Microbial carbohydrate active enzyme (CAZyme) genes and diversity from Menagesha Suba natural forest soils of Ethiopia as revealed by shotgun metagenomic sequencing.

BACKGROUND: The global over-reliance on non-renewable fossil fuels has led to the emission of greenhouse gases, creating a critical global environmental challenge. There is an urgent need for alternative solutions like biofuels. Advanced biofuel is a renewable sustainable energy generated from lignocellulosic plant materials, which can significantly contribute to mitigating CO2 emissions. Microbial Carbohydrate Active Enzymes (CAZymes) are the most crucial enzymes for the generation of sustainable biofuel energy. The present study designed shotgun metagenomics approaches to assemble, predict, and annotate, aiming to gain an insight into the taxonomic diversity, annotate CAZymes, and identify carbohydrate hydrolyzing CAZymes from microbiomes in Menagesha suba forest soil for the first time. RESULTS: The microbial diversity based on small subunit (SSU) rRNA analysis revealed the dominance of the bacterial domain representing 81.82% and 92.31% in the studied samples. Furthermore, the phylum composition result indicated the dominance of the phyla Proteobacteria (23.08%, 27.27%), Actinobacteria (11.36%, 20.51%), and Acidobacteria (10.26%, 15.91%). The study also identified unassigned bacteria which might have a unique potential for biopolymer hydrolysis. The metagenomic study revealed that 100,244 and 65,356 genes were predicted from the two distinct samples. A total number of 1806 CAZyme genes were identified, among annotated CAZymes, 758 had a known enzyme assigned to CAZymes. Glycoside hydrolases (GHs) CAZyme family contained most of the CAZyme genes with known enzymes such as ß-glucosidase, endo-ß-1,4-mannanase, exo-ß-1,4-glucanase, α-L-arabinofuranosidase and oligoxyloglucan reducing end-specific cellobiohydrolase. On the other hand, 1048 of the identified CAZyme genes were putative CAZyme genes with unknown enzymatical activity and the majority of which belong to the GHs family. CONCLUSIONS: In general, the identified putative CAZymes genes open up an opportunity for the discovery of new enzymes responsible for hydrolyzing biopolymers utilized for biofuel energy generation. This finding is used as a first-hand piece of evidence to serve as a benchmark for further and comprehensive studies to unveil novel classes of bio-economically valuable genes and their encoded products.

Expansion of Auxiliary Activity Family 5 sequence space via biochemical characterization of six new copper radical oxidases.

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.

Metagenomic analysis reveals a dynamic rumen microbiome with diversified adaptive functions in response to dietary protein restriction and re-alimentation.

Understanding the dynamics of the rumen microbiome is crucial for optimizing ruminal fermentation to improve feed efficiency and addressing concerns regarding antibiotic resistance in the livestock production industry. This study aimed to investigate the adaptive effects of microbiome and the properties of carbohydrate-active enzymes (CAZy) and antibiotic resistance genes (ARGs) in response to dietary protein shifts. Twelve Charolais bulls were randomly divided into two groups based on initial body weight: 1) Treatment (REC), where the animals received a 7 % CP diet in a 4-week restriction period, followed by a 13 % CP diet in a 2-week re-alimentation period; 2) Control (CON), where the animals were fed the 13 % CP diet both in the restriction period and the re-alimentation period. Protein restriction decreased the concentrations of acetate, propionate, isovalerate, glutamine, glutamate, and isoleucine (P < 0.05), while protein re-alimentation increased the concentrations of arginine, methionine sulfoxide, lysine, and glutamate (P < 0.05). Protein restriction decreased the relative abundances of Bacteroidota but increased Proteobacteria, with no difference observed after re-alimentation. Protein restriction decreased relative abundances of the genera Bacteroides, Prevotella, and Bifidobacterium. Following protein recovery, Escherichia was enriched in CON, while Pusillibacter was enriched in REC, indicating that distinct microbial adaptations to protein shifts. Protein restriction increased GH97 while reducing GH94 and GT35 compared to CON. Protein restriction decreased abundances of KO genes involved in VFA production pathways, while they were recovered in the re-alimentation period. Protein restriction reduced tet(W/32/O) abundances but increased those of tet(X), nimJ, and rpoB2. Following protein re-alimentation, there was a decrease in ErmQ and tet(W/N/W), and an increase in Mef(En2) compared to CON, highlighting the impact of dietary protein on the distribution of antibiotic-resistant bacteria. Overall, comprehensive metagenomic analysis reveals the dynamic adaptability of the microbiome in response to dietary shifts, indicating its capacity to modulate carbohydrate metabolism and ARGs in response to protein availability.

