Wolinella Succinogenes Research Articles

Characterization of Mycobacterium smegmatis Glutaminase-Free Asparaginase (MSMEG_3173).

l-asparaginase is an enzyme catalyzing the hydrolysis of l-asparagine into l-aspartate and ammonia, which is of great therapeutic importance in tumor treatment. However, commercially available enzymes are associated with adverse effects, and searching for a new l-asparaginase with better pharmaceutical properties was the aim of this work. The coding sequence for Mycobacterium smegmatisl-asparaginase (MsA) was cloned and expressed. The recombinant protein showed high activity toward l-asparagine, whereas none was detected for l-glutamine. The enzymatic properties (K m = 1.403 ± 0.24 mM and k cat = 708.1 ± 25.05 s-1) indicate that the enzyme would be functional within the expected blood l-asparagine concentration, with good activity, as shown by k cat. The pH and temperature profiles suggest its use as a biopharmaceutical in humans. Molecular dynamics analysis of the MsA model reveals the formation of a hydrogen bond network involving catalytic residues with l-asparagine. However, the same is not observed with l-glutamine, mainly due to steric hindrance. Additionally, the structural feature of residue 119 being a serine rather than a proline has significant implications. These findings help explain the low glutaminase activity observed in MsA, like what is described for the Wolinella succinogenes enzyme. This establishes mycobacterial asparaginases as key scaffolds to develop biopharmaceuticals against acute lymphocytic leukemia.

Effect of pH on the thermostability and redox properties of cytochrome c552 from Wolinella succinogenes

Effect of pH on the thermostability and redox properties of cytochrome c552 from Wolinella succinogenes

Biosynthesis and function of microbial methylmenaquinones.

The membranous quinone/quinol pool is essential for the majority of life forms and its composition has been widely used as a biomarker in microbial taxonomy. The most abundant quinone is menaquinone (MK), which serves as an essential redox mediator in various electron transport chains of aerobic and anaerobic respiration. Several methylated derivatives of MK, designated methylmenaquinones (MMKs), have been reported to be present in members of various microbial phyla possessing either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). Due to their low redox midpoint potentials, MMKs have been proposed to be specifically involved in appropriate electron transport chains of anaerobic respiration. The class C radical SAM methyltransferases MqnK, MenK and MenK2 have recently been shown to catalyse specific MK methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesise 8-MMK, 7-MMK and 7,8-dimethylmenaquinone (DMMK). MqnK, MenK and MenK2 from organisms such as Wolinella succinogenes, Adlercreutzia equolifaciens, Collinsella tanakaei, Ferrimonas marina and Syntrophus aciditrophicus have been functionally produced in Escherichia coli, enabling extensive quinone/quinol pool engineering of the native MK and 2-demethylmenaquinone (DMK). Cluster and phylogenetic analyses of available MK and MMK methyltransferase sequences revealed signature motifs that allowed the discrimination of MenK/MqnK/MenK2 family enzymes from other radical SAM enzymes and the identification of C-7-specific menaquinone methyltransferases of the MenK2 subfamily. It is envisaged that this knowledge will help to predict the methylation status of the menaquinone/menaquinol pool of any microbial species (or even a microbial community) from its (meta)genome.

Perchlorate reduction kinetics and genome-resolved metagenomics identify metabolic interactions in acclimated saline lake perchlorate-reducing consortia

Perchlorate is a widely detected environmental contaminant in surface and underground water, that seriously impacts human health by inhibiting the uptake of thyroidal radioiodine. Perchlorate reduction due to saline lake microorganisms is not as well understood as that in marine environments. In this study, we enriched a perchlorate-reducing microbial consortium collected from saline lake sediments and found that the perchlorate reduction kinetics of the enriched consortium fit the Michaelis–Menten kinetics well, with a maximum specific substrate reduction rate (qmax) of 0.596 ± 0.001 mg ClO4−/mg DW/h and half-saturation constant (Ks) of 16.549 ± 0.488 mg ClO4−/L. Furthermore, we used improved metagenome binning to reconstruct high-quality metagenome-assembled genomes from the metagenomes of the microbial consortia, including the perchlorate-reducing bacteria (PRB) Dechloromonas agitata and Wolinella succinogenes, with the genome of W. succinogenes harboring complete functional genes for perchlorate reduction being the first recovered. Given that the electrons were directly transferred to the electronic carrier cytochrome c-553 from the quinone pool, the electron transfer pathway of W. succinogenes was shorter and more efficient than the canonical pattern. This finding provides a theoretical basis for microbial remediation of sites contaminated by high concentrations of perchlorate. Metagenomic binning and metatranscriptomic analyses revealed the gene transcription variation of perchlorate reductase pcr and chlorite dismutase cld by PRB and the synergistic metabolic mechanism.

