A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.

Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against ß-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward ß-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

Do rumen Bacteroidetes utilize an alternative mechanism for cellulose degradation?

Uncultured and therefore uncharacterized Bacteroidetes lineages are ubiquitous in many natural ecosystems which specialize in lignocellulose degradation. However, their metabolic contribution remains mysterious, as well-studied cultured Bacteroidetes have been shown to degrade only soluble polysaccharides within the human distal gut and herbivore rumen. We have interrogated a reconstructed genome from an uncultured Bacteroidetes phylotype that dominates a switchgrass-associated community within the cow rumen. Importantly, this characterization effort has revealed the first preliminary evidence for polysaccharide utilization locus (PUL)-catalyzed conversion of cellulose. Based on these findings, we propose a further expansion of the PUL paradigm and the saccharolytic capacity of rumen Bacteroidetes species to include cellulose, the most abundant terrestrial polysaccharide on Earth. Moreover, the perspective of a cellulolytic PUL lays the foundation for PULs to be considered an alternative mechanism for cellulose degradation, next to cellulosomes and free-enzyme systems.

Two SusD-like proteins encoded within a polysaccharide utilization locus of an uncultured ruminant Bacteroidetes phylotype bind strongly to cellulose.

We demonstrate that two characteristic Sus-like proteins encoded within a polysaccharide utilization locus (PUL) bind strongly to cellulosic substrates and interact with plant primary cell walls. This shows associations between uncultured Bacteroidetes-affiliated lineages and cellulose in the rumen and thus presents new PUL-derived targets to pursue regarding plant biomass degradation.

Effects of temperature, ultraviolet radiation and pectin methyl esterase on aerobic methane release from plant material.

This study examines the effects of different irradiance types on aerobic methane (CH(4)) efflux rates from terrestrial plant material. Furthermore, the role of the enzyme pectin methyl esterase (PME) on CH(4) efflux potential was also examined. Different types of plant tissue and purified pectin were incubated in glass vials with different combinations of irradiation and/or temperature. Purified dry pectin was incubated in solution, and with or without PME. Before and after incubation, the concentration of CH(4) was measured with a gas chromatograph. Rates of CH(4) emission were found to depend exponentially on temperature and linearly on UV-B irradiance. UV-B had a greater stimulating effect than UV-A, while visible light had no effect on emission rates. PME was found to substantially reduce the potential for aerobic CH(4) emissions upon demethylation of pectin.

Identification of amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to a polystyrene surface using phage display with competitive elution.

AIMS: To identify the main amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to the polystyrene surface PolySorp. METHODS AND RESULTS: Using a combination of phage display and competitive elution revealed that 12-mer peptides of phages from competitive panning with S. cerevisiae FLO11 wild-type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11Delta mutant (TBR5) cells, suggesting that the wild-type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more abundant in the peptides of phages from competitive elution with FLO11 cells than in those from competitive elution with flo11Delta cells. Furthermore, two phages with hydrophobic peptides containing 1 or 2 tryptophan, and 3 or 5 proline, residues inhibited the adhesion of FLO11 cells to PolySorp more than a phage with a hydrophobic peptide containing no tryptophan and only two proline residues. CONCLUSIONS: Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study may contribute to the development of novel strategies to limit yeast infections in hospitals and other medical environments.

Pectin: cell biology and prospects for functional analysis.

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.

In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana.

Pectic polysaccharides are a complex set of macromolecules of the primary cell wall matrix with distinct structural domains. The biosynthesis, organisation and function of these domains within cell wall matrices are poorly understood. An immersion immunofluorescence labelling technique was developed for the in-situ analysis of pectic polysaccharides at the surface of seeds and seedlings of Arabidopsis thaliana (L.) Heynh., and used to investigate the occurrence of pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) epitopes. Seed mucilage appeared to consist of two regions: a highly methyl-esterified HG was a major component throughout the mucilage, while an inner region with relatively low porosity was stabilized by calcium-based HG cross-linking. The small size and transparency of Arabidopsis roots allowed the occurrence of pectic HG and RG-I epitopes at root surfaces to be directly determined on whole-mount preparations. Pectic epitopes were not distributed evenly over root surfaces and were notably absent from lateral root apices and from the surface of root hairs. The use of defined antibody probes in the immersion immunolabelling protocol will be useful for the analysis of the influence of growth conditions and genetic factors on pectic polysaccharides in Arabidopsis.

Altered middle lamella homogalacturonan and disrupted deposition of (1-->5)-alpha-L-arabinan in the pericarp of Cnr, a ripening mutant of tomato.

