ABSTRACT
1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.
Subject(s)
1-Naphthylamine , Pseudomonas , Pseudomonas/enzymology , Pseudomonas/genetics , Pseudomonas/metabolism , Substrate Specificity , 1-Naphthylamine/analogs & derivatives , 1-Naphthylamine/metabolism , Biodegradation, Environmental , Dioxygenases/metabolism , Dioxygenases/genetics , Dioxygenases/chemistry , Metabolic Networks and Pathways , Multigene Family , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistryABSTRACT
Compound-specific isotope analysis (CSIA) is an established tool to track the in situ transformation of organic chemicals at contaminated sites. In this work, we evaluated the potential of multi-element CSIA to assess biodegradation of 2,3-dichloroaniline (2,3-DCA), which is a major industrial feedstock. Using controlled laboratory experiments, we determined, for the first time, negligible carbon (<0.5) and hydrogen (<10) isotope fractionation and a significant inverse nitrogen isotope fractionation (>10) during aerobic 2,3-DCA biodegradation by a mixed enrichment culture. The tentative identification of a glutamate conjugate of 2,3-DCA as a reaction intermediate indicates that the initial multistep enzymatic reaction may be rate-limiting. The formation of the glutamate adduct would increase the bond energy at the N atom, thus likely explaining the observed inverse N isotope fractionation. The corresponding nitrogen enrichment factor was +6.8 ± 0.6. This value was applied to investigate the in situ 2,3-DCA biodegradation at a contaminated site where the carbon and nitrogen isotope signatures from field samples suggested similar aerobic processes by native microorganisms. Under the assumption of the applicability of the Rayleigh model in a pilot wetland treating contaminated groundwater, the extent of biodegradation was estimated to be up to 80-90%. This study proposes multi-element CSIA as a novel application to study 2,3-DCA fate in groundwater and surface water and provides insights into biodegradation pathways.
Subject(s)
Biodegradation, Environmental , Aniline Compounds/metabolism , Carbon Isotopes , Nitrogen Isotopes , Aerobiosis , Nitrogen/metabolismABSTRACT
Nitroanisoles are used widely as synthetic intermediates and explosives. Although bacteria have been reported to degrade 4-nitroanisole (4NA) under aerobic conditions, the key enzymes and the catalytic mechanism have remained elusive. Rhodococcus sp. strain JS3073 was isolated for its ability to grow on 4NA as the sole carbon and energy source. In this study, whole cell biotransformation experiments indicated that 4NA degradation is initiated by O-demethylation to form 4-nitrophenol (PNP), which undergoes subsequent degradation by a previously established pathway involving formation of 1,2,4-benzenetriol and release of nitrite. Based on comparative transcriptomics and heterologous expression, a novel three-component cytochrome P450 system encoded by pnaABC initiates the O-demethylation of 4NA to yield formaldehyde and PNP. The pnaABC genes encode a phthalate dioxygenase type reductase (PnaA), a cytochrome P450 monooxygenase (PnaB), and an EthD family protein (PnaC) with putative function similar to ferredoxins. This unusual P450 system also has a broad substrate specificity for nitroanisole derivatives. Sequence analysis of PnaAB revealed high identity with multiple self-sufficient P450s of the CYP116B subfamily. The findings revealed the molecular basis of the catabolic pathway for 4NA initiated by an unusual O-demethylase PnaABC and extends the understanding of the diversity among P450s and their electron transport chains.
Subject(s)
Rhodococcus , Rhodococcus/metabolism , Cytochrome P-450 Enzyme System/metabolism , Anisoles/metabolism , BiotransformationABSTRACT
4-Nitroaniline (4NA), the starting material for the first synthesized azo dye, is a toxic compound found in industrial wastewaters. Several bacterial strains capable of 4NA biodegradation were previously reported but the details of the catabolic pathway were not established. To search for novel metabolic diversity, we isolated a Rhodococcus sp. Strain JS360 by selective enrichment from 4NA-contaminated soil. When grown on 4NA the isolate accumulated biomass released stoichiometric amounts of nitrite and released less than stoichiometric amounts of ammonia, indicating that 4NA was used as sole carbon and nitrogen source to support growth and mineralization. Enzyme assays coupled with respirometry provided preliminary evidence that the first and second steps of 4NA degradation involve monooxygenase-catalyzed reactions followed by ring cleavage prior to deamination. Sequencing and annotation of the whole genome revealed candidate monooxygenases that were subsequently cloned and expressed in E.coli. Heterologously expressed 4NA monooxygenase (NamA) and 4-aminophenol (4AP) monooxygenase (NamB) transformed 4NA to 4AP and 4AP to 4-aminoresorcinol (4AR) respectively. The results revealed a novel pathway for nitroanilines and defined two monooxygenase mechanisms likely to be involved in the biodegradation of similar compounds.
