Anaerobic degradation of 2-chloro-4-nitroaniline by Geobacter sp. KT7 and Thauera aromatica KT9

ABSTRACT 2-chloro-4-nitroaniline is a nitroaromatic compound widely used in industrial and agricultural sectors, causing serious environmental problems. This compound and some of its analogs were utilized by two Fe3+-reducing microbial strains Geobacter sp. KT7 and Thauera aromatica KT9 isolated from contaminated sediment as sole carbon and nitrogen sources under anaerobic conditions. The anaerobic degradation of 2-chloro-4-nitroaniline by the mixed species was increased approximately by 45% compared to that of individual strains. The two isolates’ crossfeeding, nutrient sharing and cooperation in the mixed culture accounted for the increase in degradation rates. The determination of degradation pathways showed that Geobacter sp. KT7 transformed the nitro group in 2-chloro-4-nitroaniline to the amino group following by the dechlorination process, while T. aromatica KT9 dechlorinated the compound before removing the nitro group and further transformed it to aniline. This study provided an intricate network of 2-chloro-4-nitroaniline degradation in the bacterial mixture and revealed two parallel routes for the substrate catabolism.

Influence of changes in microbial cell membrane composition on isotopic fractionation of nitrate during denitrification

Pure cultures of Thauera aromatica were stressed by the addition of the toxic solvents 1-octanol and 4-chlorophenol causing adaptive modifications in their cell-membrane fatty acid composition. At the same time, we investigated the isotopic fractionation in δ15N-NO3- during denitrification. We observed a change in the degree of saturation (DoS) of the bacteria as a reaction to solvent stress and a higher degree of saturation was directly correlated to the concentration of solvents in the cell membrane. The enrichment factor ε15N-NO3- during denitrification showed a linear dependency to the DoS as well as to the membrane solvent concentration. Denitrifying bacteria thus may express less negative ε15N-NO3- when they are exposed to environmental stress that changes their cell membrane composition.

DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in theThauera aromaticaAR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway

ABSTRACTThe facultative anaerobeThauera aromaticastrain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. ThedbdRgene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR forp-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (Porf20) was only active in the presence of 3,5-DHB, the other two (PdbhLand Porf18) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the PdbdRpromoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway inThauera aromaticaAR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCEThauera aromaticaAR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

ATP-Dependent C–F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen

ABSTRACTComplete biodegradation of the abundant and persistent fluoroaromatics requires enzymatic cleavage of an arylic C–F bond, probably the most stable single bond of a biodegradable organic molecule. While in aerobic microorganisms defluorination of fluoroaromatics is initiated by oxygenases, arylic C–F bond cleavage has never been observed in the absence of oxygen. Here, an oxygen-independent enzymatic aryl fluoride bond cleavage is described during the complete degradation of 4-fluorobenzoate or 4-fluorotoluene to CO2and HF in the denitrifyingThauera aromatica: the ATP-dependent defluorination of 4-fluorobenzoyl-coenzyme A (4-F-BzCoA) to benzoyl-coenzyme A (BzCoA) and HF, catalyzed by class I BzCoA reductase (BCR). Adaptation to growth with the fluoroaromatics was accomplished by the downregulation of a promiscuous benzoate-CoA ligase and the concomitant upregulation of 4-F-BzCoA-defluorinating/dearomatizing BCR on the transcriptional level. We propose an unprecedented mechanism for reductive arylic C–F bond cleavage via a Birch reduction-like mechanism resulting in a formal nucleophilic aromatic substitution. In the proposed anionic 4-fluorodienoyl-CoA transition state, fluoride elimination to BzCoA is favored over protonation to a fluorinated cyclic dienoyl-CoA.IMPORTANCEOrganofluorides are produced as pesticides, pharmaceuticals, and other chemicals and comprise approximately one quarter of all organic compounds in the pharmaceutical and agricultural sectors; they are considered a growing class of environmentally relevant persistent pollutants. Especially in the case of fluoroaromatics, biodegradation is hampered by the extreme stability of the arylic C–F bond. In aerobic microorganisms, degradation proceeds via oxygenase-dependent C–F bond cleavage reactions, whereas the enzymes involved in the degradation of fluoroaromatics at anoxic sites are unknown. Here we report a strategy for the complete biodegradation of a fluoroaromatic to CO2and HF in a denitrifying bacterium via activation to a CoA ester, followed by oxygen-independent arylic C–F bond cleavage catalyzed by an ATP-dependent enzyme. This reaction, in conjunction with a transcriptional adaptation to fluorinated growth substrates, is essential for the anoxic biodegradation of 4-fluorobenzoate/4-F-toluene and probably other fluoroaromatics.

Identification of the Gene Cluster for the Anaerobic Degradation of 3,5-Dihydroxybenzoate (α-Resorcylate) in Thauera aromatica Strain AR-1

ABSTRACTThauera aromaticastrain AR-1 degrades 3,5-dihydroxybenzoate (3,5-DHB) with nitrate as an electron acceptor. Previous biochemical studies have shown that this strain converts 3,5-DHB to hydroxyhydroquinone (1,2,4-trihydroxybenzene) through water-dependent hydroxylation of the aromatic ring and subsequent decarboxylation, and they suggest a pathway homologous to that described for the anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) byAzoarcus anaerobius. Southern hybridization of aT. aromaticastrain AR-1 gene library identified a 25-kb chromosome region based on its homology withA. anaerobiusmain pathway genes. Sequence analysis defined 20 open reading frames. Knockout mutations of the most relevant genes in the pathway were generated by reverse genetics. Physiological and biochemical analyses identified the genes for the three main steps in the pathway which were homologous to those described inA. anaerobiusand suggested the function of several auxiliary genes possibly involved in enzyme maturation and intermediate stabilization. However,T. aromaticastrain AR-1 had an additional enzyme to metabolize hydroxyhydroquinone, a putative cytoplasmic quinone oxidoreductase. In addition, a specific tripartite ATP-independent periplasmic (TRAP) transport system was required for efficient growth on 3,5-DHB. Reverse transcription-PCR (RT-PCR) analysis showed that the pathway genes were organized in five 3,5-DHB-inducible operons, three of which have been shown to be under the control of a single LysR-type transcriptional regulator, DbdR. Despite sequence homology, the genetic organizations of the clusters inT. aromaticastrain AR-1 andA. anaerobiusdiffered substantially.

Anaerobic degradation of aromatic amino acids by the hyperthermophilic archaeon Ferroglobus placidus

Ferroglobus placidus was discovered to oxidize completely the aromatic amino acids tyrosine, phenylalanine and tryptophan when Fe(III) oxide was provided as an electron acceptor. This property had not been reported previously for a hyperthermophilic archaeon. It appeared that F. placidus follows a pathway for phenylalanine and tryptophan degradation similar to that of mesophilic nitrate-reducing bacteria, Thauera aromatica and Aromatoleum aromaticum EbN1. Phenylacetate, 4-hydroxyphenylacetate and indole-3-acetate were formed during anaerobic degradation of phenylalanine, tyrosine and tryptophan, respectively. Candidate genes for enzymes involved in the anaerobic oxidation of phenylalanine to phenylacetate (phenylalanine transaminase, phenylpyruvate decarboxylase and phenylacetaldehyde : ferredoxin oxidoreductase) were identified in the F. placidus genome. In addition, transcription of candidate genes for the anaerobic phenylacetate degradation, benzoyl-CoA degradation and glutaryl-CoA degradation pathways was significantly upregulated in microarray and quantitative real-time-PCR studies comparing phenylacetate-grown cells with acetate-grown cells. These results suggested that the general strategies for anaerobic degradation of aromatic amino acids are highly conserved amongst bacteria and archaea living in both mesophilic and hyperthermophilic environments. They also provided insights into the diverse metabolism of Archaeoglobaceae species living in hyperthermophilic environments.

Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators?

ABSTRACT The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO2 as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.