Meta-cleavage Pathway Research Articles

Biodegradation of acenaphthene by Sphingobacterium sp. strain RTSB involving trans-3-carboxy-2-hydroxybenzylidenepyruvic acid as a metabolite

A gram-negative bacterium designated as RTSB was isolated from a petroleum-contaminated soil competent of utilizing acenaphthene as the solitary source of carbon and energy. The strain RTSB was identified as a Sphingobacterium species based on the morphological, nutritional and biochemical features of the organism as well as 16S rRNA sequence analysis. By a combination of chromatographic and spectrometric techniques, different metabolites of the acenaphthene degradation pathway by the strain RTSB were isolated and identified, which indicate a novel acenaphthene degradation pathway involving 1-naphthoic acid. Characterization of different metabolites suggested transformation of acenaphthene to 1-naphthoic acid through 1-acenaphthenol, acenaphthenequinone and naphthalene-1,8-dicarboxylic acid in the upper pathway of degradation; while in the later, 1-naphthoic acid was processed via a novel meta-cleavage pathway, leading to the formation of trans-3-carboxy-2-hydroxybenzylidenepyruvic acid, and then to salicylic acid and catechol entering into the TCA cycle intermediates. This detailed study of acenaphthene degradation by a Sphingobacterium species describes a distinct pathway of acenaphthene degradation involving the novel metabolite trans-3-carboxy-2-hydroxybenzylidenepyruvic acid.

Bioconversion of toxic micropollutant triclosan to 2,4-dichlorophenol using a wastewater isolate Pseudomonas aeruginosa KS2002

Triclosan, a commonly available pesticide, has emerged as a ubiquitous pollutant posing a major threat to the environment. Here we have isolated a wastewater microorganism, Pseudomonas aeruginosa KS2002, capable of converting triclosan to 2,4-dichlorophenol within 96 h of incubation. The confirmation of the end product was done using Fourier transform infrared spectroscopy and mass spectroscopy. Different minimal media were investigated to establish a suitable media supporting maximum triclosan degradation. Spectral analysis showed that this bacterial isolate degraded 99.89% ± 0.3 of 2 g/L of triclosan spiked in an M9 minimal salt medium. This isolate utilized fructose and glycerol as a co-substrate to enhance degradation process. The cell-free extract of Pseudomonas aeruginosa KS2002 showed the activity of catechol 2,3-dioxygenase enzyme (specific enzyme activity = 0.161 U/mg). In the presence of 3-fluorocatechol, a meta-cleavage enzyme inhibitor, triclosan degradation was ceased suggesting a meta-cleavage pathway for triclosan degradation. Keeping in view the observations recorded, we proposed a pathway for partial triclosan degradation using this isolate.

Delftia sp. LCW, a strain isolated from a constructed wetland shows novel properties for dimethylphenol isomers degradation

BackgroundDimethylphenols (DMP) are toxic compounds with high environmental mobility in water and one of the main constituents of effluents from petro- and carbochemical industry. Over the last few decades, the use of constructed wetlands (CW) has been extended from domestic to industrial wastewater treatments, including petro-carbochemical effluents. In these systems, the main role during the transformation and mineralization of organic pollutants is played by microorganisms. Therefore, understanding the bacterial degradation processes of isolated strains from CWs is an important approach to further improvements of biodegradation processes in these treatment systems.ResultsIn this study, bacterial isolation from a pilot scale constructed wetland fed with phenols led to the identification of Delftia sp. LCW as a DMP degrading strain. The strain was able to use the o-xylenols 3,4-DMP and 2,3-DMP as sole carbon and energy sources. In addition, 3,4-DMP provided as a co-substrate had an effect on the transformation of other four DMP isomers. Based on the detection of the genes, proteins, and the inferred phylogenetic relationships of the detected genes with other reported functional proteins, we found that the phenol hydroxylase of Delftia sp. LCW is induced by 3,4-DMP and it is responsible for the first oxidation of the aromatic ring of 3,4-, 2,3-, 2,4-, 2,5- and 3,5-DMP. The enzyme may also catalyze both monooxygenation reactions during the degradation of benzene. Proteome data led to the identification of catechol meta cleavage pathway enzymes during the growth on ortho DMP, and validated that cleavage of the aromatic rings of 2,5- and 3,5-DMPs does not result in mineralization. In addition, the tolerance of the strain to high concentrations of DMP, especially to 3,4-DMP was higher than that of other reported microorganisms from activated sludge treating phenols.ConclusionsLCW strain was able to degraded complex aromatics compounds. DMPs and benzene are reported for the first time to be degraded by a member of Delftia genus. In addition, LCW degraded DMPs with a first oxidation of the aromatic rings by a phenol hydroxylase, followed by a further meta cleavage pathway. The higher resistance to DMP toxicity, the ability to degrade and transform DMP isomers and the origin as a rhizosphere bacterium from wastewater systems, make LCW a suitable candidate to be used in bioremediation of complex DMP mixtures in CWs systems.

