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1.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638775

RESUMO

Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl-CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.


Assuntos
Adaptação Fisiológica , Biomassa , Dioxanos/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Xanthobacter/metabolismo , Xanthobacter/genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-34410902

RESUMO

A novel, aerobic nitrogen-fixing methylotrophic bacterium, strain 29kT, was enriched and isolated from sludge generated during wastewater treatment at a paper mill in Baikal, Russian Federation. Cells were Gram-stain-variable. The cell wall was of the negative Gram-type. Cells were curved oval rod-shaped, 0.5-0.7×1.7-3.4 µm and formed yellow-coloured colonies. Cells tended to be pleomorphic if grown on media containing succinate or coccoid if grown in the presence of methyl alcohol as the sole carbon source. Cells were non-motile, non-spore-forming and contained retractile (polyphosphate) and lipid (poly-ß-hydroxybutyrate) bodies. The major respiratory quinone was ubiquinone Q-10 and the predominant cellular fatty acids were C18:1 ω7, C19:0 cyclo and C16:0. The genomic DNA G+C content was 67.95 mol%. Strain 29kT was able to grow at 4-37 °C (optimum, 30 °C), at pH 6.0-8.5 (optimum, pH 6.5-7.0) and at salinities of 0-0.5% (w/v) NaCl (optimum, 0% NaCl). Catalase and oxidase were positive. Strain 29kT could grow chemolithoautotrophically in mineral media under an atmosphere of H2, O2 and CO2 as well as chemoorganoheterotrophically on methanol, ethanol, n-propanol, n-butanol and various organic acids. The carbohydrate utilization spectrum is limited by glucose and raffinose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the newly isolated strain was a member of the genus Xanthobacter with Xanthobacter autotrophicus 7cT (99.9% similarity) and Xanthobacter viscosus 7dT (99.4 % similarity) as closest relatives among species with validly published names. The average nucleotide identity and digital DNA-DNA hybridization values of 92.7 and 44.9%, respectively, of the 29kT to the genome of the most closely related species, X. autotrophicus 7cT, were below the species cutoffs. Based on genotypic, phenotypic and chemotaxonomic characteristics, it is proposed that the isolate represents a novel species, Xanthobacter oligotrophicus sp. nov. The type strain is 29kT (=KCTC 72777T=VKM B-3453T).


Assuntos
Filogenia , Esgotos , Xanthobacter , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Pigmentação , RNA Ribossômico 16S/genética , Federação Russa , Análise de Sequência de DNA , Esgotos/microbiologia , Ubiquinona/análogos & derivados , Ubiquinona/química , Xanthobacter/classificação , Xanthobacter/isolamento & purificação
3.
J Biol Chem ; 297(2): 100961, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34265301

RESUMO

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe-His. Here, we propose that this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.


Assuntos
Dipeptídeos , Cetona Oxirredutases , Mesna , Carboxiliases/metabolismo , Domínio Catalítico , Oxirredutases/metabolismo , Especificidade por Substrato , Xanthobacter/metabolismo
4.
Curr Microbiol ; 78(3): 992-1005, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33547937

RESUMO

1,4-Dioxane is a highly toxic and carcinogenic pollutant found worldwide in groundwater and soil environments. Several microorganisms have been isolated by their ability to grow on 1,4-dioxane; however, low 1,4-dioxane tolerance and slow degradation kinetics remain obstacles for their use in 1,4-dioxane bioremediation. We report here the isolation and characterization of a new strain, Xanthobacter sp. YN2, capable of highly efficient 1,4-dioxane degradation. High degradation efficiency and high tolerance to 1,4-dioxane make this new strain an ideal candidate for the biodegradation of 1,4-dioxane in various treatment facilities. The maximum degradation rate of 1,4-dioxane was found to be 1.10 mg-1,4-dioxane/h mg-protein. Furthermore, Xanthobacter sp. YN2 was shown to grow in the presence of higher than 3000 mg/L 1,4-dioxane with little to no degradation inhibition. In addition, Xanthobacter sp. YN2 could grow on and degrade 1,4-dioxane at pH ranges 5 to 8 and temperatures between 20 and 40 °C. Xanthobacter sp. YN2 was also found to be able to grow on a variety of other substrates including several analogs of 1,4-dioxane. Genome sequence analyses revealed the presence of two soluble di-iron monooxygenase (SDIMO) gene clusters, and regulation studies determined that all of the genes in these two clusters were upregulated in the presence of 1,4-dioxane. This study provides insights into the bacterial stress response and the highly efficient biodegradation of 1,4-dioxane as well as the identification of a novel Group-2 SDIMO.


