Pseudomonas nunensis sp. nov. isolated from a suppressive potato field in Greenland.

The bacterial strain In5T was previously isolated from a suppressive potato field in southern Greenland and has been characterized and described as Pseudomonas fluorescens. However, the results of new polyphasic analyses coupled with those of phenotypic, phylogenetic and genomic analyses reported here demonstrate that the affiliation to the species P. fluorescens was incorrect. The strain is Gram-stain-negative, rod-shaped, aerobic and displays growth at 4-28 °C (optimum temperature 20-25 °C) and at pH 5-9 (optimum pH 6-7). Major fatty acids were C16 : 0 (38.2 %), a summed feature consisting of C16 : 1ω6c and/or C16 : 1ω7c) (20.7 %), C17 : 0cyclo ω7c (14.3 %) and a summed feature consisting of C18 : 1ω6c and/or C18 : 1ω7c (11.7 %). The respiratory quinones were determined to be Q9 (95.5 %) and Q8 (4.5 %) and major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was determined to be 59.4 mol%. The results of phylogenetic analysis based on the 16S rRNA gene and multi-locus sequence analysis (MLSA; concatenated 16S rRNA, gyrB, rpoB and rpoD sequences) indicated that In5T was affiliated with the Pseudomonas mandelii subgroup within the genus Pseudomonas. Comparison of the genome sequence of In5T and those of related type strains of species of the genus Pseudomonas revealed an average nucleotide identity (ANI) of 87.7 % or less and digital DNA-DNA hybridization (dDDH) of less than 34.5 % relatedness, respectively. Two more strains, In614 and In655, isolated from the same suppressive soil were included in the genome analysis. The ANI and dDDH of In614 and In655 compared with In5T were ANI: 99.9 and 97.6 and dDDH (GGDC) 99.9 and 79.4, respectively, indicating that In5T, In614 and In655 are representatives of the same species. The results of the phenotypic, phylogenetic and genomic analyses support the hypothesis that strain In5T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nunensis sp. nov. is proposed. The type strain is In5T(=LMG 32653T=NCIMB 15428T).

Three novel Erwinia billingiae phages isolated from organic waste represent three new genera.

Despite the ecological significance of viral communities, phages remain insufficiently studied. Current genomic databases lack high-quality phage genome sequences linked to specific bacteria. Bacteria of the genus Erwinia are known to colonize the phyllosphere of plants, both as commensals and as pathogens. We isolated three Erwinia billingiae phages-Zoomie, Pecta, and Snitter-from organic household waste. Based on sequence similarity to their closest relatives, we propose that they represent three new genera: "Pectavirus" within the family Zobellviridae, "Snittervirus" in the subfamily Tempevirinae, family Drexlerviridae, and "Zoomievirus" within the family Autographiviridae, which, together with the genus Limelightvirus, may constitute a new subfamily.

Four novel Curtobacterium phages isolated from environmental samples.

Despite Curtobacterium spp. often being associated with the plant phyllosphere, i.e., the areal region of different plant species, only one phage targeting a member of the genus Curtobacterium has been isolated so far. In this study, we isolated four novel plaque-forming Curtobacterium phages, Reje, Penoan, Parvaparticeps, and Pize, with two novel Curtobacterium strains as propagation hosts. Based on the low nucleotide intergenomic similarity (<32.4%) between these four phages and any phage with a genome sequence in the NCBI database, we propose the establishment of the four genera, "Rejevirus", "Pizevirus", "Penoanvirus", and "Parvaparticepsvirus", all in the class of Caudoviricetes.

Back to our roots: exploring the role of root morphology as a mediator of beneficial plant-microbe interactions.

Plant breeding for belowground traits that have a positive impact on the rhizosphere microbiome is a promising strategy to sustainably improve crop yields. Root architecture and morphology are understudied plant breeding targets despite their potential to significantly shape microbial community structure and function in the rhizosphere. In this review, we explore the relationship between various root architectural and morphological traits and rhizosphere interactions, focusing on the potential of root diameter to impact the rhizosphere microbiome structure and function while discussing the potential biological and ecological mechanisms underpinning this process. In addition, we propose three future research avenues to drive this research area in an effort to unravel the effect of belowground traits on rhizosphere microbiology. This knowledge will pave the way for new plant breeding strategies that can be exploited for sustainable and high-yielding crop cultivars.