Biological remediation and removal of radioactive metals and complex aromatic compounds from nuclear and radioactive waste

In pursuing carbon emission free technologies in the energy industry, interest in nuclear energy has grown. However, technology for handling radioactive waste containing organic materials are lacking. Methods are required for biodegradation of often recalcitrant irradiated organic compounds which are persistent in radioactive waste from the nuclear industry. In this study, a bioseparation process for extraction of 14C and de-radiation of nuclear graphite was investigated in a continuous flow sequencing-batch/biofilm reactor. Dehalogenation and mineralisation of aromatic halogenated compounds in the waste stream of a pebble bed manufacturing process was simulated by the photocatalytic/biological hybrid system in the 50 to 1,000 4-CP mg.L-1. The efficiency of degradation of the irradiated compounds was compared to conventional degradation using PAH degrading microorganisms. The Pathway of degradation was established for both systems by evaluating the intermediates of degradation of radiolabelled phenolic compounds with ortho-13C-ortho and para-13C phenol. Phenol degradation under photocatalytic conditions was shown to follow the catechol( hydroquinone( hydroxyhydroquinone(benzene-1,2,3-triol pathway, whereas the degradation in the biological stage followed the meta-cleavage pathway via catechol( 2-hydromuconic semialdehyde( pyruvate under aerobic conditions. This study demonstrates the potential of efficiently managing the organic component of nuclear waste using a cleaner, environmentally friendly biological process.

Elucidation of 2, 4-Dichlorophenol degradation by Bacillus licheniformis strain SL10

ABSTRACT2,4-Dichlorophenol (2,4-DCP) is a priority pollutant according to US Environmental Protection Agency. Its use in various chemical industries and its presence in the effluent necessitate effective removal studies. The present study focuses on degradation of 2,4-DCP by phenol adapted bacteria Bacillus licheniformis strain SL10 (MTCC 25059) at a relatively faster rate. The organism exhibited tolerance to 150 ppm of 2,4-DCP and showed a linear relationship between the growth and substrate concentration (µmax 0.022/h) and the inhibitory concentration was 55.74 mg/L. The degradation efficiency of the organism was 74% under optimum conditions but increased to 97% when the growth medium containing nil sodium chloride. The degradation of 2,4-DCP was effected by the action of extracellular cocktail enzyme containing Catechol 2, 3 dioxygenase (C23DO), phenol hydroxylase and Catechol, 1,2 dioxygenase (C12DO). In vitro enzymatic degradation studies exhibit 98% degradation of 50 ppm of 2,4-DCP within 2 h. Analyses of degradation products infer that the chosen organism followed a meta-cleavage pathway while degrading 2,4-DCP. In conclusion, the bacteria Bacillus licheniformis strain SL10 finds potential application in the remediation of 2,4-DCP.

Functional genomic analysis of phthalate acid ester (PAE) catabolism genes in the versatile PAE-mineralising bacterium Rhodococcus sp. 2G

Microbial degradation is considered the most promising method for removing phthalate acid esters (PAEs) from polluted environments; however, a comprehensive genomic understanding of the entire PAE catabolic process is still lacking. In this study, the repertoire of PAE catabolism genes in the metabolically versatile bacterium Rhodococcus sp. 2G was examined using genomic, metabolic, and bioinformatic analyses. A total of 4930 coding genes were identified from the 5.6 Mb genome of the 2G strain, including 337 esterase/hydrolase genes and 48 transferase and decarboxylase genes that were involved in hydrolysing PAEs into phthalate acid (PA) and decarboxylating PA into benzoic acid (BA). One gene cluster (xyl) responsible for transforming BA into catechol and two catechol-catabolism gene clusters controlling the ortho (cat) and meta (xyl &mhp) cleavage pathways were also identified. The proposed PAE catabolism pathway and some key degradation genes were validated by intermediate-utilising tests and real-time quantitative polymerase chain reaction. Our results provide novel insight into the mechanisms of PAE biodegradation at the molecular level and useful information on gene resources for future studies.

