Complete genome sequence of Pseudomonas corrugata strain RM1-1-4, a stress protecting agent from the rhizosphere of an oilseed rape bait plant

10.1601/nm.2592 strain RM1-1-4 is a rhizosphere colonizer of oilseed rape. A previous study has shown that this motile, Gram-negative, non-sporulating bacterium is an effective stress protecting and biocontrol agent, which protects their hosts against abiotic and biotic stresses. Here, we announce and describe the complete genome sequence of P. corrugata RM1-1-4 consisting of a single 6.1 Mb circular chromosome that encodes 5189 protein coding genes and 85 RNA-only encoding genes. Genome analysis revealed genes predicting functions such as detoxifying mechanisms, stress inhibitors, exoproteases, lipoproteins or volatile components as well as rhizobactin siderophores and spermidine. Further analysis of its genome will help to identify traits promising for stress protection, biocontrol and plant growth promotion properties.


Introduction
Pseudomonas corrugata Roberts and Scarlett (1981) emend. Sutra belongs to the genus Pseudomonas sensu stricto and it is one of the few non fluorescent Pseudomonas species. P. corrugata strain RM1-1-4 was isolated from the oilseed rape rhizosphere grown in the greenhouse, whose seeds were treated with the microbial community associated with the moss Sphagnum magellanicum [1]. Sphagnum mosses form bog ecosystems under low-nutrient and extreme conditions supported by their microbiota [2]. RM1-1-4 was selected as stress protecting agent coping high salt concentrations, reactive oxygen species and desiccation [1]. As it has a broad antagonistic spectrum exhibiting antifungal activity against phytopathogenic fungi (Ascomycota: Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum, Verticillium dahliae and Basidiomycota: Rhizoctonia solani AG2-2IIIB, Sclerotium rolfsii), it is a promising candidate for biocontrol purposes. The activity putatively base on the production of exoenzymes and the emission of antimicrobial volatile organic compounds.
In this report, we summarize the complete genome sequence and annotation of RM1-1-4. We also describe its genomic properties revealing multifaceted plant beneficial features. The genome sequence of RM1-1-4 and its comparison with related published genomes will provide a framework for further functional studies of its abiotic and biotic stress protecting effectiveness in plant and rhizosphere competence.

Organism information
Classification and features P. corrugata RM1-1-4 is a motile, Gram-negative, nonsporulating rod in the order Pseudomonadales of the class Gammaproteobacteria. The rod-shaped cells are approximately 0.5 μm in width and 1.5-2.0 μm in length ( Fig. 1 left). The strain is moderately fast-growing, forming 1 mm colonies within 1-2 days at 25°C. Colonies formed on nutrient broth II (NBII) agar plates [1] are yellow opaque shining, domed and moderately mucoid with smooth margins (Fig. 1 right). No fluorescence of the cells was visualized under UV light (312 nm) when grown on King's B agar. RM1-1-4 was isolated from the roots of healthy oilseed rape plants cv. Traviata KWS (KWS SAAT SE, Einbeck, Germany), whose seeds were treated with a microbial suspension of Sphagnum magellanicum [1].
Even though the optimal growth temperature is 30°C, RM1-1-4 can also slowly replicate at 5°C in liquid Luria Bertani (LB). Growth was observed at 37°C and slightly at 40°C in this culturing medium and on solidified medium after 24 h. The strain grows in complex media (LB, NBII), but not in Standard Succinate Medium (pH 7.0). Optimum pH for growth in LB is pH 6.0. It does not cause any deleterious effect on its original host (oilseed rape) or maize, sorghum and sugar beet [1]. Strain RM1-1-4 has natural resistance to gentamycin (10 μg mL −1 ), trimethoprim (50 μg mL −1 ) and is able to develop spontaneous rifampicin-resistance.
Minimum Information about the Genome Sequence (MIGS) of P. corrugata RM1-1-4 is summarized in Table 1. The phylogenetic relationship of P. corrugata RM1-1-4 to other species within the genus Pseudomonas is visualized in a 16S rRNA based tree [3] and a tree based on the oligopeptide content of the complete protein sequence by using a Composition Vector Tree (CVTree) approach [4,5] (Fig. 2a, b).

