Glycoside Hydrolase Genes Research Articles

Genetic diversity detection and gene discovery of novel glycoside hydrolase family 48 from soil environmental genomic DNA

Sequence diversity within a family of functional enzymes provides a platform for novel gene development and protein engineering to improve the properties of these enzymes for further applications. Glycoside hydrolase family 48 (GH48) is an important group of microbial cellulases. However, the genetic diversity and gene discovery of GH48 enzyme in natural environments are rarely reported. In this study, the genetic diversity of GH48 from Changbai Mountain soil was evaluated by building a clone library via a culture-independent molecular method for the first time. Results showed that the genetic diversity of GH48 in Changbai Mountain soil was different from that in thermophilic compost and marine sediment libraries, and more than 80% of the sequences exhibited the highest identity with cellulase genes from Chloroflexi. Novel GH48 genes were also cloned, and the recombinants Cel48_hm01 and Cel48_hm02 were prokaryotically expressed, purified, and characterized. Characterization results suggested that they were probably endocellulases that adopted a catalytic mechanism similar to the GH48 cellulase from Clostridium. This study revealed the genetic distribution of glycoside hydrolases in soil environment, described Changbai Mountain soil as a valuable source for glycoside hydrolase gene screening, and presented supplementary property data on novel GH48 from natural soil environments.

Abundance of Secreted Proteins of Trichoderma reesei Is Regulated by Light of Different Intensities.

In Trichoderma reesei light is an important factor in the regulation of glycoside hydrolase gene expression. We therefore investigated the influence of different light intensities on cellulase activity and protein secretion. Differentially secreted proteins in light and darkness as identified by mass spectrometry included members of different glycoside hydrolase families, such as CBH1, Cel3A, Cel61B, XYN2, and XYN4. Several of the associated genes showed light-dependent regulation on the transcript level. Deletion of the photoreceptor genes blr1 and blr2 resulted in a diminished difference of protein abundance between light and darkness. The amount of secreted proteins including that of the major exo-acting beta-1,4-glucanases CBH1 and CBH2 was generally lower in light-grown cultures than in darkness. In contrast, cbh1 transcript levels increased with increasing light intensity from 700 to 2,000 lux but dopped at high light intensity (5,000 lux). In the photoreceptor mutants Δblr1 and Δblr2 cellulase activity in light was reduced compared to activity in darkness, showing a discrepancy between transcript levels and secreted cellulase activity. Furthermore, evaluation of different light sensitivities revealed an increased light tolerance with respect to cellulase expression of QM9414 compared to its parental strain QM6a. Investigation of one of the differentially expressed proteins between light and darkness, CLF1, revealed its function as a factor involved in regulation of secreted protease activity. T. reesei secretes a different set of proteins in light compared to darkness, this difference being mainly due to the function of the major known photoreceptors. Moreover, cellulase regulation is adjusted to light intensity and improved light tolerance was correlated with increased cellulase production. Our findings further support the hypothesis of a light intensity dependent post-transcriptional regulation of cellulase gene expression in T. reesei.

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction.

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile.

Succession and Fermentation Products of Grass Carp (Ctenopharyngodon idellus) Hindgut Microbiota in Response to an Extreme Dietary Shift.

Dietary intake affects the structure and function of microbes in host intestine. However, the succession of gut microbiota in response to changes in macronutrient levels during a long period of time remains insufficiently studied. Here, we determined the succession and metabolic products of intestinal microbiota in grass carp (Ctenopharyngodon idellus) undergoing an abrupt and extreme diet change, from fish meal to Sudan grass (Sorghum sudanense). Grass carp hindgut microbiota responded rapidly to the diet shift, reaching a new equilibrium approximately within 11 days. In comparison to animal-diet samples, Bacteroides, Lachnospiraceae and Erysipelotrichaceae increased significantly while Cetobacterium decreased significantly in plant-diet samples. Cetobacterium was negatively correlated with Bacteroides, Lachnospiraceae and Erysipelotrichaceae, while Bacteroides was positively correlated with Lachnospiraceae. Predicted glycoside hydrolase and polysaccharide lyase genes in Bacteroides and Lachnospiraceae from the Carbohydrate-Active enZymes (CAZy) database might be involved in degradation of the plant cell wall polysaccharides. However, none of these enzymes was detected in the grass carp genome searched against dbCAN database. Additionally, a significant decrease of short chain fatty acids levels in plant-based samples was observed. Generally, our results suggest a rapid adaption of grass carp intestinal microbiota to dietary shift, and that microbiota are likely to play an indispensable role in nutrient turnover and fermentation.