Metagenomic and culture-dependent analysis of Rhinopithecius bieti gut microbiota and characterization of a novel genus of Sphingobacteriaceae.

Culture-dependent and metagenomic binning techniques were employed to gain an insight into the diversification of gut bacteria in Rhinopithecius bieti, a highly endangered primate endemic to China. Our analyses revealed that Bacillota_A and Bacteroidota were the dominant phyla. These two phyla species are rich in carbohydrate active enzymes, which could provide nutrients and energy for their own or hosts' survival under different circumstances. Among the culturable bacteria, one novel bacterium, designated as WQ 2009T, formed a distinct branch that had a low similarity to the known species in the family Sphingobacteriaceae, based on the phylogenetic analysis of its 16S rRNA gene sequence or phylogenomic analysis. The ANI, dDDH and AAI values between WQ 2009T and its most closely related strains S. kitahiroshimense 10CT, S. pakistanense NCCP-246T and S. faecium DSM 11690T were significantly lower than the accepted cut-off values for microbial species delineation. All results demonstrated that WQ 2009T represent a novel genus, for which names Rhinopithecimicrobium gen. nov. and Rhinopithecimicrobium faecis sp. nov. (Type strain WQ 2009T = CCTCC AA 2021153T = KCTC 82941T) are proposed.

Microbe Profile: Cellvibrio japonicus: living the sweet life via biomass break-down.

Cellvibrio japonicus is a saprophytic bacterium proficient at environmental polysaccharide degradation for carbon and energy acquisition. Genetic, enzymatic, and structural characterization of C. japonicus carbohydrate active enzymes, specifically those that degrade plant and animal-derived polysaccharides, demonstrated that this bacterium is a carbohydrate-bioconversion specialist. Structural analyses of these enzymes identified highly specialized carbohydrate binding modules that facilitate activity. Steady progress has been made in developing genetic tools for C. japonicus to better understand the function and regulation of the polysaccharide-degrading enzymes it possesses, as well as to develop it as a biotechnology platform to produce renewable fuels and chemicals.

Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides.

BACKGROUND: Marine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses. RESULTS: Prominent active 0.2-3 µm free-living clades comprised Aurantivirga, "Formosa", Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3-10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria. CONCLUSIONS: Particle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events. Video Abstract.

Current models in bacterial hemicellulase-encoding gene regulation.

The discovery and characterization of bacterial carbohydrate-active enzymes is a fundamental component of biotechnology innovation, particularly for renewable fuels and chemicals; however, these studies have increasingly transitioned to exploring the complex regulation required for recalcitrant polysaccharide utilization. This pivot is largely due to the current need to engineer and optimize enzymes for maximal degradation in industrial or biomedical applications. Given the structural simplicity of a single cellulose polymer, and the relatively few enzyme classes required for complete bioconversion, the regulation of cellulases in bacteria has been thoroughly discussed in the literature. However, the diversity of hemicelluloses found in plant biomass and the multitude of carbohydrate-active enzymes required for their deconstruction has resulted in a less comprehensive understanding of bacterial hemicellulase-encoding gene regulation. Here we review the mechanisms of this process and common themes found in the transcriptomic response during plant biomass utilization. By comparing regulatory systems from both Gram-negative and Gram-positive bacteria, as well as drawing parallels to cellulase regulation, our goals are to highlight the shared and distinct features of bacterial hemicellulase-encoding gene regulation and provide a set of guiding questions to improve our understanding of bacterial lignocellulose utilization. KEY POINTS: • Canonical regulatory mechanisms for bacterial hemicellulase-encoding gene expression include hybrid two-component systems (HTCS), extracytoplasmic function (ECF)-σ/anti-σ systems, and carbon catabolite repression (CCR). • Current transcriptomic approaches are increasingly being used to identify hemicellulase-encoding gene regulatory patterns coupled with computational predictions for transcriptional regulators. • Future work should emphasize genetic approaches to improve systems biology tools available for model bacterial systems and emerging microbes with biotechnology potential. Specifically, optimization of Gram-positive systems will require integration of degradative and fermentative capabilities, while optimization of Gram-negative systems will require bolstering the potency of lignocellulolytic capabilities.

