Including Glycoside Hydrolases Research Articles

192 Effects of inclusion of a probiotic and/or a postbiotic in diets containing a phytogenic feed additive on growth performance and fecal microbiome composition and function of nursery pigs

Abstract This study investigated the effects of inclusion of a Bacillus-based probiotic and a postbiotic from Lactobacillus acidophilus fermentation (Dia-V Nursery, Diamond V, IA) in diets containing a phytogenic feed additive (Fresta Protect, Delacon, Austria) on growth performance and fecal microbiome of pigs. Weaned pigs [n = 1,840; 19 d old; initial body weight (BW) = 6.1 ± 0.2 kg] were allotted to 16 replicate pens/ treatment (23 pigs/pen) in 2 barns. Pens were blocked by location within barn and randomly allotted to 5 treatments: 1) Basal diet (NC), 2) NC without specialty fats (soy oil blend and medium chain fatty acid blend) + 0.1% phytogenic (PC), 3) PC + 0.05% probiotic (1.1 x 106 CFU/kg of complete feed; PC+Prob), 4) PC + 0.2% postbiotic (PC+Post), and 5) PC + 0.05% probiotic + 0.2% postbiotic (PC+Prob+Post). Pigs were fed experimental diets during phase 1 (d 0-10) and 2 (d 10-23). Pig and feed weights were recorded at the beginning and end of each phase. Fecal samples were collected from 2 pigs/pen (n = 32/treatment) on d 7 and 25 post-wean and subjected to DNA extraction and shotgun metagenomics to evaluate gut microbiome. Performance data were analyzed using the lme4 package of R with treatment as fixed effect and barn and pen location within barn as random effects. Microbiome data were center-log-ratio transformed and analyzed for alpha- and beta-diversity, differentially abundant taxa and genes, microbial network, and carbohydrate active enzymes (CAZy). No differences on performance, mortality or removals were observed during phase 1, phase 2, or overall among treatments (Table 1). Microbiome features were similar between NC and PC. The PC+Prob did not affect alpha- and beta-diversities of microbiome. The PC+Post reduced (P < 0.05) alpha-diversity on d 7 compared with PC and affected (P < 0.05) beta-diversity on both timepoints. The PC+Post had the greatest number of differentially abundant taxa (P < 0.05) compared with PC both on d 7 (27 taxa) and 25 (14 taxa). The PC+Post had greater (P < 0.05) presence of CAZy including glycoside hydrolases, glycosyltransferases and carbohydrate-binding modules on both timepoints. Inclusion of probiotic and postbiotic separately or in combination reduced (P < 0.05) the number of positive connections within the microbial network suggesting these additives promoted a more competitive microbial ecosystem against opportunistic microorganisms compared with PC. Inclusion of postbiotic speeded up the progression of microbiome toward a more mature community. This resulted in reduction of diversity but increased carbohydrate enzyme capability making microbial community leaner and more effective. In conclusion, replacement of specialty fats with a phytogenic did not affect piglet performance or microbiome under non-challenge conditions and no differences in performance were observed between PC pigs and those supplemented with a probiotic or postbiotic despite the effects observed in microbiome.

Plant immunity suppression by an β-1,3-glucanase of the maize anthracnose pathogen Colletotrichum graminicola

BackgroundMany phytopathogens secrete a large number of cell wall degrading enzymes (CWDEs) to decompose host cell walls in order to penetrate the host, obtain nutrients and accelerate colonization. There is a wide variety of CWDEs produced by plant pathogens, including glycoside hydrolases (GHs), which determine the virulence, pathogenicity, and host specificity of phytopathogens. The specific molecular mechanisms by which pathogens suppress host immunity remain obscure.ResultIn this study, we found that CgEC124 encodes a glycosyl hydrolase with a signal peptide and a conserved Glyco_hydro_cc domain which belongs to glycoside hydrolase 128 family. The expression of CgEC124 was significantly induced in the early stage of Colletotrichum graminicola infection, especially at 12 hpi. Furthermore, CgEC124 positively regulated the pathogenicity, but it did not impact the vegetative growth of mycelia. Ecotopic transient expression of CgEC124 decreased the disease resistance and callose deposition in maize. Moreover, CgEC124 exhibited the β-1,3-glucanase activity and suppresses glucan-induced ROS burst in maize leaves.ConclusionsOur results indicate that CgEC124 is required for full virulence of C. graminicola but not for vegetative growth. CgEC124 increases maize susceptibility by inhibiting host reactive oxygen species burst as well as callose deposition. Meanwhile, our data suggests that CgEC124 explores its β-1,3-glucanase activity to prevent induction of host defenses.

