HomeMolecular Plant-Microbe Interactions®Vol. 35, No. 8Complete Genome Sequences of Five Gram-Negative Bacterial Strains Comprising Synthetic Bacterial Consortium “The Great Five” with Antagonistic Activity Against Plant-Pathogenic Pectobacterium spp. and Dickeya spp. PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseComplete Genome Sequences of Five Gram-Negative Bacterial Strains Comprising Synthetic Bacterial Consortium “The Great Five” with Antagonistic Activity Against Plant-Pathogenic Pectobacterium spp. and Dickeya spp.Tomasz Maciag, Dorota M. Krzyzanowska, Lukasz Rabalski, Sylwia Jafra, and Robert CzajkowskiTomasz MaciagLaboratory of Plant Microbiology, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, PolandSearch for more papers by this author, Dorota M. KrzyzanowskaLaboratory of Plant Microbiology, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, PolandSearch for more papers by this author, Lukasz RabalskiLaboratory of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, PolandSearch for more papers by this author, Sylwia Jafra†Corresponding authors: S. Jafra; E-mail Address: sylwia.jafra@ug.edu.pl, and R. Czajkowski; E-mail Address: robert.czajkowski@ug.edu.plhttp://orcid.org/0000-0002-4194-6180Laboratory of Plant Microbiology, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, PolandSearch for more papers by this author, and Robert Czajkowski†Corresponding authors: S. Jafra; E-mail Address: sylwia.jafra@ug.edu.pl, and R. Czajkowski; E-mail Address: robert.czajkowski@ug.edu.plhttp://orcid.org/0000-0001-9641-5603Laboratory of Biologically Active Compounds, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, PolandSearch for more papers by this authorAffiliationsAuthors and Affiliations Tomasz Maciag1 Dorota M. Krzyzanowska1 Lukasz Rabalski2 Sylwia Jafra1 † Robert Czajkowski3 † 1Laboratory of Plant Microbiology, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, Poland 2Laboratory of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, Poland 3Laboratory of Biologically Active Compounds, Intercollegiate Faculty of Biotechnology UG and MUG, University of Gdansk, Antoniego Abrahama 58, 80-307 Gdansk, Poland Published Online:25 May 2022https://doi.org/10.1094/MPMI-01-22-0020-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleGenome AnnouncementThere is a growing interest in using synthetic microbial consortia as biological control agents (biopesticides) in agricultural applications (Arora et al. 2016; Mehnaz 2016). This interest is manifested because microbial consortia can offer higher reproducibility of biological control under various environmental conditions and provide a broader array of modes of action than any individual biological control agent applied alone against the given pathogens (Mishra and Arora 2016).However, despite the global demand for better-performing, more environmentally friendly crop protection systems in agriculture, there still are very few biopesticides on the market comprising more than one active microbial biological control agent. The reasons for that situation are diverse (Vishwakarma et al. 2020); however, two critical challenges may be identified. First, there are unresolved issues with the registration and marketing of such bioproducts, which limit their potential use in modern agriculture on a large scale. Second, the difficulties in understanding the specific roles of each component of a microbial consortium and their biological activity may limit the predicted final protective effect on the crop (Czajkowski et al. 2020). Due to the above, more research is required concerning microbial consortia with biological control activity and their feasibility in agricultural applications (Xu et al. 2011). To address this issue, we sequenced the genomes of bacterial strains comprising the “Great Five” (GF) synthetic microbial consortium effective against potato soft rot disease caused by Pectobacterium and Dickeya spp. (Krzyzanowska et al. 2019; Maciag et al. 2020).This GF synthetic bacterial consortium comprises five strains: Lellilottia amnigena (former Enterobacter amnigenus) strain A167, Serratia plymuthica strain A294, Rahnella aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469 (Krzyzanowska et al. 2019). Strains of L. amnigena A167 and S. plymuthica A294 were isolated from the potato rhizosphere (Jafra et al. 2006). In contrast, strains of R. aquatilis H145 and S. rubidaea strains H440 and H469 were isolated from the inside of hyacinth bulbs (Jafra et al. 2009). When combined into the GF consortium, the five bacterial strains were shown to suppress soft rot symptoms on potato tubers in storage, even under high pathogen pressure and conditions favoring disease progression (Krzyzanowska et al. 2019). Furthermore, the GF consortium has been successfully formulated into a stable product that can be readily applied in potato production systems (Maciag et al. 2020). The latter is essential in transferring to an agricultural setting (Bashan et al. 2014).The complete genome sequences of the strains comprising the GF synthetic consortium can help to identify features essential for the biocontrol activity of the consortium, interactions between the strains, and strain compatibility, as well as the safety of use in agricultural applications (Deising et al. 2017; Loper et al. 2012; Stockwell et al. 2011).For genomic studies, bacterial DNA was isolated using the Wizard Genomic DNA purification KIT (Promega Corp.) and additionally purified with the Clean NA kit (GC Biotech b.v.) according to the instructions provided by the manufacturers. Genome sequencing was performed in parallel by two platforms: Illumina Mini-Seq and Oxford Nanopore Technology. The raw reads of each strain of the consortium produced during genome sequencings were deposited in the NCBI Sequence Read Archive under BioProject PRJNA557569 and accession number SRP363301. Data were de novo assembled using Unicycler v0.4.8, with a final mean coverages of 287.2× for A167, 199.8× for H145, 271.8× for A294, 258.9× for H440, and 259.8× for H469. Initial genome polishing was conducted by a tool integrated into the assembler (Racoon polishing script). Pilon 1.23 was used to further correct the errors. After that, Illumina reads were mapped to contigs from previous steps. Visual inspection was done on mapped files using Geneious Prime 2020. The procedure was done to check for any drop in coverage that could happen in repeated regions (manual validation) and to close any open contig that was not correctly assembled due to the multiplication of the same sequence on both contig ends (manual curation).Each time, the combined procedure produced a single contig (Table 1). The GF genomes were annotated with the NCBI Procaryotic Genome Annotation Pipeline (Tatusova et al. 2016). The obtained genome sequences of 4.5 to 5.5 Mbp in length were deposited in the NCBI GenBank database. The GenBank accession numbers, genome sizes, and GC contents of each GF strain are given in Table 1. Interestingly, we found that R. aquatilis H145 possesses three plasmids: two of 500 kbp and one of 115 kbp. L. amnigena A167 has one 109-kbp plasmid, and each S. rubidaea strain has one 3.5-kbp plasmid (Table 1). The four genomes H145, A167, H440, and H469, contained plasmids in addition to the main replicon.Table 1. General features of the genomes of five antagonistic strains belonging to the “The Great Five” consortiumClusters involved in the synthesis of secondary metabolitesdSpecies, strain, source, accessionSize (bp)aGC content (%)CDSbTotal (metabolite)cNRPSPKSSidBactLellilottia amnigena, A167, potato rhizosphereCP0423614,520,65952.94,2292 (arylopyene)1000CP042362 (plasmid)109,78764.2140−−−−−Rahnella aquatilis, H145, hyacinth bulbCP0423575,033,52451.74,6335 (desferrioxamine E, xanthoferrin)1020CP042358 (plasmid)536,32251.7486−−−−−CP042359 (plasmid)467,53349.6467−−−−−CP042360 (plasmid)111,44552.9115−−−−−Serratia plymuthica, A294, potato rhizosphereCP0423635,534,59556.25,08514 (sodorifen, zeamine)6120S. rubidaea, H440, hyacinth bulbCP0423554,952,31659.24,59612 (pyrrolnitrin)4142CP042356 (plasmid)3,46145.05−−−−−S. rubidaea, H469, hyacinth bulbCP0423534,952,53459.24,59512 (pyrrolnitrin)4142CP042354 (plasmid)3,46145.05−−−−−aGenome size. Accession numbers of replicons are given according to size from chromosome to the smallest plasmids for each strain.bCoding sequence (CDS) counts.cTotal (predicted secondary metabolite. Only clusters with score ≥50% (similarity in amino acid sequence to known clusters ≥50%) are listed in the table.dClusters responsible for secondary metabolism were assigned with antiSMASH 6.0.1. NRPS = nonribosomal peptide synthetase, PKS = poliketyde synthase, Sid = siderophore, and Bact = bacteriocins. Analyses were performed with KnownClusterBlast, ClusterBlast, SubClasterBlast, ActiveSiteFinder, Cluster Pfam analysis, and Pfam-based gene ontology term annotation features turned on. Some clusters can be assigned to more than one category; for example, NRPS siderophores.Table 1. General features of the genomes of five antagonistic strains belonging to the “The Great Five” consortiumView as image HTML The sequenced genomes were compared based on the composition of the Cluster of Orthologous Groups assigned by eggNOG (Huerta-Cepas et al. 2017). All strains had approximately 40% of genes involved in general (primary) metabolism and 20% in each of the following groups: cellular processes and