Discovery, structural characterization, and functional insights into a novel apiosidase from the GH140 family, isolated from a lignocellulolytic-enriched mangrove microbial community.

OBJECTIVES: Apiosidases are enzymes that cleave the glycosidic bond between the monosaccharides linked to apiose, a branched chain furanose found in the cell walls of vascular plants and aquatic monocots. There is biotechnological interest in this enzyme group because apiose is the flavor-active compound of grapes, fruit juice, and wine, and the monosaccharide is found to be a plant secondary metabolite with pharmaceutical properties. However, functional and structural studies of this enzyme family are scarce. Recently, a glycoside hydrolase family member GH140 was isolated from Bacteroides thetaiotaomicron and identified as an endo-apiosidase. RESULTS: The structural characterization and functional identification of a second GH140 family enzyme, termed MmApi, discovered through mangrove soil metagenomic approach, are described. Among the various substrates tested, MmApi exhibited activity on an apiose-containing oligosaccharide derived from the pectic polysaccharide rhamnogalacturonan-II. While the crystallographic model of MmApi was similar to the endo-apiosidase from Bacteroides thetaiotaomicron, differences in the shape of the binding sites indicated that MmApi could cleave apioses within oligosaccharides of different compositions. CONCLUSION: This enzyme represents a novel tool for researchers interested in studying the physiology and structure of plant cell walls and developing biocatalytic strategies for drug and flavor production.

Enzymatic and biophysical characterization of a novel modular cellulosomal GH5 endoglucanase multifunctional from the anaerobic gut fungus Piromyces finnis.

Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat ß-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley ß-glucan, therefore displaying multi-functionality. For oat ß-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with Km of 7.1 µM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.

arfA antisense RNA regulates MscL excretory activity.

Excretion of cytoplasmic protein (ECP) is a commonly observed phenomenon in bacteria, and this partial extracellular localisation of the intracellular proteome has been implicated in a variety of stress response mechanisms. In response to hypoosmotic shock and ribosome stalling in Escherichia coli, ECP is dependent upon the presence of the large-conductance mechanosensitive channel and the alternative ribosome-rescue factor A gene products. However, it is not known if a mechanistic link exists between the corresponding genes and the respective stress response pathways. Here, we report that the corresponding mscL and arfA genes are commonly co-located on the genomes of Gammaproteobacteria and display overlap in their respective 3' UTR and 3' CDS. We show this unusual genomic arrangement permits an antisense RNA-mediated regulatory control between mscL and arfA, and this modulates MscL excretory activity in E. coli These findings highlight a mechanistic link between osmotic, translational stress responses and ECP in E. coli, further elucidating the previously unknown regulatory function of arfA sRNA.

Structural and functional insights of the catalytic GH5 and Calx-ß domains from the metagenome-derived endoglucanase CelE2.

Cellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular architectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-ß domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-ß domain. In this study, the GH5 and the Calx-ß domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 °C for the catalytic domain (CelE21-381) compared to CelE2 full-length. The CD analyses revealed that the Calx-ß domain (CelE2382-477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21-381 was determined at 2.1 Å resolution, showing a typical (α/ß)8-barrel fold and a narrow active site compared to other cellulases from the same family. The biochemical characterization showed that the deletion of the Calx-ß domain increased more than 3-fold the activity of the catalytic domain CelE21-381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21-381 protein solvency. This work contributed to fundamental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production.

Integrative omics analyses of the ligninolytic Rhodosporidium fluviale LM-2 disclose catabolic pathways for biobased chemical production.

BACKGROUND: Lignin is an attractive alternative for producing biobased chemicals. It is the second major component of the plant cell wall and is an abundant natural source of aromatic compounds. Lignin degradation using microbial oxidative enzymes that depolymerize lignin and catabolize aromatic compounds into central metabolic intermediates is a promising strategy for lignin valorization. However, the intrinsic heterogeneity and recalcitrance of lignin severely hinder its biocatalytic conversion. In this context, examining microbial degradation systems can provide a fundamental understanding of the pathways and enzymes that are useful for lignin conversion into biotechnologically relevant compounds. RESULTS: Lignin-degrading catabolism of a novel Rhodosporidium fluviale strain LM-2 was characterized using multi-omic strategies. This strain was previously isolated from a ligninolytic microbial consortium and presents a set of enzymes related to lignin depolymerization and aromatic compound catabolism. Furthermore, two catabolic routes for producing 4-vinyl guaiacol and vanillin were identified in R. fluviale LM-2. CONCLUSIONS: The multi-omic analysis of R. fluviale LM-2, the first for this species, elucidated a repertoire of genes, transcripts, and secreted proteins involved in lignin degradation. This study expands the understanding of ligninolytic metabolism in a non-conventional yeast, which has the potential for future genetic manipulation. Moreover, this work unveiled critical pathways and enzymes that can be exported to other systems, including model organisms, for lignin valorization.