Host-pathogen interaction between pitaya and Neoscytalidium dimidiatum reveals the mechanisms of immune response associated with defense regulators and metabolic pathways

BackgroundUnderstanding how plants and pathogens regulate each other's gene expression during their interactions is key to revealing the mechanisms of disease resistance and controlling the development of pathogens. Despite extensive studies on the molecular and genetic basis of plant immunity against pathogens, the influence of pitaya immunity on N. dimidiatum metabolism to restrict pathogen growth is poorly understood, and how N. dimidiatum breaks through pitaya defenses. In this study, we used the RNA-seq method to assess the expression profiles of pitaya and N. dimidiatum at 4 time periods after interactions to capture the early effects of N. dimidiatum on pitaya processes.ResultsThe study defined the establishment of an effective method for analyzing transcriptome interactions between pitaya and N. dimidiatum and to obtain global expression profiles. We identified gene expression clusters in both the host pitaya and the pathogen N. dimidiatum. The analysis showed that numerous differentially expressed genes (DEGs) involved in the recognition and defense of pitaya against N. dimidiatum, as well as N. dimidiatum’s evasion of recognition and inhibition of pitaya. The major functional groups identified by GO and KEGG enrichment were responsible for plant and pathogen recognition, phytohormone signaling (such as salicylic acid, abscisic acid). Furthermore, the gene expression of 13 candidate genes involved in phytopathogen recognition, phytohormone receptors, and the plant resistance gene (PG), as well as 7 effector genes of N. dimidiatum, including glycoside hydrolases, pectinase, and putative genes, were validated by qPCR. By focusing on gene expression changes during interactions between pitaya and N. dimidiatum, we were able to observe the infection of N. dimidiatum and its effects on the expression of various defense components and host immune receptors.ConclusionOur data show that various regulators of the immune response are modified during interactions between pitaya and N. dimidiatum. Furthermore, the activation and repression of these genes are temporally coordinated. These findings provide a framework for better understanding the pathogenicity of N. dimidiatum and its role as an opportunistic pathogen. This offers the potential for a more effective defense against N. dimidiatum.

Genome mining reveals chitin degradation potential of \(\textit{Streptomyces parvulus}\) VCCM 22513

The genus Streptomyces is not only known as a natural producer of antibiotics but also a prolific source of chitinolytic enzymes that digest recalcitrant chitin to chitooligosaccharides. However, only a few reports have used whole-genome sequencing to study chitin degradation of Streptomyces to date. In the present study, out of 22 Streptomyces strains, Streptomyces parvulus VCCM 22513 produced the highest chitinase activity. Time courses of incubation revealed that the maximum chitinase (0.91 ± 0.04 U/mL) of this strain was observed after 96 hours in the yeast extract salts medium supplemented with 10.0 g/L colloidal chitin. Additional genomic analysis of VCCM 22513 was also conducted to discover the genomic information related to chitin degradation. The VCCM 22513 genome consists of 341 CAZy genes divided into 6 families including glycoside hydrolase (134 genes), carbohydrate-binding module (88 genes), glycosyl transferase (87 genes), carbohydrate esterase (18 genes), polysaccharide lyase (7 genes), and auxiliary activity (7 genes). Further genome mining revealed the presence of 10 chitinases, 4 lytic polysaccharide monooxygenases, and 14 β-N-acetylhexosaminidases, which mainly contribute to the degradation of chitin polymers. This is the first report revealing the mechanism underlying the chitin degradation of S. parvulus. Further investigations are required to characterize chitinolytic enzymes found in this study for the bioeconomic production of high-quality chitooligosaccharides from chitin food wastes.

