Specificity of O-demethylation in extracts of the homoacetogenic Holophaga foetida and demethylation kinetics measured by a coupled photometric assay

The kinetics and specificity of O-demethylation were studied in cell-free extracts of the strictly anaerobic, methanethiol- and dimethylsulfide-producing homoacetogen Holophaga foetida strain TMBS4 with methanethiol and tetrahydrofolate (H₄folate) as methyl acceptors. Extracts of cells grown with 3,4,5-trimethoxybenzoate contained an enzyme system that demethylated various phenyl methyl ethers with at least one ortho-positioned hydroxyl or methoxyl group (the ortho system) and also contained a decarboxylase. Extracts of cells grown with 3,5-dihydroxyanisole contained an enzyme system with a novel specificity that demethylated only the meta-hydroxylated compounds 3,5-dihydroxyanisole and 3-hydroxyanisole (the meta system) and lacked a decarboxylase. H₄folate-dependent demethylation produced CH₃-H₄folate. For a photometric in vitro assay of the meta system, the NADPH-consuming phloroglucinol reductase (PR) reaction was coupled to the phloroglucinol-yielding demethylation of 3,5-dihydroxyanisole. The kinetics of the indicator enzyme PR were studied. The cell extract had a high and stable specific PR activity. PR was inhibited by phloroglucinol (substrate inhibition) and the substrate analogue 3,5-dihydroxyanisole. Doubling the PR activity of the coupled enzyme assay by additions of a PR-enriched fraction had no effect, showing that the PR activity supplied by cell extract did not limit reaction rates. Demethylation activity of the meta system with either methyl acceptor increased with the square of the protein concentration. With H₄folate, the in vivo activity could be attained. Kinetic parameters for the methyl acceptors were determined. Received: 8 November 1996 / Accepted: 13 January 1997     

Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium

A strictly anaerobic gram-positive, rod-shaped bacterium, strain LuPhet1, was isolated from sewage sludge with phenoxyethanol as sole carbon and energy source, and was assigned to the genus Acetobacterium. The new isolate fermented the alkylaryl ether compound phenoxyethanol stoichiometrically to phenol and acetate, whereas phenoxyacetic acid was not degraded. In cell-free extracts of strain LuPhet1, cleavage of the ether linkage was shown, and acetaldehyde was detected as reaction product. Coenzyme A-dependent acetaldehyde: acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results indicate that the ether linkage of phenoxyethanol is cleaved by a shift of the hydroxyl group to the subterminal carbon atom, analogous to a corrinoid-dependent diol dehydratase reaction, to form an unstable hemiacetal that releases phenol and acetaldehyde. Obviously, phenoxyethanol is degraded by the same strategy as in anaerobic degradation of the alkyl ether polyethylene glycol.     

Fermentative degradation of triethanolamine by a homoacetogenic bacterium

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9±1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B₁₂) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.Abbreviation NTA nitrilotriacetate     

Isolation and characterization of a methyl chloride utilizing,strictly anaerobic bacterium

From sludge obtained from the sewage digester plant in Stuttgart-Möhringen a strictly anaerobic bacterium was enriched and isolated with methyl chloride as the energy source. The isolate, which was tentatively called strain MC, was nonmotile, gram-positive, and occurred as elongated cocci arranged in chains. Cells of strain MC formed about 3 mol of acetate per 4 mol of CH₃Cl consumed, indicating that the organism was a homoacetogenic bacterium fermenting methyl chloride plus CO₂ according to: The organism grew with 2–3% methyl chloride in the gas phase at a doubling time of near 30 h. Dichloromethane was not utilized. The bacterium also grew on carbon monoxide, H₂ plus CO₂, and methoxylated aromatic compounds. Optimal growth with methyl chloride was observed at 25°C and pH 7.3–7.7. The G+C-content of the DNA was 47.5±1.5%. The methyl chloride conversion appeared to be inducible, since H₂ plus CO₂-grown cells lacked this ability. From the morphological and physiological characteristics, the isolate could not be affiliated to a known species.     

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria

Abstract Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h⁻¹ (mg protein)⁻¹ to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO₂. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii , and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.     