Cellulomonas alba sp. nov. and Cellulomonas edaphi sp. nov., isolated from wetland soils.

Two novel strains were isolated from wetland soils in Goyang, Republic of Korea. The two Gram-stain-positive, facultatively anaerobic, rod-shaped bacterial-type strains were designated MW4T and MW9T. Phylogenomic analysis based on whole-genome sequences suggested that both strains belonged to the genus Cellulomonas. The cells of strain MW4T were non-motile and grew at 20-40 °C (optimum, 35 °C), at pH 6.0-10.0 (optimum, pH 8.0) and in the presence of 0-1.0% NaCl (optimum, 0 %). The cells of strain MW9T were non-motile and grew at 20-40 °C (optimum, 35 °C), at pH 5.0-9.0 (optimum, pH 8.0) and in the presence of 0-1.0% NaCl (optimum, 0 %). The average nucleotide identity (77.1-88.1 %) and digital DNA-DNA hybridization values (21.0-34.8 %) between the two novel strains and with their closely related strains fell within the range for the genus Cellulomonas. The novel strains MW4T and MW9T and reference strains possessed alkane synthesis gene clusters (oleA, oleB, oleC and oleD). Phylogenomic, phylogenetic, average nucleotide identity, digital DNA-DNA hybridization, physiological and biochemical data indicated that the novel strains were distinct from other members of the family Cellulomonadaceae. We propose the names Cellulomonas alba sp. nov. (type strain MW4T=KACC 23260T=TBRC 17645T) and Cellulomons edaphi sp. nov. (type strain MW9T=KACC 23261T=TBRC 17646T) for the two strains.

Rumen microbes, enzymes, metabolisms, and application in lignocellulosic waste conversion - A comprehensive review.

The rumen of ruminants is a natural anaerobic fermentation system that efficiently degrades lignocellulosic biomass and mainly depends on synergistic interactions between multiple microbes and their secreted enzymes. Ruminal microbes have been employed as biomass waste converters and are receiving increasing attention because of their degradation performance. To explore the application of ruminal microbes and their secreted enzymes in biomass waste, a comprehensive understanding of these processes is required. Based on the degradation capacity and mechanism of ruminal microbes and their secreted lignocellulose enzymes, this review concentrates on elucidating the main enzymatic strategies that ruminal microbes use for lignocellulose degradation, focusing mainly on polysaccharide metabolism-related gene loci and cellulosomes. Hydrolysis, acidification, methanogenesis, interspecific H2 transfer, and urea cycling in ruminal metabolism are also discussed. Finally, we review the research progress on the conversion of biomass waste into biofuels (bioethanol, biohydrogen, and biomethane) and value-added chemicals (organic acids) by ruminal microbes. This review aims to provide new ideas and methods for ruminal microbe and enzyme applications, biomass waste conversion, and global energy shortage alleviation.

Gut flora metagenomic analysis coupled with metabolic and deep immune profiling in chronic kidney disease.

