Aerobic Degradation Of Aromatic Compounds Research Articles

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids.

Improved purified terephthalic acid wastewater treatment using combined UAFB-SBR system: At mesophilic and ambient temperature

In this study, a combined UAFB-SBR process was introduced to improve the treatment efficiency of PTA wastewater. The techno-economic feasibility of the process was evaluated in terms of organic removal efficiencies under mesophilic (37 °C) and ambient temperature (15–25 °C) during the long-term run. The lab-scale study revealed that all organic compounds present in the PTA wastewater could be efficiently removed under both mesophilic and ambient temperature, and p-toluic acid is probably the critical pollutant regulating the overall process performance in anaerobic stage, which should be seriously considered. The Miseq Sequencing results suggested that, along with the system temperature variation from mesophilic to ambient temperature, greater effects on bacterial community than archaeal community were detected in the UAFB reactor, while only slight variations were observed in the SBR reactor. Further taxonomy analysis demonstrated that within the UAFB reactor, the syntrophic partnership of Syntrophorhabdus, Syntrophus and Desulfovibrio with hydrogenotrophic methanogens were the main impetus for aromatic organics reduction. In the meanwhile, the intensively identified Thauera and Azoarcus groups were speculated of important roles in the aerobic degradation of aromatic compounds.

Manipulating intradiol dioxygenases by C-terminus truncation

Intradiol dioxygenases (EC 1.13.11.1) are bacterial enzymes that catalyze the ring cleavage of catechols which is a central step in the aerobic degradation of aromatic compounds. Some members of this enzyme group have a C-terminus which is 4–5% longer (an additional 13–18 amino acids) compared to the majority of known sequences. The longer C-terminus itself is not highly conserved and appears to be poorly integrated in the protein structural models developed for representative intradiol dioxygenases. Using a protein engineering approach variant intradiol dioxygenases were produced by truncating the C-terminus to a size comparable to the shorter versions of the enzyme. Three enzymes were selected and were originally described from the model organisms; Burkholderia xenovorans LB400, Pseudomonas putida KT2440 and Acinetobacter baylyi ADP1. The activity of the truncated enzymes were compared to the unmodified enzymes which revealed that truncation of the C-terminus could alter the enzyme activity; increasing the LB400 enzyme activity by as much as five fold, but reducing the activity of the intradiol dioxygenases from KT2440 and ADP1. The difference in effect is explained by the presence of a greater number of amino acid residues that can contribute to forming stable protein structures in the KT2440 and ADP1 enzymes. It is hypothesized that C-terminal truncation could in some cases provide a useful strategy for increasing intradiol dioxygenase activity for biotechnological production of muconic and adipic acids.

Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation.

A culture-independent function-based screening approach was used to assess the microbial aerobic catabolome for polycyclic aromatic hydrocarbons degradation of a soil subjected to 12 years of in situ bioremediation. A total of 422750 fosmid clones were screened for key aromatic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Most of the genes encoding ring-cleavage enzymes on the 768 retrieved positive fosmids could not be identified using primer-based approaches and, thus, 205 fosmid inserts were sequenced. Nearly two hundred extradiol dioxygenase encoding genes of three different superfamilies could be identified. Additional key genes of aromatic metabolic pathways were identified, including a high abundance of Rieske non-heme iron oxygenases that provided detailed information on enzymes activating aromatic compounds and enzymes involved in activation of the side chain of methylsubstituted aromatics. The gained insights indicated a complex microbial network acting at the site under study, which comprises organisms similar to recently identified Immundisolibacter cernigliae TR3.2 and Rugosibacter aromaticivorans Ca6 and underlined the great potential of an approach that combines an activity-screening, a cost-effective high-throughput sequencing of fosmid clones and a phylogenomic-routed and manually curated database to carefully identify key proteins dedicated to aerobic degradation of aromatic compounds.

Degradation of Diphenyl Ether in Sphingobium phenoxybenzoativorans SC_3 Is Initiated by a Novel Ring Cleavage Dioxygenase.

Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenyl ether (DE) or 2-carboxy-DE as its sole carbon and energy source. In this study, we report the degradation of DE and 2-carboxy-DE initiated by a novel ring cleavage angular dioxygenase (diphenyl ether dioxygenase [Dpe]) in the strain. Dpe functions at the angular carbon and its adjacent carbon (C-1a, C-2) of a benzene ring in DE (or the 2-carboxybenzene ring in 2-carboxy-DE) and cleaves the C-1a-C-2 bond (decarboxylation occurs simultaneously for 2-carboxy-DE), yielding 2,4-hexadienal phenyl ester, which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is a type IV Rieske non-heme iron oxygenase (RHO) and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a glutathione reductase (GR)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in the degradation and utilization of DE and 2-carboxy-DE in S. phenoxybenzoativorans SC_3. Enzymatic study showed that transformation of 1 molecule of DE needs two molecules of oxygen and two molecules of NADH, supporting the assumption that the cleavage of DE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygenated at C-1a and C-2 to form a hemiacetal-like intermediate, which is further deoxygenated, resulting in the cleavage of the C-1a-C-2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledge of the mode and mechanism of ring cleavage of aromatic compounds.IMPORTANCE Benzene ring cleavage, catalyzed by dioxygenase, is the key and speed-limiting step in the aerobic degradation of aromatic compounds. As previously reported, in the ring cleavage of DEs, the benzene ring needs to be first dihydroxylated at a lateral position and subsequently dehydrogenated and opened through extradiol cleavage. This process requires three enzymes (two dioxygenases and one dehydrogenase). In this study, we identified a novel angular dioxygenase (Dpe) in S. phenoxybenzoativorans SC_3. Under Dpe-mediated catalysis, the benzene ring of DE is dioxygenated at the angular position (C-1a, C-2), resulting in the cleavage of the C-1a-C-2 bond to generate a novel product, 2,4-hexadienal phenyl ester. This process needs only one angular dioxygenase, Dpe. Thus, the ring cleavage catalyzed by Dpe represents a novel mechanism of benzene ring cleavage.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments.

The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

Mimicking the Aromatic-Ring-Cleavage Activity of Gentisate-1,2-Dioxygenase by a Nonheme Iron Complex.

Gentisate-1,2-dioxygenase (GDO), a nonheme iron enzyme in the cupin superfamily, catalyzes the cleavage of the aromatic-ring of 2,5-dihydroxybenzoic acid (gentisic acid) to form maleylpyruvic acid in the microbial aerobic degradation of aromatic compounds. To develop a functional model of GDO, we have isolated a nonheme iron(II) complex, [(TpPh2 )FeII (DHN-H)] (TpPh2 =hydrotris(3,5-diphenylpyrazole-1-yl)borate, DHN-H=1,4-dihydroxy-2-naphthoate). In the reaction with O2 , the biomimetic complex oxidatively cleaves the aromatic ring of the coordinated substrate with the incorporation of both the oxygen atoms from molecular oxygen into the cleavage product. The presence of para-hydroxy group on the substrate plays a crucial role in directing the aromatic-ring cleaving reaction.

Mimicking the Aromatic‐Ring‐Cleavage Activity of Gentisate‐1,2‐Dioxygenase by a Nonheme Iron Complex

AbstractGentisate‐1,2‐dioxygenase (GDO), a nonheme iron enzyme in the cupin superfamily, catalyzes the cleavage of the aromatic‐ring of 2,5‐dihydroxybenzoic acid (gentisic acid) to form maleylpyruvic acid in the microbial aerobic degradation of aromatic compounds. To develop a functional model of GDO, we have isolated a nonheme iron(II) complex, [(TpPh2)FeII(DHN‐H)] (TpPh2=hydrotris(3,5‐diphenylpyrazole‐1‐yl)borate, DHN‐H=1,4‐dihydroxy‐2‐naphthoate). In the reaction with O2, the biomimetic complex oxidatively cleaves the aromatic ring of the coordinated substrate with the incorporation of both the oxygen atoms from molecular oxygen into the cleavage product. The presence of para‐hydroxy group on the substrate plays a crucial role in directing the aromatic‐ring cleaving reaction.

