university of copenhagen Whole-genome sequence of Pseudomonas fluorescens EK007-RG4, a promising biocontrol agent against a broad range of bacteria, including the fire blight bacterium Erwinia amylovora Habibi, Roghayeh; Tarighi, Saeed; Behravan, Javad; Taheri, Parissa; Kjøller, Annelise Helene; Brejnrod, Asker Daniel; Madsen, Jonas Stenløkke; Sørensen, Søren Johannes Published in: Genome Announcements DOI: 10.1128/genomeA.00026-17 Publication date: 2017 Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Habibi, R., Tarighi, S., Behravan, J., Taheri, P., Kjøller, A. H., Brejnrod, A. D., ... Sørensen, S. J. (2017). Wholegenome sequence of Pseudomonas fluorescens EK007-RG4, a promising biocontrol agent against a broad range of bacteria, including the fire blight bacterium Erwinia amylovora. Genome Announcements, 5(13), [e00026-17]. https://doi.org/10.1128/genomeA.00026-17 Download date: 13. Jun. 2020 PROKARYOTES crossm Roghayeh Habibi,a,b Saeed Tarighi,a Javad Behravan,c Parissa Taheri,a Annelise Helene Kjøller,b Asker Brejnrod,b Jonas Stenløkke Madsen,b Søren Johannes Sørensenb Section of Phytopathology, Department of Plant Protection, Ferdowsi University of Mashhad, Mashhad, Irana; Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmarkb; Section of Pharmaceutical Biotechnology, Department of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iranc Here, we report the first draft whole-genome sequence of Pseudomonas fluorescens strain EK007-RG4, which was isolated from the phylloplane of a pear tree. P. fluorescens EK007-RG4 displays strong antagonism against Erwinia amylovora, the causal agent for fire blight disease, in addition to several other pathogenic and nonpathogenic bacteria. ABSTRACT P seudomonas fluorescens is a Gram negative, rod-shaped bacterium that is widely distributed in various environments (1, 2). The P. fluorescens group is one of the most diverse within the Pseudomonas genus, currently including more than 50 named species (3). Many strains belonging to the P. fluorescens group are plant commensals with efficient biocontrol properties (1, 3). P. fluorescens EK007-RG4 was isolated from the phyllosphere of a pear tree in northern Iran. The strain produces compounds that are highly inhibitory against Erwinia amylovora, the common causal agent of fire blight disease, in addition to a diverse array of Gram-negative and -positive bacteria. The genome of EK007-RG4 was sequenced to further the understanding of the antibacterial properties of the strain. Genomic DNA was extracted using the DNA blood and tissue kit from Qiagen. The whole-genome sequencing library was prepared using the Nextera XT DNA library preparation kit (Illumina) and quantified by a fragment analyzer (Advanced Analytical Technologies, Inc.). Sequencing was completed with 2 ⫻ 250-bp paired-end reads using the Illumina MiSeq platform (Illumina). Standard protocols were used for all of the above kits, as provided by the manufacturers. The reads were cleaned and trimmed (trim sequences tool; settings: ambiguous limit ⫽ 2, quality limit ⫽ 0.05) using CLC Genomics Workbench version 7 (CLC bio). Next, quality-filtered reads were assembled into contigs (de novo assembly tool; default settings, including both paired and orphaned reads; minimum contig length ⫽ 500 bp). The RAST and SEED (4) servers were used to annotate the resulting contigs. The draft genome of P. fluorescens EK007-RG4 was 5,970,564 bp long, with an average coverage of 207⫻, and was assembled into 27 contigs with an average G⫹C content of 60.2% and 67 RNAs. RNAmmer analysis predicted 21 copies of the 5s and three copies each of the 23s and 16s rRNAs. The results of sequence annotation revealed 5,318 coding regions with 53% assigned to a SEED subsystem. Volume 5 Issue 13 e00026-17 Received 9 January 2017 Accepted 12 January 2017 Published 30 March 2017 Citation Habibi R, Tarighi S, Behravan J, Taheri P, Kjøller AH, Brejnrod A, Madsen JS, Sørensen SJ. 2017. Whole-genome sequence of Pseudomonas fluorescens EK007-RG4, a promising biocontrol agent against a broad range of bacteria, including the fire blight bacterium Erwinia amylovora. Genome Announc 5:e00026-17. https://doi.org/10.1128/genomeA.00026-17. Copyright © 2017 Habibi et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Søren Johannes Sørensen, sjs@bio.ku.dk. genomea.asm.org 1 Downloaded from http://genomea.asm.org/ on August 3, 2017 by KB, Københavns Universitetsbibliotek Whole-Genome Sequence of Pseudomonas fluorescens EK007-RG4, a Promising Biocontrol Agent against a Broad Range of Bacteria, Including the Fire Blight Bacterium Erwinia amylovora Habibi et al. ACKNOWLEDGMENTS We thank Karin P. Vestberg and Luma George Odish for technical assistance in the laboratory. We also thank Martin Asser Hansen and Waleed Abu Al-Soud for assistance with DNA sequencing and bioinformatics. This work was supported by the Danish Council for Independent Research grant 11106571. The funders had no role in study design, data collection and analysis, or preparation of the manuscript. REFERENCES 1. Loper JE, Hassan KA, Mavrodi DV, Davis EW, Lim CK, Shaffer BT, Elbourne LD, Stockwell VO, Hartney SL, Breakwell K, Henkels MD, Tetu SG, Rangel LI, Kidarsa TA, Wilson NL, van de Mortel JE, Song C, Blumhagen R, Radune D, Hostetler JB, Brinkac LM, Durkin AS, Kluepfel DA, Wechter WP, Anderson AJ, Kim YC, Pierson LS, Pierson EA, Lindow SE, Kobayashi DY, Raaijmakers JM, Weller DM, Thomashow LS, Allen AE, Paulsen IT. 2012. Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. 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Nucleic Acids Res 44: D1113–D1118. https://doi.org/10.1093/nar/gkv1143. genomea.asm.org 2 Downloaded from http://genomea.asm.org/ on August 3, 2017 by KB, Københavns Universitetsbibliotek Genome comparison showed closest similarity between EK007-RG4 and the biocontrol agents P. fluorescens A506 and P. fluorescens SS101. Unique genes homologous to the nonribosomal peptide synthase genes massA, massB, and massC (5) were identified in the EK007-RG4 genome. These genes code for enzymes that biosynthesize cyclic lipopeptides (CLPs), which are biosurfactants with antimicrobial properties (6). A protein BLAST search of the translated massA, massB, and massC genes of EK007-RG4 showed 96%, 97%, and 97% similarity, respectively, to any GenBank sequences. The core structure of the putative mass CLP produced by EK007-RG4 was predicted as (lipid tail)-Leu-Asp-Thr-Ile-Leu-Ser-Leu-Ser-Ile using antiSMASH (7). This predicted CLP is different from any known nonribosomal peptides in the Norine database (8). The potentially new EK007-RG4 CLP is likely a type of viscosin. However, the putative EK007-RG4 CLP is unique in that the second amino acid is Asp, while it is consistently Glu among known viscosins. Accession number(s). This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number MRST00000000. The version described in this paper is the first version, MRST01000000.