A CRE1- regulated cluster is responsible for light dependent production of dihydrotrichotetronin in Trichoderma reesei.

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide). Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.

Role of carbon source in the shift from oxidative to hydrolytic wood decomposition by Postia placenta

Brown rot fungi initiate wood decay using oxidative pretreatments to improve access for cellulolytic enzymes. These pretreatments are incompatible with enzymes, and we recently showed that Postia placenta overcomes this issue by delaying glycoside hydrolase (GH) gene upregulation briefly (<48h) until expression of oxidoreductases (ORs) is repressed. This implies an inducible cellulase system rather than a constitutive system, as often reported, and it remains unclear what cues this transition. To address this, we grew P. placenta along wood wafers and spatially mapped expression (via quantitative PCR) of twelve ORs and GHs targeted using functional genomics analyses. By layering expression patterns over solubilized sugar data (via HPLC) from wood, we observed solubilization of wood glucose, cellobiose, mannose, and xylose coincident with the OR-GH transition. We then tested effects of these soluble sugars, plus polymeric carbon sources (spruce powder, cellulose), on P. placenta gene expression in liquid cultures. Expression of ORs was strictly (aox1, cro5) or progressively repressed over time (qrd1, lcc1) by all soluble sugars, including cellobiose, but not by polymeric sources. Simple sugars repressed hemicellulase gene expression over time, but these sugars did not repress cellulases. Cellulase genes were upregulated, however, along with hemicellulases in the presence of soluble cellobiose and in the presence of polymeric carbon sources, relative to starvation (carbon-free). This verifies an inducible cellulase system in P. placenta that lacks carbon catabolite repression (CCR), and it suggests that brown rot fungi use soluble sugars, particularly cellobiose, to cue a critical oxidative-hydrolytic transition.

Prebiotic effect of xylooligosaccharides produced from birchwood xylan by a novel fungal GH11 xylanase

A fungal endoxylanase belonging to the glycoside hydrolase gene family 11 (GH11) was obtained from the ascomycete Talaromyces amestolkiae. The enzyme was purified, characterized and used to produce a mixture of xylooligosaccharides (XOS) from birchwood xylan. A notable yield of neutral XOS was obtained (28.8%) upon enzyme treatment and the mixture contained a negligible amount of xylose, having xylobiose, xylotriose and xylotetraose as its main components. The prebiotic potential of this mixture was demonstrated upon analyzing the variations in microorganisms’ composition and organic acids profile in breast-fed child faeces fermentations. The strong production of acetic and lactic acid, the decrease of potentially pathogenic bacteria and the increase of bifidobacteria, and possible beneficial commensals, confirmed the prebiotic value of these xylooligosaccharides.

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

Characterization of Potential Polysaccharide Utilization Systems in the Marine Bacteroidetes Gramella Flava JLT2011 Using a Multi-Omics Approach.

