Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

Metatranscriptomics—gene express profiling via DNA sequencing—is a powerful tool to identify genes that are actively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial community (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhouse gas twenty times more potent than carbon dioxide, and is of general importance for the physiological wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socioeconomically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accurate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-incubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with previously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.

Washed microbiota transplantation for Crohn’s disease:A metagenomic,metatranscriptomic,and metabolomic-based study

BACKGROUND Fecal microbiota transplantation(FMT)is a promising therapeutic approach for treating Crohn’s disease(CD).The new method of FMT,based on the automatic washing process,was named as washed microbiota transplantation(WMT).Most existing studies have focused on observing the clinical phenomena.However,the mechanism of action of FMT for the effective management of CD-particularly in-depth multi-omics analysis involving the metagenome,metatranscriptome,and metabolome-has not yet been reported.AIM To assess the efficacy of WMT for CD and explore alterations in the microbiome and metabolome in response to WMT.METHODS We conducted a prospective,open-label,single-center clinical study.Eleven CD patients underwent WMT.Their clinical responses(defined as a decrease in their CD Activity Index score of>100 points)and their microbiome(metagenome,metatranscriptome)and metabolome profiles were evaluated three months after the procedure.RESULTS Seven of the 11 patients(63.6%)showed an optimal clinical response three months post-WMT.Gut microbiome diversity significantly increased after WMT,consistent with improved clinical symptoms.Comparison of the metagenome and metatranscriptome analyses revealed consistent alterations in certain strains,such as Faecalibac-terium prausnitzii,Roseburia intestinalis,and Escherichia coli.In addition,metabolomics analyses demonstrated that CD patients had elevated levels of various amino acids before treatment compared to the donors.However,levels of vital amino acids that may be associated with disease progression(e.g.,L-glutamic acid,gamma-glutamyl-leucine,and prolyl-glutamine)were reduced after WMT.CONCLUSION WMT demonstrated therapeutic efficacy in CD treatment,likely due to the effective reconstruction of the patient’s microbiome.Multi-omics techniques can effectively help decipher the potential mechanisms of WMT in treating CD.

Reconstruction and Dynamics of the Human Intestinal Microbiome Observed In Situ

The human gut microbiome has primarily been studied through the use of fecal samples,a practice that has generated vital knowledge on the composition and functional capacities of gastrointestinal microbial communities.However,this reliance on fecal materials limits the investigation of microbial dynamics in other locations along the gastrointestinal tract(in situ),and the infrequent availability of fecal samples prevents analysis at finer temporal scales(e.g.,hours).In our study,we utilized colonic transendoscopic enteral tubing,a technology originally developed for fecal microbiota transplantation,to sample the ileocecal microbiome twice daily;metagenomic and metatranscriptomic analyses were then conducted on these samples.A total of 43 ileocecal and 28 urine and fecal samples were collected from five healthy volunteers.The ileocecal and fecal microbiomes,as profiled in the five volunteers,were found to be similar in metagenomic profiling,yet their active genes(metatranscriptome)were found to be highly distinct.Both microbiomes were perturbed after laxative exposure;over time,they exhibited reduced dissimilarity to their pre-treatment state,thereby demonstrating resilience as an innate property of the gut microbiome,although they did not fully recover within our observation time window.Sampling of the ileocecal microbiome during the day and at night revealed the existence of diurnal rhythms in a series of bacterial species and functional pathways,particularly those related to short-chain fatty acid production,such as Propionibacterium acnes and coenzyme A biosynthesis Ⅱ.Autocorrelation analysis and fluctuations decomposition further indicated the significant periodicity of the diurnal oscillations.Metabolomic profiling in the fecal and urine samples mirrored the perturbance and recovery in the gut microbiome,indicating the crucial contribution of the gut microbiome to many key metabolites involved in host health.This study provides novel insights into the human gut microbiome and its inner resilience and diurnal rhythms,as well as the potential consequences of these to the host.