Cnr (colorless non-ripening) is a pleiotropic tomato (Lycopersicon esculentum) fruit ripening mutant with altered tissue properties including weaker cell-to-cell contacts in the pericarp (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). Whereas the genetic basis of the Cnr mutation is being identified by molecular analyses, here we report the identification of cell biological factors underlying the Cnr texture phenotype. In comparison with wild type, ripe-stage Cnr fruits have stronger, non-swollen cell walls (CW) throughout the pericarp and extensive intercellular space in the inner pericarp. Using electron energy loss spectroscopy imaging of calcium-binding capacity and anti-homogalacturonan (HG) antibody probes (PAM1 and JIM5) we demonstrate that maturation processes involving middle lamella HG are altered in Cnr fruit, resulting in the absence or a low level of HG-/calcium-based cell adhesion. We also demonstrate that the deposition of (1-->5)-alpha-L-arabinan is disrupted in Cnr pericarp CW and that this disruption occurs prior to fruit ripening. The relationship between the disruption of (1-->5)-alpha-L-arabinan deposition in pericarp CW and the Cnr phenotype is discussed.

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion.

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Cell wall antibodies without immunization: generation and use of de-esterified homogalacturonan block-specific antibodies from a naive phage display library.

Homogalacturonan (HG) is a multi-functional pectic polysaccharide of primary cell walls involved in calcium cross-linking and gel formation, and the regulation of ionic status and porosity of the cell wall matrix, and is a source of oligosaccharins functioning in development and defence. Phase display monoclonal antibodies with specificity for de-esterified stretches ('blocks') of pectic HG have been isolated from a naive phage display library without the need for immunization of animals or conjugation of an oligosaccharide to protein. These antibodies, designated PAM1 and PAM2, bind specifically to de-esterified and un-substituted HG. Assays with a series of pectins de-esterified by the action of plant or fungal pectin methyl esterases indicated that the antibodies were specific to de-esterified blocks resulting from the blockwise action of plant pectin methyl esterases. Analysis of antibody binding to a series of oligogalacturonides indicated that optimal binding required in the region of 30 de-esterified GalA residues. The recognition of such a large epitope by these antibodies allows the HG block architecture of primary cell walls to be identified and localized for the first time. Furthermore, we have demonstrated that monoclonal antibodies with high specificity and avidity to cell wall epitopes can be generated using a 'single pot' phage display approach.

Immunoprofiling of pectic polysaccharides.

An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.

Generation of monoclonal antibody specific to (1-->5)-alpha-L-arabinan.

A neoglycoprotein (a heptasaccharide of (1-->5)-alpha-L-linked-arabinosyl residues linked to bovine serum albumin) has been used to generate a rat monoclonal antibody specific to a linear chain of (1-->5)-alpha-L-arabinan which is a structural feature of the side chains of pectins. The antibody, designated LM6, detected 100 ng of debranched sugar beet arabinan in an immunodot binding assay and 1 microgram of commercial citrus pectin in a similar assay. Hapten inhibition studies indicated that the antibody recognized 5-6 Ara residues and 50% inhibition of antibody binding in a competitive inhibition ELISA was achieved with ca. 2ng (21 nM) of (1-->5)-alpha-L-Arabinohexaose. The antibody will be useful for the localization of arabinans in plant tissue and will have uses in the analyses of pectin structure. We report here on the localization of the arabinan epitope in lemon fruits using tissue printing.

A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of beta-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana.

Seedlings of Arabidopsis thaliana were germinated and grown in medium containing beta-glucosyl Yariv reagent (beta GlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of beta GlcY on the seedlings was to reduce the overall growth of both the root and the shoot. beta GlcY only accumulated in the root tissues and the reduced growth of the shoot appeared to be an indirect effect of impaired root growth. Reduced root growth was a consequence of a reduction in cell elongation during the postproliferation phase of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. beta GlcY penetrated roots as far as the endodermis and it is suggested that the interaction of beta GlcY with AGPs in the load-bearing cell layers inhibited root elongation. When beta GlcY was added to carrot suspension-cultured cells that had been induced to elongate rather than proliferate, cell elongation was inhibited. The AGP-unreactive alpha-galactosyl Yariv reagent (alpha GalY) had no biological activity in either of these systems.

The Immunofluorescent Localization of Subventral Pharyngeal Gland Epitopes of Preparasitic Juveniles of Heterodera glycines Using Laser Scanning Confocal Microscopy.

Laser scanning confocal microscopy (LSCM) was used to localize the reactivity of a monoclonal antibody (Sv2) that binds to the subventral pharyngeal glands of preparasitic juveniles of Heterodera glycines. The greater resolution, magnification, and image analysis of LSCM compared with conventional epifluorescent microscopy enabled Sv2 binding to be localized much more precisely to the periphery of the secretory granules. A linear increase of about 55% in fluorescent intensity was found over a 23-mum length of subventral pharyngeal gland just distal to the terminal ampullae. LSCM is a rapid and effective technique for precise immunolocalization of epitopes.