Ultrahigh resolution MS1/MS2-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action.

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS1/data independent-MS2 method to track the simultaneous incorporation of the heavy isotopes 13C and 15N into the moieties of purine/pyrimidine nucleotides in mammalian cells. Ultrahigh resolution-MS1 resolves and counts multiple tracer atoms in intact metabolites, while data independent-tandem MS (MS2) determines isotopic enrichment in their moieties without concern for the numerous mass isotopologue source ions to be fragmented. Together, they enabled rigorous MS-based reconstruction of metabolic networks at specific enzyme levels. We have expanded this approach to trace the labeled atom fate of [13C6]-glucose in 3D A549 spheroids in response to the anticancer agent selenite and that of [13C5,15N2]-glutamine in 2D BEAS-2B cells in response to arsenite transformation. We deduced altered activities of specific enzymes in the Krebs cycle, pentose phosphate pathway, gluconeogenesis, and UDP-GlcNAc synthesis pathways elicited by the stressors. These metabolic details help elucidate the resistance mechanism of 3D versus 2D A549 cultures to selenite and metabolic reprogramming that can mediate the transformation of BEAS-2B cells by arsenite.

Genome-Wide and Functional View of Proteolytic and Lipolytic Bacteria for Efficient Biogas Production through Enhanced Sewage Sludge Hydrolysis.

In this study, we used a multifaceted approach to select robust bioaugmentation candidates for enhancing biogas production and to demonstrate the usefulness of a genome-centric approach for strain selection for specific bioaugmentation purposes. We also investigated the influence of the isolation source of bacterial strains on their metabolic potential and their efficiency in enhancing anaerobic digestion. Whole genome sequencing, metabolic pathway reconstruction, and physiological analyses, including phenomics, of phylogenetically diverse strains, Rummeliibacillus sp. POC4, Ochrobactrum sp. POC9 (both isolated from sewage sludge) and Brevundimonas sp. LPMIX5 (isolated from an agricultural biogas plant) showed their diverse enzymatic activities, metabolic versatility and ability to survive under varied growth conditions. All tested strains display proteolytic, lipolytic, cellulolytic, amylolytic, and xylanolytic activities and are able to utilize a wide array of single carbon and energy sources, as well as more complex industrial by-products, such as dairy waste and molasses. The specific enzymatic activity expressed by the three strains studied was related to the type of substrate present in the original isolation source. Bioaugmentation with sewage sludge isolates-POC4 and POC9-was more effective for enhancing biogas production from sewage sludge (22% and 28%, respectively) than an approach based on LPMIX5 strain (biogas production boosted by 7%) that had been isolated from an agricultural biogas plant, where other type of substrate is used.

Reconstitution of Metabolic Pathway in Nicotiana benthamiana.

Reconstitution of metabolite biosynthesis pathway plays a pivotal role in functional characterization of biosynthesis enzymes and metabolite bioengineering. Traditionally, metabolic pathways are reconstituted in bacteria or yeast due to their ease for genetic manipulation and transformation. Many plant metabolite pathways involve multiple enzyme complexes channeled on plant endomembrane system, which is absent in bacteria and yeast. Nicotiana benthamiana is particularly suitable for reconstitution plant metabolite pathway involving enzymes associated with plant endomembrane systems. Compared with other plants, N. benthamiana can be easily transiently transformed by multiple genes simultaneously by a procedure called leaf agroinfiltration. The results of transient transformation can be analyzed in several days, compared with several months with other stable transformation procedures. In this chapter, we present a protocol for multiple-gene transformation by agroinfiltration, followed by UPLC MS analysis.

Isolation of a Novel Thermophilic Methanogen and the Evolutionary History of the Class Methanobacteria.

Methanogens can produce methane in anaerobic environments via the methanogenesis pathway, and are regarded as one of the most ancient life forms on Earth. They are ubiquitously distributed across distinct ecosystems and are considered to have a thermophilic origin. In this study, we isolated, pure cultured, and completely sequenced a single methanogen strain DL9LZB001, from a hot spring at Tengchong in Southwest China. DL9LZB001 is a thermophilic and hydrogenotrophic methanogen with an optimum growth temperature of 65 °C. It is a putative novel species, which has been named Methanothermobacter tengchongensis-a Class I methanogen belonging to the class Methanobacteria. Comparative genomic and ancestral analyses indicate that the class Methanobacteria originated in a hyperthermal environment and then evolved to adapt to ambient temperatures. This study extends the understanding of methanogens living in geothermal niches, as well as the origin and evolutionary history of these organisms in ecosystems with different temperatures.

The Role of Petrimonas mucosa ING2-E5AT in Mesophilic Biogas Reactor Systems as Deduced from Multiomics Analyses.