Subject(s)
Rhodococcus , Rhodococcus/metabolism , Biodegradation, Environmental , Aniline Compounds/metabolism , Mixed Function Oxygenases/metabolismABSTRACT
Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) enter the environment from natural sources and anthropogenic activities. To date, microorganisms able to mineralize nitro-PAHs have not been reported. Here, Sphingobium sp. strain JS3065 was isolated by selective enrichment for its ability to grow on 1-nitronaphthalene as the sole carbon, nitrogen, and energy source. Analysis of the complete genome of strain JS3065 indicated that the gene cluster encoding 1-nitronaphthalene catabolism (nin) is located on a plasmid. Based on the genetic and biochemical evidence, the nin genes share an origin with the nag-like genes encoding naphthalene degradation in Ralstonia sp. strain U2. The initial step in degradation of 1-nitronaphthalene is catalyzed by a three-component dioxygenase, NinAaAbAcAd, resulting in formation of 1,2-dihydroxynaphthalene which is also an early intermediate in the naphthalene degradation pathway. Introduction of the ninAaAbAcAd genes into strain U2 enabled its growth on 1-nitronaphthalene. Phylogenic analysis of NinAc suggested that an ancestral 1-nitronaphthalene dioxygenase was an early step in the evolution of nitroarene dioxygenases. Based on bioinformatic analysis and enzyme assays, the subsequent assimilation of 1,2-dihydroxynaphthalene seems to follow the well-established pathway for naphthalene degradation by Ralstonia sp. strain U2. This is the first report of catabolic pathway for 1-nitronaphthalene and is another example of how expanding the substrate range of Rieske type dioxygenase enables bacteria to grow on recalcitrant nitroaromatic compounds. IMPORTANCE Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have been widely detected in the environment and they are more toxic than their corresponding parent PAHs. Although biodegradation of many PAHs has been extensively described at genetic and biochemical levels, little is known about the microbial degradation of nitro-PAHs. This work reports the isolation of a Sphingobium strain growing on 1-nitronaphthalene and the genetic basis for the catabolic pathway. The pathway evolved from an ancestral naphthalene catabolic pathway by a remarkably small modification in the specificity of the initial dioxygenase. Data presented here not only shed light on the biochemical processes involved in the microbial degradation of globally important nitrated polycyclic aromatic hydrocarbons, but also provide an evolutionary paradigm for how bacteria evolve a novel catabolic pathway with minimal alteration of preexisting pathways for natural organic compounds.
Subject(s)
Dioxygenases , Polycyclic Aromatic Hydrocarbons , Sphingomonadaceae , Naphthalenes/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Dioxygenases/genetics , Dioxygenases/metabolism , Biodegradation, Environmental , Sphingomonadaceae/genetics , Sphingomonadaceae/metabolismABSTRACT
Insensitive munitions compounds (IMCs), such as 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO), are replacing conventional explosives in munitions formulations. Manufacture and use of IMCs generate waste streams in manufacturing plants and load/assemble/pack facilities. There is a lack of practical experience in executing biodegradation strategies to treat IMCs waste streams. This study establishes a proof-of-concept that bacterial consortia can be designed to mineralize IMCs and co-occurring nitroaromatics in waste streams. First, DNAN, 4-nitroanisole (4-NA), and 4-chloronitrobenzene (4-CNB) in a synthetic DNAN-manufacturing waste stream were biodegraded using an aerobic fluidized-bed reactor (FBR) inoculated with Nocardioides sp. JS 1661 (DNAN degrader), Rhodococcus sp. JS 3073 (4-NA degrader), and Comamonadaceae sp. LW1 (4-CNB degrader). No biodegradation was detected when the FBR was operated under anoxic conditions. Second, DNAN and NTO were biodegraded in a synthetic load/assemble/pack waste stream during a sequential treatment comprising: (i) aerobic DNAN biodegradation in the FBR; (ii) anaerobic NTO biotransformation to 3-amino-1,2,4-triazol-5-one (ATO) by an NTO-respiring enrichment; and (iii) aerobic ATO mineralization by an ATO-oxidizing enrichment. Complete biodegradation relied on switching redox conditions. The results provide the basis for designing consortia to treat mixtures of IMCs and related waste products by incorporating microbes with the required catabolic capabilities.