Homology modeling and virtual characterization of cytochrome c nitrite reductase (NrfA) in three model bacteria responsible for short-circuit pathway, DNRA in the terrestrial nitrogen cycle.

NrfA is the molecular marker for dissimilatory nitrate reduction to ammonium (DNRA) activity, catalysing cytochrome c nitrite reductase enzyme. However, the limited study has been made so far to understand the structural homology modeling of NrfA protein in DNRA bacteria. Therefore, three model DNRA bacteria (Escherechia coli, Wolinella succinogenes and Shewanella oneidensis) were chosen in this study for in-silico protein modeling of NrfA which roughly consists of similar length of amino acids and molecular weight and they belong to two contrasting taxonomicfamilies (γ-proteobacteria with nrfABCDEFG and ε-proteobacteria with nrfHAIJoperon). Multiple bioinformatic tools were used to examine the primary, secondary, and tertiary structure of NrfA protein using three distinct homology modeling pipelines viz., Phyre2, Swiss model and Modeller. The results indicated that NrfA protein in E. coli, W. succinogenes and S. oneidensis was mostly periplasmic and hydrophilic. Four conserved Cys-X1-X2-Cys-His motifs, one Cys-X1-X2-Cys-Lys haem-binding motif and Ca ligand were also identified in NrfA protein irrespective of three model bacteria. Moreover, 11 identical conserved amino acids sequence was observed for the first time between serine and proline in NrfA protein. Secondary structure of NrfA revealed that α-helices were observed in 77.9%, 73.4%, and 77.4% in E. coli, W. succinogenes and S. oneidensis, respectively. Ramachandran plot showed that number of residue in favored region in E. coli, W. succinogenes and S. oneidensis was 97.03%, 97.01% and 97.25%, respectively. Our findings also revealed that among three pipelines, Modeller was considered the best in-silico tool for prediction of NrfA protein. Overall, significant findings of this study may aid in the identification of future unexplored DNRA bacteria containing cytochrome c nitrite reductase. The NrfA system, which is linked to respiratory nitrite ammonification, provides an analogous target for monitoring less studied N-retention processes, particularly in agricultural ecosystems. Furthermore, one of the challenging research tasks for the future is to determine how the NrfA protein responds to redox status in the microbial cells.

A Cytoplasmic NAD(P)H-Dependent Polysulfide Reductase with Thiosulfate Reductase Activity from the Hyperthermophilic Bacterium Thermotoga maritima.

ABSTRACTThermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H2 by fermenting carbohydrates. High concentration of H2 inhibits the growth of T. maritima, and S0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx, yeeE, and rnf genes when the strain grew in S0- or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments.IMPORTANCE The reduction of S0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S0, polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S0- or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

EVALUATION OF STRUCTURAL, MOLECULAR INTERACTION AND SIMULATION STUDY OF L-ASPARAGINASE OF Streptomyces koyangensis

A vital step in drug discovery is structural and interaction assessment. L-asparaginase is a highly used compound in various treatment regimens which includes combinational therapies. The application is not only limited to treating Acute lymphoblastic leukemia, where it is also found effective for myeloid leukemia and canine lymphoma. The study details about L-asparaginase of Streptomyces koyangensis (WP_203215894). Structural insights are obtained by evaluation with GalaxyTBM. The constructed 3D-model is based on L-asparaginase models belonging to Escherichia coli, Wolinella succinogenes and Helicobacter pylori. The Ramachandran assessment corresponds to 91.4% of residues in the favourable region while ProSA infers to -8.9 Z-score. Further, QMEAN4 score of -0.46, 92.903 ERRAT score as well as 100% of residues in agreement with Verify3D theory describe the quality of the protein to be good and acceptable. The protein is identified to be secreted in the cytoplasm as per subsequent CELLO version2.5. Docking studies involving L-asparaginase (WP_203215894) molecule with L-asparagine resulted in the lowest energy of -4.5 kcal/mol along with prominent active sites recognized by PyRx. Similar prediction in addition to -4.6 and -4.8 kcal/mol is also resulted by CB-Dock. Further, the binding conformation referring to affinity of -4.5 kcal/mol is also supported by the interpretation of GalaxySite with identification of the same residues involved in ligand interaction. High values of affinity correspond to the analysis by SwissDock referring to -9.42 kcal/mol. The RMSF plot generated as part of protein flexibility analysis by CABS flex 2.0 depicted varied fluctuations however, the crucial residues interacting with the ligand are computed to have RMSF value < 2 represents rigidity. The therapeutic effectiveness of the enzyme has created interest in terms of tracing prospective sources and conducting computational analysis. The anticipated structure of WP_203215894 and derived assumptions are helpful and contribute valuable information for future in-vitro findings on L-asparaginase.