Members of the genera Proteiniphilum and Petrimonas were speculated to represent indicators reflecting process instability within anaerobic digestion (AD) microbiomes. Therefore, Petrimonas mucosa ING2-E5AT was isolated from a biogas reactor sample and sequenced on the PacBio RSII and Illumina MiSeq sequencers. Phylogenetic classification positioned the strain ING2-E5AT in close proximity to Fermentimonas and Proteiniphilum species (family Dysgonomonadaceae). ING2-E5AT encodes a number of genes for glycosyl-hydrolyses (GH) which are organized in Polysaccharide Utilization Loci (PUL) comprising tandem susCD-like genes for a TonB-dependent outer-membrane transporter and a cell surface glycan-binding protein. Different GHs encoded in PUL are involved in pectin degradation, reflecting a pronounced specialization of the ING2-E5AT PUL systems regarding the decomposition of this polysaccharide. Genes encoding enzymes participating in amino acids fermentation were also identified. Fragment recruitments with the ING2-E5AT genome as a template and publicly available metagenomes of AD microbiomes revealed that Petrimonas species are present in 146 out of 257 datasets supporting their importance in AD microbiomes. Metatranscriptome analyses of AD microbiomes uncovered active sugar and amino acid fermentation pathways for Petrimonas species. Likewise, screening of metaproteome datasets demonstrated expression of the Petrimonas PUL-specific component SusC providing further evidence that PUL play a central role for the lifestyle of Petrimonas species.

Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter.

In the thermophilic biogas-producing microbial community, the genus Methanothermobacter was previously described to be frequently abundant. The aim of this study was to establish and analyze the genome sequence of the archaeal strain Methanothermobacter wolfeii SIV6 originating from a thermophilic industrial-scale biogas fermenter and compare it to related reference genomes. The circular chromosome has a size of 1,686,891 bases, featuring a GC content of 48.89%. Comparative analyses considering three completely sequenced Methanothermobacter strains revealed a core genome of 1494 coding sequences and 16 strain specific genes for M. wolfeii SIV6, which include glycosyltransferases and CRISPR/cas associated genes. Moreover, M. wolfeii SIV6 harbors all genes for the hydrogenotrophic methanogenesis pathway and genome-centered metatranscriptomics indicates the high metabolic activity of this strain, with 25.18% of all transcripts per million (TPM) belong to the hydrogenotrophic methanogenesis pathway and 18.02% of these TPM exclusively belonging to the mcr operon. This operon encodes the different subunits of the enzyme methyl-coenzyme M reductase (EC: 2.8.4.1), which catalyzes the final and rate-limiting step during methanogenesis. Finally, fragment recruitment of metagenomic reads from the thermophilic biogas fermenter on the SIV6 genome showed that the strain is abundant (1.2%) within the indigenous microbial community. Detailed analysis of the archaeal isolate M. wolfeii SIV6 indicates its role and function within the microbial community of the thermophilic biogas fermenter, towards a better understanding of the biogas production process and a microbial-based management of this complex process.

Proteiniphilum saccharofermentans str. M3/6T isolated from a laboratory biogas reactor is versatile in polysaccharide and oligopeptide utilization as deduced from genome-based metabolic reconstructions.

Proteiniphilum saccharofermentans str. M3/6T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, ß-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6T and its closest described relative P. acetatigenes str. DSM 18083T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars.

Characterization of Bathyarchaeota genomes assembled from metagenomes of biofilms residing in mesophilic and thermophilic biogas reactors.

BACKGROUND: Previous studies on the Miscellaneous Crenarchaeota Group, recently assigned to the novel archaeal phylum Bathyarchaeota, reported on the dominance of these Archaea within the anaerobic carbohydrate cycle performed by the deep marine biosphere. For the first time, members of this phylum were identified also in mesophilic and thermophilic biogas-forming biofilms and characterized in detail. RESULTS: Metagenome shotgun libraries of biofilm microbiomes were sequenced using the Illumina MiSeq system. Taxonomic classification revealed that between 0.1 and 2% of all classified sequences were assigned to Bathyarchaeota. Individual metagenome assemblies followed by genome binning resulted in the reconstruction of five metagenome-assembled genomes (MAGs) of Bathyarchaeota. MAGs were estimated to be 65-92% complete, ranging in their genome sizes from 1.1 to 2.0 Mb. Phylogenetic classification based on core gene sets confirmed their placement within the phylum Bathyarchaeota clustering as a separate group diverging from most of the recently known Bathyarchaeota clusters. The genetic repertoire of these MAGs indicated an energy metabolism based on carbohydrate and amino acid fermentation featuring the potential for extracellular hydrolysis of cellulose, cellobiose as well as proteins. In addition, corresponding transporter systems were identified. Furthermore, genes encoding enzymes for the utilization of carbon monoxide and/or carbon dioxide via the Wood-Ljungdahl pathway were detected. CONCLUSIONS: For the members of Bathyarchaeota detected in the biofilm microbiomes, a hydrolytic lifestyle is proposed. This is the first study indicating that Bathyarchaeota members contribute presumably to hydrolysis and subsequent fermentation of organic substrates within biotechnological biogas production processes.