Diverse functional genes harboured in extracellular vesicles from environmental and human microbiota.

Exchange of mobile functional genes within microbiota benefits the microbial community. However, the status of the mobile gene pool in environment is still largely unclear, impeding the understanding on the process of gene transfer in natural microbial communities. The release of extracellular vesicles (EVs) by diverse organisms has been proposed to be a vital way in the complex networks of interactions between microbes and their habitats. In this study, we hypothesized that microbial EVs encapsulating functional DNA are widely distributed in the environmental matrix. The prevalence, source and DNA cargoes of EVs in three types of typical microbial habitats were studied. High abundance of EVs comparable to the bacterial concentration was found in human faeces, wastewater and soil. Metagenomic analysis showed the diverse and differential taxonomy of EVs-associated DNA compared to source microbiome. An array of efficient EVs producing species was identified. A wide variety of mobile genes including glycoside hydrolase family 25 were enriched. Antibiotic resistance genes co-localizing with mobile genetic elements were abundant in the EVs. This study provides novel insights into the prevalent EVs as a reservoir for the mobile functional genes in the natural environment.

Exploring recycled agricultural wastes for high-rate removal of nitrogen in wastewater: Emphasizing on the investigation of the inner driving force and comparison with conventional liquid carbon sources

In this study, four typical recycled agricultural wastes (AWs), corn cob, wheat straw, sawdust and walnut shells (named AW1, AW2, AW3 and AW4, respectively), were selected as external solid carbon sources to enhance the removal of nitrogen in wastewater, and specifically, the driving mechanism was thoroughly investigated. The leaching experiments showed that the dissolved organic carbon (DOC) release capacity followed the order of AW1>AW2>AW3>AW4, ranging from 6.21 to 31.92 mg/g. DOC released from AWs mainly consisted of protein-like substances, fulvic acid-like substances and humic-like substances. AW1 and AW2 achieved comparable NOx−-N removal performance with a liquid carbon source of sodium acetate (SA) during the long-term denitrification experiments (>94.2%) but not for the other two AWs (only 16.8%-38.1%). Denitrification performance relied on DOC released from AWs at the beginning, while the enrichment of the functional CAZymes (including glycoside hydrolase and carbohydrate esterase) involved in cellulose and hemicellulose decomposition of AWs and functional genes (GAPDH, gap 2, PK, etc.) related to glycolysis were the inner driving force, which guaranteed the continuous supply of electron donors for denitrification. The relatively high abundances of napAB, narGHI, nirKS, norBC and nosZ, which encode nitrate reductase, nitrite reductase, NO reductase and N2O reductase, assured the better denitrification performance in the SA, AW1 and AW2 groups. In addition to denitrification-related functional genes, the relative abundances of nirBD and nrfAH associated with dissimilatory nitrate reduction were much higher in AW1 and AW2 groups than in SA group, implying that the nitrogen removal mechanism should be different in liquid carbon source and AW-based solid carbon source systems. In addition, GLU, gltBD and glnA, which participate in ammonia assimilation were the highest in the AW2 group, resulting in a large amount of organic nitrogen accumulation (peak concentration of approximately 24.5 mg/L), and this finally ruled it out as an alternative external carbon source. The abovementioned microbial mechanism was verified based on the correlation analysis of nutrient removal and functional genes combined with host bacterial analysis. Our study can provide valuable information for understanding the mechanism of using AWs as alternative external carbon sources to promote the removal of nitrogen in wastewater.