BACKGROUND: Perturbation of gut microbiota has been linked to chronic kidney disease (CKD), which was correlated with a sophisticated milieu of metabolic and immune dysregulation. METHODS: To clarify the underlying host-microbe interaction in CKD, we performed multi-omics measurements, including systems-level gut microbiome, targeted serum metabolome and deep immunotyping, in a cohort of patients and non-CKD controls. RESULTS: Our analyses on functional profiles of the gut microbiome showed a decrease in the diversity and abundance of carbohydrate-active enzyme (CAZyme) genes but an increase in the abundance of antibiotic resistance, nitrogen cycling enzyme and virulence factor genes in CKD. Moreover, models generated using measurements of serum metabolites (amino acids, bile acids and short-chain fatty acids) or immunotypes were predictive of renal impairment but less so than many of the functional profiles derived from gut microbiota, with the CAZyme genes being the top-performing model to accurately predict the early stage of diseases. In addition, co-occurrence analyses revealed coordinated host-microbe relationships in CKD. Specifically, the highest fractions of significant correlations were identified with circulating metabolites by several taxonomic and functional profiles of gut microbiome, while immunotype features were moderately associated with the abundance of microbiome-encoded metabolic pathways and serum levels of amino acids (e.g. B cell cluster tryptophan and B cell cluster tryptophan metabolism). CONCLUSION: Overall, our multi-omics integration revealed several signatures of systems-level gut microbiome in robust associations with host-microbe co-metabolites and renal function, which may have aetiological and diagnostic implications in CKD.

Independent, cooperative regulation of cellulolytic genes by paralogous transcription factors ClbR and ClbR2 in Aspergillus aculeatus.

The cellobiose-responsive regulator ClbR, a Zn(II)2Cys6 binuclear-cluster transcription factor, is a positive regulator of carbohydrate-active enzyme (CAZyme) genes responsive to cellulose in Aspergillus aculeatus. Because Zn(II)2Cys6 transcription factors tend to dimerize with proteins of the same family, we searched for a counterpart of ClbR and identified ClbR2, which is 42% identical to ClbR, as an interacting partner of ClbR by yeast two-hybrid screening. Genetic analyses suggested that ClbR and ClbR2 cooperatively regulate the expression of CAZyme genes in response to cellulose and 1,4-ß-mannobiose in A. aculeatus. CAZyme genes under the control of the transcription factor ManR were regulated by ClbR and ClbR2, whereas those controlled by the transcription factor XlnR were regulated by ClbR, but not ClbR2. These findings suggest that ClbR participates in multiple regulatory pathways in A. aculeatus by altering an interacting factor.

Metagenomic analysis fecal microbiota of dysentery-like diarrhoea in a pig farm using next-generation sequencing.

Porcine enteric diseases including swine dysentery involves a wide range of possible aetiologies and seriously damages the intestine of pigs of all ages. Metagenomic next-generation sequencing is commonly used in research for detecting and analyzing pathogens. In this study, the feces of pigs from a commercial swine farm with dysentery-like diarrhea was collected and used for microbiota analysis by next-generation sequencing. While Brachyspira spp. was not detected in diarrheal pig fecal samples, indicating that the disease was not swine dysentery. The quantity of microbial population was extremely lowered, and the bacterial composition was altered with a reduction in the relative abundance of the probiotics organisms, Firmicutes and Bacteroidetes, with an increase in pathogens like Fusobacterium and Proteobacteria, in which the specific bacteria were identified at species-level. Viral pathogens, porcine circovirus type 2, porcine lymphotropic herpesviruses 1, and porcine mastadenovirus A were also detected at pretty low levels. Carbohydrate-active enzymes (CAZy) analysis indicated that the constitute of Firmicutes and Bacteroidete were also changed. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) alignment analysis indicated that the microbiota of diarrheal pigs had a lower ability in utilizing energy sources but were enriched in multi-drug resistance pathways. Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factors of Pathogenic Bacteria (VFDB) analysis indicated that genes for elfamycin and sulfonamide resistance and the iron uptake system were enriched in diarrheal pigs. This revealed potential bacterial infection and can guide antibiotic selection for treating dysentery. Overall, our data suggested that alterations in both the population and functional attributes of microbiota in diarrheal pigs with decreased probiotic and increased pathogenic microorganisms. These results will help elucidate the mechanism of dysentery-like diarrhea and the development of approaches to control the disease.

The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase.

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the WT enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using small-angle X-ray scattering, NMR, molecular dynamics simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the WT and a serine-rich variant, which have the same linker length, demonstrated better performance compared with those with either a shorter linker or a longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, whereas the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass-processing enzymes is more complex than mere structural tethering.

Screening and genome-wide analysis of lignocellulose-degrading bacteria from humic soil.