Deciphering the black box of microbial community of common effluent treatment plant through integrated metagenomics: Tackling industrial effluent

Identifying the microbial community and their functional potential from different stages of common effluent treatment plants (CETP) can enhance the efficiency of wastewater treatment systems. In this study, wastewater metagenomes from 8 stages of CETP were screened for microbial diversity and gene profiling along with their corresponding degradation activities. The microbial community displayed 98.46% of bacterial species, followed by Eukarya (0.10%) and Archaea 0.02%. At the Phylum level, Proteobacteria (28.8%) was dominant, followed by Bacteroidetes (16.1%), Firmicutes (11.7%), and Fusobacteria (6.9%) which are mainly capable of degrading the aromatic compounds. Klebsiella pneumoniae, Wolinella succinogenes, Pseudomonas stutzeri, Desulfovibrio vulgaris, and Clostridium sticklandii were the most prevalent species. The functional analysis further demonstrated the presence of enzymes linked with genes/pathways known to be involved in the degradation/metabolization of aromatic compounds like benzoate, bisphenol, 1,2-dichloroethane phenylalanine. This information was further validated with the whole genome analysis of the bacteria isolated from the CETP. We anticipate that integrating both shotgun and whole-genome analyses can reveal the rich reservoir for novel enzymes and genes present in CETP effluent that can contribute to designing efficient bioremediation strategies for the environment in general CETP system, in particular.

Prevalence of Hepatitis B in Blood Groups and Level of Education of Blood Donors in Al-Najaf Governorate

Advancement in sequencing techniques and transformative progress in metagenomics provides an unprecedented platform for functional and taxonomic characterization of the enormous microbial diversity inhabiting and governing various biochemical processes of the freshwater sources. Metagenomic analysis of freshwater resources has led to the discovery and identification of novel microbial genes and an understanding of how microorganisms mediate energy and carbon. In this study, we report the taxonomical classification of bacterial sequences obtained from 6 dam reservoir sites in Pune city, Maharashtra, India. The analysis was performed using two different alignment tools: BLAST and Kaiju. The bacterial diversity was dominated by the presence of Vogecella indigofera, uncultured Proteobacterium, Wolinella Succinogenes, Chromobacterium violaceum, and Heliobacter billis. It was further observed that, despite an identical bacterial composition over various reservoir sites, there were nominal differences in the relative abundance of the inhabitant species. Almost all reservoirs were dominated by Vogecella indigofera (~29%) and uncultured Proteobacterium (~15%). A seasonal analysis performed using BLAST resulted in a number of species exclusive to the season and the site of their growth. A high proportion of unidentified sequences were also reported which demands sequential identification. The results obtained through BLAST and Kaiju, were significantly different, suggesting inconsistencies and inaccuracies in existing metagenomic reads comparison.

Prevalence of Hepatitis B in Blood Groups and Level of Education of Blood Donors in Al-Najaf Governorate

Advancement in sequencing techniques and transformative progress in metagenomics provides an unprecedented platform for functional and taxonomic characterization of the enormous microbial diversity inhabiting and governing various biochemical processes of the freshwater sources. Metagenomic analysis of freshwater resources has led to the discovery and identification of novel microbial genes and an understanding of how microorganisms mediate energy and carbon. In this study, we report the taxonomical classification of bacterial sequences obtained from 6 dam reservoir sites in Pune city, Maharashtra, India. The analysis was performed using two different alignment tools: BLAST and Kaiju. The bacterial diversity was dominated by the presence of Vogecella indigofera, uncultured Proteobacterium, Wolinella Succinogenes, Chromobacterium violaceum, and Heliobacter billis. It was further observed that, despite an identical bacterial composition over various reservoir sites, there were nominal differences in the relative abundance of the inhabitant species. Almost all reservoirs were dominated by Vogecella indigofera (~29%) and uncultured Proteobacterium (~15%). A seasonal analysis performed using BLAST resulted in a number of species exclusive to the season and the site of their growth. A high proportion of unidentified sequences were also reported which demands sequential identification. The results obtained through BLAST and Kaiju, were significantly different, suggesting inconsistencies and inaccuracies in existing metagenomic reads comparison.

