Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分的纯化和性质

Methylomonassp．GYJ3菌株中经DEAE-SepharoseCL-6B阴离子交换层析和SephacrylS300凝胶层析分离纯化出甲烷加氧酶羟基化酶组分．经HPLC分析,纯度大于90％,分子量为240kD,纯化倍数为3．9,比活为225nmol环氧丙烷每分钟毫克蛋白．SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD．ICPAES测定羟基化酶的Fe含量为2.1molFe每摩尔蛋白．HPLC法用于甲烷单加氧酶羟基化酶组分的纯化,纯化的羟基化酶组分比活为541nmol（环氧丙烷）每分钟毫克蛋白,是两步LC法纯化的羟基化酶的两倍,Fe含量为3．78molFe每摩尔蛋白．催化性质研究表明羟基化酶能够被化学还原剂还原为还原态羟基化酶,还原态的羟基化酶单独存在时表现出MMO活性,说明它是MMO活性中心,天然态的羟基化酶单独存在时无MMO活性,加入粗酶液中MMO活性明显增加,说明GYJ3菌中MMO是一个复合酶系．     

Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading Methylomonas sp. Strains and Detection of Methanotrophs during In Situ Bioremediation

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.     

Effects of Temperature on Methane Consumption in a Forest Soil and in Pure Cultures of the Methanotroph Methylomonas rubra

Methane oxidation in soil cores from a mixed hardwood-coniferous forest varied relatively little as a function of incubation temperatures from −1 to 30°C. The increase in oxidation rate was proportional to T^2.4 (in kelvins). This relationship was consistent with limitation of methane transport through a soil gas phase to a subsurface zone of consumption by diffusion. The Q₁₀ for CO₂ production, 3.4, was substantially higher than that for methane oxidation, 1.1, and indicated that the response of soil respiration to temperature was limited by enzymatic processes and not diffusion of either organic substrates or molecular oxygen. When grown under conditions of phase-transfer limitation, cultures of Methylomonas rubra showed a minimal response to temperature changes between 19 and 38°C, as indicated by methane oxidation rates; in the absence of phase-transfer limitations, M. rubra oxidized methane at rates strongly dependent on temperature.     

Purification and Properties of a Soluble Methane Monooxygenase from Methylocystis sp. M

A soluble methane monooxygenase (sMMO: EC 1.14.13.25) was purified from a type II obligate methanotroph, Methylocystis sp. M. Ion exchange chromatography elution separated the sMMO into three components, I, II, and III. Components II and III were purified to homogeneity and were essential for the sMMO activity. Components II and III had molecular masses of approximately 233,000 and 39,000, respectively. Component II consisted of three subunits with molecular masses of 55,000, 44,000, and 21,000, which appeared to be present in stoichiometric amounts, suggesting a (αβγ)₂ configuration in the native protein. Component II contained 1–4 mol of iron and was considered to be a hydroxylase. Component III was a flavoprotein, which contained 1 mol of FAD as well as 1–2mol of iron. It catalyzed the reduction of K₃Fe(CN)₆ and 2,6-dichloroindophenol by NADH. Component I, which was partially purified and not essential for sMMO activity, stimulated the activity by about 11-fold. Its stimulation could be replaced by addition of Fe²⁺. The molecular mass of the partially purified component I was estimated to be from 35,000 to 40,000 based on gel filtration, which suggested the presence of a new type of regulatory protein of sMMO.     

Oxidation of Trichloroethylene, 1,1-Dichloroethylene, and Chloroform by Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE (3.3 μM), 1,1-DCE (1.25 μM), and chloroform (6.3 μM) at initial rates of 3.1, 3.6, and 1.6 nmol/(min · mg of protein), respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization (2.6, 1.5, and 2.3 Cl⁻ atoms per molecule of TCE, 1,1-DCE, and chloroform, respectively). Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.     

Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium Methylocystis sp. Strain WI 14

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α₂β₂γ₂ structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg²⁺ ions (with a total loss of enzyme activity at 0.01 mM Hg²⁺) and Cu²⁺, Zn²⁺, and Ni²⁺ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).     

Production of Cytochrome c by an Obligate Methylotroph,Methylomonas sp. YK 1

An obligate methanol-utilizing bacterium, Methylomonas sp. YK 1, was isolated and used as a cytochrome c producer. The strain was mutagenized so as to be resistant to metabolic inhibitors related to the function of cytochrome c. The strain, YK 56, which was derived as a KCN-resistant mutant contained 3 times the cellular level of cytochrome c compared to the parent strain. Optimization of the culture conditions for the mutant to enhance the cytochrome c productivity was performed. Peptone, succinate, l-malate or FeSO₄ · 7H₂O increased the productivity when added to the culture medium. Under the optimal culture conditions, strain YK 56 produced about 60 mg cytochrome c per liter when methanol and peptone were fed to the medium during the cultivation.     

Production of D-[1-13C]Fructose 6-Phosphate by a Formaldehyde-fixing System in Methylomonas aminofaciens 77a

Restriction patterns of plasmids isolated from ten Japanese wild strains of Agrobacterium rhizogenes were analyzed and the homology of the plasmid digestion patterns within these strains is described. From the size of homologous fragment against the EcoRI digested 7.5 kb T-DNA of pRi1724, these strains could be divided into two groups which was consistent with opine types.     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

[1-14C] Acetate assimilation by obligate methylotrophs,Pseudomonas methanica and Methylosinus trichosporium

The oxidation of one carbon compounds (methane, methanol, formaldehyde, formate) and primary alcohols (ethanol, propanol, butanol) supported the assimilation of [1-¹⁴C]acetate by cell suspensions of type I obligate methylotroph; Pseudomonas methanica, Texas strain, and type II obligate methylotroph, Methylosinus trichosporium, strain PG. The amount of oxygen consumed and substrate oxidized correlated with the amount of [1-¹⁴C]acetate assimilated during oxidation of C-1 compounds and primary alcohols.Oxidation of methanol, formaldehyde, and primary alcohols in extracts of Pseudomonas methanica, Texas strain, and Methylosinus trichosporium, strain PG, was catalyzed by a phenazine methosulfate linked, ammonium ion dependent methanol dehydrogenase. The oxidation of aldehydes was catalyzed by a phenazine methosulfate linked, ammonium ion independent aldehyde dehydrogenase. Formate was oxidized by a NAD⁺ linked formate dehydrogenase.Deceased.This work was supported by Grant GB 8173 from the National Science Foundation and by a grant from the Robert A. Welch Foundation.     

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme,Active on C2 to C4 Alkanes and Alkenes

Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc²-155. Cells of mc²-155(pSmo) metabolized C₂ to C₄ alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc²-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc²-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc²-155(pSmo) provides a new model for studying sMMO-like monooxygenases.     

Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.     

p53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling

Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling.     

SRSF1 Facilitates Cytosolic DNA-Induced Production of Type I Interferons Recognized by RIG-I

BackgroundEvidence has shown that psoriasis is closely associated with infection; however, the mechanism of this association remains unclear. In mammalian cells, viral or bacterial infection is accompanied by the release of cytosolic DNA, which in turn triggers the production of type-I interferons (IFNs). Type I IFNs and their associated genes are significantly upregulated in psoriatic lesions. RIG-I is also highly upregulated in psoriatic lesions and is responsible for IFN production. However, RIG-I mediated regulatory signaling in psoriasis is poorly understood.MethodsWe screened a cDNA library and identified potential RIG-I interacting partners that may play a role in psoriasis.ResultsWe found that serine/arginine-rich splicing factor 1 (SRSF1) could specifically interact with RIG-I to facilitate RIG-I mediated production of type-I IFN that is triggered by cytosolic DNA. We found SRSF1 associates with RNA polymerase III and RIG-I in a DNA-dependent manner. In addition, treatment with a TNFα inhibitor downregulated SRSF1 expression in peripheral blood mononuclear cells (PBMCs) from psoriasis vulgaris patients.DiscussionBased on the abundance of pathogenic cytosolic DNA that is detected in psoriatic lesions, our finding that RIG-I interacts with SRSF1 to regulate type-I IFN production reveals a critical link regarding how cytosolic DNA specifically activates aberrant IFN expression. These data may provide new therapeutic targets for the treatment of psoriasis.