Crop straw contains huge amounts of exploitable energy, and efficient biomass degradation measures have attracted worldwide attention. Mining strains with high yields of cellulose-degrading enzymes is of great significance for developing clean energy and industrial production of related enzymes. In this study, we reported a high-quality genome sequence of Bacillus velezensis SSF6 strain using high-throughput sequencing technology (Illumina PE150 and PacBio) and assessed its lignocellulose degradation potential. The results demonstrated that the genome of B. velezensis SSF6 was 3.89 Mb and contained 4,015 genes, of which 2,972, 3,831 and 158 genes were annotated in the COGs (Clusters of Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes) and CAZyme (Carbohydrate-Active enZymes) databases, respectively, and contained a large number of genes related to carbohydrate metabolism. Furthermore, B. velezensis SSF6 has a high cellulose degradation capacity, with a filter paper assay (FPA) and an exoglucanase activity of 64.48 ± 0.28 and 78.59 ± 0.42 U/mL, respectively. Comparative genomic analysis depicted that B. velezensis SSF6 was richer in carbohydrate hydrolase gene. In conclusion, the cellulose-degrading ability of B. velezensis SSF6 was revealed by genome sequencing and the determination of cellulase activity, which laid a foundation for further cellulose degradation and bioconversion.

Surface Plasmon Resonance Analysis for Quantifying Protein-Carbohydrate Interactions.

During the past two decades, surface plasmon resonance (SPR) analysis has emerged as an important tool for studying protein-carbohydrate interactions, with several commercial instruments available. Binding affinities in the nM to mM range can be determined; however, there are pitfalls that require careful experimental design to avoid. Here we give an overview of each step in the SPR analysis from immobilization to data analysis, providing key points of consideration that will allow practitioners to achieve reliable and reproducible results.

Identification of Genes Involved in the Degradation of Lignocellulose Using Comparative Transcriptomics.

Lignocellulosic biomass represents an abundant, renewable resource that can be used to produce biofuels, low-cost livestock feed, and high-value chemicals. The potential of this bioresource has led to intensive research efforts to develop cost-effective methods to break down lignocellulose. The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well recognized and in recent years has received renewed interest. Transcriptomics has been used to identify enzymes that are expressed by these fungi and are involved in the degradation of a range of lignocellulose feedstocks. The transcriptome is the entire complement of coding and non-coding RNA transcripts that are expressed by a cell under a particular set of conditions. Monitoring changes in gene expression can provide fundamental information about the biology of an organism. Here we outline a general methodology that will enable researchers to conduct comparative transcriptomic studies with the goal of identifying enzymes involved in the degradation of the plant cell wall. The method described will include growth of fungal cultures, isolation and sequencing of RNA, and a basic description of data analysis for bioinformatic identification of differentially expressed transcripts.

Differential effects of Bacillus subtilis- and Bacillus licheniformis-fermented products on growth performance, intestinal morphology, intestinal antioxidant and barrier function gene expression, cecal microbiota community, and microbial carbohydrate-active enzyme composition in broilers.

This study investigated the differential effects of Bacillus subtilis-fermented products (SFPs) and Bacillus licheniformis-fermented products (LFPs) on the growth performance, intestinal morphology, intestinal gene expression, cecal microbiota community, and microbial carbohydrate-active enzyme composition of broilers. In total, 160 one-day-old unsexed Arbor Acres broiler chicks were randomly allocated to 4 treatment groups (with 8 replicates per group and 5 chicks per replicate): control (CON), enramycin (ENM), SFP, and LFP groups, which were fed a basal diet, basal diet supplemented with 10 mg/kg ENM, basal diet supplemented with 108 colony-forming units (CFU) of B. subtilis spores per gram of feed, and basal diet supplemented with 108 CFU of B. licheniformis spores per gram of feed, respectively. LFP treatment resulted in a higher (P < 0.01) body weight at the age of 35 d and higher average daily gain over 15 to 35 (P < 0.05) and 1 to 35 (P < 0.05) d of age than did the CON and SFP treatments. The average villus heights in the jejunum were longer (P < 0.05) in the LFP group than those in the SFP group. Moreover, the LFP group exhibited a higher jejunal barrier function gene expression (mucin 2, zonula occludens-1, and occludin) and cecal antioxidant gene expression (superoxide dismutase) than did the CON group (P < 0.05). A principal coordinate analysis of cecal microbiota and carbohydrate-active enzyme composition demonstrated distinct clustering among the groups. Lactobacillus crispatus abundance in the cecal digesta was higher (P < 0.01) in the SFP group than in the CON and LFP groups. Finally, microbial glycosyltransferase 2 gene expression in the cecal digesta was higher in the LFP group than in the CON and ENM groups. In conclusion, LFPs can improve the growth performance, increase intestinal barrier function and antioxidant gene expression, and modulate cecal microflora composition and carbohydrate-active enzyme composition of broilers. The overall effect of LFPs on growth promotion in broilers was superior to that of SFPs.