Genome project history
The genome of P. corrugata strain RM1-1-4 was selected for sequencing based on its ability to exert stress protecting abilities against abiotic and biotic stresses and to promote plant growth. The strain was isolated from the rhizosphere of an oilseed rape plant that was subjected to a bait plant strategy: oilseed rape seeds were treated with the microbial community of Sphagnum magellanicum [1], where RM1-1-4 was originally identified as P. fluorescens. After Average Nucleotide Identity (ANI) [6] comparison of the genome sequence against the genomes of the type strains and proxytype strains that are already in GenBank, RM1-1-4 showed 99.585% identity to the type genome of P. corrugata with 97% coverage of the genome. To clarify the taxonomic affiliation we performed a systematic method of inferring evolutionary relatedness of microbial organisms from the 16S rRNA gene region (Fig. 2a) and the oligopeptide content of their complete protein sequences by using CVTree showing its phylogenetic positioning (Fig. 2b) [3][4][5]. The genome project is deposited in the NCBI BioProject PRJNA309490 database with the Biosample SAMN04453325. This whole genome shotgun project has been deposited in the NCBI database under the accession no. CP014262 (Table 2).
Growth conditions and genomic DNA preparation P. corrugata strain RM1-1-4 was grown in 50 mL of NBII (Sifin, Berlin, Germany) medium and incubated for 20 h at 30°C. 0.5 mL were centrifuged at 2500 x g for 5 min at 4°C and genomic DNA was extracted using the MasterPure DNA purification kit (Epicentre, Madison, WI, USA). DNA quality and quantity were checked by agarose gel electrophoresis and spectrophotometry using a UV-Vis spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific, Waltham, MA USA). In total, 91 μg genomic DNA (3.1 μg μL −1 ) was sent on dry ice to the sequencing service. PacBio RS libraries with inserts of 8 to 20 kb were constructed and sequenced at GATC Biotech (Konstanz, Germany).

Genome annotation
Automatic annotation was conducted on the RAST Web server (version 36) using RAST gene calling based on FIGfam version Release70 [7,8], and additional annotation for using the automated assignment of COGfunctions to protein-coding genes was completed on the BASys web server using Glimmer gene prediction [9,10]. Pseudogenes were predicted using the NCBI Prokaryotic Genome Annotation Pipeline. Signal peptides and transmembrane helices were predicted using SignalP [11,12].

Genome properties
The genome of RM1-1-4 is composed of one circular chromosome consisting of 6,124,363 bp with an average GC content of 60.7% (Table 3 and Fig. 3), which is comparable to that of other P. corrugata strains. Among the 5335 predicted genes, 5189 were identified as protein coding genes of which 4110 (79.2%) were assigned as putative function, while the other 1079 (20.8%) were designated as hypothetical proteins. The classification of CDSs into functional categories according to the COG (Clusters of Orthologous Groups) [13,14] database is summarized in Table 4 on BASys gene prediction. Beside the predicted genes, the genome annotation revealed 65 tRNA, five rRNA loci (5S, 16S, 23S) with one additional 5S rRNA, four ncRNAs and 284 predicted SEED subsystem features.