Multi-omics analysis of the metabolism of phenolic compounds in tea leaves by Aspergillus luchuensis during fermentation of pu-erh tea

Aspergillus fungi are extensively used in traditional food fermentation, so their functions, mechanisms, and safety risks are worth exploring. In this study, a dominant fungal strain (P1) was isolated from a fermented pu-erh tea and identified as A. luchuensis by phylogenetic analysis of fungal internally-transcribed spacer sequencing, partial β-tubulin and calmodulin genes. A pure-strain fermentation of tea leaves was developed, and tea compounds were analyzed by widely-targeted metabolomics, using high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). The mycotoxins, aflatoxin (B1, B2, M1 and M2), fumonisin B1 and B2, ochratoxin A, citrinin, were not detected in fermented tea leaves using methods in the National Standard of the Peoples’ Republic of China. The genome of 36.60 Mb with 11,836 protein-coding genes was sequenced by PacBio sequencing and annotated. Expression of fungal genes during fermentation was analyzed by Illumina HiSeq 2500; genes encoding enzymes including glycoside hydrolases, phenolic acid esterases, laccases, tyrosinases, dehydrogenases, peroxidases, dioxygenases, monooxygenases, decarboxylases and O-methyltransferases were identified. These enzymes catalyze hydrolysis, oxidation, ring cleavage, hydroxylation, decarboxylation and O-methylation of phenolic compounds , significantly (p < 0.05) changing the phenolic compound composition. While, phenolic compounds were degraded through degradation of aromatic compounds pathways and xenobiotics biodegradation and metabolism pathways. These findings advance knowledge of the functions and mechanisms of action of Aspergillus in traditional food fermentation.

Comparative Genome Analyses of Plant Rust Pathogen Genomes Reveal a Confluence of Pathogenicity Factors to Quell Host Plant Defense Responses.

Switchgrass rust caused by Puccinia novopanici (P. novopanici) has the ability to significantly affect the biomass yield of switchgrass, an important biofuel crop in the United States. A comparative genome analysis of P. novopanici with rust pathogen genomes infecting monocot cereal crops wheat, barley, oats, maize and sorghum revealed the presence of larger structural variations contributing to their genome sizes. A comparative alignment of the rust pathogen genomes resulted in the identification of collinear and syntenic relationships between P. novopanici and P. sorghi; P. graminis tritici 21–0 (Pgt 21) and P. graminis tritici Ug99 (Pgt Ug99) and between Pgt 21 and P. triticina (Pt). Repeat element analysis indicated a strong presence of retro elements among different Puccinia genomes, contributing to the genome size variation between ~1 and 3%. A comparative look at the enriched protein families of Puccinia spp. revealed a predominant role of restriction of telomere capping proteins (RTC), disulfide isomerases, polysaccharide deacetylases, glycoside hydrolases, superoxide dismutases and multi-copper oxidases (MCOs). All the proteomes of Puccinia spp. share in common a repertoire of 75 secretory and 24 effector proteins, including glycoside hydrolases cellobiohydrolases, peptidyl-propyl isomerases, polysaccharide deacetylases and protein disulfide-isomerases, that remain central to their pathogenicity. Comparison of the predicted effector proteins from Puccinia spp. genomes to the validated proteins from the Pathogen–Host Interactions database (PHI-base) resulted in the identification of validated effector proteins PgtSR1 (PGTG_09586) from P. graminis and Mlp124478 from Melampsora laricis across all the rust pathogen genomes.

Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber

While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome’s functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin. We found that the microbiome of mice subjected to a high fiber diet high in soluble fiber had increased functional activity of multiple proteins, including glycoside hydrolases, polysaccharide lyases, and sugar transport proteins from diverse taxa. The results point to an increase in activity of the Bifidobacterium shunt metabolic pathway in the microbiome of mice fed high fiber diets. In those subjected to a low fiber diet, we identified a shift from the degradation of dietary fibers to that of gut mucins, in particular by the recently isolated taxon “Musculibacterium intestinale”, which experienced dramatic growth in response to fiber deprivation. When combined with metabolomics and shotgun metagenomics analyses, our findings provide a functional investigation of dietary fiber metabolism in the gut microbiome and demonstrates the power of a combined ABPP-multiomics approach for characterizing the response of the gut microbiome to perturbations.