Recombinant expression, purification and characterization of an active bacterial feruloyl-CoA synthase with potential for application in vanillin production.

The ferulic acid (FA) represents a high-value molecule with applications in the cosmetic and pharmaceutical industries. This aromatic molecule is derived from lignin and can be enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. This process starts with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases. Therefore, here, we report the successfully expression, purification as well as the initial structural and biochemical characterization of a stable, correctly folded, and catalytically active bacterial feruloyl-CoA synthase (here named FCS3) isolated from a lignin-degrading microbial consortium. The purification of recombinant FCS3 to near homogeneity was achieved using affinity chromatography. The FCS3 structure is composed of a mixture of α and ß secondary structures and most likely forms stable homodimers in solution. The FCS3 presented a notable structural stability at alkaline pH values and it was able to convert FA and coenzyme A (CoA) into feruloyl-CoA complex at room temperature. This study should provide a useful basis for future biotechnological applications of FCS3, especially in the field of conversion of lignin-derived FA into high value compounds.

Unique properties of a Dictyostelium discoideum carbohydrate-binding module expand our understanding of CBM-ligand interactions.

Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallographic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-ß-1,4-glucanase (glycoside hydrolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) polysaccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glucomannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional characterization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein-carbohydrate interactions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glycoside hydrolase accessory modules.

Applying biochemical and structural characterization of hydroxycinnamate catabolic enzymes from soil metagenome for lignin valorization strategies.

The biocatalytic production of fuels and chemicals from plant biomass represents an attractive alternative to fossil fuel-based refineries. In this context, the mining and characterization of novel biocatalysts can promote disruptive innovation opportunities in the field of lignocellulose conversion and valorization. In the present work, we conducted the biochemical and structural characterization of two novel hydroxycinnamic acid catabolic enzymes, isolated from a lignin-degrading microbial consortium, a feruloyl-CoA synthetase, and a feruloyl-CoA hydratase-lyase, named LM-FCS2 and LM-FCHL2, respectively. Besides establishing the homology model structures for novel FCS and FCHL members with unique characteristics, the enzymes presented interesting biochemical features: LM-FCS2 showed stability in alkaline pHs and was able to convert a wide array of p-hydroxycinnamic acids to their respective CoA-thioesters, including sinapic acid; LM-FCHL2 efficiently converted feruloyl-CoA and p-coumaroyl-CoA into vanillin and 4-hydroxybenzaldehyde, respectively, and could produce vanillin directly from ferulic acid. The coupled reaction of LM-FCS2 and LM-FCHL2 produced vanillin, not only from commercial ferulic acid but also from a crude lignocellulosic hydrolysate. Collectively, this work illuminates the structure and function of two critical enzymes involved in converting ferulic acid into high-value molecules, thus providing valuable concepts applied to the development of plant biomass biorefineries. KEY POINTS: • Comprehensive characterization of feruloyl-CoA synthetase from metagenomic origin. • Novel low-resolution structures of hydroxycinnamate catabolic enzymes. • Production of vanillin via enzymatic reaction using lignocellulosic hydrolysates.

Characterization and in vitro testing of newly isolated lytic bacteriophages for the biocontrol of Pseudomonas aeruginosa.

Aim: Two lytic phages were isolated using P. aeruginosa DSM19880 as host and fully characterized. Materials & methods: Phages were characterized physicochemically, biologically and genomically. Results & conclusion: Host range analysis revealed that the phages also infect some multidrug-resistant (MDR) P. aeruginosa clinical isolates. Increasing MOI from 1 to 1000 significantly increased phage efficiency and retarded bacteria regrowth, but phage ph0034 (reduction of 7.5 log CFU/ml) was more effective than phage ph0031 (reduction of 5.1 log CFU/ml) after 24 h. Both phages belong to Myoviridae family. Genome sequencing of phages ph0031 and ph0034 showed that they do not carry toxin, virulence, antibiotic resistance and integrase genes. The results obtained are highly relevant in the actual context of bacterial resistance to antibiotics.

