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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

Draft Genome Sequence of a Novel Serratia sp. Strain with Antifungal Activity.

This report describes the draft genome sequence of Serratia sp. strain S40, isolated from potato; it contains 5,383,735 bp and a G+C content of 55.9% and harbors 4,875 predicted coding sequences across 29 contigs. The genomic data provide insight into the genetics underpinning the antifungal activity of this strain.

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia.

The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

A Novel Enzyme Portfolio for Red Algal Polysaccharide Degradation in the Marine Bacterium Paraglaciecola hydrolytica S66T Encoded in a Sizeable Polysaccharide Utilization Locus.

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both ß-carrageenan and κ/ß-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/ß-type carrageenan by P. hydrolytica S66T. The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

A Screening Method for the Isolation of Bacteria Capable of Degrading Toxic Steroidal Glycoalkaloids Present in Potato.

Potato juice, a by-product of starch processing, is a potential high-value food ingredient due to its high protein content. However, conversion from feed to human protein requires the removal of the toxic antinutritional glycoalkaloids (GAs) α-chaconine and α-solanine. Detoxification by enzymatic removal could potentially provide an effective and environmentally friendly process for potato-derived food protein production. While degradation of GAs by microorganisms has been documented, there exists limited knowledge on the enzymes involved and in particular how bacteria degrade and metabolize GAs. Here we describe a series of methods for the isolation, screening, and selection of GA-degrading bacteria. Bacterial cultures from soils surrounding greened potatoes, including the potato peels, were established and select bacterial isolates were studied. Screening of bacterial crude extracts for the ability to hydrolyze GAs was performed using a combination of thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and liquid chromatography mass spectrometry (LC-MS). Analysis of the 16S rRNA sequences revealed that bacteria within the genus Arthrobacter were among the most efficient GA-degrading strains.

A Microplate Reader-Based System for Visualizing Transcriptional Activity During in vivo Microbial Interactions in Space and Time.

Here, we report the development of a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. A bacterium expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. This novel technique enables real-time live imaging of samples by time-series multi-channel automatic recordings using a microplate reader as both an incubator and image recorder of general use to researchers. The technique can aid in deciding when to destructively sample for other methods e.g. transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during the interaction.

Transcriptomic profiling of microbe-microbe interactions reveals the specific response of the biocontrol strain P. fluorescens In5 to the phytopathogen Rhizoctonia solani.

BACKGROUND: Few studies to date report the transcriptional response of biocontrol bacteria toward phytopathogens. In order to gain insights into the potential mechanism underlying the antagonism of the antimicrobial producing strain P. fluorescens In5 against the phytopathogens Rhizoctonia solani and Pythium aphanidermatum, global RNA sequencing was performed. METHODS: Differential gene expression profiling of P. fluorescens In5 in response to either R. solani or P. aphanidermatum was investigated using transcriptome sequencing (RNA-seq). Total RNA was isolated from single bacterial cultures of P. fluorescens In5 or bacterial cultures in dual-culture for 48 h with each pathogen in biological triplicates. RNA-seq libraries were constructed following a default Illumina stranded RNA protocol including rRNA depletion and were sequenced 2 × 100 bases on Illumina HiSeq generating approximately 10 million reads per sample. RESULTS: No significant changes in global gene expression were recorded during dual-culture of P. fluorescens In5 with any of the two pathogens but rather each pathogen appeared to induce expression of a specific set of genes. A particularly strong transcriptional response to R. solani was observed and notably several genes possibly associated with secondary metabolite detoxification and metabolism were highly upregulated in response to the fungus. A total of 23 genes were significantly upregulated and seven genes were significantly downregulated with at least respectively a threefold change in expression level in response to R. solani compared to the no fungus control. In contrast, only one gene was significantly upregulated over threefold and three transcripts were significantly downregulated over threefold in response to P. aphanidermatum. Genes known to be involved in synthesis of secondary metabolites, e.g. non-ribosomal synthetases and hydrogen cyanide were not differentially expressed at the time points studied. CONCLUSION: This study demonstrates that genes possibly involved in metabolite detoxification are highly upregulated in P. fluorescens In5 when co-cultured with plant pathogens and in particular the fungus R. solani. This highlights the importance of studying microbe-microbe interactions to gain a better understanding of how different systems function in vitro and ultimately in natural systems where biocontrol agents can be used for the sustainable management of plant diseases.