Biochemical pathways and enhanced degradation of di-n-octyl phthalate (DOP) in sequencing batch reactor (SBR) by Arthrobacter sp. SLG-4 and Rhodococcus sp. SLG-6 isolated from activated sludge

Two bacterial strains designated as Arthrobacter sp. SLG-4 and Rhodococcus sp. SLG-6, capable of utilizing di-n-octyl phthalate (DOP) as sole source of carbon and energy, were isolated from activated sludge. The analysis of DOP degradation intermediates indicated Arthrobacter sp. SLG-4 could completely degrade DOP. Whereas DOP could not be mineralized by Rhodococcus sp. SLG-6 and the final metabolic product was phthalic acid (PA). The proposed DOP degradation pathway by Arthrobacter sp. SLG-4 was that strain SLG-4 initially transformed DOP to PA via de-esterification pathway, and then PA was metabolized to protocatechuate acid and eventually converted to tricarboxylic acid (TCA) cycle through meta-cleavage pathway. Accordingly, Phthalate 3,4-dioxygenase genes (phtA) responsible for PA degradation were successfully detected in Arthrobacter sp. SLG-4 by real-time quantitative PCR (q-PCR). q-PCR analysis demonstrated that the quantity of phthalate 3,4-dioxygenase was positively correlated to DOP degradation in SBRs. Bioaugmentation by inoculating DOP-degrading bacteria effectively shortened the start-up of SBRs and significantly enhanced DOP degradation in bioreactors. More than 91% of DOP (500mgL-1) was removed in SBR bioaugmented with bacterial consortium, which was double of the control SBR. This study suggests bioaugmentation is an effective and feasible technique for DOP bioremediation in practical engineering.

Metagenomic Analysis of a Biphenyl-Degrading Soil Bacterial Consortium Reveals the Metabolic Roles of Specific Populations.

Polychlorinated biphenyls (PCBs) are widespread persistent pollutants that cause several adverse health effects. Aerobic bioremediation of PCBs involves the activity of either one bacterial species or a microbial consortium. Using multiple species will enhance the range of PCB congeners co-metabolized since different PCB-degrading microorganisms exhibit different substrate specificity. We have isolated a bacterial consortium by successive enrichment culture using biphenyl (analog of PCBs) as the sole carbon and energy source. This consortium is able to grow on biphenyl, benzoate, and protocatechuate. Whole-community DNA extracted from the consortium was used to analyze biodiversity by Illumina sequencing of a 16S rRNA gene amplicon library and to determine the metagenome by whole-genome shotgun Illumina sequencing. Biodiversity analysis shows that the consortium consists of 24 operational taxonomic units (≥97% identity). The consortium is dominated by strains belonging to the genus Pseudomonas, but also contains betaproteobacteria and Rhodococcus strains. whole-genome shotgun (WGS) analysis resulted in contigs containing 78.3 Mbp of sequenced DNA, representing around 65% of the expected DNA in the consortium. Bioinformatic analysis of this metagenome has identified the genes encoding the enzymes implicated in three pathways for the conversion of biphenyl to benzoate and five pathways from benzoate to tricarboxylic acid (TCA) cycle intermediates, allowing us to model the whole biodegradation network. By genus assignment of coding sequences, we have also been able to determine that the three biphenyl to benzoate pathways are carried out by Rhodococcus strains. In turn, strains belonging to Pseudomonas and Bordetella are the main responsible of three of the benzoate to TCA pathways while the benzoate conversion into TCA cycle intermediates via benzoyl-CoA and the catechol meta-cleavage pathways are carried out by beta proteobacteria belonging to genera such as Achromobacter and Variovorax. We have isolated a Rhodococcus strain WAY2 from the consortium which contains the genes encoding the three biphenyl to benzoate pathways indicating that this strain is responsible for all the biphenyl to benzoate transformations. The presented results show that metagenomic analysis of consortia allows the identification of bacteria active in biodegradation processes and the assignment of specific reactions and pathways to specific bacterial groups.