Assuntos
Dioxanos , Xanthobacter , Bactérias , Biodegradação Ambiental
5.
J Am Chem Soc ; 142(43): 18407-18421, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33075224

RESUMO

Polyphosphates (polyPs) are ubiquitous polymers in living organisms from bacteria to mammals. They serve a wide variety of biological functions, ranging from energy storage to stress response. In the last two decades, polyPs have been primarily viewed as linear polymers with varying chain lengths. However, recent biochemical data show that small metaphosphates, cyclic oligomers of [PO3](-), can bind to the enzymes ribonuclease A and NAD kinase, raising the question of whether metaphosphates can occur naturally as products of biological activity. Before the 1980s, metaphosphates had been reported in polyPs extracted from various organisms, but these results are considered artifactual due to the extraction and purification protocols. Here, we employ nondestructive 31P solid-state NMR spectroscopy to investigate the chemical structure of polyphosphates in whole cells as well as insoluble fractions of the bacterium Xanthobacter autotrophicus. Isotropic and anisotropic 31P chemical shifts of hydrated whole cells indicate the coexistence of linear and cyclic phosphates. Under our cell growth conditions and the concentrated conditions of the solid-state NMR samples, we found substantial amounts of cyclic phosphates in X. autotrophicus, suggesting that in fresh cells metaphosphate concentrations can be significant. The cellular metaphosphates are identified by comparison with the 31P chemical shift anisotropy of synthetic metaphosphates of known structures. In X. autotrophicus, the metaphosphates have a chemical shift anisotropy that is consistent with an average size of 3-8 phosphate units. These metaphosphates are enriched in insoluble and electron-dense granules. Exogenous hexametaphosphate added to X. autotrophicus cell extracts is metabolized to trimetaphosphates, supporting the presence and biological role of metaphosphates in cells. The definitive evidence for the presence of metaphosphates, reported here in whole bacterial cells for the first time, opens the path for future investigations of the biological function of metaphosphates in many organisms.


Assuntos
Espectroscopia de Ressonância Magnética , Polifosfatos/química , Fósforo/química , Polifosfatos/metabolismo , Xanthobacter/metabolismo
6.
Chemosphere ; 260: 127514, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32688309

RESUMO

The main aim of this study was to evaluate the performance of an air membrane bioreactor (aMBR) for the treatment of gas-phase methanol. A laboratory-scale hollow fiber aMBR was operated for 150 days, at inlet methanol concentrations varying between 2 and 30 g m-3 and at empty bed residence times (EBRT) of 30, 10 and 5 s. Under steady-state conditions, a maximum methanol removal efficiency (RE) of 98% was obtained at an EBRT of 30 s and a decrease in RE of methanol was observed at lower EBRTs. On increasing the inlet loading rate, some portion of gas-phase MeOH was stripped into the liquid phase due to its solubility in water. Under transient conditions, the MeOH removal efficiency dropped from an average value of 95%-90% after 5 h of 10-fold shock load and dropped from an average value of 95%-88% under 5-fold increase in shock load. During transient-state tests, the aMBR performed well under different upset loading conditions and a drop in RE of ∼ 5-10% was observed. However, the aMBR performance was restored within 1-2 days when pre-shock conditions were restored. The results from microbial structure analysis revealed a big shift of the dominant methanol degrader, from Candida boidinii strain TBRC 217 to Xanthobacter sp. and Fusicolla sp., respectively.


Assuntos
Poluentes Atmosféricos/análise , Reatores Biológicos/microbiologia , Membranas Artificiais , Metanol/análise , Biodegradação Ambiental , Biofilmes/crescimento & desenvolvimento , Candida/crescimento & desenvolvimento , Desenho de Equipamento , Filtração/métodos , Xanthobacter/crescimento & desenvolvimento
7.
J Biol Chem ; 294(30): 11536-11548, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31182484

