Complete genome of a new Firmicutes species belonging to the dominant human colonic microbiota (‘Ruminococcus bicirculans’) reveals two chromosomes and a selective capacity to utilize plant glucans.
Wegmann, U., Louis, P., Goesmann, A., Henrissat, B., Duncan, S. H. & Flint, H. J. (2014). Environmental Microbiology, 16(9), 2879–2890.
The recently isolated bacterial strain 80/3 represents one of the most abundant 16S rRNA phylotypes detected in the healthy human large intestine and belongs to the Ruminococcaceae family of Firmicutes. The completed genome sequence reported here is the first for a member of this important family of bacteria from the human colon. The genome comprises two large chromosomes of 2.24 and 0.73 Mbp, leading us to propose the name Ruminococcus bicirculans for this new species. Analysis of the carbohydrate active enzyme complement suggests an ability to utilize certain hemicelluloses, especially β-glucans and xyloglucan, for growth that was confirmed experimentally. The enzymatic machinery enabling the degradation of cellulose and xylan by related cellulolytic ruminococci is however lacking in this species. While the genome indicated the capacity to synthesize purines, pyrimidines and all 20 amino acids, only genes for the synthesis of nicotinate, NAD+, NADP+ and coenzyme A were detected among the essential vitamins and co-factors, resulting in multiple growth requirements. In vivo, these growth factors must be supplied from the diet, host or other gut microorganisms. Other features of ecological interest include two type IV pilins, multiple extracytoplasmic function-sigma factors, a urease and a bile salt hydrolase.
Crystallization and preliminary X-ray analysis of the rhamnogalacturonan lyase YesW from Bacillus subtilis strain 168, a member of polysaccharide lyase family 11.
Ochiai, A., Yamasaki, M., Itoh, T., Mikami, B., Hashimoto, W. & Murata, K. (2006). Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 62(5), 438-440.
Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a β-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P21 and diffract to 2.40 Å resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 Å, β = 94.9°. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase.
Degradation of different pectins by fungi: correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin.
Benoit, I., Coutinho, P. M., Schols, H. A., Gerlach, J. P., Henrissat, B. & de Vries, R. P. (2012). BMC Genomics, 13(1), 321.
Background: Pectins are diverse and very complex biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. Results: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. Conclusions: This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.
Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization.
Ochiai, A., Itoh, T., Kawamata, A., Hashimoto, W. & Murata, K. (2007). Applied and Environmental Microbiology, 73(12), 3803-3813.
Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.
Generation of a monoclonal antibody specific to (1→5)-α-L-arabinan.
Willats, W. G. T., Marcus, S. E. & Knox, J. P. (1998). Carbohydrate Research, 308(1), 149-152.
A neoglycoprotein (a heptasaccharide of (1→5)-α-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)-α-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 µg 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. 2 ng (21 nM) of (1→5)-α-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.
Functional identification of two nonredundant Arabidopsis α(1,2) fucosyltransferases specific to arabinogalactan proteins.
Wu, Y., Williams, M., Bernard, S., Driouich, A., Showalter, A. M. & Faik, A. (2010). Journal of Biological Chemistry, 285(18), 13638-13645.
Virtually nothing is known about the mechanisms and enzymes responsible for the glycosylation of arabinogalactan proteins (AGPs). The glycosyltransferase 37 family contains plant-specific enzymes, which suggests involvement in plant-specific organs such as the cell wall. Our working hypothesis is that AtFUT4 and AtFUT6 genes encode α(1,2)fucosyltransferases (FUTs) for AGPs. Multiple lines of evidence support this hypothesis. First, overexpression of the two genes in tobacco BY2 cells, known to contain nonfucosylated AGPs, resulted in a staining of transgenic cells with eel lectin, which specifically binds to terminal α-linked fucose. Second, monosaccharide analysis by high pH anion exchange chromatography and electrospray ionization mass spectrometry indicated the presence of fucose in AGPs from transgenic cell lines but not in AGPs from wild type cells. Third, detergent extracts from microsomal membranes prepared from transgenic lines were able to fucosylate, in vitro, purified AGPs from BY2 wild type cells. Susceptibility of [14C]fucosylated AGPs to α(1,2)fucosidase, and not to α(1,3/4)fucosidase, indicated that an α(1,2) linkage is formed. Furthermore, dearabinosylated AGPs were not substrate acceptors for these enzymes, indicating that arabinosyl residues represent the fucosylation sites on these molecules. Testing of several polysaccharides, oligosaccharides, and glycoproteins as potential substrate acceptors in the fucosyl transfer reactions indicated that the two enzymes are specific for AGPs but are not functionally redundant because they differentially fucosylate certain AGPs. AtFUT4 and AtFUT6 are the first enzymes to be characterized for AGP glycosylation and further our understanding of cell wall biosynthesis.
Expression, purification and characterization of pectate lyase A from Aspergillus nidulans in Escherichia coli.
Zhao, Q., Yuan, S., Zhang, Y., Zhu, H., Dai, C., Yang, F. & Han, F. (2007). World Journal of Microbiology and Biotechnology, 23(8), 1057-1064.
Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni2+-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml-1 medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn2+, Ca2+, Fe2+, Mg2+ and Fe3+ ions stimulated the pectate lyase activity, but Cu2+ and Zn2+ inhibited it. The recombinant PelA had a Vmax of 77 µmol min-1 mg-1 and an apparent Km of 0.50 mg ml-1 for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.
Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro.
Zhao, Q., Yuan, S., Wang, X., Zhang, Y., Zhu, H. & Lu, C. (2008). Plant Physiology, 147(4), 1874-1885.
Mature plant cell walls lose their ability to expand and become unresponsive to expansin. This phenomenon is believed to be due to cross-linking of hemicellulose, pectin, or phenolic groups in the wall. By screening various hydrolytic enzymes, we found that pretreatment of nongrowing, heat-inactivated, basal cucumber (Cucumis sativus) hypocotyls with pectin lyase (Pel1) from Aspergillus japonicus could restore reconstituted exogenous expansin-induced extension in mature cell walls in vitro. Recombinant pectate lyase A (PelA) and polygalacturonase (PG) from Aspergillus spp. exhibited similar capacity to Pel1. Pel1, PelA, and PG also enhanced the reconstituted expansin-induced extension of the apical (elongating) segments of cucumber hypocotyls. However, the effective concentrations of PelA and PG for enhancing the reconstituted expansin-induced extension were greater in the apical segments than in the basal segments, whereas Pel1 behaved in the opposite manner. These data are consistent with distribution of more methyl-esterified pectin in cell walls of the apical segments and less esterified pectin in the basal segments. Associated with the degree of esterification of pectin, more calcium was found in cell walls of basal segments compared to apical segments. Pretreatment of the calcium chelator EGTA could also restore mature cell walls' susceptibility to expansin by removing calcium from mature cell walls. Because recombinant pectinases do not hydrolyze other wall polysaccharides, and endoglucanase, xylanase, and protease cannot restore the mature wall's extensibility, we can conclude that the pectin network, especially calcium-pectate bridges, may be the primary factor that determines cucumber hypocotyl mature cell walls' unresponsiveness to expansin.