Transcriptional control of the phenol hydroxylase gene phe of Corynebacterium glutamicum by the AraC-type regulator PheR

Corynebacterium glutamicum can degrade phenol by a meta-cleavage pathway, which depends on ncgl2588 (phe) of the phe operon encoding phenol hydroxylase. An additional gene, ncgl2587 (pheR), is located upstream of phe. The pheR encodes an AraC/XylR-type regulator protein with 377 amino acid residues and is transcribed in the same direction as phe. Disruption of pheR by homologous recombination resulted in the accumulation of phenol in C. glutamicum. PheR demonstrates a low type of constitutive expression where phenol induces phe expression. PheR shares 75% sequence identity with AraC-type regulator of Corynebacterium lubricantis and 37 conserved residues, characteristic of AraC family, were located. A constructed pK18mobsacB-Pphe:lacZ transcriptional fusion plasmid was transformed into the wild-type, ΔpheR, and ΔpheR+ strains, and the results indicated that PheR activates the expression of phe encoding phenol hydroxylase. Electrophoretic mobility shift assay (EMSA) demonstrated a direct interaction of PheR with the phe promoter region and binding site of PheR on the Pphe was located 109-bp upstream of phe, as indicated by foot printing analysis. Our research provides deep insight into PheR expression and its regulatory function on Phe in C. glutamicum.

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s-1 and 2.5 ± 0.2 × 107 m-1 s-1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

Aerobic degradation of fenvalerate by a Gram-positive bacterium, Bacillus flexus strain XJU-4.

Synthetic pyrethroid-fenvalerate-is one of the most widespread toxic pollutants and has adverse effect on living systems. However, little is known about its biotransformation mechanism in different microorganisms. To elucidate the pathway that might be involved in the catabolism of fenvalerate, we used Bacillus flexus strain XJU-4 (3-nitrobenzoate degrading organism) as an ideal fenvalerate degrading bacterium. Thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry analysis results revealed that 3-phenoxybenzoate, protocatechuate, and catechol are the three main by-products of fenvalerate metabolism. Additionally, the bacterial cell-free enzymes showed the activities of fenvalerate hydrolyzing esterase, 3-phenoxybenzaldehyde dehydrogenase, 3-phenoxybenzoate dioxygenase, phenol hydroxylase, protocatechuate 2,3-dioxygenase and catechol-2,3-dioxygenase. Thus, in strain XJU-4, protocatechuate and catechol were further metabolized through meta-cleavage pathway. Moreover, laboratory-scale soil experiments results suggest that B. flexus strain XJU-4 is a suitable contender for bioremediation of pyrethroid fenvalerate-contaminated sites.

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches.

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31–43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM).

Synergistic degradation of pyrene by five culturable bacteria in a mangrove sediment-derived bacterial consortium

A pyrene-degrading microbial consortium was obtained after enrichment with mangrove sediment collected from Thailand. Five cultivable bacteria (Mycobacterium spp. PO1 and PO2, Novosphingobium pentaromativorans PY1, Ochrobactrum sp. PW1, and Bacillus sp. FW1) were successfully isolated from the consortium. Draft genomes of them showed that two different morphotypes of Mycobacterium (PO1 and PO2), possessed a complete gene set for pyrene degradation. PY1 contained genes for phthalate assimilation via protocatechuate, a central intermediate, by meta-cleavage pathway, and PW1 possessed genes for protocatechuate degradation via ortho-cleavage pathway. The occurrence of biosurfactant-producing genes in FW1 suggests the involvement in enhancing the pyrene bioavailability. Biotransformation experiments revealed that Mycobacterium completely degraded 100mgL−1 pyrene within six days, whereas no significant degradation was observed with the others. Notably, PY1 and PW1 exhibited higher activity for protocatechuate degradation than the others. The artificially reconstructed consortia containing Mycobacterium with the other three strains (PY1, PW1 and FW1) showed three-fold higher degradation rate for pyrene than the individual Mycobacterium. The enhanced pyrene biodegradation achieved in the consortium was due to the cooperative interaction of bacterial mixture. Our findings showing that synergistic degradation of pyrene in the consortium will facilitate the application of the defined bacterial consortium in bioremediation.