Fungal-Associated Molecules Induce Key Genes Involved in the Biosynthesis of the Antifungal Secondary Metabolites Nunamycin and Nunapeptin in the Biocontrol Strain Pseudomonas fluorescens In5.

Pseudomonas fluorescens In5 synthesizes the antifungal cyclic lipopeptides (CLPs) nunamycin and nunapeptin, which are similar in structure and genetic organization to the pseudomonas-derived phytotoxins syringomycin and syringopeptin. Regulation of syringomycin and syringopeptin is dependent on the two-component global regulatory system GacS-GacA and the SalA, SyrF, and SyrG transcription factors, which activate syringomycin synthesis in response to plant signal molecules. Previously, we demonstrated that a specific transcription factor, NunF, positively regulates the synthesis of nunamycin and nunapeptin in P. fluorescens In5 and that the nunF gene is upregulated by fungal-associated molecules. This study focused on further unravelling the complex regulation governing CLP synthesis in P. fluorescens In5. Promoter fusions were used to show that the specific activator NunF is dependent on the global regulator of secondary metabolism GacA and is regulated by fungal-associated molecules and low temperatures. In contrast, GacA is stimulated by plant signal molecules leading to the hypothesis that P. fluorescens is a hyphosphere-associated bacterium carrying transcription factor genes that respond to signals indicating the presence of fungi and oomycetes. Based on these findings, we present a model for how synthesis of nunamycin and nunapeptin is regulated by fungal- and oomycete-associated molecules.IMPORTANCE Cyclic lipopeptide (CLP) synthesis gene clusters in pseudomonads display a high degree of synteny, and the structures of the peptides synthesized are very similar. Accordingly, the genomic island encoding the synthesis of syringomycin and syringopeptin in P. syringae pv. syringae closely resembles that of P. fluorescens In5, which contains genes coding for synthesis of the antifungal and anti-oomycete peptides nunamycin and nunapeptin, respectively. However, the regulation of syringomycin and syringopeptin synthesis is different from that of nunamycin and nunapeptin synthesis. While CLP synthesis in the plant pathogen P. syringae pv. syringae is induced by plant signal molecules, such compounds do not significantly influence synthesis of nunamycin and nunapeptin in P. fluorescens In5. Instead, fungal-associated molecules positively regulate antifungal peptide synthesis in P. fluorescens In5, while the synthesis of the global regulator GacA in P. fluorescens In5 is positively regulated by plant signal molecules but not fungal-associated molecules.

Serratia inhibens sp. nov., a new antifungal species isolated from potato (Solanum tuberosum).

A novel bacterial strain, S40T, with strong antifungal activity was isolated from the rhizosphere of green potato collected from Zealand, Denmark. Polyphasic analysis with a combined phenotypic, phylogenetic and genomic approach was used to characterize S40T. Phylogenetic analysis based on the 16S rRNA gene and MLSA (concatenated gyrB, rpoD, infB and atpD sequences) showed that strain S40T was affiliated with the genus Serratia and with Serratia plymuthica PRI-2C as the closest related strain [average nucleotide identity (ANI), 99.26 %; DNA-DNA hybridization (dDDH), 99.20%]. However, whole genome sequence analyses revealed that S40T and S. plymuthica PRI-2C genomes displayed lower similarities when compared to all other S. plymuthica strains (ANI ≤94.34 %; dDDH ≤57.6 % relatedness). The DNA G+C content of strain S40T was determined to be 55.9 mol%. Cells of the strain were Gram-negative, rod-shaped, facultative anaerobic and displayed growth at 10-37 °C (optimum, 25-30 °C) and at pH 6-9 (optimum, pH 6-7). Major fatty acids were C16 : 0 (27.9 %), summed feature (C16 : 1 ω6c/C16 : 1 ω7c; 18.0 %) and C17 : 0 cyclo (15.1 %). The respiratory quinone was determined to be Q8 (94 %) and MK8 (95 %) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The results of phenotypic, phylogenetic and genomic analyses support the hypothesis that strain S40T represents a novel species of the genus Serratia, for which the name Serratia inhibens sp. nov. is proposed. The type strain is S40T (=LMG 31467T=NCIMB 15235T). In addition, we propose that S. plymuthica PRI-2C is reclassified and transferred to the species S. inhibens as S. inhibens PRI-2C.