Lignocellulose degradation by rumen bacterial communities: New insights from metagenome analyses.

Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment for the storage and microbial fermentation of ingested feeds dominated by plant materials. The rumen microbiota has acquired diverse and functionally overlapped enzymes for the degradation of plant cell wall polysaccharides. In rumen Bacteroidetes, enzymes involved in degradation are clustered into polysaccharide utilization loci to facilitate coordinated expression when target polysaccharides are available. Firmicutes use free enzymes and cellulosomes to degrade the polysaccharides. Fibrobacters either aggregate lignocellulose-degrading enzymes on their cell surface or release them into the extracellular medium in membrane vesicles, a mechanism that has proven extremely effective in the breakdown of recalcitrant cellulose. Based on current metagenomic analyses, rumen Bacteroidetes and Firmicutes are categorized as generalist microbes that can degrade a wide range of polysaccharides, while other members adapted toward specific polysaccharides. Particularly, there is ample evidence that Verrucomicrobia and Spirochaetes have evolved enzyme systems for the breakdown of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin. It is concluded that diversity in degradation mechanisms is required to ensure that every component in feeds is efficiently degraded, which is key to harvesting maximum energy by host animals.

A holobiont approach towards polysaccharide degradation by the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis.

BACKGROUND: Termites are among the most successful insects on Earth and can feed on a broad range of organic matter at various stages of decomposition. The termite gut system is often referred to as a micro-reactor and is a complex structure consisting of several components. It includes the host, its gut microbiome and fungal gardens, in the case of fungi-growing higher termites. The digestive tract of soil-feeding higher termites is characterised by radial and axial gradients of physicochemical parameters (e.g. pH, O2 and H2 partial pressure), and also differs in the density and structure of residing microbial communities. Although soil-feeding termites account for 60% of the known termite species, their biomass degradation strategies are far less known compared to their wood-feeding counterparts. RESULTS: In this work, we applied an integrative multi-omics approach for the first time at the holobiont level to study the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis. We relied on 16S rRNA gene community profiling, metagenomics and (meta)transcriptomics to uncover the distribution of functional roles, in particular those related to carbohydrate hydrolysis, across different gut compartments and among the members of the bacterial community and the host itself. We showed that the Labiotermes gut was dominated by members of the Firmicutes phylum, whose abundance gradually decreased towards the posterior segments of the hindgut, in favour of Bacteroidetes, Proteobacteria and Verrucomicrobia. Contrary to expectations, we observed that L. labralis gut microbes expressed a high diversity of carbohydrate active enzymes involved in cellulose and hemicelluloses degradation, making the soil-feeding termite gut a unique reservoir of lignocellulolytic enzymes with considerable biotechnological potential. We also evidenced that the host cellulases have different phylogenetic origins and structures, which is possibly translated into their different specificities towards cellulose. From an ecological perspective, we could speculate that the capacity to feed on distinct polymorphs of cellulose retained in soil might have enabled this termite species to widely colonise the different habitats of the Amazon basin. CONCLUSIONS: Our study provides interesting insights into the distribution of the hydrolytic potential of the highly compartmentalised higher termite gut. The large number of expressed enzymes targeting the different lignocellulose components make the Labiotermes worker gut a relevant lignocellulose-valorising model to mimic by biomass conversion industries.