RESUMO

An engineered metabolic pathway consisting of reactions that convert fatty acids to aldehydes and eventually alkanes would provide a means to produce biofuels from renewable energy sources. The enzyme aldehyde-deformylating oxygenase (ADO) catalyzes the conversion of aldehydes and oxygen to alkanes and formic acid and uses oxygen and a cellular reductant such as ferredoxin (Fd) as co-substrates. In this report, we aimed to increase ADO-mediated alkane production by converting an unused by-product, formate, to a reductant that can be used by ADO. We achieved this by including the gene (fdh), encoding formate dehydrogenase from Xanthobacter sp. 91 (XaFDH), into a metabolic pathway expressed in Escherichia coli Using this approach, we could increase bacterial alkane production, resulting in a conversion yield of ∼50%, the highest yield reported to date. Measuring intracellular nicotinamide concentrations, we found that E. coli cells harboring XaFDH have a significantly higher concentration of NADH and a higher NADH/NAD+ ratio than E. coli cells lacking XaFDH. In vitro analysis disclosed that ferredoxin (flavodoxin):NADP+ oxidoreductase could use NADH to reduce Fd and thus facilitate ADO-mediated alkane production. As formic acid can decrease the cellular pH, the addition of formate dehydrogenase could also maintain the cellular pH in the neutral range, which is more suitable for alkane production. We conclude that this simple, dual-pronged approach of increasing NAD(P)H and removing extra formic acid is efficient for increasing the production of renewable alkanes via synthetic biology-based approaches.


Assuntos
Alcanos/metabolismo , Formiato Desidrogenases/metabolismo , Engenharia Metabólica/métodos , Xanthobacter/metabolismo , Biocombustíveis , Catálise , Clonagem Molecular , Escherichia coli/genética , Ácidos Graxos/metabolismo , Formiato Desidrogenases/genética , NAD/metabolismo , Oxirredução , Xanthobacter/enzimologia
8.
Appl Microbiol Biotechnol ; 103(11): 4525-4538, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993384

RESUMO

Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition-based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose.


Assuntos
Biofilmes/crescimento & desenvolvimento , Consórcios Microbianos , Ramnose/metabolismo , Xanthobacter/crescimento & desenvolvimento , Xanthobacter/metabolismo , Carbono/metabolismo , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/metabolismo , Glucose/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Ramnose/isolamento & purificação , Análise de Sequência de DNA , Esgotos/microbiologia
9.
Mol Biotechnol ; 61(7): 477-488, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919327

RESUMO

The application of native enzymes may not be economical owing to the stability factor. A smaller protein molecule may be less susceptible to external stresses. Haloalkane dehalogenases (HLDs) that act on toxic haloalkanes may be incorporated as bioreceptors to detect haloalkane contaminants. Therefore, this study aims to develop mini proteins of HLD as an alternative bioreceptor which was able to withstand extreme conditions. Initially, the mini proteins were designed through computer modeling. Based on the results, five designed mini proteins were deemed to be viable stable mini proteins. They were then validated through experimental study. The smallest mini protein (model 5) was chosen for subsequent analysis as it was expressed in soluble form. No dehalogenase activity was detected, thus the specific binding interaction of between 1,3-dibromopropane with mini protein was investigated using isothermal titration calorimetry. Higher binding affinity between 1,3-dibromopropane and mini protein was obtained than the native. Thermal stability study with circular dichroism had proven that the mini protein possessed two times higher Tm value at 83.73 °C than the native at 43.97 °C. In conclusion, a stable mini protein was successfully designed and may be used as bioreceptors in the haloalkane sensor that is suitable for industrial application.


Assuntos
Proteínas de Bactérias/química , Simulação por Computador , Hidrolases/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/síntese química , Hidrolases/biossíntese , Hidrolases/síntese química , Conformação Proteica , Especificidade por Substrato , Xanthobacter/enzimologia
10.
J Biol Chem ; 294(13): 5137-5145, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696768

RESUMO

NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.


Assuntos
Dióxido de Carbono/metabolismo , Cetona Oxirredutases/metabolismo , Xanthobacter/enzimologia , Domínio Catalítico , Cetona Oxirredutases/química , Cinética , Modelos Moleculares , NADP/metabolismo , Oxirredução , Especificidade por Substrato , Xanthobacter/química , Xanthobacter/metabolismo
11.
ACS Synth Biol ; 7(12): 2758-2764, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30433765

RESUMO

The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ∼10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus. Furthermore, engineering an enzyme in the CoA detour, the Lactobacillus brevis CoA-acylating aldehyde dehydrogenase, allowed stringent product selectivity. Targeted production of 3-methyl-1-butanol (3-MB) in E. coli mediated by the CoA detour showed a 3-MB:side-product (isobutanol) ratio of >20, an increase over the ratios previously achieved using the conventional Ehrlich pathway.