Members of phylum Bacteroidetes are distributed across diverse marine niches and Flavobacteria is often the predominant bacterial class decomposing algae-derived polysaccharides. Here, we report the complete genome of Gramella flava JLT2011 (Flavobacteria) isolated from surface water of the southeastern Pacific. A remarkable genomic feature is that the number of glycoside hydrolase (GH) genes in the genome of G. flava JLT2011 is more than 2-fold higher than that of other Gramella species. The functional profiles of the GHs suggest extensive variation in Gramella species. Growth experiments revealed that G. flava JLT2011 has the ability to utilize a wide range of polysaccharides for growth such as xylan and homogalacturonan in pectin. Nearly half of all GH genes were located on the multi-gene polysaccharide utilization loci (PUL) or PUL-like systems in G. flava JLT2011. This species was also found to harbor the two xylan PULs and a pectin PUL, respectively. Gene expression data indicated that more GHs and sugar-specific outer-membrane susC-susD systems were found in the presence of xylan than in the presence of pectin, suggesting a different strategy for heteropolymeric xylan and homoglacturonan utilization. Multi-omics data (transcriptomics, proteomics, and metabolomics) indicated that xylan PULs and pectin PUL are respectively involved in the catabolism of their corresponding polysaccharides. This work presents a comparison of polysaccharide decomposition within a genus and expands current knowledge on the diversity and function of PULs in marine Bacteroidetes, thereby deepening our understanding of their ecological role in polysaccharide remineralization in the marine system.

A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomic

The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.

Identification of differentially expressed genes from Trichoderma atroviride strain SS003 in the presence of cell wall of Cronartium ribicola

The fungal genus Trichoderma has been extensively studied due to its role in the mycoparasitism, and thus developed as biocontrol agent against various plant pathogens. Although the mycoparasitic processes of several Trichoderma species have already been well understood, the information about the mycoparasitic mechanisms of Trichoderma strains resulted from different growth conditions or interacting with different phytopathogens is still limited. In this study, we utilized transcriptome sequencing to identify the differentially expressed genes (DEGs) at 0, 24, 72 and 120 h from T. atroviride strain SS003, growing on an induced-medium with cell walls of Pinus armandii pathogen Cronartium ribicola (CRCW). In total, 86,155,316 reads were obtained with 43,077,658 clean reads. Further, 10,422 genes were identified from four transcriptomes and accounted for 93.89% of annotated genes in T. atroviride IMI 206040 genome, reflecting high-quality sequencing and assembly. In each pairwise comparison, a large number of DEGs were identified with different numbers of genes for up- and down-regulation, respectively. In the presence of CRCW, expression of two main glycoside hydrolase gene families (i.e. chitinase and glucosidase) was induced. Most of 14 secreted enzymes by quantitative real time PCR (qPCR) analysis exhibited a consistent expression pattern with that by RNA-Seq data. This comparative study leads to the identification of phase-specific genes in the interactions of T. atroviride SS003 with C. ribicola, and provides potential molecular targets for improved biocontrol strategies.

The Different Roles of Penicillium oxalicum LaeA in the Production of Extracellular Cellulase and β-xylosidase.

Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ΔlaeA (deletion of laeA), OEclrB (clrB overexpression), OEclrBΔlaeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnRΔlaeA (xlnR overexpression with deletion of laeA), ΔcreA (deletion of creA), and ΔcreAΔlaeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ΔlaeA, OEclrBΔlaeA, OExlnRΔlaeA, and ΔcreAΔlaeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ΔcreA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular β-xylosidase formation was negatively regulated by LaeA. The extracellular β-xylosidase activities improved over 5-fold in the OExlnRΔlaeA mutant compared with that of WT, and the expression of prominent β-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more β-xylosidase.

Composting-Like Conditions Are More Efficient for Enrichment and Diversity of Organisms Containing Cellulase-Encoding Genes than Submerged Cultures.

Cost-effective biofuel production from lignocellulosic biomass depends on efficient degradation of the plant cell wall. One of the major obstacles for the development of a cost-efficient process is the lack of resistance of currently used fungal enzymes to harsh conditions such as high temperature. Adapted, thermophilic microbial communities provide a huge reservoir of potentially interesting lignocellulose-degrading enzymes for improvement of the cellulose hydrolysis step. In order to identify such enzymes, a leaf and wood chip compost was enriched on a mixture of thermo-chemically pretreated wheat straw, poplar and Miscanthus under thermophile conditions, but in two different set-ups. Unexpectedly, metagenome sequencing revealed that incubation of the lignocellulosic substrate with compost as inoculum in a suspension culture resulted in an impoverishment of putative cellulase- and hemicellulase-encoding genes. However, mimicking composting conditions without liquid phase yielded a high number and diversity of glycoside hydrolase genes and an enrichment of genes encoding cellulose binding domains. These identified genes were most closely related to species from Actinobacteria, which seem to constitute important players of lignocellulose degradation under the applied conditions. The study highlights that subtle changes in an enrichment set-up can have an important impact on composition and functions of the microcosm. Composting-like conditions were found to be the most successful method for enrichment in species with high biomass degrading capacity.

