archive at EMBL-EBI under accession GCA_921293885, and BioProject accession number PRJEB48832.AcknowledgmentsWe thank CENIBANANO, AUGURA, and the PECET group for helping with the phage propagation and purification and J. Correa and the APOLO Scientific Computing Center at EAFIT University for providing the computational resources.Author-Recommended Internet ResourcesBBMap v.38.87 package: https://sourceforge.net/projects/bbmap/FastQC v.0.11.9: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/Masked human reference genome (hg19): https://zenodo.org/record/4116107Phenol:Chloroform extraction method: https://cpt.tamu.edu/wordpress/wp-content/uploads/2018/09/Phage-DNA-Extraction-by-PhenolChloroform-Protocol.pdfThe author(s) declare no conflict of interest.Literature CitedAziz, R. K., Bartels, D., Best, A. 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This is an open access article distributed under the CC BY-NC-ND 4.0 International license.The corresponding author was changed on November 7, 2022.DetailsFiguresLiterature CitedRelated Vol. 35, No. 6 June 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Article History Issue Date: 20 May 2022Published: 8 Apr 2022Accepted: 27 Feb 2022 Pages: 496-499 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY-NC-ND 4.0 International license.FundingGovernment of Antioquia and "Ministerio de Ciencia, Tecnología e Innovación" (MINCIENCIAS)Grant/Award Number: 826-2018Keywordsbiocontrolgenome resourcesgenomicsphagesphage therapyRalstonia solanacearumThe author(s) declare no conflict of interest.PDF downloadCited byA Capsid Structure of Ralstonia solanacearum podoviridae GP4 with a Triangulation Number T = 91 November 2022 | Viruses, Vol. 14, No. 11
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