Genetic diversity of RNA viruses infecting invertebrate pests of rice

Invertebrate species are a natural reservoir of viral genetic diversity,and invertebrate pests are widely distributed in crop fields.However,information on viruses infecting invertebrate pests of crops is limited.In this report,we describe the deep metatranscriptomic sequencing of 88 invertebrate samples covering all major invertebrate pests in rice fields.We identified 296 new RNA viruses and 13 known RNA viruses.These viruses clustered within 31 families,with many highly divergent viruses constituting potentially new families and genera.Of the identified viruses,13 RNA viruses clustered within the Fiersviridae family of bacteriophages,and 48 RNA viruses clustered within families and genera of mycoviruses.We detected known rice viruses in novel invertebrate hosts at high abundances.Furthermore,some novel RNA viruses have genome structures closely matching to known plant viruses and clustered within genera of several plant virus species.Fortyfive potential insect pathogenic RNA viruses were detected in invertebrate species.Our analysis revealed that host taxonomy plays a major role and geographical location plays an important role in structuring viral diversity.Cross-species transmission of RNA viruses was detected between invertebrate hosts.Newly identified viral genomes showed extensive variation for invertebrate viral families or genera.Together,the large-scale metatranscriptomic analysis greatly expands our understanding of RNA viruses in rice invertebrate species,the results provide valuable information for developing efficient strategies to manage insect pests and virus-mediated crop diseases.

Unraveling the skatole biodegradation process in an enrichment consortium using integrated omics and culture-dependent strategies

3-Methylindole(skatole)is regarded as one of the most offensive compounds in odor emission.Biodegradation is feasible for skatole removal but the functional species and genes responsible for skatole degradation remain enigmatic.In this study,an efficient aerobic skatole-degrading consortium was obtained.Rhodococcus and Pseudomonas were identified as the two major and active populations by integrated metagenomic and metatranscriptomic analyses.Bioinformatic analyses indicated that the skatole downstream degradation wasmainly via the catechol pathway,and upstream degradation was likely catalyzed by the aromatic ring-hydroxylating oxygenase and flavin monooxygenase.Genome binning and gene analyses indicated that Pseudomonas,Pseudoclavibacter,and Raineyella should cooperate with Rhodococcus for the skatole degradation process.Moreover,a pure strain Rhodococcus sp.DMU1 was successfully obtained which could utilize skatole as the sole carbon source.Complete genome sequencing showed that strain DMU1 was the predominant population in the consortium.Further crude enzyme and RT-qPCR assays indicated that strain DMU1 degraded skatole through the catechol ortho-cleavage pathway.Collectively,our results suggested that synergistic degradation of skatole in the consortium should be performed by diverse bacteria with Rhodococcus as the primary degrader,and the degradation mainly proceeded via the catechol pathway.

Exploration of the metabolic flexibility of glycogen accumulating organisms through metatranscriptome analysis and metabolic characterization

Glycogen accumulating organisms(GAOs) are closely related to the deterioration of enhanced biological phosphorus removal systems. However, the metabolic mechanisms that drive GAOs remain unclear. Here, the two-thirds supernatant of a reactor were decanted following the anaerobic period to enrich GAOs. Long-term monitoring demonstrated that the system was stable and exhibited typical characteristics of GAOs metabolism. Acetate was completely consumed after 60 min of the anaerobic phase. The level of glycogen decreased from 0.20 to 0.14 g/gSS during the anaerobic phase, whereas the level of glycogen significantly increased to 0.21g/gSS at the end of the aerobic period. Moreover, there was almost no phosphate release and absorption in the complete periods, thus confirming the successful construction of a GAOs enrichment system. Microbial community analysis demonstrated that Ca. Contendobacter was among the core functional genera and showed the highest activity among all of the communities. Furthermore, our study is the first to identify the involvement of the ethyl-malonyl-CoA pathway in the synthesis of polyhydroxyvalerate via croR, ccr, ecm, mcd, mch and mcl genes. The Embden-Meyerhof-Parnas(EMP) pathway was preferentially used via glgP. Furthermore, the glyoxylate cycle was the main source of ATP under anaerobic conditions, whereas the tricarboxylic acid cycle provided ATP under aerobic conditions. aceA and mdh appeared to be major modulators of the glyoxylate pathway for controlling energy flow. Collectively, our findings not only revealed the crucial metabolic mechanisms in a GAOs enrichment system but also provided insights into the potential application of Ca. Contendobacter for wastewater treatment.