Consecutive monoculture regimes differently affected the diversity of the rhizosphere soil viral community and accumulated soil-borne plant viruses

Viruses interact with nearly all organisms on Earth, thereby influencing biogeochemical cycles, agriculture, and health. However, less is known about the composition and function of viruses in agricultural rhizosphere soils, especially under consecutive monoculture regimes. Our qRT-PCR analysis in this study showed that consecutive monoculture regimes significantly increased the content of pathogenic Broad bean wilt virus 2, Turnip mosaic virus, and Cucumber mosaic virus in Radix pseudostellariae roots at different growth stages. Metatranscriptome analysis indicated that consecutive monoculture altered the rhizosphere soil viral community structure, composition, and habitat niche breadth and accumulated pathogenic plant viruses in the rhizosphere soil. In addition, 54 unique carbohydrate metabolism-related open reading frames (ORFs) were detected as encoding carbohydrate-active enzymes (CAZymes) in viromes, including glycoside hydrolases and glycosyl-transferase activities. Our results also showed that the soil viral richness and the Shannon index displayed a negative correlation with bacterial abundance and a positive correlation with fungal abundance. Soil pH, total nitrogen, available phosphorus, and phenolic acids were the key abiotic drivers of the soil viral community composition. Our study provides new insights into how consecutive monoculture regimes shape the soil viral community composition and further influence plant viral diseases.

Transcriptome and protein networks to elucidate the mechanism underlying nitrite degradation by Lactiplantibacillus plantarum

Excessive nitrite residue is one of the bottlenecks in the production of many fermented foods. Lactiplantibacillus plantarum PK25 obtained from traditional Chinese pickles exhibited excellent nitrite degradation ability. Here, transcriptome, protein-protein interaction networks, and phenotype were performed to evaluate systematically the mechanism of nitrite degradation of L. plantarum PK25. The results demonstrated that genes expression varied considerably at key time points for nitrite degradation. 553 (upregulated: 366, downregulated: 187) and 767 (upregulated: 425, downregulated: 342) differentially expressed genes were identified at 6 h and 24 h, respectively. The hub genes were mainly enriched in carbohydrate metabolism, energy metabolism, and nucleotide synthesis. PK25 expanded its carbon source utilizing profile and improved glycolysis to produce more ATP to counteract environmental stress. The related enzymes including glycoside hydrolase, sugar ABC transporter protein, and PTS sugar transporter were 5.714, 5.885, and 3.578-fold upregulated at the transcriptional level. For strain to sustain energy levels and acid generation, pyruvate metabolism was critical, with the result that phosphoenolpyruvate synthase and pyruvate oxidase were up-regulated to accelerate the pyruvate transition. To repair DNA lesions induced by nitrite, both base excision repair mechanism and recombinational DNA repair pathway were exploited, such as endodeoxyribonuclease upregulated 5.314 and 19.687-fold at the two moments. The results provided a theoretical reference and practical possibility to reduce nitrite residue and improve safety during food fermented products.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

A novel fungal metal-dependent \u03b1-l-arabinofuranosidase of family 54 glycoside hydrolase shows expanded substrate specificity

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme’s biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

Isolation and sequence-based characterization of a koala symbiont: Lonepinella koalarum.

