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.

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.

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.

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.

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.

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.

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.

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.

Draft Genome Sequence of Pseudomonas sp. Strain In5 Isolated from a Greenlandic Disease Suppressive Soil with Potent Antimicrobial Activity.

Pseudomonas sp. In5 is an isolate of disease suppressive soil with potent activity against pathogens. Its antifungal activity has been linked to a gene cluster encoding nonribosomal peptide synthetases producing the peptides nunamycin and nunapeptin. The genome sequence will provide insight into the genetics behind the antimicrobial activity of this strain.

The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.

Draft genome sequences of two protease-producing strains of arsukibacterium, isolated from two cold and alkaline environments.

Arsukibacterium ikkense GCM72(T) and a close relative, Arsukibacterium sp. MJ3, were isolated from two cold and alkaline environments as producers of extracellular proteolytic enzymes active at high pH and low temperature. This report describes the two draft genome sequences, which may serve as sources of future industrial enzymes.

Microbial diversity in a permanently cold and alkaline environment in Greenland.

The submarine ikaite columns located in the Ikka Fjord in Southern Greenland represent a unique, permanently cold (less than 6°C) and alkaline (above pH 10) environment and are home to a microbial community adapted to these extreme conditions. The bacterial and archaeal community inhabiting the ikaite columns and surrounding fjord was characterised by high-throughput pyrosequencing of 16S rRNA genes. Analysis of the ikaite community structure revealed the presence of a diverse bacterial community, both in the column interior and at the surface, and very few archaea. A clear difference in overall taxonomic composition was observed between column interior and surface. Whereas the surface, and in particular newly formed ikaite material, was primarily dominated by Cyanobacteria and phototrophic Proteobacteria, the column interior was dominated by Proteobacteria and putative anaerobic representatives of the Firmicutes and Bacteroidetes. The results suggest a stratification of the ikaite columns similar to that of classical soda lakes, with a light-exposed surface inhabited by primary producers and an anoxic subsurface. This was further supported by identification of major taxonomic groups with close relatives in soda lake environments, including members of the genera Rhodobaca, Dethiobacter, Thioalkalivibrio and Tindallia, as well as very abundant groups related to uncharacterised environmental sequences originally isolated from Mono Lake in California.

Nonribosomal peptides, key biocontrol components for Pseudomonas fluorescens In5, isolated from a Greenlandic suppressive soil.

UNLABELLED: Potatoes are cultivated in southwest Greenland without the use of pesticides and with limited crop rotation. Despite the fact that plant-pathogenic fungi are present, no severe-disease outbreaks have yet been observed. In this report, we document that a potato soil at Inneruulalik in southern Greenland is suppressive against Rhizoctonia solani Ag3 and uncover the suppressive antifungal mechanism of a highly potent biocontrol bacterium, Pseudomonas fluorescens In5, isolated from the suppressive potato soil. A combination of molecular genetics, genomics, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) revealed an antifungal genomic island in P. fluorescens In5 encoding two nonribosomal peptides, nunamycin and nunapeptin, which are key components for the biocontrol activity by strain In5 in vitro and in soil microcosm experiments. Furthermore, complex microbial behaviors were highlighted. Whereas nunamycin was demonstrated to inhibit the mycelial growth of R. solani Ag3, but not that of Pythium aphanidermatum, nunapeptin instead inhibited P. aphanidermatum but not R. solani Ag3. Moreover, the synthesis of nunamycin by P. fluorescens In5 was inhibited in the presence of P. aphanidermatum. Further characterization of the two peptides revealed nunamycin to be a monochlorinated 9-amino-acid cyclic lipopeptide with similarity to members of the syringomycin group, whereas nunapeptin was a 22-amino-acid cyclic lipopeptide with similarity to corpeptin and syringopeptin. IMPORTANCE: Crop rotation and systematic pest management are used to only a limited extent in Greenlandic potato farming. Nonetheless, although plant-pathogenic fungi are present in the soil, the farmers do not experience major plant disease outbreaks. Here, we show that a Greenlandic potato soil is suppressive against Rhizoctonia solani, and we unravel the key biocontrol components for Pseudomonas fluorescens In5, one of the potent biocontrol bacteria isolated from this Greenlandic suppressive soil. Using a combination of molecular genetics, genomics, and microbial imaging mass spectrometry, we show that two cyclic lipopeptides, nunamycin and nunapeptin, are important for the biocontrol activity of P. fluorescens In5 both in vitro and in microcosm assays. Furthermore, we demonstrate that the synthesis of nunamycin is repressed by the oomycete Pythium aphanidermatum. Overall, our report provides important insight into interkingdom interference between bacteria and fungi/oomycetes.

Draft Genome Sequence of the Psychrophilic and Alkaliphilic Rhodonellum psychrophilum Strain GCM71T.

Rhodonellum psychrophilum GCM71(T), isolated from the cold and alkaline submarine ikaite columns in the Ikka Fjord in Greenland, displays optimal growth at 5 to 10°C and pH 10. Here, we report the draft genome sequence of this strain, which may provide insight into the mechanisms of adaptation to these extreme conditions.

Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation.

Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and the cysteine at position 130 (Cys130) within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

Comprehensive survey of redox sensitive starch metabolising enzymes in Arabidopsis thaliana.

In chloroplasts, the ferredoxin/thioredoxin pathway regulates enzyme activity in response to light by reduction of regulatory disulfides in target enzymes, ensuring coordination between photosynthesis and diurnal metabolism. Although earlier studies have suggested that many starch metabolic enzymes are similarly regulated, redox regulation has only been verified for a few of these in vitro. Using zymograms and enzyme assays, we performed a comprehensive analysis of the redox sensitivity of known starch metabolising enzymes in extracts of Arabidopsis thaliana. Manipulation of redox potentials revealed that several enzymatic activities where activated by reduction at physiologically relevant potentials. Among these where the isoamylase complex AtISA1/AtISA2, the limit dextrinase AtLDA, starch synthases AtSS1 and AtSS3, and the starch branching enzyme AtBE2. The reversibility of the redox reaction was confirmed by enzyme assays for AtLDA, AtSS1 and AtSS3. Analysis of an AtBAM1 knock-out mutant identified an additional redox sensitive ß-amylase activity, which was most likely AtBAM3. A similar requirement for reducing conditions was observed for recombinant chloroplastic α-amylase (AtAMY3) activity. This study adds further candidates to the list of reductively activated starch metabolising enzymes and supports the view that redox regulation plays a role in starch metabolism.

Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.

Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.

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.

A CBM20 low-affinity starch-binding domain from glucan, water dikinase.

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

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.