Assuntos
Coenzima A/metabolismo , Engenharia Metabólica/métodos , Pentanóis/metabolismo , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Escherichia coli/metabolismo , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Lactobacillus brevis/enzimologia , Pentanóis/química , Plasmídeos/genética , Plasmídeos/metabolismo , Xanthobacter/enzimologia
12.
J Biol Chem ; 293(29): 11505-11512, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29858243

RESUMO

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.


Assuntos
Cumarínicos/metabolismo , Hidrolases/metabolismo , Xanthobacter/enzimologia , Domínio Catalítico , Cumarínicos/química , Cristalografia por Raios X , Dicloretos de Etileno/metabolismo , Halogenação , Hidrolases/química , Cinética , Metilação , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por Substrato , Xanthobacter/química , Xanthobacter/metabolismo
13.
J Biol Chem ; 293(14): 5236-5246, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29414784

RESUMO

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze ß-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes ß-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing ß-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate ß-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.


Assuntos
Mesna/metabolismo , Fosfatos/metabolismo , Xanthobacter/metabolismo , Aspartato Amônia-Liase/metabolismo , Bactérias/metabolismo , Biologia Computacional/métodos , Cristalografia por Raios X , Fumarato Hidratase/metabolismo , Fumaratos , Fosfoenolpiruvato/metabolismo , Ácidos Fosfóricos , Monoéster Fosfórico Hidrolases , Proteômica , Fosfato de Piridoxal
14.
J Oleo Sci ; 66(11): 1247-1256, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29021488

RESUMO

It is important to construct microbiological treatment systems for organic solvent-contaminated water. We developed a continuous culture supplemented with a biostimulation agent named BD-C, which is formulated from canola oil, and Xanthobacter autotrophicus strain GJ10 for an aerobic dichloromethane (DCM)-dechlorinating microorganism. The continuous culture was a chemostat constructed using a 1 L screw-capped bottle containing artificial wastewater medium with 2.0 mM DCM and 1.0% (v/v) BD-C. The expression of genes for DCM metabolism in the dechlorinating aerobe was monitored and analyzed by reverse transcription-quantitative PCR. Strain GJ10 was able to dechlorinate approximately 74% of the DCM in medium supplemented with BD-C during 12 days of incubation. The DCM dechlorination rate was calculated to be 0.11 mM/day. The ΔΔCT method showed that expression of haloalkane dehalogenase increased 5.4 times in the presence of BD-C. Based on batch culture growth tests conducted with mineral salt medium containing three DCM concentrations (0.07, 0.20, 0.43 and 0.65 mM) with BD-C, the apparent maximum specific consumption rate (νmax) and the saturation constant (Ks) determined for DCM degradation in this test were 19.0 nmol/h/CFU and 0.44 mM, respectively. In conclusion, BD-C enhanced the aerobic degradation of DCM by strain GJ10.


Assuntos
Detergentes , Ácidos Graxos , Cloreto de Metileno/metabolismo , Óleo de Brassica napus , Xanthobacter/metabolismo , Acetatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Halogenação , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Xanthobacter/genética
15.
Proc Natl Acad Sci U S A ; 114(25): 6450-6455, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28588143

RESUMO

We demonstrate the synthesis of NH3 from N2 and H2O at ambient conditions in a single reactor by coupling hydrogen generation from catalytic water splitting to a H2-oxidizing bacterium Xanthobacter autotrophicus, which performs N2 and CO2 reduction to solid biomass. Living cells of X. autotrophicus may be directly applied as a biofertilizer to improve growth of radishes, a model crop plant, by up to ∼1,440% in terms of storage root mass. The NH3 generated from nitrogenase (N2ase) in X. autotrophicus can be diverted from biomass formation to an extracellular ammonia production with the addition of a glutamate synthetase inhibitor. The N2 reduction reaction proceeds at a low driving force with a turnover number of 9 × 109 cell-1 and turnover frequency of 1.9 × 104 s-1⋅cell-1 without the use of sacrificial chemical reagents or carbon feedstocks other than CO2 This approach can be powered by renewable electricity, enabling the sustainable and selective production of ammonia and biofertilizers in a distributed manner.


Assuntos
Ciclo do Nitrogênio/fisiologia , Nitrogênio/metabolismo , Amônia/metabolismo , Biomassa , Catálise , Hidrogênio/metabolismo , Fixação de Nitrogênio/fisiologia , Nitrogenase/metabolismo , Temperatura , Água/metabolismo , Xanthobacter/metabolismo
16.
Mikrobiologiia ; 86(1): 107-13, 2017.
Artigo em Chinês | MEDLINE | ID: mdl-30207149

RESUMO

During the summer period (15­25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 µg/mL) and are probably plant symbionts.