Does regulation hold the key to optimizing lipopeptide production in Pseudomonas for biotechnology?

Lipopeptides (LPs) produced by Pseudomonas spp. are specialized metabolites with diverse structures and functions, including powerful biosurfactant and antimicrobial properties. Despite their enormous potential in environmental and industrial biotechnology, low yield and high production cost limit their practical use. While genome mining and functional genomics have identified a multitude of LP biosynthetic gene clusters, the regulatory mechanisms underlying their biosynthesis remain poorly understood. We propose that regulation holds the key to unlocking LP production in Pseudomonas for biotechnology. In this review, we summarize the structure and function of Pseudomonas-derived LPs and describe the molecular basis for their biosynthesis and regulation. We examine the global and specific regulator-driven mechanisms controlling LP synthesis including the influence of environmental signals. Understanding LP regulation is key to modulating production of these valuable compounds, both quantitatively and qualitatively, for industrial and environmental biotechnology.

The potential of Pseudomonas fluorescens SBW25 to produce viscosin enhances wheat root colonization and shapes root-associated microbial communities in a plant genotype-dependent manner in soil systems.

Microorganisms interact with plant roots through colonization of the root surface, i.e., the rhizoplane or the surrounding soil, i.e., the rhizosphere. Beneficial rhizosphere bacteria such as Pseudomonas spp. can promote plant growth and protect against pathogens by producing a range of bioactive compounds, including specialized metabolites like cyclic lipopeptides (CLPs) known for their biosurfactant and antimicrobial activities. However, the role of CLPs in natural soil systems during bacteria-plant interactions is underexplored. Here, Pseudomonas fluorescens SBW25, producing the CLP viscosin, was used to study the impact of viscosin on bacterial root colonization and microbiome assembly in two cultivars of winter wheat (Heerup and Sheriff). We inoculated germinated wheat seeds with SBW25 wild type or a viscosin-deficient mutant and grew the plants in agricultural soil. After 2 weeks, enhanced root colonization of SBW25 wild type compared to the viscosin-deficient mutant was observed, while no differences were observed between wheat cultivars. In contrast, the impact on root-associated microbial community structure was plant-genotype-specific, and SBW25 wild type specifically reduced the relative abundance of an unclassified oomycete and Phytophthora in Sheriff and Heerup, respectively. This study provides new insights into the natural role of viscosin and specifically highlights the importance of viscosin in wheat root colonization under natural soil conditions and in shaping the root microbial communities associated with different wheat cultivars. Furthermore, it pinpoints the significance of microbial microdiversity, plant genotype, and microbe-microbe interactions when studying colonization of plant roots. IMPORTANCE: Understanding parameters governing microbiome assembly on plant roots is critical for successfully exploiting beneficial plant-microbe interactions for improved plant growth under low-input conditions. While it is well-known from in vitro studies that specialized metabolites are important for plant-microbe interactions, e.g., root colonization, studies on the ecological role under natural soil conditions are limited. This might explain the often-low translational power from laboratory testing to field performance of microbial inoculants. Here, we showed that viscosin synthesis potential results in a differential impact on the microbiome assembly dependent on wheat cultivar, unlinked to colonization potential. Overall, our study provides novel insights into factors governing microbial assembly on plant roots, and how this has a derived but differential effect on the bacterial and protist communities.

Complete Genome Sequence of Sphingopyxis sp. Strain PET50, a Potential Polyethylene Terephthalate (PET)-Degrading Bacterium Isolated from Compost.

We report the complete genome sequence of a potential polyethylene terephthalate (PET)-degrading bacterium, Sphingopyxis sp. strain PET50, isolated from compost.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

The cytokinin-producing plant beneficial bacterium Pseudomonas fluorescens G20-18 primes tomato (Solanum lycopersicum) for enhanced drought stress responses.

