Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato.

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.

Ballast water treatment and bacteria: Analysis of bacterial activity and diversity after treatment of simulated ballast water by electrochlorination and UV exposure.

Effects of ballast water (BW) treatment by ultra-violet (UV) light and electrochlorination (EC) on survival, activity and diversity of marine bacterioplankton and release of organic matter from cell damage were examined at discharge in a large-scale BW test facility (250 m3 tanks) at Hundested harbour, Denmark. The tests were performed in accordance with the requirements for type approval testing by International Maritime Organization (IMO) and US Coast Guard. After treatment, the water was held in the tanks for one day (EC) before discharge, or 6 days (UV, including also a final UV re-treatment) before discharge. In the discharged and treated water, numbers of viable bacteria and bacterial growth rate had decreased significantly relative to the untreated water, but the total number of bacteria only was reduced in the EC-treated water. After additional storage for up to 10 days in small-scale laboratory incubations, significant regrowth of bacteria was observed after either treatment. Sequencing of 16S rRNA gene amplicons demonstrated that α-Proteobacteria initially were dominant, but γ-Proteobacteria dominated after regrowth. Bacteria used to document BW treatment efficiency (E. coli, Vibrio spp., enterococci) survived both treatments; neither treatment reduced the risk of pathogen dispersal. Concentrations of amino acids in the water were used as indicators of treatment-induced cell damage and demonstrated higher concentrations at discharge, but only after the EC treatments. Our results indicate that activity of bacteria, rather than their abundances, should be used when examining effects by ballast water treatment on microorganisms and that none of the examined treatment technologies could eliminate pathogenic 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.

Discovery and screening of novel metagenome-derived GH107 enzymes targeting sulfated fucans from brown algae.

Sulfated fucans, often denoted as fucoidans, are highly variable cell wall polysaccharides of brown algae, which possess a wide range of bioactive properties with potential pharmaceutical applications. Due to their complex architecture, the structures of algal fucans have until now only been partly determined. Enzymes capable of hydrolyzing sulfated fucans may allow specific release of defined bioactive oligosaccharides and may serve as a tool for structural elucidation of algal walls. Currently, such enzymes include only a few hydrolases belonging to the glycoside hydrolase family 107 (GH107), and little is known about their mechanistics and the substrates they degrade. In this study, we report the identification and recombinant production of three novel GH107 family proteins derived from a marine metagenome. Activity screening against a large substrate collection showed that all three enzymes degraded sulfated fucans from brown algae in the order Fucales. This is in accordance with a hydrolytic activity against α-1,4-fucosidic linkages in sulfated fucans as reported for previous GH107 members. Also, the activity screening gave new indications about the structural differences in brown algal cell walls. Finally, sequence analyses allowed identification of the proposed catalytic residues of the GH107 family. The findings presented here form a new basis for understanding the GH107 family of enzymes and investigating the complex sulfated fucans from brown algae. DATABASE: The assembled metagenome and raw sequence data is available at EMBL-EBI (Study number: PRJEB28480). Sequences of the GH107 fucanases (Fp273, Fp277, and Fp279) have been deposited in GenBank under accessions MH755451-MH755453.

An extra-plastidial alpha-glucan, water dikinase from Arabidopsis phosphorylates amylopectin in vitro and is not necessary for transient starch degradation.

Starch phosphorylation catalysed by the alpha-glucan, water dikinases (GWD) has profound effects on starch degradation in plants. The Arabidopsis thaliana genome encodes three isoforms of GWD, two of which are localized in the chloroplast and are involved in the degradation of transient starch. The third isoform, termed AtGWD2 (At4g24450), was heterologously expressed and purified and shown to have a substrate preference similar to potato GWD. Analyses of AtGWD2 null mutants did not reveal any differences in growth or starch and sugar levels, when compared to the wild type. Subcellular localization studies in Arabidopsis leaves and in vitro chloroplast import assays indicated that AtGWD2 was not targeted to the chloroplasts. The AtGWD2 promoter showed a highly restricted pattern of activity, both spatially and temporally. High activity was observed in the companion cells of the phloem, with expression appearing just before the onset of senescence. Taken together, these data indicate that, although AtGWD2 is capable of phosphorylating alpha-glucans in vitro, it is not directly involved in transient starch degradation.

Constructing and Screening a Metagenomic Library of a Cold and Alkaline Extreme Environment.

Natural cold or alkaline environments are common on Earth. A rare combination of these two extremes is found in the permanently cold (less than 6 °C) and alkaline (pH above 10) ikaite columns in the Ikka Fjord in Southern Greenland. Bioprospecting efforts have established the ikaite columns as a source of bacteria and enzymes adapted to these conditions. They have also highlighted the limitations of cultivation-based methods in this extreme environment and metagenomic approaches may provide access to novel extremophilic enzymes from the uncultured majority of bacteria. Here, we describe the construction and screening of a metagenomic library of the prokaryotic community inhabiting the ikaite columns.

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.

Draft Genome Sequence of a Novel Marine Bacterium, Paraglaciecola sp. Strain S66, with Hydrolytic Activity against Seaweed Polysaccharides.

A novel agarolytic gammaproteobacterium, ITALIC! Paraglaciecolasp. S66, was isolated from marine samples of eelgrass ( ITALIC! Zosterasp.) and sequenced. The draft genome contains a large number of enzyme-encoding genes with predicted function against several complex polysaccharides found in the cell walls of algae.

In situ Dynamics of O2, pH, Light, and Photosynthesis in Ikaite Tufa Columns (Ikka Fjord, Greenland)-A Unique Microbial Habitat.

The Ikka Fjord (SW Greenland) harbors a unique microbial habitat in the form of several hundred submarine tufa columns composed of ikaite, a special hexahydrate form of calcium carbonate that precipitates when alkaline phosphate- and carbonate-enriched spring water seeping out of the sea floor meets cold seawater. While several unique heterotrophic microbes have been isolated from the tufa columns, the microbial activity, and the boundary conditions for microbial growth in ikaite have remained unexplored. We present the first detailed in situ characterization of the physico-chemical microenvironment and activity of oxygenic phototrophs thriving within the ikaite columns. In situ underwater microsensor measurements of pH, temperature, and irradiance in the porous ikaite crystal matrix, revealed an extreme microenvironment characterized by low temperatures, strong light attenuation, and gradients of pH changing from pH 9 at the outer column surface to above pH 10 over the first 1-2 cm of the ikaite. This outer layer of the freshly deposited ikaite matrix contained densely pigmented yellow and green zones harboring a diverse phototrophic community dominated by diatoms and cyanobacteria, respectively, as shown by amplicon sequencing. In situ O2 measurements, as well as underwater variable chlorophyll fluorescence measurements of photosynthetic activity, demonstrated high levels of oxygenic photosynthesis in this extreme gradient environment with strong irradiance-driven O2 dynamics ranging from anoxia to hyperoxic conditions in the ikaite matrix, albeit the local formation of gas bubbles buffered the day-night dynamics of O2 in the tufa columns. The microbial phototrophs in the ikaite matrix are embedded in exopolymers forming endolithic biofilms that may interact with mineral formation and cementing of ikaite crystals.

Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases.

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography-tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin-like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20 °C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity.