Utilization of aromatic compounds by phototrophic purple nonsulfur bacteria

Biodegradation of aromatic compounds byRhodopseudomonas blastica andRhodospirillum rubrum appears to be lacking in the literature. The above species grew phototrophically (illuminated anaerobic conditions) on a variety of organic compounds. They were found to degrade benzoate, benzyl alcohol, 4-hydroxy-3,5-dimethoxybenzoate (Syringate) and 4-hydroxy-3-methoxybenzoate (vanillate). The ability of the above species to photocatabolize aromatic compounds indicates that these organisms may be ecologically significant as scavengers of aromatic derivatives in illuminated anaerobic habitats in nature.     

Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields

Anaerobic enrichments with methoxylated aromatic compounds as substrates (vanillate, syringate, trimethoxycinnamate) were inoculated from freshwater mud and sewage sludge samples. In 12 out of 16 cultures the same type of rod-shaped, motile bacteria was selectively enriched. Two strains, NZva16 and NZva24, were isolated in pure culture and recognized as Acetobacterium woodii by comparison with the type strain (DSM 1030).All three Acetobacterium strains were able to grow with all 10 of the tested aromatic compounds containing methoxyl groups. In the presence of bicarbonate, these substrates were used as sole organic electron donors and carbon sources. UV-absorption spectra revealed that the aromatic rings were not degraded, and that the corresponding hydroxy derivatives of the methoxylated compounds were formed. The only further fermentation product formed was acetate. When equimolar concentrations of the methoxylated benzoic acid derivatives were applied, the growth yields were proportional to the number of methoxyl groups per molecule. Methoxyl groups or methanol were metabolized by homoacetate fermentation: in the presence of bicarbonate 4 mol of acetate. In case of the methoxylated cinnamic acid derivatives less acetate was formed and the corresponding hydroxy derivatives of phenylpropionic acid appeared as a result of the double bond reduction in the acrylate side chain. In comparison to the benzoate derivatives with the same number of methoxyl groups, higher growth yields were obtained with the cinnamate derivatives.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

Reduction of 3-chlorobenzoate, 3-bromobenzoate, and benzoate to corresponding alcohols by Desulfomicrobium escambiense, isolated from a 3-chlorobenzoate-dechlorinating coculture.

An anaerobic bacterial coculture which dechlorinated 3-chlorobenzoate (3CB) to benzoate was obtained by single-colony isolation from an anaerobic bacterial consortium which completely degraded 3CB in defined medium. Of 29 additional halogenated aromatic compounds tested, the coculture removed the meta halogen from 2,3- and 2,5-dichlorobenzoate, 3-bromobenzoate (3BB), 5-chlorovanillate (5CV), and 3-chloro-4-hydroxybenzoate. Dechlorinating activity in the coculture required the presence of pyruvate. 5CV was also O-demethoxylated. The coculture contained two cell types: a short, straight gram-negative rod and a long, thin, curved gram-positive rod. The short rod, Desulfomicrobium escambiense, was recently isolated and identified as a new sulfate-reducing bacterial species (B. R. Sharak Genthner, S. D. Friedman, and R. Devereux, Int. J. Syst. Bacteriol. 47:889-892, 1997; B. R. Sharak Genthner, G. Mundfrom, and R. Devereux, Arch. Microbiol. 161:215-219, 1994). D. escambiense did not dehalogenate any of the compounds dehalogenated by the coculture, nor dit it O-demethoxylate 5CV or vanillate. However, D. escambiense reduced 3CB, EBB, and benzoate to their respective benzyl alcohols. Reduction to alcohols required the presence of pyruvate, which was transformed to acetate, lactate, and succinate in the presence of absence of 3CB, 3BB, or benzoate. Alcohol formation did not occur in pyruvate-sulfate medium. Under these conditions, sulfate was preferentially reduced. Other electron donors that supported the growth of D. escambiense during sulfate reduction did not support benzoate reduction to benzyl alcohol.     

Obligate Sulfide-Dependent Degradation of Methoxylated Aromatic Compounds and Formation of Methanethiol and Dimethyl Sulfide by a Freshwater Sediment Isolate, Parasporobacterium paucivorans gen. nov., sp. nov.

Methanethiol (MT) and dimethyl sulfide (DMS) have been shown to be the dominant volatile organic sulfur compounds in freshwater sediments. Previous research demonstrated that in these habitats MT and DMS are derived mainly from the methylation of sulfide. In order to identify the microorganisms that are responsible for this type of MT and DMS formation, several sulfide-rich freshwater sediments were amended with two potential methyl group-donating compounds, syringate and 3,4,5-trimethoxybenzoate (0.5 mM). The addition of these methoxylated aromatic compounds resulted in excess accumulation of MT and DMS in all sediment slurries even though methanogenic consumption of MT and DMS occurred. From one of the sediment slurries tested, a novel anaerobic bacterium was isolated with syringate as the sole carbon source. The strain, designated Parasporobacterium paucivorans, produced MT and DMS from the methoxy groups of syringate. The hydroxylated aromatic residue (gallate) was converted to acetate and butyrate. Like Sporobacterium olearium, another methoxylated aromatic compound-degrading bacterium, the isolate is a member of the XIVa cluster of the low-GC-content Clostridiales group. However, the new isolate differs from all other known methoxylated aromatic compound-degrading bacteria because it was able to degrade syringate in significant amounts only in the presence of sulfide.     

2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min⁻¹ mg of protein⁻¹. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.     

Formation of dimethylsulfide and methanethiol from methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic bacteria

Formation of gas and of methylated sulfur compounds was observed in anaerobic enrichment cultures with methoxylated aromatic compounds as substrates. Via direct dilution of mud samples in defined reduced media supplemented with trimethoxybenzoate or syringate two new strains of anaerobic homoacetogenic bacteria (strain TMBS4 and strain SA2) were obtained in pure culture. Both strains produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. Growth tests and determination of stoichiometries demonstrated that the volatile sulfur compounds were formed from the methyl group at the aromatic ring and the sulfide added as reducing agent to the medium (R = aromatic residue): 2 R - O - CH₃ + H₂ S 2 R - OH + (CH₃)₂SDimethylsulfide was the major organic sulfur compound formed, whereas methanethiol appeared only as intermediate in small quantities. The isolates grew also with trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol without formation of methylated sulfur compounds. The aromatic compounds were degraded to acetate. The freshwater strain TMBS4 also fermented pyruvate. Other aliphatic or aromatic compounds were not utilized. External electron acceptors (sulfate, nitrate, fumarate) were not reduced. Both strains were mesophilic and formed rod-shaped, non-motile, Gram-negative cells. Spore formation was not observed. Tentatively, both isolates can be affiliated to the genus Pelobacter.Abbreviations TMB 3,4,5-trimethoxybenzoate - MT methanethiol - DMS dimethylsulfide     

Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.

Toluene and related aromatic compounds can be mineralized to CO₂ under anoxic conditions. Oxidation requires new dehydrogenase-type enzymes and water as oxygen source, as opposed to the aerobic enzymatic attack by oxygenases, which depends on molecular oxygen. We studied the anaerobic process in the denitrifying bacterium Thauera sp. strain K172. Toluene and a number of its fluoro-, chloro- and methyl-analogues were transformed to benzoate and the respective analogues by whole cells and by cell extracts. The transformation of xylene isomers to methylbenzoate isomers suggests that xylene degradation is similarly initiated by oxidation of one of the methyl groups. Toluene oxidation was strongly, but reversibly inhibited by benzyl alcohol. The in vitro oxidation of the methyl group was coupled to the reduction of nitrate, required glycerol for activity, and was inhibited by oxygen. Cells also contained benzyl alcohol dehydrogenase (NAD⁺), benzaldehyde dehydrogenase (NADP⁺), benzoate-CoA ligase (AMP-forming), and benzoyl-CoA reductase (dearomatizing). The toluene-oxidizing activity was induced when cells were grown anaerobically with toluene and also with benzyl alcohol or benzaldehyde, suggesting that benzyl alcohol or benzaldehyde acts as inducer. The other enzymes were similarly active in cells grown with toluene, benzyl alcohol, benzaldehyde, or benzoate. This is the first in vitro study of anaerobic oxidation of an aromatic hydrocarbon and of the whole-cell regulation of the toluene-oxidizing enzyme.Dedicated to Prof. Achim Trebst     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Cloning and Sequencing of the Sphingomonas (Pseudomonas) paucimobilis Gene Essential for the O Demethylation of Vanillate and Syringate

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5′-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2′,3′-trihydroxy-3-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.     

Antagonistic control of a dual-input mammalian gene switch by food additives

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.     

Bidirectional transformation of aromatic aldehydes by Desulfovibrio desulfuricans under nitrate-dissimilating conditions

M. PAREKH, H.L. DRAKE AND S.L. DANIEL 1996. Desulfovibrio desulfuricans ATCC 27774 was screened for reactivity against aromatic compounds during lactate-dependent, nitrate-dissimilating growth. Only aromatic aldehydes (benzaldehyde, 2-hydroxybenzaldehyde, 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, vanillin, iso -vanillin and o -vanillin) were reactive and, with the exception of 2-hydroxybenzaldehyde, were stimulatory to lactate-dependent growth. Aromatic aldehydes were transformed to their corresponding benzoate and benzyl alcohol derivatives, with the ratio of benzoate-to-benzyl alcohol derivatives being dependent upon lactate availability. In presence of lactate, aromatic aldehydes were primarily reduced to their corresponding benzyl alcohol derivatives; in the absence of lactate, aromatic aldehydes were mainly oxidized to their corresponding benzoate derivatives. In the absence of nitrate, 3-hydroxybenzaldehyde was neither reduced nor oxidized. These results indicate that D. desulfuricans is competent in the bidirectional transformation of aromatic aldehydes under nitrate-dissimilating conditions and that the direction of transformation (i.e. reduction or oxidation) is regulated by reductant availability.     

Purification of glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate

Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).     

Photometabolism of aromatic compounds byRhodopseudomonas palustris

The phototrophic bacteriumRhodopseudomonas palustris has been reported to be versatile in photometabolism of aromatic compounds. However, the kinetics of degradation of aromatic compounds byR. palustris appears not to have been reported in the literature. In this laboratory a photosynthetic bacterium that was identified asRhodopseudomonas palustris(bp) was isolated from a sample collected from a canal where refinery wastewater was discharged. The paper presents the results of the growth of the isolate on different aromatic compounds, their catabolic pathways, and the kinetics of their degradation. The doubling time of the isolate was found to be 23, 22, 17, 27, 20, 24, and 26 h for benzoate,p-hydroxybenzoate, cinnamate,p-coumarate, phenyl valerate, phenyl acetate, and cinnamyl alcohol respectively. It was also found that cinnamate, phenyl valerate, phenyl acetate, and cinnamyl alcohol were converted to benzoate, whilep-coumarate was converted top-hydroxybenzoate. The substrate inhibition constant (Ki) was found to be 1082, 1178, 1362, 1260, 3098, 1347, and 322 mg/L for benzoate,p-hydroxybenzoate, cinnamate,p-coumarate, phenyl valerate, phenyl acetate, and cinnamyl alcohol respectively.     

Isolation and characterization of a new spore-forming sulfate-reducing bacterium growing by complete oxidation of catechol

A new mesophilic sulfate-reducing bacterium, strain Groll, was isolated from a benzoate enrichment culture inoculated with black mud from a freshwater ditch. The isolate was a spore-forming, rod-shaped, motile, gram-positive bacterium. This isolate was able of complete oxidation of several aromatic compounds including phenol, catechol, benzoate, p-and m-cresol, benzyl alcohol and vanillate. With hydrogen and carbon dioxide, formate or O-methylated aromatic compounds, autotrophic growth during sulfate reduction or homoacetogenesis was demonstrated. Lactate was not used as a substrate. SO _inf4 ^sup2- , SO _inf3 ^sup2- , and S₂O _inf3 ^sup2- were utilized as electron acceptors. Although strain Groll originated from a freshwater habitat, salt concentrations of up to 30 g·l^-1 were tolerated. The optimum temperature for growth was 35–37°C. The G+C content of DNA was 42.1 mol%. This isolate is described as a new species of the genus Desulfotomaculum.     

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1

Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O₂ or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O₂. The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.     

Enzymatic release of halogens or methanol from some substituted protocatechuic acids.

Four strains of gram-negative bacteria capable of growing at the expense of 5-chlorovanillate were isolated from soil, and the metabolism of one strain was studied in particular detail. In the presence of alpha, alpha'-bipyridyl, a suspension of 5-chlorovanillate-grown cells accumulated 5-chloroprotocatechuate from 5-chlorovanillate; in the absence of inhibitor these compounds, and various other 5-substituted protocatechuates and vanillates, were oxidized to completion. Cell suspensions of this strain grown on 5-chlorovanillate or vanillate released chloride quantitatively from 5-chlorovanillate and released methanol from syringate. Extracts of cells grown with 4-hydroxybenzoate, vanillate, or syringate possessed high levels of both protocatechuate 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate hydrolase; extracts from acetate-grown cells did not. Protocatechuate 4,5-dioxygenase, purified from strains that could grow with 5-chlorovanillate, oxidized 5-halogeno-protocatechuates and 3-O-methylgallate with the formation of 2-pyrone-4,6-dicarboxylate. A crude extract converted 5-chloroprotocatechuate into pyruvate plus oxaloacetate. On the basis of these observations, a meta-fission reaction sequence is proposed for the bacterial degradation of vanillate and protocatechuate substituted at C-5 of the benzene ring with halogen or methoxyl.