Crystallization and Preliminary X-ray Diffraction Study of a Mutant of L-Asparaginase from Wolinella succinogenes

The double mutant of Wolinella succinogenes L-asparaginase (Was72) with the V23Q and K24T substitutions in the C-terminal region of the N-terminal loop enclosing the active site was crystallized in the apo form and in complexes with L-aspartic and L-glutamic acids. This mutant exhibits glutaminase activity eight times lower compared to the wild-type enzyme. Crystals of the apo enzyme were grown in two modifications (sp. gr. P21 and sp. gr. P22121). Crystals grown in the presence of both aspartic and glutamic acids belong to the same space group (P21) but have different unit cell parameters. The X-ray diffraction data sets were collected from all types of crystals at 1.65–2.00 A resolution. All X-ray diffraction data sets are suitable for the determination of the three-dimensional structure of the enzyme.

Diverse hydrogen production and consumption pathways influence methane production in ruminants

Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H2 metabolism. Two-thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyse H2 production or consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g., Ruminococcus) accounted for half of all hydrogenase transcripts. Various H2 uptake pathways were also expressed, including methanogenesis (Methanobrevibacter), fumarate and nitrite reduction (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis-related transcripts predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic fumarate reducer Wolinella succinogenes in co-culture compared with pure culture. We conclude that H2 metabolism is a more complex and widespread trait among rumen microorganisms than previously recognised. There is evidence that alternative hydrogenotrophs, including acetogenic and respiratory bacteria, can prosper in the rumen and effectively compete with methanogens for H2. These findings may help to inform ongoing strategies to mitigate methane emissions by increasing flux through alternative H2 uptake pathways, including through animal selection, dietary supplementation and methanogenesis inhibitors.

Improvement of catalytic efficiency and thermal stability of l-asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus

Abstract Herein, the catalytic efficiency and thermal stability of l -Asparaginase (ASN, EC 3.5.1.1) from Bacillus subtilis 168 were enhanced by modifying its N-terminus highly flexible loop. A continuous non-conserved region (residues20–29) within the N-terminal loop of the ASN was identified and substituted with the structurally equivalent region of ASNs from Escherichia coli, Helicobacter pylori, Wolinella succinogenes, Pseudomonas aeruginosa, Erwinia chrysanthemi, and Pectobacterium carotovorum, yielding the ASN loop variants L1, L2, L3, L4, L5, and L6, respectively. In contrast to the wild-type enzyme, L6 exhibited the highest increase (2.1-fold) in specific activity. Molecular dynamics analysis suggested a negative correlation between the flexibility of the continuous non-conserved region and specific activity. Saturation mutagenesis and complex mutation on flexible residues Ala26 and Gly29 of L6 obtained a double variant L6-A26N/G29F with 3.44-fold and 7.76-fold increases in specific activity and half-life at 65 °C in comparison with wild-type enzyme, respectively. As suggested by structural analysis, the reduced flexibility of the continuous non-conserved region benefit the correct orientation of the active site Thr16 and closure of the substrate pocket. Our results demonstrated that reducing the flexibility of the highly flexible loop was an efficient method to enhance the catalytic efficiency and thermal stability of ASN.

Significance of MccR, MccC, MccD, MccL and 8-methylmenaquinone in sulfite respiration of Wolinella succinogenes

Reduction of sulfite to sulfide is an essential step in the biogeochemical sulfur cycle. The Epsilonproteobacterium Wolinella succinogenes uses the copper-containing octahaem cytochrome c sulfite reductase MccA to respire sulfite. MccA is encoded by the first gene of the mcc gene cluster, whose transcription is apparently induced by the two-component regulatory system MccRS. It has been proposed that the iron‑sulfur protein MccC, the putative quinol dehydrogenase MccD, the copper chaperone MccL as well as menaquinone-6 (MK6) and/or 8-methylmenaquinone-6 (8-MMK6) are involved in the electron transport chain of W. succinogenes sulfite respiration. Here, non-polar W. succinogenes mutants were constructed that lacked MccC, MccD, MccL or the 8-MMK6-producing MK6 methyltransferase MqnK. Each mutant possessed a frameshift-corrected mccR gene, thus inducing mcc expression in the presence of a mixture of fumarate and sulfite as terminal electron acceptors. Under these conditions, growth by sulfite respiration of cells lacking MccA, MccC or MccD was found to be abolished. However, cells lacking MccL or 8-MMK6 still coupled formate oxidation to sulfite reduction and grew by sulfite respiration to some extent. The results indicate that MccR, MccC, MccD, MccL and 8-MMK6 are essential or significant components of W. succinogenes sulfite respiration.