Plant growth-promoting rhizobacteria (PGPR) are known for exerting beneficial effects on plant growth and tolerance to plant pathogens. However, their specific role in mediating protection against abiotic stress remains underexplored. The aim of this study was to characterise the ability of the cytokinin-producing beneficial bacterium Pseudomonas fluorescens G20-18 to enhance tomato growth and boost tolerance to drought stress. Tomato seedlings were root inoculated and their growth and physiological and molecular responses assessed under well-watered conditions and also in response to progressive drought stress and a subsequent recovery period. Root inoculation with G20-18 had a significant positive impact on tomato growth. Furthermore, G20-18 inoculated and drought-stressed plants showed higher leaf chlorophyll and abscisic acid (ABA) content and stomatal closure than non-inoculated controls. Root inoculation also increased the activity of different carbohydrate metabolism enzymes, which are important for root and leaf growth and development in drought stressed plants. A significant increase in the activity of different antioxidant enzymes and total antioxidant capacity correlated with elevated levels of relevant secondary metabolites, such as phenolics, anthocyanins and flavonoids. RNA sequencing revealed distinct qualitative and quantitative differences in gene regulation in response to G20-18. Notably, the number of genes differentially regulated in response to G20-18 was approximately sevenfold higher during drought stress, indicating that root inoculation with the bacteria primed the plants for a much stronger transcriptionally regulated systemic drought stress response. The regulated genes are related to phenylalanine metabolism and other key processes linked to plant growth, development and drought stress resilience. A role of the ability of G20-18 to produce the plant hormone cytokinin for interaction with tomato was established by the cytokinin-deficient biosynthesis mutants CNT1 and CNT2. In comparison with G20-18, the inoculation of plants with CNT1 resulted in a reduced number of differentially regulated genes. The relative change was most prominent under well-watered conditions with a 85 % reduction, corresponding to 462 genes. However, under drought conditions the absolute number of differentially regulated genes was reduced by even 2219 in response to the CNT1 mutant. The relevance of the ability of G20-18 to produce cytokinins for interaction with plants was also evident from differences in growth and specific cell and ecophysiological parameters in response to CNT1 and CNT2. These findings provide novel insights about G20-18's ability to improve drought stress responses and the role of interkingdom signalling by bacterial-derived cytokinins, and contribute to enhance the robustness of the practical application of these microorganisms to improve crop resilience in agricultural production.

Microbial Diversity in Four Rhizocompartments (Bulk Soil, Rhizosphere, Rhizoplane, and Endosphere) of Four Winter Wheat Varieties at the Fully Emerged Flag Leaf Growth Stage.

Community composition and recruitment are important elements of plant-microbe interactions and may provide insights for plant development and resilience. The results of 16S rRNA amplicon sequencing from four rhizocompartments for four wheat cultivars grown under controlled conditions and sampled after flag leaf emergence are provided. Data demonstrate differences in microbial communities according to rhizocompartment.

Diversity and Structure of Bacterial Communities in Different Rhizocompartments (Rhizoplane, Rhizosphere, and Bulk) at Flag Leaf Emergence in Four Winter Wheat Varieties.

Understanding basic interactions at the plant-soil interphase is critical if we are to exploit natural microbial communities for improved crop resilience. We report here 16S amplicon sequencing data from 3 rhizocompartments of 4 wheat cultivars grown under controlled greenhouse conditions. We observed that rhizocompartments and cultivar affect the community composition.

Complete Genome Sequence of the Cytokinin-Producing Biocontrol Strain Pseudomonas fluorescens G20-18.

Here, we report the complete genome sequence of the cytokinin-producing plant growth-promoting strain Pseudomonas fluorescens G20-18. The complete genome assembly resulted in a single, circular chromosome of 6.48 Mbp and harbors several secondary metabolite biosynthesis gene clusters that are potentially involved in its plant growth-promoting function.

Complete Genome Sequence of Paenibacillus sp. Strain 37, a Plant Growth-Promoting Bacterium Isolated from the Rhizosphere of Abies nordmanniana (Nordmann Fir).

We report the complete genome sequence of Paenibacillus sp. strain 37, a plant growth-promoting bacterium (PGPB) isolated from the rhizosphere of Abies nordmanniana (Stev.) Spach; it contains a single chromosome of 7.08 Mbp and one plasmid of 54.33 kbp, including 6,445 protein-coding genes, 107 tRNAs, and 13 rRNA loci.