Microbial enrichment and meta-omics analysis identify CAZymes from mangrove sediments with unique properties.

Although lignocellulose is the most abundant and renewable natural resource for biofuel production, its use remains under exploration because of its highly recalcitrant structure. Its deconstruction into sugar monomers is mainly driven by carbohydrate-active enzymes (CAZymes). To develop highly efficient and fast strategies to discover biomass-degrading enzymes for biorefinery applications, an enrichment process combined with integrative omics approaches was used to identify new CAZymes. The lignocellulolytic-enriched mangrove microbial community (LignoManG) established on sugarcane bagasse (SB) was enriched with lignocellulolytic bacteria and fungi such as Proteobacteria, Bacteroidetes, Basidiomycota, and Ascomycota. These microbial communities were able to degrade up to 55 % of the total SB, indicating the production of lignocellulolytic enzymes. Metagenomic analysis revealed that the LignoManG harbors 18.042 CAZyme sequences such as of cellulases, hemicellulases, carbohydrate esterases, and lytic polysaccharide monooxygenase. Similarly, our metaproteomic analysis depicted several enzymes from distinct families of different CAZy families. Based on the LignoManG data, a xylanase (coldXynZ) was selected, amplified, cloned, expressed, and biochemically characterized. The enzyme displayed psicrofilic properties, with the highest activity at 15 °C, retaining 77 % of its activity when incubated at 0 °C. Moreover, molecular modeling in silico indicated that coldXynZ is composed of a TIM barrel, which is a typical folding found in the GH10 family, and displayed similar structural features related to cold-adapted enzymes. Collectively, the data generated in this study represent a valuable resource for lignocellulolytic enzymes with potential biotechnological applications.

Multi-omics analysis provides insights into lignocellulosic biomass degradation by Laetiporus sulphureus ATCC 52600.

BACKGROUND: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. RESULTS: The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative-hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. CONCLUSION: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

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.

Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium.

Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial factors economically challenging to its use. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge, enrichment strategies can be applied to develop microbial communities with an efficient CAZyme arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of the ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S rRNA sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of the ERAC disclosed 41 metagenome-assembled genomes (MAGs) harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The amino acid sequences of the majority of the predicted CAZymes (60% of the total) shared less than 90% identity with the sequences found in public databases. Additionally, a clostridial MAG identified in this study produced proteins during consortium development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism.IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries.

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.

Lignolytic-consortium omics analyses reveal novel genomes and pathways involved in lignin modification and valorization.

BACKGROUND: Lignin is a heterogeneous polymer representing a renewable source of aromatic and phenolic bio-derived products for the chemical industry. However, the inherent structural complexity and recalcitrance of lignin makes its conversion into valuable chemicals a challenge. Natural microbial communities produce biocatalysts derived from a large number of microorganisms, including those considered unculturable, which operate synergistically to perform a variety of bioconversion processes. Thus, metagenomic approaches are a powerful tool to reveal novel optimized metabolic pathways for lignin conversion and valorization. RESULTS: The lignin-degrading consortium (LigMet) was obtained from a sugarcane plantation soil sample. The LigMet taxonomical analyses (based on 16S rRNA) indicated prevalence of Proteobacteria, Actinobacteria and Firmicutes members, including the Alcaligenaceae and Micrococcaceae families, which were enriched in the LigMet compared to sugarcane soil. Analysis of global DNA sequencing revealed around 240,000 gene models, and 65 draft bacterial genomes were predicted. Along with depicting several peroxidases, dye-decolorizing peroxidases, laccases, carbohydrate esterases, and lignocellulosic auxiliary (redox) activities, the major pathways related to aromatic degradation were identified, including benzoate (or methylbenzoate) degradation to catechol (or methylcatechol), catechol ortho-cleavage, catechol meta-cleavage, and phthalate degradation. A novel Paenarthrobacter strain harboring eight gene clusters related to aromatic degradation was isolated from LigMet and was able to grow on lignin as major carbon source. Furthermore, a recombinant pathway for vanillin production was designed based on novel gene sequences coding for a feruloyl-CoA synthetase and an enoyl-CoA hydratase/aldolase retrieved from the metagenomic data set. CONCLUSION: The enrichment protocol described in the present study was successful for a microbial consortium establishment towards the lignin and aromatic metabolism, providing pathways and enzyme sets for synthetic biology engineering approaches. This work represents a pioneering study on lignin conversion and valorization strategies based on metagenomics, revealing several novel lignin conversion enzymes, aromatic-degrading bacterial genomes, and a novel bacterial strain of potential biotechnological interest. The validation of a biosynthetic route for vanillin synthesis confirmed the applicability of the targeted metagenome discovery approach for lignin valorization strategies.