A pilot study demonstrating the altered gut microbiota functionality in stable adults with Cystic Fibrosis

Cystic Fibrosis (CF) and its treatment result in an altered gut microbiota composition compared to non-CF controls. However, the impact of this on gut microbiota functionality has not been extensively characterised. Our aim was to conduct a proof-of-principle study to investigate if measurable changes in gut microbiota functionality occur in adult CF patients compared to controls. Metagenomic DNA was extracted from faecal samples from six CF patients and six non-CF controls and shotgun metagenomic sequencing was performed on the MiSeq platform. Metabolomic analysis using gas chromatography-mass spectrometry was conducted on faecal water. The gut microbiota of the CF group was significantly different compared to the non-CF controls, with significantly increased Firmicutes and decreased Bacteroidetes. Functionality was altered, with higher pathway abundances and gene families involved in lipid (e.g. PWY 6284 unsaturated fatty acid biosynthesis (p = 0.016)) and xenobiotic metabolism (e.g. PWY-5430 meta-cleavage pathway of aromatic compounds (p = 0.004)) in CF patients compared to the controls. Significant differences in metabolites occurred between the two groups. This proof-of-principle study demonstrates that measurable changes in gut microbiota functionality occur in CF patients compared to controls. Larger studies are thus needed to interrogate this further.

Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast

Phenol is reported to be one of the most toxic environmental pollutants present in the discharge of various industrial effluents causing a serious threat to the existing biome. Biodegradation of phenol by oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was found to degrade 1000mg/l phenol. The pathways for phenol degradation by both ortho and meta-cleavage were proposed by the identification of metabolites and enzymatic assays of ring cleavage enzymes in the cell extracts. Results suggest that this oleaginous yeast degrade phenol via meta-cleavage pathway and accumulates a high quantity of lipid content (64.92%; wt/wt) as compared to control glucose synthetic medium (GSM). Meta-cleavage pathway of phenol degradation leads to formation of pyruvate and acetaldehyde. Both these end products feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway which causes accumulation of TAG in the lipid droplets (LD) of 6.12±0.78μm grown on phenol while 2.38±0.52μm observed on GSM. This was confirmed by fluorescence microscopic images of BODIPY505-515nm stained live yeast cells. GC–MS analysis of extracted total lipid showed enhanced amount of monounsaturated fatty acid (MUFA) which was as 51.87%, 58.33% and 62.98% in presence of 0.5, 0.75 and 1g/l of phenol.

Salicylate degradation by a cold-adapted Pseudomonas sp.

Pseudomonas sp. strain MC1 was characterized as a cold-adapted, naphthalene-degrading bacterium that is able to grow in a broad temperature range of 5–30°C. MC1 harbors a catabolic plasmid, designated pYIC1, which is almost identical to the archetypal NAH7 plasmid from the mesophilic bacterium Pseudomonas putida G7. On pYIC1, the catabolic genes for naphthalene degradation are clustered in two operons: nahAa-Ab-Ac-Ad-B-F-C-Q-E-D encoding the conversion of naphthalene to salicylate, and nahG-T-H-I-N-L-O-M-K-J encoding the conversion of salicylate through meta-cleavage pathway to pyruvate and acetyl CoA. NahH, the bona fide extradiol dioxygenase in MC1 salicylate metabolism, is thermolabile and is a cold-adapted enzyme. The thermal profiles of the NahH enzyme activities expressed in different hosts indicate the presence of a factor(s) or mechanism(s) to protect the thermolabile NahH enzyme (100% aa identity with MC1 counterpart) in G7. Overall, the results reported in the present work suggest that the thermolabile NahH might be a product of the cold-adaptation process of MC1 and thus contribute to the survival and growth ability of MC1 on salicylate and naphthalene in cold environments.

Cometabolic degradation of chloramphenicol via a meta-cleavage pathway in a microbial fuel cell and its microbial community

The performance of a microbial fuel cell (MFC) in terms of degradation of chloramphenicol (CAP) was investigated. Approximately 84% of 50mg/L CAP was degraded within 12h in the MFC. A significant interaction of pH, temperature, and initial CAP concentration was found on removal of CAP, and a maximum degradation rate of 96.53% could theoretically be achieved at 31.48°C, a pH of 7.12, and an initial CAP concentration of 106.37mg/L. Moreover, CAP was further degraded through a ring-cleavage pathway. The antibacterial activity of CAP towards Escherichia coli ATCC 25922 and Shewanella oneidensis MR-1 was largely eliminated by MFC treatment. High-throughput sequencing analysis indicated that Azonexus, Comamonas, Nitrososphaera, Chryseobacterium, Azoarcus, Rhodococcus, and Dysgonomonas were the predominant genera in the MFC anode biofilm. In conclusion, the MFC shows potential for the treatment of antibiotic residue-containing wastewater due to its high rates of CAP removal and energy recovery.

Biological degradation of 4-chlorobenzoic acid by a PCB-metabolizing bacterium through a pathway not involving (chloro)catechol.