Mechanism of anaerobic degradation of triethanolamine by a homoacetogenic bacterium

Triethanolamine (TEA) is converted into acetate and ammonia by a strictly anaerobic, gram-positive Acetobacterium strain LuTria3. Fermentation experiments with resting cell suspensions and specifically deuterated substrates indicate that in the acetate molecule the carboxylate and the methyl groups correspond to the alcoholic function and to its adjacent methylene group, respectively, of the 2-hydroxyethyl unit of TEA. A 1,2 shift of a hydrogen (deuterium) atom from -CH2-O- to =N-CH2- without exchange with the medium was observed. This fact gives evidence that a radical mechanism occurs involving the enzyme and/or coenzyme molecule as a hydrogen carrier. Such a biodegradation appears analogous to the conversion of 2-phenoxyethanol into acetate mediated by another strain of the anaerobic homoacetogenic bacterium Acetobacterium.     

Genome sequence of the homoacetogenic bacterium Holophaga foetida type strain (TMBS4(T))

Holophaga foetida Liesack et al. 1995 is a member of the phylum Acidobacteria and is of interest for its ability to anaerobically degrade aromatic compounds and for its production of volatile sulfur compounds through a unique pathway. The genome of H. foetida strain TMBS4(T) is the first to be sequenced for a representative of the class Holophagae. Here we describe the features of this organism, together with the complete genome sequence (improved high quality draft), and annotation. The 4,127,237 bp long chromosome with its 3,615 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay

An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD⁺ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε₃₅₀ was about 29.5 mM^–1cm^–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995     

Fermentation of mandelate to benzoate and acetate by a homoacetogenic bacterium

A strictly anaerobic, Gram-positive, rod-shaped bacterium, strain AmMan1, was isolated from freshwater sediment with mandelate (-hydroxy-phenylacetate) as sole carbon and energy source, and was assigned to the genus Acetobacterium. Only the d-enantiomer of mandelate was degraded, and was fermented to acetate and benzoate. Non-aromatic growth substrates (pyruvate, lactate, malate, glycerol, ethylene glycol, and H₂/CO₂) were fermented to acetate as sole product. Methoxylated aromatics were demethoxylated to the corresponding phenols. The guanine-plus-cytosine content of the DNA was 36.5±1.5%. Carbon monoxide dehydrogenase, dichlorophenol indophenol-reducing lactate dehydrogenase, NAD-dependent mandelate dehydrogenase, phosphate acetyl transferase, acetate kinase, and pyruvate- or phenylglyoxylate-dependent benzylviologen reductase were measured in mandelate-and/or lactate-grown cells, respectively. A pathway of the homoacetogenic fermentation of mandelate is suggested as another example of incomplete substrate oxidation by homoacetogenic bacteria.     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997     

Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum

A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).     

Thermodynamics of methylenetetrahydrofolate reduction to methyltetrahydrofolate and its implications for the energy metabolism of homoacetogenic bacteria

The thermodynamics of the methylenetetrahydrofolate reduction to 5-methyltetrahydrofolate was studied with the methylenetetrahydrofolate reductase purified from the homoacetogenic bacterium Peptostreptococcus productus. The equilibrium constants were determined for the forward and backward reactions of methylenetetrahydrofolate reduction with NADH or acetylpyridine adenine dinucleotide (APADH), respectively, as the electron donors. From the equilibrium constants and the known standard redox potentials at pH 7 (E ^o ) of the couples NAD⁺/NADH or APAD⁺/APADH the E ^o of the couple methylene-/methyltetrahydrofolate was determined to be about-200mV. This value is different from values reported before for this couple. The implications for the mechanism of energy conservation of homoacetogens is discussed.Abbreviations FH₄ tetrahydrofolate - CH₂=FH₄ 5,10-methylenetrahydrofolate - CH₃-FH₄ 5-methyltetrahydrofolate - K _eq equilibrium constant - G ^o Gibb's free energy change under standard conditions (all concentrations of reactants = 1 M) - G ^o G ^o at pH 7 ([H⁺]=10^-7 M) - E ^o standard redox potential - G ^o standard redox potential difference of two reactants - E ^o E ^o at pH 7 - R gas constant - F Faraday constant - APAD acetylpyridine adenine dinucleotide (NAD⁺-analogue)     

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

一株敌草胺降解菌的分离鉴定及降解性能

为探讨酰胺类除草剂敌草胺的微生物降解机理和土壤中敌草胺降解的生物强化性能,从长期使用敌草胺的烟田土壤中分离到一株敌草胺降解细菌菌株,命名为LGY06.经形态特征、生理生化特性及16S rDNA序列分析将其鉴定为蜡状芽孢杆菌.在纯培养条件下,菌株LGY06对敌草胺的降解符合一级动力学特征,7 d内对50 mg·L^-1敌草胺降解率达到75.7%;LGY06降解敌草胺的最适温度和最适pH值分别为35 ℃和8.0.通过气相色谱-质谱鉴定了菌株LGY06降解敌草胺的降解产物并分析了其降解途径,α-萘酚和丙酰胺是主要的降解产物,LGY06对敌草胺的作用方式主要有脱烷基、氧化(或水解),为矿化过程.室内模拟条件下,LGY06能有效促进土壤中敌草胺残留的降解,与未接种灭菌土、非根际土和根际土相比,敌草胺在接种LGY06土壤中的降解半衰期分别缩短了79.5%、36.6%和41.1%.     

Comparative evaluation of the metabolic potentials of different strains of Peptostreptococcus productus: utilization and transformation of aromatic compounds

Three strains of Peptostreptococcus productus were tested for growth at the expense of methoxylated aromatic compounds. Strain M8A-18 (human fecal isolate) was unable to utilize methoxylated aromatic compounds. While the type strain ATCC 27340 (human septicemia isolate) was capable of minimal growth with methoxylated aromatic compounds, ATCC 35244 (sewage sludge isolate) displayed significant growth on methoxylated aromatic compounds. Methoxylated phenols, benzoates, benzyl alcohol and phenylacrylates supported the growth of ATCC 35244 and were O-demethylated to their respective hydroxylated derivatives. During O-methyl- or CO-dependent growth, the double bond of the acrylate side chain of certain methoxylated and non-methoxylated phenylacrylates was reduced. Although other aromatic substituent groups (-COOH and -CH3) were transformed during CO-dependent growth, in short-term growth studies, the aromatic ring was not subject to reduction or degradation. Of the three strains tested, only strain M8A-18 failed to grow at the expense of carbon monoxide (CO).     

Aerobic degradation of mercaptosuccinate by the gram-negative bacterium Variovorax paradoxus strain B4

The Gram-negative bacterium Variovorax paradoxus strain B4 was isolated from soil under mesophilic and aerobic conditions to elucidate the so far unknown catabolism of mercaptosuccinate (MS). During growth with MS this strain released significant amounts of sulfate into the medium. Tn5::mob-induced mutagenesis was successfully employed and yielded nine independent mutants incapable of using MS as a carbon source. In six of these mutants, Tn5::mob insertions were mapped in a putative gene encoding a molybdenum (Mo) cofactor biosynthesis protein (moeA). In two further mutants the Tn5::mob insertion was mapped in the gene coding for a putative molybdopterin (MPT) oxidoreductase. In contrast to the wild type, these eight mutants also showed no growth on taurine. In another mutant a gene putatively encoding a 3-hydroxyacyl-coenzyme A dehydrogenase (paaH2) was disrupted by transposon insertion. Upon subcellular fractionation of wild-type cells cultivated with MS as sole carbon and sulfur source, MPT oxidoreductase activity was detected in only the cytoplasmic fraction. Cells grown with succinate, taurine, or gluconate as a sole carbon source exhibited no activity or much lower activity. MPT oxidoreductase activity in the cytoplasmic fraction of the Tn5::mob-induced mutant Icr6 was 3-fold lower in comparison to the wild type. Therefore, a new pathway for MS catabolism in V. paradoxus strain B4 is proposed: (i) MPT oxidoreductase catalyzes the conversion of MS first into sulfinosuccinate (a putative organo-sulfur compound composed of succinate and a sulfino group) and then into sulfosuccinate by successive transfer of oxygen atoms, (ii) sulfosuccinate is cleaved into oxaloacetate and sulfite, and (iii) sulfite is oxidized to sulfate.     