Subject(s)
Explosive Agents , Nitro Compounds , Anisoles/metabolism , Bacteria/metabolism , Biodegradation, Environmental , Biotransformation , Explosive Agents/metabolism , Nitro Compounds/metabolism , Triazoles/metabolismABSTRACT
Insensitive munitions compounds (IMCs) are emerging nitroaromatic contaminants developed by the military as safer-to-handle alternatives to conventional explosives. Biotransformation of nitroaromatics via microbial respiration has only been reported for a limited number of substrates. Important soil microorganisms can respire natural organic matter (NOM) by reducing its quinone moieties to hydroquinones. Thus, we investigated the NOM respiration combined with the abiotic reduction of nitroaromatics by the hydroquinones formed. First, we established nitroaromatic concentration ranges that were nontoxic to the quinone respiration. Then, an enrichment culture dominated by Geobacter anodireducens could indirectly reduce a broad array of nitroaromatics by first respiring NOM components or the NOM surrogate anthraquinone-2,6-disulfonate (AQDS). Without quinones, no nitroaromatic tested was reduced except for the IMC 3-nitro-1,2,4-triazol-5-one (NTO). Thus, the quinone respiration expanded the spectrum of nitroaromatics susceptible to transformation. The system functioned with very low quinone concentrations because NOM was recycled by the nitroaromatic reduction. A metatranscriptomic analysis demonstrated that the microorganisms obtained energy from quinone or NTO reduction since respiratory genes were upregulated when AQDS or NTO was the electron acceptor. The results indicated microbial NOM respiration sustained by the nitroaromatic-dependent cycling of quinones. This process can be applied as a nitroaromatic remediation strategy, provided that a quinone pool is available for microorganisms.
Subject(s)
Hydroquinones , Soil Microbiology , Benzoquinones , Oxidation-Reduction , Quinones , RespirationABSTRACT
Halonitrobenzenes are toxic chemical intermediates used widely for industrial synthesis of dyes and pesticides. Bacteria able to degrade 2- and 4-chloronitrobenzene have been isolated and characterized; in contrast, no natural isolate has been reported to degrade meta-halonitrobenzenes. In this study, Diaphorobacter sp. strain JS3051, previously reported to degrade 2,3-dichloronitrobenzene, grew readily on 3-chloronitrobenzene and 3-bromonitrobenzene, but not on 3-fluoronitrobenzene, as sole sources of carbon, nitrogen, and energy. A Rieske nonheme iron dioxygenase (DcbAaAbAcAd) catalyzed the dihydroxylation of 3-chloronitrobenzene and 3-bromonitrobenzene, resulting in the regiospecific production of ring-cleavage intermediates 4-chlorocatechol and 4-bromocatechol. The lower activity and relaxed regiospecificity of DcbAaAbAcAd toward 3-fluoronitrobenzene is likely due to the higher electronegativity of the fluorine atom, which hinders it from interacting with E204 residue at the active site. DccA, a chlorocatechol 1,2-dioxygenase, converts 4-chlorocatechol and 4-bromocatechol into the corresponding halomuconic acids with high catalytic efficiency, but with much lower Kcat/Km values for fluorocatechol analogues. The results indicate that the Dcb and Dcc enzymes of Diaphorobacter sp. strain JS3051 can catalyze the degradation of 3-chloro- and 3-bromonitrobenzene in addition to 2,3-dichloronitrobenzene. The ability to utilize multiple substrates would provide a strong selective advantage in a habitat contaminated with mixtures of chloronitrobenzenes. IMPORTANCE Halonitroaromatic compounds are persistent environmental contaminants, and some of them have been demonstrated to be degraded by bacteria. Natural isolates that degrade 3-chloronitrobenzene and 3-bromonitrobenzene have not been reported. In this study, we report that Diaphorobacter sp. strain JS3051 can degrade 2,3-dichloronitrobenzene, 3-chloronitrobenzene, and 3-bromonitrobenzene using the same catabolic pathway, whereas it is unable to grow on 3-fluoronitrobenzene. Based on biochemical analyses, it can be concluded that the initial dioxygenase and lower pathway enzymes are inefficient for 3-fluoronitrobenzene and even misroute the intermediates, which is likely responsible for the failure to grow. These results advance our understanding of how the broad substrate specificities of catabolic enzymes allow bacteria to adapt to habitats with mixtures of xenobiotic contaminants.
Subject(s)
Comamonadaceae , Dioxygenases , Biodegradation, Environmental , Comamonadaceae/metabolism , Dioxygenases/genetics , Dioxygenases/metabolism , NitrobenzenesABSTRACT
Alkanes are ubiquitous in marine ecosystems and originate from diverse sources ranging from natural oil seeps to anthropogenic inputs and biogenic production by cyanobacteria. Enzymes that degrade cyanobacterial alkanes (typically C15-C17 compounds) such as the alkane monooxygenase (AlkB) are widespread, but it remains unclear whether or not AlkB variants exist that specialize in degradation of crude oil from natural or accidental spills, a much more complex mixture of long-chain hydrocarbons. In the present study, large-scale analysis of available metagenomic and genomic data from the Gulf of Mexico (GoM) oil spill revealed a novel, divergent AlkB clade recovered from genomes with no cultured representatives that was dramatically increased in abundance in crude-oil impacted ecosystems. In contrast, the AlkB clades associated with biotransformation of cyanobacterial alkanes belonged to 'canonical' or hydrocarbonoclastic clades, and based on metatranscriptomics data and compared to the novel clade, were much more weakly expressed during crude oil biodegradation in laboratory mesocosms. The absence of this divergent AlkB clade in metagenomes of uncontaminated samples from the global ocean survey but not from the GoM as well as its frequent horizontal gene transfer indicated a priming effect of the Gulf for crude oil biodegradation likely driven by natural oil seeps.
Subject(s)
Biodegradation, Environmental , Cyanobacteria , Cytochrome P-450 CYP4A , Petroleum , Alkanes/metabolism , Cyanobacteria/enzymology , Cytochrome P-450 CYP4A/genetics , Cytochrome P-450 CYP4A/metabolism , Ecosystem , Petroleum/metabolism , PhylogenyABSTRACT
Diaphorobacter sp. strain JS3051 utilizes 2,3-dichloronitrobenzene (23DCNB), a toxic anthropogenic compound, as the sole carbon, nitrogen, and energy source for growth, but the metabolic pathway and its origins are unknown. Here, we establish that a gene cluster (dcb), encoding a Nag-like dioxygenase, is responsible for the initial oxidation of the 23DCNB molecule. The 2,3-dichloronitrobenzene dioxygenase system (DcbAaAbAcAd) catalyzes conversion of 23DCNB to 3,4-dichlorocatechol (34DCC). Site-directed mutagenesis studies indicated that residue 204 of DcbAc is crucial for the substrate specificity of 23DCNB dioxygenase. The presence of glutamic acid at position 204 of 23DCNB dioxygenase is unique among Nag-like dioxygenases. Genetic, biochemical, and structural evidence indicate that the 23DCNB dioxygenase is more closely related to 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 than to the 34DCNB dioxygenase from Diaphorobacter sp. strain JS3050, which was isolated from the same site as strain JS3051. A gene cluster (dcc) encoding the enzymes for 34DCC catabolism, homologous to a clc operon in Pseudomonas knackmussii strain B13, is also on the chromosome at a distance of 2.5 Mb from the dcb genes. Heterologously expressed DccA catalyzed ring cleavage of 34DCC with high affinity and catalytic efficiency. This work not only establishes the molecular mechanism for 23DCNB mineralization, but also enhances the understanding of the recent evolution of the catabolic pathways for nitroarenes. IMPORTANCE Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. But their phylogeny and how they evolved in response to novel nitroaromatic compounds are largely unknown. Elucidation of the molecular basis for 23DCNB degradation revealed that the catabolic pathways of two DCNB isomers in different isolates from the same site were derived from different recent origins. Integrating structural models of catalytic subunits and enzymatic activities data provided new insight about how recently modified enzymes were selected depending on the structure of new substrates. This study enhances understanding and prediction of adaptive evolution of catabolic pathways in bacteria in response to new chemicals.