Genome Sequence of Salegentibacter salarius KCTC 12974, Isolated from a Marine Solar Saltern of the Yellow Sea in South Korea.

Salegentibacter salarius KCTC 12974 is isolated from a marine solar saltern of the Yellow Sea in South Korea. Here, we report the draft genome sequence of Salegentibacter salarius KCTC 12974. Various glycoside hydrolase genes in even numbers in the genome reflect the ecological adaption of KCTC 12974 to its habitat.

Biodegradation of nanocrystalline cellulose by two environmentally-relevant consortia.

Nanocellulose is growing in popularity due to its versatile properties and applications. However, there is a void of knowledge regarding the environmental fate of nanocellulose and the response of environmental microbial communities that are historically adapted to non-nano cellulose forms. Given its distinction in terms of size and chemical and physical properties, nanocellulose could potentially resist biodegradation and/or pose a xenobiotic influence on microbial communities during wastewater treatment or in receiving environments. In this study, biodegradation of H2SO4 hydrolyzed nanocrystalline cellulose (HNC) was compared with that of microcrystalline cellulose using two distinct anaerobic cellulose-degrading microbial consortia initially sourced from anaerobic digester (AD) and wetland (W) inocula. Equivalent cellulose masses were dosed and monitored with time by measurement of liberated glucose. HNC biodegraded at slightly faster rate than microcrystalline cellulose (1st order decay constants: 0.62 ± 0.08 wk−1 for HNC versus 0.39 ± 0.05 wk−1 for microcrystalline cellulose for the AD consortium; 0.69 ± 0.04 wk−1for HNCversus 0.58 ± 0.05 wk−1 for microcrystalline cellulose for the W consortium). 16S rRNA (total bacteria) and cel48 (glycoside hydrolase gene family 48, indicative of cellulose-degrading potential) genes were observed to be more enriched in the HNC condition for both consortia. According to Illumina amplicon sequencing of 16S rRNA genes, the composition of the consortia underwent distinct shifts in concert with HNC versus microcrystalline cellulose degradation. This study demonstrates that the biodegradation of cellulose is not inhibited in the nano-size range, particularly in the crystalline form, though the microbes and pathways involved likely differ.

Comprehensive detection of bacterial carbohydrate-active enzyme coding genes expressed in cow rumen.

To find the abundant and characteristic fibrolytic enzyme-coding gene expressed in fiber-associating microbiota, a metatranscriptomic data set was obtained from fiber-associating microbiota, and it was compared with that of rumen fluid-floating microbiota and two metagenomic data sets. Fibrolytic rumen bacteria associate with plant polysaccharide and hydrolyze it in the rumen. We obtained a metatranscriptomic assembly from fiber-associating microbiota in three ruminally fistulated Holstein cows fed timothy (Phleum pratense) hay. Each metatranscriptomic data set involved over a thousand of the glycoside hydrolase (GH) gene transcripts that accounted for about 1% of total protein coding gene transcripts. Three-quarters of the total GH gene transcripts were dominated by non-structural oligosaccharide-acting hydrolase gene transcripts. In the fiber-associating microbiota, endo-cellulase coding gene families, especially GHs 9 and 5, were abundantly detected, and GHs 9, 11, 30 and 43, carbohydrate esterase 8 and carbohydrate-binding module 6 were characteristically detected. Most fibrolytic gene transcripts assigned to Fibrobacter succinogenes were detected in fiber-associating sections, and GHs 45, 44, 74, 11, 30 and 16 were Fibrobacter-characteristically detected. The metatranscriptomic assembly highlighted the characteristic fibrolytic enzymes expressed in the fiber-associated rumen microbiota and offered access to the fibrolytic activities in each fibrolytic bacteria.

