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Chembiochem : a European Journal of... Mar 2020Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in...
Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH ) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF-CH ) reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-p-aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl-CH and THF. Altogether, the THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.
Topics: Bacterial Proteins; Betaine; Biocatalysis; Crystallography, X-Ray; Desulfitobacterium; Methylation; Models, Molecular; Molecular Structure; Tetrahydrofolates
PubMed: 31518049
DOI: 10.1002/cbic.201900515 -
MBio Aug 2019Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. JR...
Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis. Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.
Topics: Cell Membrane; Cytochrome c Group; Electricity; Electron Transport; Electrons; Gram-Positive Bacteria; Heme; Kinetics; Metals; Oxidation-Reduction; Oxidoreductases; Peptococcaceae; Protein Conformation
PubMed: 31431546
DOI: 10.1128/mBio.01210-19 -
Frontiers in Microbiology 2019() functions as a probiotic in animals, but the underlying mechanisms remain unclear. We aim to evaluate the protective effects and definite mechanism by which orally...
() functions as a probiotic in animals, but the underlying mechanisms remain unclear. We aim to evaluate the protective effects and definite mechanism by which orally administered prevents D-galactosamine (D-GalN)-induced liver injury in rats. Twenty-one Sprague-Dawley rats were equally assigned into three groups ( = 7 animals per group). ATCC11778 (2 × 10 colony-forming units/ml) was administered to the group via gavage, and phosphate-buffered saline was administered to the positive control (PC) and negative control (NC) groups for 2 weeks. The PC and groups received 1.1 g/kg D-GalN via an intraperitoneal injection to induce liver injury. The blood, terminal ileum, liver, kidney and mesenteric lymph nodes (MLNs) were collected for histological examinations and to evaluate bacterial translocation. Liver function was also determined. Fecal samples were collected for deep sequencing of the 16S rRNA on an Illumina MiSeq platform. significantly attenuated D-GalN-induced liver injury and improved serum alanine aminotransferase (ALT) and serum cholinesterase levels ( < 0.05 and < 0.01, respectively). modulated cytokine secretion, as indicated by the elevated levels of the anti-inflammatory cytokine interleukin-10 (IL-10) in both the liver and plasma ( < 0.05 and < 0.01, respectively) and the substantially decreased levels of the cytokine IL-13 in the liver ( < 0.05). Pretreatment with attenuated anoxygenic bacterial translocation in the veins ( < 0.05) and liver ( < 0.05) and upregulated the expression of the tight junction protein 1. The gut microbiota from the group clustered separately from that of the PC group, with an increase in species of the and families and a decrease in those of the , , and families. The potential probiotic attenuated liver injury by restoring the gut flora balance and enhancing the intestinal barrier function.
PubMed: 31417535
DOI: 10.3389/fmicb.2019.01751 -
International Journal of Environmental... Aug 2019Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H) for various electron-accepting processes....
Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H) for various electron-accepting processes. This deficiency often results in the accumulation of persistent organic pollutants, where bioremediation using organic compounds often leads to secondary contamination. This study involves the reductive dechlorination of pentachlorophenol (PCP) by dechlorinators that do not use H directly, but rather through a reduced state of humin-a solid-phase humic substance-as the extracellular electron donor, which requires an organic donor such as formate, lactate, etc. This shortcoming was addressed by the development of an anaerobic mixed culture that was capable of reductively dechlorinating PCP using humin under autotrophic conditions induced by homoacetogenesis. Here, H was used for carbon-dioxide fixation to acetate; the acetate produced was used for the reduction of humin; and consequently used for dechlorination through reduced humin. The 16SrRNA gene sequencing analysis showed Dehalobacter and Dehalobacterium as the possible dechlorinators, while Clostridium and Oxobacter were identified as the homoacetogens. Thus, this work contributes to the development of an anaerobic consortium that balanced H dependency, where efficiency of humin reduction extends the applicability of anaerobic microbial remediation in aquifers through autotrophy, syntrophy, and reductive dechlorination.
Topics: Anaerobiosis; Autotrophic Processes; Biodegradation, Environmental; Clostridium; Electrons; Groundwater; Halogenation; Humic Substances; Microbiota; Oxidation-Reduction; Pentachlorophenol; Peptococcaceae; Water Pollutants, Chemical
PubMed: 31405258
DOI: 10.3390/ijerph16162873 -
Applied Microbiology and Biotechnology Jun 2019Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide...
Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens.
Topics: Clostridium; Culture Media; Desulfotomaculum; Gene Expression; Genes, Bacterial; Metabolic Engineering; Metabolic Networks and Pathways; Recombinant Proteins; Vitamins
PubMed: 30972463
DOI: 10.1007/s00253-019-09763-6 -
Extremophiles : Life Under Extreme... Jul 2019A thermophilic and hydrogenogenic carboxydotroph, Carboxydothermus pertinax, performs hydrogenogenic CO metabolism in which CODH-II couples with distally encoded ECH. To...
A thermophilic and hydrogenogenic carboxydotroph, Carboxydothermus pertinax, performs hydrogenogenic CO metabolism in which CODH-II couples with distally encoded ECH. To enhance our knowledge of its hydrogenogenic CO metabolism, we performed whole transcriptome analysis of C. pertinax grown under 100% CO or 100% N using RNA sequencing. Of the 2577 genes, 36 and 64 genes were differentially expressed genes (DEGs) with false discovery rate adjusted P value < 0.05 when grown under 100% CO or 100% N, respectively. Most of the DEGs were components of 23 gene clusters, suggesting switch between metabolisms via intensive expression changes in a relatively low number of gene clusters. Of the 9 significantly expressed gene clusters under 100% CO, CODH-II and ECH gene clusters were found. Only the ECH gene cluster was regulated by the CO-responsive transcriptional factor CooA, suggesting that others were separately regulated in the same transcriptional cascade as the ECH gene cluster. Of the 14 significantly expressed gene clusters under 100% N, ferrous iron transport gene cluster involved in anaerobic respiration and prophage region were found. Considering that the expression of the temperate phage was strictly repressed under 100% CO, hydrogenogenic CO metabolism might be stable for C. pertinax.
Topics: Aldehyde Oxidoreductases; Bacterial Proteins; Carbon Monoxide; Hydrogen; Multienzyme Complexes; Peptococcaceae; Thermotolerance; Transcription Factors; Transcriptome
PubMed: 30941583
DOI: 10.1007/s00792-019-01091-x -
The ISME Journal Aug 2019An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where...
An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where it formed a single-species ecosystem fuelled by hydrogen from water radiolysis. It was believed that in situ conditions CDA relied on scarce energy supply and did not divide for hundreds to thousand years. We have isolated CDA strain BYF from a 2-km-deep aquifer in Western Siberia and obtained a laboratory culture growing with a doubling time of 28.5 h. BYF uses not only H but also various organic electron donors for sulfate respiration. Growth required elemental iron, and ferrous iron did not substitute for it. A complex intracellular organization included gas vesicles, internal membranes, and electron-dense structures enriched in phosphorus, iron, and calcium. Genome comparison of BYF with the South African CDA revealed minimal differences mostly related to mobile elements and prophage insertions. Two genomes harbored <800 single-nucleotide polymorphisms and had nearly identical CRISPR loci. We suggest that spores with the gas vesicles may facilitate global distribution of CDA followed by colonization of suitable subsurface environments. Alternatively, a slow evolution rate in the deep subsurface could result in high genetic similarity of CDA populations at two sites spatially separated for hundreds of millions of years.
Topics: Ecosystem; Evolution, Molecular; Genomics; Groundwater; Iron; Peptococcaceae; Phylogeny; Siberia; South Africa; Sulfates
PubMed: 30899075
DOI: 10.1038/s41396-019-0402-3 -
MBio Feb 2019Microbial diversity in the environment is mainly concealed within the rare biosphere (all species with <0.1% relative abundance). While dormancy explains a low-abundance...