Hydroquinone degradation via reductive dehydroxylation of gentisyl-CoA by a strictly anaerobic fermenting bacterium

Anaerobic degradation of hydroquinone was studied with the fermenting bacterium strain HQGö1. The rate of hydroquinone degradation by dense cell suspensions was dramatically accelerated by addition of NaHCO₃. During fermentation of hydroquinone in the presence of ¹⁴C-Na₂CO₃ benzoate was formed as a labelled product, indicating an initial ortho-carboxylation of hydroquinone to gentisate. Gentisate was activated to the corresponding CoA-ester in a CoA ligase reaction at a specific activity of 0.15 mol x min^–1 x mg protein^–1. Gentisyl-CoA was reduced to benzoyl-CoA with reduced methyl viologen as electron donor by simultaneous reductive elimination of both the ortho and meta hydroxyl group. The specific activity of this novel gentisyl-CoA reductase was 17 nmol x min^–1 x mg protein^–1. Further degradation to acetate was catalyzed by enzymes which occur also in other bacteria degrading aromatic compounds via benzoyl-CoA.     

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacterium Cupriavidus necator JMP134

Cupriavidus necator JMP134 is a model for chloroaromatics biodegradation, capable of mineralizing 2,4-D, halobenzoates, chlorophenols and nitrophenols, among other aromatic compounds. We performed the metabolic reconstruction of aromatics degradation, linking the catabolic abilities predicted in silico from the complete genome sequence with the range of compounds that support growth of this bacterium. Of the 140 aromatic compounds tested, 60 serve as a sole carbon and energy source for this strain, strongly correlating with those catabolic abilities predicted from genomic data. Almost all the main ring-cleavage pathways for aromatic compounds are found in C. necator : the β-ketoadipate pathway, with its catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage branches; the (methyl)catechol meta ring-cleavage pathway; the gentisate pathway; the homogentisate pathway; the 2,3-dihydroxyphenylpropionate pathway; the (chloro)hydroxyquinol pathway; the (amino)hydroquinone pathway; the phenylacetyl-CoA pathway; the 2-aminobenzoyl-CoA pathway; the benzoyl-CoA pathway and the 3-hydroxyanthranilate pathway. A broad spectrum of peripheral reactions channel substituted aromatics into these ring cleavage pathways. Gene redundancy seems to play a significant role in the catabolic potential of this bacterium. The literature on the biochemistry and genetics of aromatic compounds degradation is reviewed based on the genomic data. The findings on aromatic compounds biodegradation in C. necator reviewed here can easily be extrapolated to other environmentally relevant bacteria, whose genomes also possess a significant proportion of catabolic genes.     

Role of electron-donating cosubstrates in the anaerobic biotransformation of chlorophenoxyacetates to chlorophenols by a bacterial consortium enriched on phenoxyacetate

A bacterial consortium that anaerobically mineralized phenoxyacetate, with transient production of phenol as an intermediate, was obtained from a methanogenic aquifer site near the Norman, OK municipal landfill. This consortium was able to convert the eight halogenated chlorophenoxyacetates tested to the corresponding chlorophenols. The chlorophenols were not subsequently metabolized. The addition of reduced substrates increased the rate of degradation of all chlorophenoxyacetates, with 78% of mono- and di-chlorinated substrates being transformed to chlorophenols in butyrate-amended cultures, compared to less than 37% transformed in unsupplemented cultures. Butyrate increased the transformation of 2,4,5-trichlorophenoxyacetate from 10% to 20%. An experiment evaluating the effects of several compounds on the side-chain cleavage reaction of 3-chlorophenoxyacetate showed that addition of compounds with readily act as hydrogen donors (butyrate, crotonate, ethanol, propionate, and hydrogen) resulted in 2 to 5 times the amount of 3-chlorophenoxyacetate transformed compared to controls with no amendment, formate had a slight stimulatory effect, and acetate and methanol had no effect. Butyrate addition also increased the rate of phenoxyacetate degradation, resulting in transient phenol accumulation not observed in butyrate-unamended controls. These results support the hypothesis that the side-chain cleavage of phenoxyacetate is a reductive process that is stimulated by the oxidation of reduced cosubstrates.