Subject(s)
Comamonadaceae/genetics , Comamonadaceae/metabolism , Metabolic Networks and Pathways/genetics , Multigene Family , Nitrobenzenes/metabolism , Comamonadaceae/enzymology , Genome, Bacterial , Nitrobenzenes/chemistry , Substrate SpecificityABSTRACT
4-Nitrophenol, a priority pollutant, is degraded by Gram-positive and Gram-negative bacteria via 1,2,4-benzenetriol (BT) and hydroquinone (HQ), respectively. All enzymes involved in the two pathways have been functionally identified. So far, all Gram-negative 4-nitrophenol utilizers are from the genera Pseudomonas and Burkholderia. But it remains a mystery why pnpG, an apparently superfluous BT 1,2-dioxygenase-encoding gene, always coexists in the catabolic cluster (pnpABCDEF) encoding 4-nitrophenol degradation via HQ. Here, the physiological role of pnpG in Burkholderia sp. strain SJ98 was investigated. Deletion and complementation experiments established that pnpG is essential for strain SJ98 growing on 4-nitrocatechol rather than 4-nitrophenol. During 4-nitrophenol degradation by strain SJ98 and its two variants (pnpG deletion and complementation strains), 1,4-benzoquinone and HQ were detected, but neither 4-nitrocatechol nor BT was observed. When the above-mentioned three strains (the wild type and complementation strains with 2,2'-dipyridyl) were incubated with 4-nitrocatechol, BT was the only intermediate detected. The results established the physiological role of pnpG that encodes BT degradation in vivo. Biotransformation analyses showed that the pnpA-deleted strain was unable to degrade both 4-nitrophenol and 4-nitrocatechol. Thus, the previously characterized 4-nitrophenol monooxygenase PnpASJ98 is also essential for the conversion of 4-nitrocatechol to BT. Among 775 available complete genomes for Pseudomonas and Burkholderia, as many as 89 genomes were found to contain the putative pnpBCDEFG genes. The paucity of pnpA (3 in 775 genomes) implies that the extension of BT and HQ pathways enabling the degradation of 4-nitrophenol and 4-nitrocatechol is rarer, more recent, and likely due to the release of xenobiotic nitroaromatic compounds. IMPORTANCE An apparently superfluous gene (pnpG) encoding BT 1,2-dioxygenase is always found in the catabolic clusters involved in 4-nitrophenol degradation via HQ by Gram-negative bacteria. Our experiments reveal that pnpG is not essential for 4-nitrophenol degradation in Burkholderia sp. strain SJ98 but instead enables its degradation of 4-nitrocatechol via BT. The presence of pnpG genes broadens the range of growth substrates to include 4-nitrocatechol or BT, intermediates from the microbial degradation of many aromatic compounds in natural ecosystems. In addition, the existence of pnpCDEFG in 11.6% of the above-mentioned two genera suggests that the ability to degrade BT and HQ simultaneously is ancient. The extension of BT and HQ pathways including 4-nitrophenol degradation seems to be an adaptive evolution for responding to synthetic nitroaromatic compounds entering the environment since the industrial revolution.
Subject(s)
Bacterial Proteins/metabolism , Burkholderia/enzymology , Catechols/metabolism , Dioxygenases/metabolism , Hydroquinones/metabolism , Nitrophenols/metabolism , Bacterial Proteins/genetics , Biotransformation , Burkholderia/genetics , Dioxygenases/genetics , Pseudomonas/enzymology , Pseudomonas/geneticsABSTRACT
The nitroheterocyclic 3-nitro-1,2,4-triazol-5-one (NTO) is an ingredient of insensitive explosives increasingly used by the military, becoming an emergent environmental pollutant. Cometabolic biotransformation of NTO occurs in mixed microbial cultures in soils and sludges with excess electron-donating substrates. Herein, we present the unusual energy-yielding metabolic process of NTO respiration, in which the NTO reduction to 3-amino-1,2,4-triazol-5-one (ATO) is linked to the anoxic acetate oxidation to CO2 by a culture enriched from municipal anaerobic digester sludge. Cell growth was observed simultaneously with NTO reduction, whereas the culture was unable to grow in the presence of acetate only. Extremely low concentrations (0.06 mg L-1) of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited NTO reduction, indicating that the process was linked to respiration. The ultimate evidence of NTO respiration was adenosine triphosphate production due to simultaneous exposure to NTO and acetate. Metagenome sequencing revealed that the main microorganisms (and relative abundances) were Geobacter anodireducens (89.3%) and Thauera sp. (5.5%). This study is the first description of a nitroheterocyclic compound being reduced by anaerobic respiration, shedding light on creative microbial processes that enable bacteria to make a living reducing NTO.