Polysaccharide utilization loci and nutritional specialization in a dominant group of butyrate-producing human colonic Firmicutes.

Firmicutes and Bacteroidetes are the predominant bacterial phyla colonizing the healthy human large intestine. Whilst both ferment dietary fibre, genes responsible for this important activity have been analysed only in the Bacteroidetes, with very little known about the Firmicutes. This work investigates the carbohydrate-active enzymes (CAZymes) in a group of Firmicutes, Roseburia spp. and Eubacterium rectale, which play an important role in producing butyrate from dietary carbohydrates and in health maintenance. Genome sequences of 11 strains representing E. rectale and four Roseburia spp. were analysed for carbohydrate-active genes. Following assembly into a pan-genome, core, variable and unique genes were identified. The 1840 CAZyme genes identified in the pan-genome were assigned to 538 orthologous groups, of which only 26 were present in all strains, indicating considerable inter-strain variability. This analysis was used to categorize the 11 strains into four carbohydrate utilization ecotypes (CUEs), which were shown to correspond to utilization of different carbohydrates for growth. Many glycoside hydrolase genes were found linked to genes encoding oligosaccharide transporters and regulatory elements in the genomes of Roseburia spp. and E. rectale, forming distinct polysaccharide utilization loci (PULs). Whilst PULs are also a common feature in Bacteroidetes, key differences were noted in these Firmicutes, including the absence of close homologues of Bacteroides polysaccharide utilization genes, hence we refer to Gram-positive PULs (gpPULs). Most CAZyme genes in the Roseburia/E. rectale group are organized into gpPULs. Variation in gpPULs can explain the high degree of nutritional specialization at the species level within this group.

A novel metagenome-derived gene cluster from termite hindgut: Encoding phosphotransferase system components and high glucose tolerant glucosidase

Functional screening of a metagenomic library of termite hindgut identified an overlapping gene cluster encoding the phosphotransferase system (PTS) components, which consisted of a glucoside specific PTS enzyme II gene (glu1923) and a glycoside hydrolase gene (glu1392). Hydrolytic experiments revealed that the combined effect of Glu1923 and Glu1392 was responsible for the utilization of glucosidic substrates in recombinant Escherichia coli (E. coli) strains. Yeast two hybrid analysis suggested that there was an interaction between Glu1923 and Glu1392, and the domain EIIA of Glu1923 played an important role for the interaction. In addition, the hydrolase Glu1392 displayed hydrolysis ability toward cellobiose and maltose, and had a very high tolerance to glucose with a Ki value of 2.25M. These properties make Glu1392 an interesting candidate in biotechnology applications after further study.

Intraspecies Genomic Diversity and Long-Term Persistence of Bifidobacterium longum.

Members of genus Bifidobacterium are Gram-positive bacteria, representing a large part of the human infant microbiota and moderately common in adults. However, our knowledge about their diversity, intraspecific phylogeny and long-term persistence in humans is still limited. Bifidobacterium longum is generally considered to be the most common and prevalent species in the intestinal microbiota. In this work we studied whole genome sequences of 28 strains of B. longum, including 8 sequences described in this paper. Part of these strains were isolated from healthy children during a long observation period (up to 10 years between isolation from the same patient). The three known subspecies (longum, infantis and suis) could be clearly divided using sequence-based phylogenetic methods, gene content and the average nucleotide identity. The profiles of glycoside hydrolase genes reflected the different ecological specializations of these three subspecies. The high impact of horizontal gene transfer on genomic diversity was observed, which is possibly due to a large number of prophages and rapidly spreading plasmids. The pan-genome characteristics of the subspecies longum corresponded to the open pan-genome model. While the major part of the strain-specific genetic loci represented transposons and phage-derived regions, a large number of cell envelope synthesis genes were also observed within this category, representing high variability of cell surface molecules. We observed the cases of isolation of high genetically similar strains of B. longum from the same patients after long periods of time, however, we didn’t succeed in the isolation of genetically identical bacteria: a fact, reflecting the high plasticity of microbiota in children.

Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu(2+) tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and Km and Vmax values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.