Microbial diversity in the environment is mainly concealed within the rare biosphere (all species with <0.1% relative abundance). While dormancy explains a low-abundance state very well, the mechanisms leading to rare but active microorganisms remain elusive. We used environmental systems biology to genomically and transcriptionally characterize " Desulfosporosinus infrequens," a low-abundance sulfate-reducing microorganism cosmopolitan to freshwater wetlands, where it contributes to cryptic sulfur cycling. We obtained its near-complete genome by metagenomics of acidic peat soil. In addition, we analyzed anoxic peat soil incubated under -like conditions for 50 days by -targeted qPCR and metatranscriptomics. The population stayed at a constant low abundance under all incubation conditions, averaging 1.2 × 10 16S rRNA gene copies per cm³ soil. In contrast, transcriptional activity of " Desulfosporosinus infrequens" increased at day 36 by 56- to 188-fold when minor amendments of acetate, propionate, lactate, or butyrate were provided with sulfate, compared to the no-substrate-control. Overall transcriptional activity was driven by expression of genes encoding ribosomal proteins, energy metabolism, and stress response but not by expression of genes encoding cell growth-associated processes. Since our results did not support growth of these highly active microorganisms in terms of biomass increase or cell division, they had to invest their sole energy for maintenance, most likely counterbalancing acidic pH conditions. This finding explains how a rare biosphere member can contribute to a biogeochemically relevant process while remaining in a zero-growth state over a period of 50 days. The microbial rare biosphere represents the largest pool of biodiversity on Earth and constitutes, in sum of all its members, a considerable part of a habitat's biomass. Dormancy or starvation is typically used to explain the persistence of low-abundance microorganisms in the environment. We show that a low-abundance microorganism can be highly transcriptionally active while remaining in a zero-growth state for at least 7 weeks. Our results provide evidence that this zero growth at a high cellular activity state is driven by maintenance requirements. We show that this is true for a microbial keystone species, in particular a cosmopolitan but permanently low-abundance sulfate-reducing microorganism in wetlands that is involved in counterbalancing greenhouse gas emissions. In summary, our results provide an important step forward in understanding time-resolved activities of rare biosphere members relevant for ecosystem functions.
Topics: Bacterial Load; Biomass; Gene Expression Profiling; Genome, Bacterial; Metagenomics; Peptococcaceae; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Soil Microbiology; Transcription, Genetic
PubMed: 30755506
DOI: 10.1128/mBio.02189-18 -
Applied and Environmental Microbiology Mar 2019Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic...
Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader " Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway. Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader " Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium and " Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.
Topics: Amino Acid Sequence; Anaerobiosis; Bacterial Proteins; Biodegradation, Environmental; Methylene Chloride; Methyltransferases; Peptococcaceae; Proteogenomics; Sequence Alignment
PubMed: 30658979
DOI: 10.1128/AEM.02768-18 -
Frontiers in Microbiology 2018Sulfate- and iron-reducing heterotrophic bacteria represented minor proportion of the indigenous microbial community of highly acidic, oligotrophic acid mine drainage...
Sulfate- and iron-reducing heterotrophic bacteria represented minor proportion of the indigenous microbial community of highly acidic, oligotrophic acid mine drainage (AMD), but they can be successfully stimulated for bioremediation of an AMD impacted soil (AIS). These anaerobic microorganisms although played central role in sulfate- and metal-removal, they remained inactive in the AIS due to the paucity of organic carbon and extreme acidity of the local environment. The present study investigated the scope for increasing the abundance and activity of inhabitant sulfate- and iron-reducing bacterial populations of an AIS from Malanjkhand Copper Project. An AIS of pH 3.5, high soluble SO (7838 mg/l) and Fe (179 mg/l) content was amended with nutrients (cysteine and lactate). Thorough geochemical analysis, 16S rRNA gene amplicon sequencing and qPCR highlighted the intrinsic metabolic abilities of native bacteria in AMD bioremediation. Following 180 days incubation, the nutrient amended AIS showed marked increase in pH (to 6.6) and reduction in soluble -SO (95%), -Fe (50%) and other heavy metals. Concomitant to physicochemical changes a vivid shift in microbial community composition was observed. Members of the present as a minor group (1.5% of total community) in AIS emerged as the single most abundant taxon (∼56%) following nutrient amendments. Organisms affiliated to , etc. known for their fermentative, iron and sulfate reducing abilities were prevailed in the amended samples. qPCR data corroborated with this change and further revealed an increase in abundance of dissimilatory sulfite reductase gene (B) and specific bacterial taxa. Involvement of these enhanced populations in reductive processes was validated by further enrichments and growth in sulfate- and iron-reducing media. Amplicon sequencing of these enrichments confirmed growth of members and proved their sulfate- and iron-reduction abilities. This study provided a better insight on ecological perspective of members within the AMD impacted sites, particularly their involvement in sulfate- and iron-reduction processes, pH management and bioremediation.
PubMed: 30619102
DOI: 10.3389/fmicb.2018.02882