The ICEXTD of Azoarcus sp. CIB, an integrative and conjugative element with aerobic and anaerobic catabolic properties.

Integrative and conjugative elements (ICE) play a major role in aerobic degradation of aromatic compounds, but they have not yet been shown to be involved in anaerobic degradation. We have characterized here the ICEXTD element which endows to the beta-proteobacterium Azoarcus sp. CIB with the ability to utilize aromatic hydrocarbons. The core region of ICEXTD , which shows a remarkable synteny with that of ICEclc-like elements, allows its own intracellular and intercellular mobility. ICEXTD integrates at the tRNAGly of the host chromosome, but it can also excise to produce a ready to transfer circular form. The adaptation modules of ICEXTD represent a unique combination of gene clusters for aerobic (tod genes) and anaerobic (bss-bbs and mbd genes) degradation of certain aromatic hydrocarbons, e.g., toluene, m-xylene and cumene. Transfer of ICEXTD to other Azoarcus strains, e.g., A. evansii, confers them the ability to degrade aromatic hydrocarbons both aerobically and anaerobically. Interestingly, ICEXTD allows Cupriavidus pinatubonensis, a bacterium unable to degrade anaerobically aromatic compounds, to grow with m-xylene under anoxic conditions. Thus, ICEXTD constitutes the first mobile genetic element able to expand the catabolic abilities of certain bacteria for the removal of aromatic hydrocarbons either in the presence or absence of oxygen.

Mechanistic Insights into Phenol Oxidation by a Copper(II) Complex of a Pyridine‐ and Amide‐Containing Copolymer in an Aqueous Medium

A CuII complex of a pyridine- and amide-containing copolymer (Cu-P1) exhibits effective activity toward phenol hydroxylation in 50 % aqueous methanol solution at apparent pH 8.0 and 25 °C. The complex shows significant first-order rate accelerations of 4.2 × 105 and 1.4 × 105 relative to phenol autoxidation in air and in the presence of 20 mM H2O2, respectively. The reaction mechanisms with H2O2 and air are different on the basis of (a) the different activity profiles for Cu binding, (b) the different deuterium kinetic isotope effects (2.8 in 20 mM H2O2 and 1.2 aerobically), and (c) the formation of a dinuclear substrate–(Cu-P1) complex with H2O2 but a mononuclear one aerobically. The mechanism in the presence of H2O2 is consistent with that of the type-3 dicopper tyrosinase. Although oxidized di-CuII tyrosinase can only use H2O2 for phenol hydroxylation, CuII-P1 can use either air or H2O2 for this process. The studies herein introduce a versatile chemical system for the further exploration of Cu–oxygen chemistry and other types of metal-centered chemistry and for the aerobic degradation of aromatic compounds in environmental and green chemistry.

Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds

Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.

The Ins and Outs of Ring-Cleaving Dioxygenases

ABSTRACTRing-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.

Iron‐reducing bacteria unravel novel strategies for the anaerobic catabolism of aromatic compounds

Although the aerobic degradation of aromatic compounds has been extensively studied in many microorganisms, the anaerobic mineralization of the aromatic ring is a more recently discovered microbial capacity on which very little information is available from facultative anaerobic bacteria. In this issue of Molecular Microbiology, Wischgoll and colleagues use proteomic and reverse-transcription polymerase chain reaction (PCR) approaches to identify for the first time the gene clusters involved in the central pathway for the catabolism of aromatic compounds in Geobacter metallireducens, a strictly anaerobic iron-reducing bacterium. This work highlights that the major difference in anaerobic benzoate metabolism of facultative and strictly anaerobic bacteria is the reductive process for dearomatization of benzoyl-CoA. The authors propose that a new type of benzoyl-CoA reductase, comprising molybdenum- and selenocysteine-containing proteins, is present in strictly anaerobic bacteria. This work paves the way to fundamental studies on the biochemistry and regulation of this new reductive process and provides the first genetic clues on the anaerobic catabolism of benzoate by strict anaerobes.