Plant-associated fungal communities in the light of meta’omics

Approaches for the cultivation-independent analysis of microbial communities are summarized as meta’omics,which predominantly includes metagenomic,-transcriptomic,-proteomic and-metabolomic studies.These have shown that endophytic,root-associated and soil fungal communities are strongly shaped by associated plant species.The impact of plant identity on the composition of its litterssociated fungal community remains to be disentangled from the impact of litter chemistry.The composition of the plant community also shapes the fungal community.Most strikingly,adjacent plant species may share mycorrhizal symbionts even if the plants usually have different types of mycorrhizal fungi associated with them(ectomycorrhizal,ericoid and arbuscular mycorrhizal fungi).Environmental parameters weakly explain fungal community composition globally,and their effect is inconsistent at local and regional scales.Decrease in similarity among communities with increasing distance(i.e.distance decay)has been reported from local to global scales.This pattern is only exceptionally caused by spatial dispersal limitation of fungal propagules,but mostly due to the inability of the fungi to establish at the particular locality(i.e.environmental filtering or competitive exclusion).Fungal communities usually undergo pronounced seasonal changes and also differ between consecutive years.This indicates that development of the communities is usually not solely cyclic.Meta’omic studies challenge the classical view of plant litter decomposition.They show that mycorrhizal and(previously)endophytic fungi may be involved in plant litter decomposition and only partly support the idea of a succession from an Ascomycota to a Basidiomycota-dominated community.Furthermore,vertical separation of saprotrophic and mycorrhizal species in soil and sequential degradation from easily accessible to‘recalcitrant’plant compounds,such as lignin,can probably not be generalized.The current models of litter decomposition may therefore have to be eventually refined for certain ecosystems and environmental conditions.To gain deeper insights into fungal ecology,a meta’omic study design is outlined which focuses on environmental processes,because fungal communities are usually taxonomically diverse,but functionally redundant.This approach would initially identify dynamics of chemical shifts in the host and/or substrate by metametabolomics.Detected shifts would be subsequently linked to microbial activity by correlation with metatranscriptomic and/or metaproteomic data.A holistic trait-based approach might finally identify factors shaping taxonomic composition in communities against the dynamics of the environmental process(es)they are involved in.

Diverse Asgard archaea including the novel phylum Gerdarchaeota participate in organic matter degradation

Asgard is an archaeal superphylum that might hold the key to understand the origin of eukaryotes, but its diversity and ecological roles remain poorly understood. Here, we reconstructed 15 metagenomic-assembled genomes from coastal sediments covering most known Asgard archaea and a novel group, which is proposed as a new Asgard phylum named as the "Gerdarchaeota".Genomic analyses predict that Gerdarchaeota are facultative anaerobes in utilizing both organic and inorganic carbon. Unlike their closest relatives Heimdallarchaeota, Gerdarchaeota have genes encoding for cellulase and enzymes involved in the tetrahydromethanopterin-based Wood–Ljungdahl pathway. Transcriptomics showed that most of our identified Asgard archaea are capable of degrading organic matter, including peptides, amino acids and fatty acids, occupying ecological niches in different depths of layers of the sediments. Overall, this study broadens the diversity of the mysterious Asgard archaea and provides evidence for their ecological roles in coastal sediments.