Discovery of a Bacterial Gene Cluster for Deglycosylation of Toxic Potato Steroidal Glycoalkaloids α-Chaconine and α-Solanine.

Potato juice is a byproduct of starch processing currently used as feed. However, potato proteins are an untapped source of high-protein food for human nutrition if harmful constituents notably glycoalkaloids (GAs) are detoxified. The two principle GAs found in potato are α-chaconine and α-solanine, both consisting of a solanidine aglycone with a carbohydrate side chain. The first step in the detoxification of these compounds is the removal of the trisaccharide. Whole-genome sequencing of a bacterial isolate, Arthrobacter sp. S41, capable of completely degrading α-chaconine and α-solanine, revealed the presence of a gene cluster possibly involved in the deglycosylation of GAs. Functional characterization confirmed the enzymatic activity of the gene cluster involved in the complete deglycosylation of both α-chaconine and α-solanine. The novel enzymes described here may find value in the bioconversion of feed proteins to food proteins suitable for human nutrition.

A Multifunctional Polysaccharide Utilization Gene Cluster in Colwellia echini Encodes Enzymes for the Complete Degradation of κ-Carrageenan, ι-Carrageenan, and Hybrid ß/κ-Carrageenan.

Algal cell wall polysaccharides constitute a large fraction in the biomass of marine primary producers and are thus important in nutrient transfer between trophic levels in the marine ecosystem. In order for this transfer to take place, polysaccharides must be degraded into smaller mono- and disaccharide units, which are subsequently metabolized, and key components in this degradation are bacterial enzymes. The marine bacterium Colwellia echini A3T is a potent enzyme producer since it completely hydrolyzes agar and κ-carrageenan. Here, we report that the genome of C. echini A3T harbors two large gene clusters for the degradation of carrageenan and agar, respectively. Phylogenetical and functional studies combined with transcriptomics and in silico structural modeling revealed that the carrageenolytic cluster encodes furcellaranases, a new class of glycoside hydrolase family 16 (GH16) enzymes that are key enzymes for hydrolysis of furcellaran, a hybrid carrageenan containing both ß- and κ-carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43-O-monosulfate [DA-(α1,3)-G4S-(ß1,4)-DA-(α1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, κ-carrageenases, ß-agarases, and ß-porphyranases. Furthermore, C. echini A3T was shown to encode κ-carrageenases, ι-carrageenases, and members of a new class of enzymes, active only on hybrid ß/κ-carrageenan tetrasaccharides. On the basis of our genomic, transcriptomic, and functional analyses of the carrageenolytic enzyme repertoire, we propose a new model for how C. echini A3T degrades complex sulfated marine polysaccharides such as furcellaran, κ-carrageenan, and ι-carrageenan.IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini, harbors a large number of enzymes enabling the bacterium to grow on κ-carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of κ-carrageenan and agar, respectively. As the first, we report on the structure/function relationship of a new class of enzymes that hydrolyze furcellaran, a partially sulfated ß/κ-carrageenan. Using an in silico model, we hypothesize a molecular structure of furcellaranases and compare structural features and active site architectures of furcellaranases with those of other GH16 polysaccharide hydrolases, such as κ-carrageenases, ß-agarases, and ß-porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 ß-galactosidases and show that this new class of enzymes is active only on hybrid ß/κ-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize ß/κ-, κ-, and ι-carrageenan.

Imaging Gene Expression Dynamics in Pseudomonas fluorescens In5 duringInteractions with the Fungus Fusarium graminearum PH-1.

Genomics, transcriptomics and metabolomics are powerful technologies for studying microbial interactions. The main drawback of these methods is the requirement for destructive sampling. We have established an alternative but complementary technique based on a microplate system combined with promoter fusions for visualizing gene expression in space and time. Here we provide a protocol for measuring spatial and temporal gene expression of a bacterial reporter strain interacting with a fungus on a solid surface.

Draft Genome Sequences of Two Glycoalkaloid-Degrading Arthrobacter Strains Isolated from Green Potato Peel.

Here, we report the genome sequences of two Arthrobacter sp. strains isolated from potato and capable of degrading the toxic potato-derived glycoalkaloids (GAs) α-chaconine and α-solanine. Information from the genome sequences will provide insight into the genetic mechanism of GA degradation by these isolates.

A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria.

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria.