Demethylation and degradation of phenylmethylethers by the sulfide-methylating homoacetogenic bacterium strain TMBS 4

Biochemical studies on anaerobic phenylme-thylether cleavage by homoacetogenic bacteria have been hampered so far by the complexity of the reaction chain involving methyl transfer to acetyl-CoA synthase and subsequent methyl group carbonylation to acetyl-CoA. Strain TMBS 4 differs from other demethylating homoacetogenic bacteria in using sulfide as a methyl acceptor, thereby forming methanethiol and dimethylsulfide. Growing and resting cells of strain TMBS 4 used alternatitively CO₂ as a precursor of the methyl acceptor CO for homoacetogenic acetate formation. Demethylation was inhibited by propyl iodide and reactivated by light, indicating involvement of a corrinoid-dependent methyltransferase. Strain TMBS 4 contained ca. 750 nmol g dry mass^-1 of a corrinoid tentatively identified as 5-hydroxybenzimidazolyl cobamide. A photometric assay for measuring the demethylation activity in cell extracts was developed based on the formation of a yellow complex of Ti³⁺ with 5-hydroxyvanillate produced from syringate by demethylation. In cell extracts, the methyltransfer reaction from methoxylated aromatic compounds to sulfide or methanethiol depended on reductive activation by Ti³⁺. ATP and Mg²⁺ together greatly stimulated this reductive activation without being necessary for the demethylation reaction itself. The specific activity of the transmethylating enzyme system increased proportionally with protein concentration up to 3 mg ml^-1 reaching a constant level of 20 nmol min^-1 mg^-1 at protein concentrations 10 mg ml^-1. The specific rate of activation increased in a non-linear manner with protein concentration. Strain TMBS 4 degraded gallate, the product of sequential demethylations, to 3 acetate through the phloroglucinol pathway as found earlier with Pelobacter acidigallici.Abbreviations BV benzyl viologen - CTAB cetyltrimethylammonium bromide - H₄folate tetrahydrofolate - MOPS 3-[N-morpholino]propanesulfonic acid - MV methyl viologen - NTA nitrilotriacetate - t_d doubling time - TMB 3,4,5-trimethoxybenzoate     

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.     

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).     

Methanogenic degradation of hydroquinone and catechol via reductive dehydroxylation to phenol

Abstract Fermentative degradation of hydroquinone, catechol, and phenol was demonstrated with nearly-homogeneous mixed methanogenic cultures obtained from freshwater sediments and sewage sludge by enrichment with the respective phenolic substrates. Gram-negative short rods predominated in these cultures, together with hydrogen- and acetate-utilizing methanogens. Acetate and methane were the only degradation products. Bacteria enriched with hydroquinone or catechol also degraded phenol and p -hydroxy-benzoate, but not resorcinol or resorcylic acids. Phenol was formed as an intermediate during catechol and hydroquinone degradation, indicating that reductive dehydroxylation was the primary event in degradation of these substrates. Inhibition experiments with bromoethanesulfonate and acetylene indicated that catechol, hydroquinone, and phenol degradation depended on a syntrophic co-operation of fermenting bacteria and hydrogen-oxidizing methanogens.     

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     

Initial steps of catabolism of trihydroxybenzenes in Pelobacter acidigallici

The initial steps of the anaerobic degradation of trihydroxylated aromatic monomers were investigated in a strain (AG2) isolated on gallic acid and identified as Pelobacter acidigallici. Kinetic studies showed that strain AG2 fermented gallic acid into acetate with a transient accumulation of pyrogallol and phloroglucinol in the medium. In addition phloroglucinol was produced from all other trihydroxylated aromatic monomers and pyrogallol from 2,3,4-trihydroxybenzoate. Although protocatechuate did not support growth of the organism, it was partially decarboxylated by resting cells of strain AG2. Cell free extract of strain AG2 catalysed the oxidation of NADPH in presence of resorcinol, 2,4,6-trihydroxybenzoate and phloroglucinol. However, comparison of activities indicated that the latter was the true physiological electron acceptor. Phloroglucinol and its reduction product dihydrophloroglucinol appeared thus to play a key role in metabolism of trihydroxybenzenes and a unified pathway, involving a decarboxylation of trihydroxybenzoates, a para transhydroxylation of pyrogallol into phloroglucinol and the formation of dihydrophloroglucinol, was proposed.     

Metabolism of cyclohexane carboxylic acid by the photosynthetic bacteriumRhodopseudomonas palustris

Cyclohexane carboxylate supported relatively rapid growth (doubling times 7–8 h) of Rhodopseudomonas palustris under oxic or photosynthetic conditions, but did not serve as a substrate for either of the known aromatic CoA ligases. A CoA ligase that thioesterifies cyclohexane carboxylate was partially purified and did not cross react immunologically with the two CoA ligases purified previously from this bacterium. Crude extracts of R. palustris cells grown with a range of aromatic or alicyclic acids contained a dehydrogenase that reacted with cyclohexane carboxyl-CoA or cyclohex-1-ene carboxyl-CoA, using 2,6-dichlorophenolindophenol or ferricenium ion as electron carrier. This activity was not detected in extracts of adipate-, glutamate-, or succinate-grown cells. No oxidation or reduction of nonesterified cyclohexane carboxylate or cyclohexene carbocylate was detected in extracts of cells grown with aromatic or aliphatic substrates, neither aerobically nor anaerobically. A constitutively expressed thioesterase that hydrolyzed cyclohexane carboxyl-CoA and also some alicyclic and aliphatic CoA derivatives was purified and characterized. The enzyme had little or no activity on benzoyl-CoA or 4-hydroxybenzoyl-CoA. The presence of a thioesterase that effectively hydrolyzes cyclohexane carboxyl-CoA suggests that transient production of cyclohexane carboxylate is a physiological response to temporary excess of reductant during metabolism of aromatic compounds. Received: 22 May 1995 / Accepted: 13 September 1995     

Fermentative degradation of monohydroxybenzoates by defined syntrophic cocultures

From anaerobic freshwater enrichment cultures with 3-hydroxybenzoate as sole substrate, a slightly curved rod-shaped bacterium was isolated in coculture with Desulfovibrio vulgaris as hydrogen scavenger. The new isolate degraded only 3-hydroxybenzoate or benzoate, and depended on syntrophic cooperation with a hydrogenoxidizing methanogen or sulfate reducer. 3-Hydroxybenzoate was degraded via reductive dehydroxylation to benzoate. With 2-hydroxybenzoate (salicylate), short coccoid rods were enriched from anaerobic freshwater mud samples, and were isolated in defined coculture with D. vulgaris. This isolate also fermented 3-hydroxybenzoate or benzoate in obligate syntrophy with a hydrogen-oxidizing anaerobe. The new isolates were both Gram-negative, non-sporeforming strict anaerobes. They fermented hydroxybenzoate or benzoate to acetate, CO₂, and, presumably, hydrogen which was oxidized by the syntrophic partner organism. With hydroxybenzoates, but not with benzoate, Acetobacterium woodii could also serve as syntrophic partner. Other substrates such as sugars, alcohols, fatty or amino acids were not fermented. External electron acceptors such as sulfate, sulfite, nitrate, or fumarate were not reduced. In enrichment cultures with 4-hydroxybenzoate, decarboxylation to phenol was the initial step in degradation which finally led to acetate, methane and CO₂.     

Anaerobic biodegradation of methyl esters byAcetobacterium woodii andEubacterium limosum

Summary The ability ofAcetobacterium woodii andEubacterium limosum to degrade methyl esters of acetate, propionate, butyrate, and isobutyrate was examined under growing and resting-cell conditions. Both bacteria hydrolyzed the esters to the corresponding carboxylates and methanol under either condition. Methanol was further oxidized to formate under growing but not resting conditions. Unlike the metabolism of phenylmethylethers, no H₂ requirement was evident for ester biotransformation. The hydrolysis of methyl carboxylates is thermodynamically favorable under standard conditions and the mixotrophic metabolism of ester/CO₂ allowed for bacterial growth. These results suggest that the degradation of methyl carboxylates may be a heretofore unrecognized nutritional option for acetogenic bacteria.     