Insights from the genome sequence
The genome encodes genes that can be linked to detoxification mechanisms of oxygen radicals, toxins and heavy metals by efflux pumps as well as to stress response by heat and cold shock proteins and the universal stress protein A (UspA). UspA with orthologues (Locus Tags AXG94_02180, AXG94_04130, AXG94_24005, AXG94_ 24695) could play a significant role in protecting RM1-1-4 cells from H 2 O 2 and low pH as found in organisms inhabiting extreme environments [15] and analysed in detail for the clinical strain Acinetobacter baumannii ATCC 17978 [16]. A water stress/hypersensitive response protein (AXG94_21760) is present, which is supposed to be transferred to symbiotic or pathogenic bacteria by horizontal gene transfer from plants and can be seen as the acquisition of a function putatively related to the cell defense [17]. The genome of RM1-1-4 contains several genes, which are important contributors to biological control. They are related to the biosynthesis of secondary metabolites or antimicrobial products that are similar to those found in the genomes of other Pseudomonads: productions of exoproteases and lipoproteins [18]. We further identified genes Phylum Proteobacteria TAS [26] Class Gammaproteobacteria TAS [27] Order Pseudomonadales TAS [28,29] Family Pseudomonadaceae TAS [26,30] Genus    most probably involved in the direct promotion of plant growth, such as biosynthesis or carrier gene clusters for aminocyclopropane-1-carboxylate deaminase suggested to be a key in the modulation of ethylene levels in plants by bacteria [19], auxin, biofilm dispersion, rhizobactin siderophores and spermidine. Genes predicting the synthesis of volatile components are present in the RM1-1-4 genome as well. Volatile components have been shown to act as antibiotics and to induce plant growth [20,21]. An example is hydrogen cyanide (HCN), an inorganic compound with antagonistic effects against soil microbes [22]. RM1-1-4 encodes a hydrogen cyanide synthase HcnA (AXG94_04380) and orthologues of genes required for the biosynthesis of other volatile components such as 2,3-butanediol (AXG94_01200) and its precursor acetoin (AXG94_01195) were annotated too. Beside the presence of specific genes and the noticeable ability of RM1-1-4 to expose stress protection, the function of particular genes needs to be clarified in further detailed studies.
The genome-wide phylogenetic analysis on Pseudomonas species [3][4][5] with the RM1-1-4 genome showed that strain RM1-1-4 clusters within the P. fluorescens group (Fig. 2a, b) and most closely to P. corrugata CFBP 5454 (DDBJ/EMBL/GenBank accession ATKI01000000). The two P. corrugata strains belong to the few non fluorescent Pseudomonas species. CFBP 5454 was originally described as the causal agent of the tomato disease called 'pith necrosis' and is yet considered as a biological resource in the fields of biocontrol of plant diseases and production of industrially promising microbial biopolymers like antimicrobial cyclic lipopeptides [23].  3 Graphical map of the chromosome. The outer scale is marked every 10 kb. Circles range from 1 (outer circle) to 7 (inner circle). Circle 1 and 2, ORFs encoded by leading and lagging strand respectively, with color code for functions: salmon, translation, ribosomal structure and biogenesis; aquamarine, RNA processing and modification; light blue, transcription; cyan, DNA replication, recombination and repair; tan, chromatin structure and dynamics; turquoise, cell division; dark orange, defense mechanisms; deep pink, post-translational modification, protein turnover and chaperones; dark olive green, cell envelope biogenesis; purple, cell motility and secretion; lavender, intracellular trafficking, secretion, and vesicular transport; forest green, inorganic ion transport and metabolism; pink, signal transduction; red, energy production; sienna, carbohydrate transport and metabolism; yellow, amino acid transport; orange, nucleotide transport and metabolism; gold, co-enzyme transport and metabolism; cornflower blue, lipid metabolism; blue, secondary metabolites, transport and catabolism; gray, general function prediction only; yellow green, unknown function; black, function unclassified or unknown. Circle 3 and 4, distributions of tRNA genes and rrn operons respectively. Circle 5, distribution of pseudogenes. Circle 6 and 7, G + C content and GC skew (G-C/G + C) respectively

Conclusions
This report described the complete genome sequence of P. corrugata strain RM1-1-4. It is a "Pseudomonadales" within the non-fluorescent P. corrugata clade that was originally isolated from the roots of moss microbiomeprimed oilseed rape seeds cv. Traviata KWS grown in a greenhouse in Graz, Austria. This strain was selected for sequencing based on its ability to protect plants from abiotic and biotic stresses and to promote plant growth. We could highlight genes encoding abiotic and biotic stress protecting factors and other well-known bacterial traits for establishment of beneficial plant-microbe interactions. The genome encodes for a collection of genes predicting biofilm dispersion, detoxifying compounds, volatile components and enzymes such as a protease and a deaminase. Such properties likely have origins in a repertoire of genes including efflux pumps, putative T2SS, T4SS and T6SS, and several genes presumably implicated in auxin, rhizobactin siderophore and spermidine production. Further functional studies and comparative genomics with related isolates will provide insights into naturally acquired plant stress protection and promotion of plant health.  The total is based on the total number of protein coding genes in the genome based on BASys gene prediction