signalling, information storage and processing, and poorly characterized. Comparing the percentage of genes from different orthologous groups, L. aminigena A167 differs the most from the other strains of the GF consortium. A167 has more genes responsible for inorganic ion transport and metabolism and fewer genes accountable for amino acid transport and metabolism (Supplementary Table S1).The AntiSMASH 6.0.1 (Blin et al. 2021) platform was used to analyze genomes for the presence of genes involved in secondary metabolism (production of antibacterial or antifungal secondary metabolites). Analyses were performed with KnownClusterBlast, ClusterBlast, SubClasterBlast, ActiveSiteFinder, Cluster Pfam analysis, and Pfam-based gene ontology term annotation features (Table 1). For S. plymuthica A294, the algorithm detected 14 clusters involved in secondary metabolism, 6 belonging to nonribosomal peptide synthetases (NRPS), 1 poliketyde synthase (PKS), and 2 siderophore clusters. In addition, two of the found clusters have a 100% similarity with clusters responsible for the synthesis of sodorifen and zeamine. For L. amnigena A167, antiSMASH 6.0.1 detected only two clusters: one NRPS cluster and one region with 100% similarity to a cluster involved in the synthesis of arylopyene. For R. aquatilis H145, five clusters were detected: one NRPS cluster, two siderophore regions, one region with 100% similarity to the cluster involved in desferrioxamine E synthesis, and another with 57% similarity to a region for the synthesis of xanthoferrin. For S. rubidaea strains H440 and H469, 12 clusters were detected: 4 NRPS, 1 PKS, 4 siderophore regions, 2 bacteriocins, and 1 region with 100% similarity to a cluster involved in the synthesis of pyrrolnitrin.We have not found clusters encoding any secondary metabolites toxic to humans and animals in the obtained genomes, suggesting that the respective GF strains do not produce such compounds. These results, however, should be experimentally confirmed.In turn, we found secondary metabolites with known antifungal activity, such as pyrronitrin (Arima et al. 1964), and antibacterial activity, such as zeamine (Hellberg et al. 2015). Therefore, the obtained data can help identify a broader use for the tested GF synthetic consortium; for example, against important potato pathogens other than Pectobacterium and Dickeya spp., including fungal pathogens such as Rhizoctonia solani (Jung et al. 2018). In addition, sodorifen produced by S. plymuthica is considered essential for interspecies communication by volatile compounds (Domik et al. 2016). Volatile-based communication may lead to changes in the profile of produced secondary metabolites and, therefore, changes in the antimicrobial activity of the GF consortium (Kai and Piechulla 2018; Schmidt et al. 2017).The complete genomes of the biological control strains provide a valuable reference for research on understanding the traits essential in the consortium's interspecies interactions and their further applications in agriculture. 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Czajkowski and by The Innovation Incubator 2.0 (project: 17/CTT/II2) implemented by the University of Gdansk in a Consortium with the Gdansk University of Technology, the Medical University of Gdansk and Excento Sp. z o. o. under the noncompetitive project “Support for management of scientific research and commercialization of the results of R&D works in scientific units and enterprises”, Intelligent Development Operational Program 2014-2020 (POIR.04.04.00-00-0004/15 and MNiSW/2019/169/DIR) to R. Czajkowski.The author(s) declare no conflict of interest. Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.DetailsFiguresLiterature CitedRelated Vol. 35, No. 8 August 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Article History Issue Date: 31 Aug 2022Published: 25 May 2022Accepted: 18 Mar 2022 Pages: 711-714 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY-NC-ND 4.0 International license.Funding Narodowe Centrum Badan i RozwojuGrant/Award Number: LIDER/450/L-6/14/NCBR/2015 The Innovation Incubator 2.0 (project: 17/CTT/II2) implemented by the University of Gdansk in a Consortium with the Gdansk University of Technology, the Medical University of Gdansk and Excento Sp. z o. o. under the noncompetitive project “Support for management of scientific research and commercialization of the results of R&D works in scientific units and enterprises”, Intelligent Development Operational Program 2014-2020 (POIR.04.04.00-00-0004/15 and MNiSW/2019/169/DIR) Keywordsagriculturebiological controlblackleggenomicsplant pathogensoft rotsyn-comsynthetic consortiaThe author(s) declare no conflict of interest.PDF download