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.     

Novel upper meta-pathway extradiol dioxygenase gene diversity in polluted soil

For the determination of the catabolic community diversity that is related to biodegradation potential, we developed a protocol for the assessment of catabolic marker genes in polluted soils. Primers specific to upper pathway extradiol dioxygenase genes were designed which amplified a 469-bp product from Sphingomonas sp. HV3. The constructed primers were used in PCR amplification of upper pathway ring cleavage genes from DNA directly isolated from a mineral oil polluted landfill site, a mineral oil landfarming site and a birch rhizosphere-associated soil that was either artificially polluted with a PAH mixture or not polluted. Amplicons were cloned and subjected to restriction fragment length polymorphism analysis dividing the HhaI-digested products into operational taxonomic units. Altogether 26 different operational taxonomic units were detected with the sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Phylogenetic analysis divided the operational taxonomic units from the polluted soils into seven clusters. Two contained exclusively sequences with no close homologues in the database, therefore representing novel catabolic genes. This large proportion of novel extradiol sequences shows that there is an extensive unknown catabolic diversity in polluted environments.     

Phloroglucinol pathway in the strictly anaerobic Pelobacter acidigallici: fermentation of trihydroxybenzenes to acetate via triacetic acid

The strictly anaerobic, fermenting bacterium Pelobacter acidigallici degrades several trihydroxybenzene derivatives to stoichiometric amounts of acetate. We now report on the enzymatic activities in cell extracts which are responsible for the fermentative degradation of these aromatic compounds, and postulate a novel phloroglucinol pathway involving triacetic acid as an unusual metabolic intermediate. Gallate is decarboxylated to pyrogallol by a specific, Mg²⁺-dependent, soluble enzyme activity, followed by conversion of pyrogallol to phloroglucinol, involving an unusual intermolecular transhydroxylation described previously. Phloroglucinol is then reduced to dihydrophloroglucinol (5-hydroxy-1,3-cyclohexanedione) by an NADPH-dependent phloroglucinol reductase. Dihydrophloroglucinol is cleaved hydrolytically to 3-hydroxy-5-oxohexanoic acid, which is then oxidized to triacetic acid (3,5-dioxohexanoic acid) by a unique, NADP⁺-dependent dehydrogenase. Triacetic acid is activated by CoA transfer from acetyl-CoA, and then converted to 3 acetyl-CoA by two subsequent β-ketothiolase reactions. ATP is generated via phosphotransacetylase and acetate kinase.     

Complete anaerobic oxidation of hydroquinone by Desulfococcus sp. strain Hy5: indications of hydroquinone carboxylation to gentisate

The sulfate-reducing strain Hy5 was able to grow with hydroquinone as sole source of carbon and energy. In experiments with dense cell suspensions, several indications were found that gentisate was the first intermediate in anaerobic degradation of hydroquinone: (1) degradation of hydroquinone was accelerated by addition of bicarbonate; (2) cell suspensions grown with hydroquinone oxidized gentisate at a rate similar to that of suspensions grown with gentisate, whereas the latter were not able to degrade hydroquinone in the presence of chloramphenicol; (3) in SDS-PAGE analysis of cell-free extracts of strain Hy5, two additional protein bands were found after growth with hydroquinone that were not detected in cells grown with gentisate, probably representing a hydroquinone carboxylating enzyme. A corresponding enzyme activity could not be detected. In cell-free extracts of hydroquinone-grown strain Hy5, the specific acyl-CoA ligase activity with gentisate as substrate was detected at 70 nmol x mg^-1 x min^-1. Gentisyl-CoA was enzymatically reduced to several unidentified nonaromatic products in the presence of dithionite-reduced methyl viologen.