Koalas (Phascolarctos cinereus) are highly specialized herbivorous marsupials that feed almost exclusively on Eucalyptus leaves, which are known to contain varying concentrations of many different toxic chemical compounds. The literature suggests that Lonepinella koalarum, a bacterium in the Pasteurellaceae family, can break down some of these toxic chemical compounds. Furthermore, in a previous study, we identified L. koalarum as the most predictive taxon of koala survival during antibiotic treatment. Therefore, we believe that this bacterium may be important for koala health. Here, we isolated a strain of L. koalarum from a healthy koala female and sequenced its genome using a combination of short-read and long-read sequencing. We placed the genome assembly into a phylogenetic tree based on 120 genome markers using the Genome Taxonomy Database (GTDB), which currently does not include any L. koalarum assemblies. Our genome assembly fell in the middle of a group of Haemophilus, Pasteurella and Basfia species. According to average nucleotide identity and a 16S rRNA gene tree, the closest relative of our isolate is L. koalarum strain Y17189. Then, we annotated the gene sequences and compared them to 55 closely related, publicly available genomes. Several genes that are known to be involved in carbohydrate metabolism could exclusively be found in L. koalarum relative to the other taxa in the pangenome, including glycoside hydrolase families GH2, GH31, GH32, GH43 and GH77. Among the predicted genes of L. koalarum were 79 candidates putatively involved in the degradation of plant secondary metabolites. Additionally, several genes coding for amino acid variants were found that had been shown to confer antibiotic resistance in other bacterial species against pulvomycin, beta-lactam antibiotics and the antibiotic efflux pump KpnH. In summary, this genetic characterization allows us to build hypotheses to explore the potentially beneficial role that L. koalarum might play in the koala intestinal microbiome. Characterizing and understanding beneficial symbionts at the whole genome level is important for the development of anti- and probiotic treatments for koalas, a highly threatened species due to habitat loss, wildfires, and high prevalence of Chlamydia infections.

Aspergillus sydowii: Genome Analysis and Characterization of Two Heterologous Expressed, Non-redundant Xylanases.

A prerequisite for the transition toward a biobased economy is the identification and development of efficient enzymes for the usage of renewable resources as raw material. Therefore, different xylanolytic enzymes are important for efficient enzymatic hydrolysis of xylan-heteropolymers. A powerful tool to overcome the limited enzymatic toolbox lies in exhausting the potential of unexplored habitats. By screening a Vietnamese fungal culture collection of 295 undiscovered fungal isolates, 12 highly active xylan degraders were identified. One of the best xylanase producing strains proved to be an Aspergillus sydowii strain from shrimp shell (Fsh102), showing a specific activity of 0.6 U/mg. Illumina dye sequencing was used to identify our Fsh102 strain and determine differences to the A. sydowii CBS 593.65 reference strain. With activity based in-gel zymography and subsequent mass spectrometric identification, three potential proteins responsible for xylan degradation were identified. Two of these proteins were cloned from the cDNA and, furthermore, expressed heterologously in Escherichia coli and characterized. Both xylanases, were entirely different from each other, including glycoside hydrolases (GH) families, folds, substrate specificity, and inhibition patterns. The specific enzyme activity applying 0.1% birch xylan of both purified enzymes were determined with 181.1 ± 37.8 or 121.5 ± 10.9 U/mg for xylanase I and xylanase II, respectively. Xylanase I belongs to the GH11 family, while xylanase II is member of the GH10 family. Both enzymes showed typical endo-xylanase activity, the main products of xylanase I are xylobiose, xylotriose, and xylohexose, while xylobiose, xylotriose, and xylopentose are formed by xylanase II. Additionally, xylanase II showed remarkable activity toward xylotriose. Xylanase I is stable when stored up to 30°C and pH value of 9, while xylanase II started to lose significant activity stored at pH 9 after exceeding 3 days of storage. Xylanase II displayed about 40% activity when stored at 50°C for 24 h. The enzymes are tolerant toward mesophilic temperatures, while acting in a broad pH range. With site directed mutagenesis, the active site residues in both enzymes were confirmed. The presented activity and stability justify the classification of both xylanases as highly interesting for further development.

Verrucomicrobia use hundreds of enzymes to digest the algal polysaccharide fucoidan.