Assuntos
Biodiversidade , Florestas , Methylobacillus , Methylobacterium , Methylophilus , Paracoccus , Xanthobacter , Methylobacillus/classificação , Methylobacillus/crescimento & desenvolvimento , Methylobacillus/isolamento & purificação , Methylobacterium/classificação , Methylobacterium/crescimento & desenvolvimento , Methylobacterium/isolamento & purificação , Methylophilus/classificação , Methylophilus/crescimento & desenvolvimento , Methylophilus/isolamento & purificação , Paracoccus/classificação , Paracoccus/crescimento & desenvolvimento , Paracoccus/isolamento & purificação , Federação Russa , Xanthobacter/classificação , Xanthobacter/crescimento & desenvolvimento , Xanthobacter/isolamento & purificação
17.
FEBS Lett ; 590(17): 2991-6, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27447465

RESUMO

The characteristic His-Glu catalytic dyad of the disulfide oxidoreductase (DSOR) family of enzymes is replaced in 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) by the residues Phe-His. 2-KPCC is the only known carboxylating member of the DSOR family and has replaced this dyad potentially to eliminate proton-donating groups at a key position in the active site. Substitution of the Phe-His by the canonical residues results in production of higher relative concentrations of acetone versus the natural product acetoacetate. The results indicate that these differences in 2-KPCC are key in discriminating between carbon dioxide and protons as attacking electrophiles.


Assuntos
Catálise , Dipeptídeos/química , Cetona Oxirredutases/química , Dióxido de Carbono/química , Domínio Catalítico , Dipeptídeos/metabolismo , Cetona Oxirredutases/metabolismo , Prótons , Especificidade por Substrato , Xanthobacter/química , Xanthobacter/enzimologia
18.
ACS Synth Biol ; 5(12): 1485-1496, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27403844

RESUMO

We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.


Assuntos
2-Propanol/metabolismo , Escherichia coli/metabolismo , Engenharia Genética/métodos , Sarina/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Óperon , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Xanthobacter/genética , Xanthobacter/metabolismo
19.
Appl Environ Microbiol ; 82(17): 5298-308, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27342553

RESUMO

UNLABELLED: 1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1 Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE: Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes.


Assuntos
Alphaproteobacteria/isolamento & purificação , Dicloretos de Etileno/metabolismo , Água Subterrânea/microbiologia , Plasmídeos/genética , Poluentes Químicos da Água/metabolismo , Xanthobacter/isolamento & purificação , Alphaproteobacteria/química , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Sequência de Aminoácidos , Austrália , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Dados de Sequência Molecular , Plasmídeos/metabolismo , Alinhamento de Sequência , Poluição Química da Água , Xanthobacter/química , Xanthobacter/genética , Xanthobacter/metabolismo
20.
Appl Environ Microbiol ; 81(22): 7833-8, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26341208

RESUMO

Two previously uncharacterized potential broad-spectrum mercury (Hg) resistance operons (mer) are present on the chromosome of the soil Alphaproteobacteria Xanthobacter autotrophicus Py2. These operons, mer1 and mer2, contain two features which are commonly found in mer operons in the genomes of soil and marine Alphaproteobacteria, but are not present in previously characterized mer operons: a gene for the mercuric reductase (MerA) that encodes an alkylmercury lyase domain typical of those found on the MerB protein, and the presence of an additional gene, which we are calling merK, with homology to glutathione reductase. Here, we demonstrate that Py2 is resistant to 0.2 µM inorganic mercury [Hg(II)] and 0.05 µM methylmercury (MeHg). Py2 is capable of converting MeHg and Hg(II) to elemental mercury [Hg(0)], and reduction of Hg(II) is induced by incubation in sub toxic concentrations of Hg(II). Transcription of the merA genes increased with Hg(II) treatment, and in both operons merK resides on the same polycistronic mRNA as merA. We propose the use of Py2 as a model system for studying the contribution of mer to Hg mobility in soil and marine ecosystems.


Assuntos
Proteínas de Bactérias/genética , Mercúrio/metabolismo , Compostos de Metilmercúrio/metabolismo , Poluentes do Solo/metabolismo , Xanthobacter/genética , Proteínas de Bactérias/metabolismo , Óperon , Oxirredução , Xanthobacter/metabolismo
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