Structural insights into the electron/proton transfer pathways in the quinol:fumarate reductase from Desulfovibrio gigas

The membrane-embedded quinol:fumarate reductase (QFR) in anaerobic bacteria catalyzes the reduction of fumarate to succinate by quinol in the anaerobic respiratory chain. The electron/proton-transfer pathways in QFRs remain controversial. Here we report the crystal structure of QFR from the anaerobic sulphate-reducing bacterium Desulfovibrio gigas (D. gigas) at 3.6 Å resolution. The structure of the D. gigas QFR is a homo-dimer, each protomer comprising two hydrophilic subunits, A and B, and one transmembrane subunit C, together with six redox cofactors including two b-hemes. One menaquinone molecule is bound near heme bL in the hydrophobic subunit C. This location of the menaquinone-binding site differs from the menaquinol-binding cavity proposed previously for QFR from Wolinella succinogenes. The observed bound menaquinone might serve as an additional redox cofactor to mediate the proton-coupled electron transport across the membrane. Armed with these structural insights, we propose electron/proton-transfer pathways in the quinol reduction of fumarate to succinate in the D. gigas QFR.

Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK6 was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK6, 8-MMK6 and 7,8-DMMK6 were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.

Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a 'missing link' in the multihaem cytochrome c family.

Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite-ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host-associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep-sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao-maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao-typical absorbance maximum at 460 nm. In most cases, the εHao-encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane-bound HaoCA assembly reminiscent of the menaquinol-oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a 'missing link' in the evolution of NrfA and Hao enzymes.

Model-based quantification of metabolic interactions from dynamic microbial-community data.

An important challenge in microbial ecology is to infer metabolic-exchange fluxes between growing microbial species from community-level data, concerning species abundances and metabolite concentrations. Here we apply a model-based approach to integrate such experimental data and thereby infer metabolic-exchange fluxes. We designed a synthetic anaerobic co-culture of Clostridium acetobutylicum and Wolinella succinogenes that interact via interspecies hydrogen transfer and applied different environmental conditions for which we expected the metabolic-exchange rates to change. We used stoichiometric models of the metabolism of the two microorganisms that represents our current physiological understanding and found that this understanding - the model - is sufficient to infer the identity and magnitude of the metabolic-exchange fluxes and it suggested unexpected interactions. Where the model could not fit all experimental data, it indicates specific requirement for further physiological studies. We show that the nitrogen source influences the rate of interspecies hydrogen transfer in the co-culture. Additionally, the model can predict the intracellular fluxes and optimal metabolic exchange rates, which can point to engineering strategies. This study therefore offers a realistic illustration of the strengths and weaknesses of model-based integration of heterogenous data that makes inference of metabolic-exchange fluxes possible from community-level experimental data.

A class C radical S‐adenosylmethionine methyltransferase synthesizes 8‐methylmenaquinone

The membranous quinone/quinol pool is essential to the majority of life forms and has been widely used as an important biomarker in microbial taxonomy. In the anaerobic world, the most important quinones are menaquinone (MK) and a methylated form of MK, designated methylmenaquinone (MMK), which is anticipated to serve specifically in low-potential electron transport chains involved in anaerobic respiration. However, it has remained unclear how MMK is generated. Here, we show that a novel enzyme homologous to class C radical SAM methyltransferases (RSMTs) synthesizes MMK using MK as substrate. Such enzymes, termed either MenK or MqnK, are present in MMK-producing bacteria (and some archaea) that possess either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). An mqnK deletion mutant of the model Epsilonproteobacterium Wolinella succinogenes was unable to produce MMK6 but its formation was restored upon genomic complementation using either the native mqnK gene or menK from the human gut bacterium Adlercreutzia equolifaciens or Shewanella oneidensis. Moreover, any of the menK genes enabled Escherichia coli cells to produce MMK8 and a methylated form of 2-demethylmenaquinone8 (DMK8 ). The results expand the knowledge on quinone synthesis and demonstrate an unprecedented function for a class C RSMT-type enzyme in primary cell metabolism.