Genomic and transcriptomic dissection of Theionarchaea in marine ecosystem

Theionarchaea is a recently described archaeal class within the Euryarchaeota.While it is widely distributed in sediment ecosystems,little is known about its metabolic potential and ecological features.Here,we used metagenomics and metatranscriptomics to characterize 12 theionarchaeal metagenome-assembled genomes,which were further divided into two subgroups,from coastal mangrove sediments of China and seawater columns of the Yap Trench.Genomic analysis revealed that apart from the canonical sulfhydrogenase,Theionarchaea harbor genes encoding heliorhodopsin,group 4[NiFe]-hydrogenase,and flagellin,in which genes for heliorhodopsin and group 4[NiFe]-hydrogenase were transcribed in mangrove sediment.Further,the theionarchaeal substrate spectrum may be broader than previously reported as revealed by metagenomics and metatranscriptomics,and the potential carbon substrates include detrital proteins,hemicellulose,ethanol,and CO_(2).The genes for organic substrate metabolism(mainly detrital protein and amino acid metabolism genes)have relatively higher transcripts in the top sediment layers in mangrove wetlands.In addition,co-occurrence analysis suggested that the degradation of these organic compounds by Theionarchaea might be processed in syntrophy with fermenters(e.g.,Chloroflexi)and methanogens.Collectively,these observations expand the current knowledge of the metabolic potential of Theionarchaea,and shed light on the metabolic strategies and roles of these archaea in the marine ecosystems.

Metatranscriptome of the protistan community in Reticufitermes flaviceps

The hindgut of lower termites harbors various symbiotic protists, which per- form varied functions in lignocellulose decomposition. As termites are social insects, the species and numbers of these flagellated protists in the termite gut vary among the different castes. Juvenile hormones （JHs） can regulate caste differentiation in termites. In this study, we used the juvenile hormone analog fenoxycarh to induce termite workers （Reticulitermesflaviceps） to differentiate into pre-soldiers. A metatranscriptomic investigation of the protistan community was then performed by 454 pyrosequencing. From a thorough analysis based on 597 312 generated reads, we found that the starch and sucrose metabolism pathway was the most abundant pathway across the metatranscriptome. The current study demonstrates that the metatranscriptome of the protistan community in termites contains an abundance of lignocellulase, which plays a vital role in termite nutrition.

Insights from metagenomic,metatranscriptomic,and molecular ecological network analyses into the effects of chromium nanoparticles on activated sludge system

The objectives of this study were to investigate the influence of chromium nanoparticles(Cr NPs)on the nitrogen and phosphorus removal performance and the bacterial structures of an activated sludge(AS)system.Also,we through molecular ecological networks(MENs)discussed the bacterial interactions.At last we researched the change of the functional genes and their expression patterns related to nitrogen and phosphorus removaT in an AS system.The results showed that long-term exposure to 1 mg/L Cr NPs significantly promoted the denitrifying process and phosphorus removal in the AS system.The relative abundance of denitrifying and phosphorus removal microorganisms,such as Denitratisoma,Thauera,Dechloromonas,and Defluviicoccus,increased significantly.Candidatus Accumulibacter,well-known as polyphosphate-accumulating organisms(PAOs),increased significantly;the relative abundance of Candidatus Competibacter,known as glycogen-accumulating organisms(GAOs),decreased significantly.Furthermore,metagenomic and metatranscriptomic analysis revealed that most of the genera related to denitrifying and phosphorus removal had greatly increased,according to the quantities of denitrifying and phosphorus genes,and the corresponding transcription likewise greatly increased.Lastly,MENs analysis showed that although the overall network became smaller and looser in the presence of Cr NPs,the microbial connections among members related to nitrogen and phosphorus removal were enhanced.The abundance increases of denitrifiers and PAOs,and their increased transcription of functional genes,together with the enhanced interactions may be associated with the promotion of the denitrifying process and phosphorus removal.