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.

Genomic characterization of Defluviitoga tunisiensis L3, a key hydrolytic bacterium in a thermophilic biogas plant and its abundance as determined by metagenome fragment recruitment.

The genome sequence of Defluviitoga tunisiensis L3 originating from a thermophilic biogas-production plant was established and recently published as Genome Announcement by our group. The circular chromosome of D. tunisiensis L3 has a size of 2,053,097bp and a mean GC content of 31.38%. To analyze the D. tunisiensis L3 genome sequence in more detail, a phylogenetic analysis of completely sequenced Thermotogae strains based on shared core genes was performed. It appeared that Petrotoga mobilis DSM 10674(T), originally isolated from a North Sea oil-production well, is the closest relative of D. tunisiensis L3. Comparative genome analyses of P. mobilis DSM 10674(T) and D. tunisiensis L3 showed moderate similarities regarding occurrence of orthologous genes. Both genomes share a common set of 1351 core genes. Reconstruction of metabolic pathways important for the biogas production process revealed that the D. tunisiensis L3 genome encodes a large set of genes predicted to facilitate utilization of a variety of complex polysaccharides including cellulose, chitin and xylan. Ethanol, acetate, hydrogen (H2) and carbon dioxide (CO2) were found as possible end-products of the fermentation process. The latter three metabolites are considered to represent substrates for methanogenic Archaea, the key organisms in the final step of the anaerobic digestion process. To determine the degree of relatedness between D. tunisiensis L3 and dominant biogas community members within the thermophilic biogas-production plant, metagenome sequences obtained from the corresponding microbial community were mapped onto the L3 genome sequence. This fragment recruitment revealed that the D. tunisiensis L3 genome is almost completely covered with metagenome sequences featuring high matching accuracy. This result indicates that strains highly related or even identical to the reference strain D. tunisiensis L3 play a dominant role within the community of the thermophilic biogas-production plant.

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.

New FeFe-hydrogenase genes identified in a metagenomic fosmid library from a municipal wastewater treatment plant as revealed by high-throughput sequencing.

A fosmid metagenomic library was constructed with total community DNA obtained from a municipal wastewater treatment plant (MWWTP), with the aim of identifying new FeFe-hydrogenase genes encoding the enzymes most important for hydrogen metabolism. The dataset generated by pyrosequencing of a fosmid library was mined to identify environmental gene tags (EGTs) assigned to FeFe-hydrogenase. The majority of EGTs representing FeFe-hydrogenase genes were affiliated with the class Clostridia, suggesting that this group is the main hydrogen producer in the MWWTP analyzed. Based on assembled sequences, three FeFe-hydrogenase genes were predicted based on detection of the L2 motif (MPCxxKxxE) in the encoded gene product, confirming true FeFe-hydrogenase sequences. These sequences were used to design specific primers to detect fosmids encoding FeFe-hydrogenase genes predicted from the dataset. Three identified fosmids were completely sequenced. The cloned genomic fragments within these fosmids are closely related to members of the Spirochaetaceae, Bacteroidales and Firmicutes, and their FeFe-hydrogenase sequences are characterized by the structure type M3, which is common to clostridial enzymes. FeFe-hydrogenase sequences found in this study represent hitherto undetected sequences, indicating the high genetic diversity regarding these enzymes in MWWTP. Results suggest that MWWTP have to be considered as reservoirs for new FeFe-hydrogenase genes.

Complete genome sequence of the novel Porphyromonadaceae bacterium strain ING2-E5B isolated from a mesophilic lab-scale biogas reactor.

In this study, the whole genome sequence of the mesophilic, anaerobic Porphyromonadaceae bacterium strain ING2-E5B (LMG 28429, DSM 28696) is reported. The new isolate belongs to the phylum Bacteroidetes and was obtained from a biogas-producing lab-scale completely stirred tank reactor (CSTR) optimized for anaerobic digestion of maize silage in co-fermentation with pig and cattle manure. The genome of strain ING2-E5B contains numerous genes encoding proteins and enzymes involved in the degradation of complex carbohydrates and proteinaceous compounds. Moreover, it possesses genes catalyzing the production of volatile fatty acids. Hence, this bacterium was predicted to be involved in hydrolysis and acidogenesis during anaerobic digestion and biomethanation.