Subject(s)
Bacteria , Nitro Compounds , Bacteria/genetics , Geobacter , Respiration , TriazolesABSTRACT
The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to ß-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment.
Subject(s)
Bacterial Proteins/metabolism , Catechols/metabolism , Comamonadaceae/metabolism , Dioxygenases/metabolism , Nitrobenzenes/metabolism , Bacterial Proteins/genetics , Biodegradation, Environmental , Comamonadaceae/enzymology , Comamonadaceae/genetics , Dioxygenases/genetics , Environmental Pollutants/metabolism , Genome, Bacterial/genetics , Metabolic Networks and Pathways/genetics , Plasmids/genetics , Plasmids/metabolismABSTRACT
Crude oil buried in intertidal sands may be exposed to alternating oxic and anoxic conditions but the effect of this tidally induced biogeochemical oscillation remains poorly understood, limiting the effectiveness of remediation and managing efforts after oil spills. Here, we used a combination of metatranscriptomics and genome-resolved metagenomics to study microbial activities in oil-contaminated sediments during oxic-anoxic cycles in laboratory chambers that closely emulated in situ conditions. Approximately 5-fold higher reductions in the total petroleum hydrocarbons were observed in the oxic as compared to the anoxic phases with a relatively constant ratio between aerobic and anaerobic oil decomposition rates even after prolonged anoxic conditions. Metatranscriptomics analysis indicated that the oxic phases promoted oil biodegradation in subsequent anoxic phases by microbially mediated reoxidation of alternative electron acceptors like sulfide and by providing degradation-limiting nitrogen through biological nitrogen fixation. Most population genomes reconstructed from the mesocosm samples represented uncultured taxa and were present typically as members of the rare biosphere in metagenomic data from uncontaminated field samples, implying that the intertidal communities are adapted to changes in redox conditions. Collectively, these results have important implications for enhancing oil spill remediation efforts in beach sands and coastal sediments and underscore the role of uncultured taxa in such efforts.
Subject(s)
Petroleum Pollution , Petroleum , Biodegradation, Environmental , Geologic Sediments , Hydrocarbons , Petroleum Pollution/analysisABSTRACT
To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+ â NH2OH â NO â NO2- â NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.
Subject(s)
Ammonium Compounds/pharmacology , Archaeal Proteins/analysis , Bacterial Proteins/analysis , DNA, Archaeal/analysis , DNA, Bacterial/analysis , Fertilizers , Microbiota/drug effects , Nitrification , RNA, Archaeal/analysis , RNA, Bacterial/analysis , Soil Microbiology , Urea/pharmacology , Archaea/drug effects , Archaea/genetics , Archaea/isolation & purification , Bacteria/drug effects , Bacteria/genetics , Bacteria/isolation & purification , Gene Expression Regulation, Archaeal/drug effects , Gene Expression Regulation, Bacterial/drug effects , Gene Ontology , Metagenomics , Nitrates/analysis , Nitrification/genetics , Nitrogen Isotopes/analysis , Oxidation-Reduction , Phylogeny , Proteomics , RNA, Ribosomal, 16S/analysis , Soil/chemistryABSTRACT
3-Nitro-1,2,4-triazol-5-one (NTO) is one of the main ingredients of many insensitive munitions, which are being used as replacements for conventional explosives. As its use becomes widespread, more research is needed to assess its environmental fate. Previous studies have shown that NTO is biologically reduced to 3-amino-1,2,4-triazol-5-one (ATO). However, the final degradation products of ATO are still unknown. We have studied the aerobic degradation of ATO by enrichment cultures derived from the soil. After multiple transfers, ATO degradation was monitored in closed bottles through measurements of inorganic carbon and nitrogen species. The results indicate that the members of the enrichment culture utilize ATO as the sole source of carbon and nitrogen. As ATO was mineralized to CO2, N2, and NH4+, microbial growth was observed in the culture. Co-substrates addition did not increase the ATO degradation rate. Quantitative polymerase chain reaction analysis revealed that the organisms that enriched using ATO as carbon and nitrogen source were Terrimonas spp., Ramlibacter-related spp., Mesorhizobium spp., Hydrogenophaga spp., Ralstonia spp., Pseudomonas spp., Ectothiorhodospiraceae, and Sphingopyxis. This is the first study to report the complete mineralization of ATO by soil microorganisms, expanding our understanding of natural attenuation and bioremediation of the explosive NTO.