Aerobic biodegradation of alkylated aromatic hydrocarbons by a bacterial community

The aerobic degradation of aromatic compounds using a bacterial community from the Arcachon Harbour (South West coast of France) was studied. The aromatic fraction was prepared from an Arabian light crude oil residue and was incubated with this bacterial community under oxic conditions for up to 7 days. The study was focussed onto the degradation of alkylated naphthalenes, dibenzothiophenes and phenanthrenes present in the aromatic fraction. The disappearance of the various aromatic compounds was followed by gas chromatography/mass spectrometry. This bacterial community used the aromatic compounds as the sole source of carbon (more than 70% of the compounds disappeared within 7 days). The degradation phenomenon was shown to follow first order kinetics and did not show any difference between non-sulphur aromatic compounds (phenanthrenes) and sulphur aromatic compounds (dibenzothiophenes). The degradation of methylated phenanthrenes and dibenzothiophenes did not show significant differences in terms of isomeric composition. On the contrary, major differences in biodegradation rates have been observed for the dimethylated derivatives.

Microbial consortia for the aerobic degradation of aromatic compounds in olive oil mill effluent

Aerobic consortia that grow on olive oil mill effluent (OOME) were obtained by enrichment. Several cultures were capable of metabolizing monoaromatic compounds, supplied as the sole carbon source at 2 g L−1. Some consortia degraded mixtures of seven aromatics (4 g L−1) after 1 week of incubation at 32°C. The consortia were also active against monoaromatics of the undiluted OOME. This reduced the inhibitory effect of phenolic compounds prior to the anaerobic digestion of OOME at batch scale. No inhibition of the anaerobic microbial populations was noticed with treated OOME. From the most active consortium, nine different bacterial strains were isolated and shown to grow on simple aromatic compounds. Removal of 50% of the initial chemical oxygen demand and degradation of almost all of the simple aromatics in undiluted OOME was obtained with reconstituted bacterial mixtures. A slight reduction in colouration was due to adsorption of coloured compounds to bacterial cells. Presumably, the consortia could not reduce and degrade the coloured compounds in OOME.

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume.

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.

CHEMILUMINESCENCE IN THE PEROXIDATION OF TANNIC ACID

Abstract— Peroxidation of tannins with alkaline H2O2 is accompanied by weak chemiluminescence in the spectral region 480–800 nm. o‐Di and tri‐hydroxy groups of polyphenols undergo oxidation by a free‐radical mechanism and a green intermediate anion‐radical with absorption Δmax= 600 nm is formed. The radical mechanism is supported by the low activation energy 14–20 kJ/mol and the quenching effect of radical scavengers. The reaction of the green intermediate with peroxy anions is the chemiluminescence rate limiting step. In the presence of a‐hydroxy‐methylperoxide formed from H2O2 and formaldehyde, the alkaline peroxidation of tannins is accompanied by strong red luminescence (420–800 nm). The base catalyzed decomposition of peroxides gives only a weak red emission (460–800 nm). Light intensity is enhanced in D2O by a factor 6.5. Quenchers of O2(1Δg) and 1,3‐di‐phenylisobenzofurane diminish light intensity in non‐aqueous solutions. The data suggest 1O2 participation in the observed chemiluminescence. Thermo‐chemical calculations give —ΔH values from 250–1000 kJ/mol for one elementary reaction step which limits the mechanism of chemi‐enereization. Chemiexcitation of tannins is relevant to biochemical mechanisms of aerobic degradation of aromatic compounds, energy utilization as well as to defense and resistance processes in plants.

Biochemistry of the bacterial catabolism of aromatic compounds in anaerobic environments.

Methods of aerobic degradation of aromatic compounds in the biosphere are well understood, but it is only relatively recently that it has been shown how some bacteria can also degrade these substrates in the absence of molecular oxygen. This occurs by photometabolism (Athiorhodaceae), nitrate respiration (Pseudomonas and Moraxella sp.) and methanogenic fermentation (a consortium) in which the benzene nucleus is first reduced and then cleaved by hydrolysis to yield aliphatic acids for cell growth. These methods may be used by microbial communities to catabolise man-made pollutants.