Cupriavidus sp. strain SK-3, previously isolated on polychlorinated biphenyl mixtures, was found to aerobically utilize a wide spectrum of substituted aromatic compounds including 4-fluoro-, 4-chloro- and 4-bromobenzoic acids as a sole carbon and energy source. Other chlorobenzoic acid (CBA) congeners such as 2-, 3-, 2,3-, 2,5-, 3,4- and 3,5-CBA were all rapidly transformed to respective chlorocatechols (CCs). Under aerobic conditions, strain SK-3 grew readily on 4-CBA to a maximum concentration of 5mM above which growth became impaired and yielded no biomass. Growth lagged significantly at concentrations above 3mM, however chloride elimination was stoichiometric and generally mirrored growth and substrate consumption in all incubations. Experiments with resting cells, cell-free extracts and analysis of metabolite pools suggest that 4-CBA was metabolized in a reaction exclusively involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoic acid, which was then hydroxylated to protocatechuic acid (PCA) and subsequently metabolized via the β-ketoadipate pathway. When strain SK-3 was grown on 4-CBA, there was gratuitous induction of the catechol-1,2-dioxygenase and gentisate-1,2-dioxygenase pathways, even if both were not involved in the metabolism of the acid. While activities of the modified ortho- and meta-cleavage pathways were not detectable in all extracts, activity of PCA-3,4-dioxygenase was over ten-times higher than those of catechol-1,2- and gentisate-1,2-dioxygenases. Therefore, the only reason other congeners were not utilized for growth was the accumulation of CCs, suggesting a narrow spectrum of the activity of enzymes downstream of benzoate-1,2-dioxygenase, which exhibited affinity for a number of substituted analogs, and that the metabolic bottlenecks are either CCs or catabolites of the modified ortho-cleavage metabolic route.

Comparative genomic analysis of isoproturon-mineralizing sphingomonads reveals the isoproturon catabolic mechanism.

The worldwide use of the phenylurea herbicide, isoproturon (IPU), has resulted in considerable concern about its environmental fate. Although many microbial metabolites of IPU are known and IPU-mineralizing bacteria have been isolated, the molecular mechanism of IPU catabolism has not been elucidated yet. In this study, complete genes that encode the conserved IPU catabolic pathway were revealed, based on comparative analysis of the genomes of three IPU-mineralizing sphingomonads and subsequent experimental validation. The complete genes included a novel hydrolase gene ddhA, which is responsible for the cleavage of the urea side chain of the IPU demethylated products; a distinct aniline dioxygenase gene cluster adoQTA1A2BR, which has a broad substrate range; and an inducible catechol meta-cleavage pathway gene cluster adoXEGKLIJC. Furthermore, the initial mono-N-demethylation genes pdmAB were further confirmed to be involved in the successive N-demethylation of the IPU mono-N-demethylated product. These IPU-catabolic genes were organized into four transcription units and distributed on three plasmids. They were flanked by multiple mobile genetic elements and highly conserved among IPU-mineralizing sphingomonads. The elucidation of the molecular mechanism of IPU catabolism will enhance our understanding of the microbial mineralization of IPU and provide insights into the evolutionary scenario of the conserved IPU-catabolic pathway.

Meta-cleavage pathway of phenol degradation by Acinetobacter sp. strain AQ5NOL 1

The characterization of bacterial enzymatic pathways of phenol metabolism is important to better understand phenol biodegradation. Phenol hydroxylase is the first enzyme involved in the oxidative metabolism of phenol, followed by further degradation via either meta- or ortho-pathways. In this study, the first known instance of phenol degradation via the meta-pathway by a member of the genus Acinetobacter (Acinetobacter sp. strain AQ5NOL 1) is reported. Phenol hydroxylase converts phenol to catechol, which is then converted via the meta-pathway to 2-hydroxymuconic semialdehyde by the catechol 2,3-dioxygenase enzyme. Phenol hydroxylase extracted from strain AQ5NOL 1 was fully purified using DEAE-Sepharose®, DEAE-Sephadex®, Q-Sepharose® and Zorbax® Bioseries GF-250 gel filtration and was demonstrated by SDS-PAGE to have a molecular weight of 50 kDa. The phenol hydroxylase was purified to about 210.51 fold. The optimum pH and temperature for enzyme activities are 20 °C and 7–7.5, respectively. The apparent K m and V max values of phenol hydroxylase with phenol as the substrate were 13.4 µM and 2.5 µmol min−1 mg−1, respectively. The enzyme was stable at −20 °C for 36 days.