Brown algae are important players in the global carbon cycle by fixing carbon dioxide into 1 Gt of biomass annually, yet the fate of fucoidan-their major cell wall polysaccharide-remains poorly understood. Microbial degradation of fucoidans is slower than that of other polysaccharides, suggesting that fucoidans are more recalcitrant and may sequester carbon in the ocean. This may be due to the complex, branched and highly sulfated structure of fucoidans, which also varies among species of brown algae. Here, we show that 'Lentimonas' sp. CC4, belonging to the Verrucomicrobia, acquired a remarkably complex machinery for the degradation of six different fucoidans. The strain accumulated 284 putative fucoidanases, including glycoside hydrolases, sulfatases and carbohydrate esterases, which are primarily located on a 0.89-megabase pair plasmid. Proteomics reveals that these enzymes assemble into substrate-specific pathways requiring about 100 enzymes per fucoidan from different species of brown algae. These enzymes depolymerize fucoidan into fucose, which is metabolized in a proteome-costly bacterial microcompartment that spatially constrains the metabolism of the toxic intermediate lactaldehyde. Marine metagenomes and microbial genomes show that Verrucomicrobia including 'Lentimonas' are abundant and highly specialized degraders of fucoidans and other complex polysaccharides. Overall, the complexity of the pathways underscores why fucoidans are probably recalcitrant and more slowly degraded, since only highly specialized organisms can effectively degrade them in the ocean.

Diversity and potential biogeochemical impacts of viruses in bulk and rhizosphere soils.

Viruses can affect microbial dynamics, metabolism and biogeochemical cycles in aquatic ecosystems. However, viral diversity and functions in agricultural soils are poorly known, especially in the rhizosphere. We used virome analysis of eight rhizosphere and bulk soils to study viral diversity and potential biogeochemical impacts in an agro-ecosystem. The order Caudovirales was the predominant viral type in agricultural soils, with Siphoviridae being the most abundant family. Phylogenetic analysis of the terminase large subunit of Caudovirales identified high viral diversity and three novel groups. Viral community composition differed significantly between bulk and rhizosphere soils. Soil pH was the main environmental driver of the viral community structure. Remarkably, abundant auxiliary carbohydrate-active enzyme (CAZyme) genes were detected in viromes, including glycoside hydrolases, carbohydrate esterases and carbohydrate-binding modules. These results demonstrate that virus-encoded putative auxiliary metabolic genes or metabolic genes that may change bacterial metabolism and indirectly contribute to biogeochemical cycling, especially carbon cycling, in agricultural soil.

Characterization of novel α-galactosidase in glycohydrolase family 97 from Bacteroides thetaiotaomicron and its immobilization for industrial application

Bacteroides thetaiotaomicron (B. thetaiotaomicron), which resides in the human intestinal tract, has a number of carbohydrate enzymes, including glycoside hydrolase (GH) family 97. Only a few GH 97 enzymes have been characterized to date. In this study, a novel α-galactosidase (Bt_3294) was cloned from B. thetaiotaomicron, expressed in Escherichia coli, and purified using affinity chromatography. This novel enzyme showed optimal activity at 60 °C and pH 7.0. Enzyme activity was reduced by 94.4% and 95.7% in the presence of 5 mM Ca2+ and Fe2+, respectively. It is interesting that Bt_3294 specifically hydrolyzed shorter α-galactosyl oligosaccharides, such as melibiose and raffinose. The D-values of Bt_3294 at 40 °C and 50 °C were about 107 and 6 min, respectively. After immobilization of Bt_3294, the D-values at 40 °C and 50 °C were about 37.6 and 29.7 times higher than those of the free enzyme, respectively. As a practical application, the immobilized Bt_3294 was used to hydrolyze raffinose family oligosaccharides (RFOs) in soy milk, decreasing the RFOs by 98.9%.

Transcriptional and Metabolomic Analyses Indicate that Cell Wall Properties are Associated with Drought Tolerance in Brachypodium distachyon.

Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for “regulated stress response”, “plant cell wall” and “oxidative stress” associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops.

Transcriptome analysis of differential gene expression in Dichomitus squalens during interspecific mycelial interactions and the potential link with laccase induction.

Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.