The completely annotated genome and comparative genomics of the Peptoniphilaceae bacterium str. ING2-D1G, a novel acidogenic bacterium isolated from a mesophilic biogas reactor.

The strictly anaerobic Peptoniphilaceae bacterium str. ING2-D1G (=DSM 28672=LMG 28300) was isolated from a mesophilic laboratory-scale completely stirred tank biogas reactor (CSTR) continuously co-digesting maize silage, pig and cattle manure. Based on 16S rRNA gene sequence comparison, the closest described relative to this strain is Peptoniphilus obesi ph1 showing 91.2% gene sequence identity. The most closely related species with a validly published name is Peptoniphilus indolicus DSM 20464T whose 16S rRNA gene sequence is 90.6% similar to the one of strain ING2-D1G. The genome of the novel strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding anaerobic digestion of biomass. The strain harbors a circular chromosome with a size of 1.6 Mb that contains 1466 coding sequences, 53 tRNA genes and 4 ribosomal RNA (rrn) operons. The genome carries a 28,261bp prophage insertion comprising 47 phage-related coding sequences. Reconstruction of fermentation pathways revealed that strain ING2-D1G encodes all enzymes for hydrogen, lactate and acetate production, corroborating that it is involved in the acido- and acetogenic phase of the biogas process. Comparative genome analyses of Peptoniphilaceae bacterium str. ING2-D1G and its closest relative Peptoniphilus obesi ph1 uncovered rearrangements, deletions and insertions within the chromosomes of both strains substantiating a divergent evolution. In addition to genomic analyses, a physiological and phenotypic characterization of the novel isolate was performed. Grown in Brain Heart Infusion Broth with added yeast extract, cells were spherical to ovoid, catalase- and oxidase-negative and stained Gram-positive. Optimal growth occurred between 35 and 37°C and at a pH value of 7.6. Fermentation products were acetate, butanoate and carbon dioxide.

Complete genome analysis of Clostridium bornimense strain M2/40(T): A new acidogenic Clostridium species isolated from a mesophilic two-phase laboratory-scale biogas reactor.

Taxonomic and functional profiling based on metagenome analyses frequently revealed that members of the class Clostridia dominate biogas reactor communities and perform different essential metabolic pathways in the biogas fermentation process. Clostridium bornimense strain M2/40(T) was recently isolated from a mesophilic two-phase lab-scale biogas reactor continuously fed with maize silage and wheat straw. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding carbohydrate active enzyme production and fermentation of organic compounds for consolidated biofuel production from biomass. The C. bornimense M2/40(T) genome consists of a chromosome (2,917,864bp in size) containing 2613 protein coding sequences, and a 699,161bp chromid (secondary replicon) harboring 680 coding sequences. Both replicons feature very similar GC-contents of approximately 29%. The complex genome comprises three prophage regions, two CRISPR-cas systems and a putative cellulosomal gene cluster that is located on the second replicon (chromid) of the strain. The overexpressed glycosyl hydrolases (GH) CelK (GH9) and CelA (GH48) encoded in the cellulosomal gene cluster were shown to be active on the substrates xylan and xyloglucan whereas XghA (GH74) is highly active on xyloglucan. Reconstruction of fermentation pathways from genome sequence data revealed that strain M2/40(T) encodes all enzymes for hydrogen, acetate, formate, lactate, butyrate, and ethanol production, leading to the classification of the isolate as acidogenic bacterium. Phylogenetic analyses uncovered that the closest characterized relative of C. bornimense is C. cellulovorans. Comparative analyses of the C. bornimense and C. cellulovorans genomes revealed considerable rearrangements within their chromosomes suggesting that both species evolved separately for a relatively long period of time and adapted to specific tasks within microbial consortia responsible for anaerobic digestion.

Comparative Biochemistry and In Vitro Pathway Reconstruction as Powerful Partners in Studies of Metabolic Diversity.

There are estimated to be >300,000 plant species, producing >200,000 metabolites. Many of these metabolites are restricted to specific plant lineages and are referred to as "specialized" metabolites. These serve varied functions in plants including defense against biotic and abiotic stresses, plant-plant and plant-microbe communication, and pollinator attraction. These compounds also have important applications in agriculture, medicine, skin care, and in diverse aspects of human culture. The specialized metabolic repertoire of plants can vary even within and between closely related species, in terms of the number and classes of specialized metabolites as well as their structural variants. This phenotypic variation can be exploited to discover the underlying variation in the metabolic enzymes. We describe approaches for using the diversity of specialized metabolites and variation in enzyme structure and function to identify novel enzymatic activities and understand the structural basis for these differences. The knowledge obtained from these studies will provide new modules for the synthetic biology toolbox.