Subject(s)
Explosive Agents , Nitro Compounds , Biodegradation, Environmental , Nuclear Family , Oxidative Stress , TriazolesABSTRACT
Dichloronitrobenzenes (DCNB) are intermediates in the production of dichloroanilines, which are key feedstocks for synthesis of diuron and other herbicides. Although DCNB is a major contaminant at certain chemical manufacturing sites, aerobic DCNB biodegradation is poorly understood and such sites have not been candidates for bioremediation. When a bench-scale aerobic fluidized- bed bioreactor was inoculated with samples from a DCNB contaminated site in Brazil 2,3-DCNB, 3,4-DCNB, 1,2-dichlorobenzene (o-DCB), and chlorobenzene (CB) were biodegraded simultaneously. Biodegradation of the mixture was complete even when the reactor was operated at high flow rates (1.6â¯h hydraulic residence time), and bacteria able to degrade the individual contaminants were isolated from the reactor by selective enrichment. The enrichments yielded 2 strains of bacteria able to degrade 3,4-DCNB and one able to degrade 2,3-DCNB. The isolates released nitrite during growth on the respective DCNB isomers under aerobic conditions. The draft genome sequence of Diaphorobacter sp. JS3050, which grew on 3,4-DCNB, revealed the presence of putative nitroarene dioxygenase genes, which is consistent with initial attack by a dioxygenase analogous to the initial steps in degradation of nitrobenzene and dinitrotoluenes. The results indicate clearly that the DCNB isomers are biodegradable under aerobic conditions and thus are candidates for natural attenuation/bioremediation.
Subject(s)
Aerobiosis , Biodegradation, Environmental , Nitrobenzenes/chemistry , Water Pollutants, Chemical/chemistry , Bioreactors , Brazil , Catalysis , Chlorobenzenes/chemistry , Comamonadaceae/metabolism , DNA, Bacterial/genetics , Genome, Bacterial , Groundwater , Nitrites/chemistry , Sewage , Water Purification/methodsABSTRACT
Modeling crude-oil biodegradation in sediments remains a challenge due in part to the lack of appropriate model organisms. Here we report the metagenome-guided isolation of a novel organism that represents a phylogenetically narrow (>97% 16S rRNA gene identity) group of previously uncharacterized, crude-oil degraders. Analysis of available sequence data showed that these organisms are highly abundant in oiled sediments of coastal marine ecosystems across the world, often comprising ~30% of the total community, and virtually absent in pristine sediments or seawater. The isolate genome encodes functional nitrogen fixation and hydrocarbon degradation genes together with putative genes for biosurfactant production that apparently facilitate growth in the typically nitrogen-limited, oiled environment. Comparisons to available genomes revealed that this isolate represents a novel genus within the Gammaproteobacteria, for which we propose the provisional name "Candidatus Macondimonas diazotrophica" gen. nov., sp. nov. "Ca. M. diazotrophica" appears to play a key ecological role in the response to oil spills around the globe and could be a promising model organism for studying ecophysiological responses to oil spills.
Subject(s)
Gammaproteobacteria/genetics , Geologic Sediments/microbiology , Hydrocarbons/metabolism , Metagenome , Petroleum/metabolism , Biodegradation, Environmental , DNA, Bacterial/genetics , Ecosystem , Gammaproteobacteria/isolation & purification , Gammaproteobacteria/physiology , Geologic Sediments/chemistry , Nitrogen Fixation , Petroleum Pollution , Phylogeny , RNA, Ribosomal, 16S/genetics , SeawaterABSTRACT
Many plants produce allelopathic chemicals, such as stilbenes, to inhibit pathogenic fungi. The degradation of allelopathic compounds by bacteria associated with the plants would limit their effectiveness, but little is known about the extent of biodegradation or the bacteria involved. Screening of tissues and rhizosphere of peanut (Arachis hypogaea) plants revealed substantial enrichment of bacteria able to grow on resveratrol and pterostilbene, the most common stilbenes produced by the plants. Investigation of the catabolic pathway in Sphingobium sp. strain JS1018, isolated from the rhizosphere, indicated that the initial cleavage of pterostilbene was catalyzed by a carotenoid cleavage oxygenase (CCO), which led to the transient accumulation of 4-hydroxybenzaldehyde and 3,5-dimethoxybenzaldehyde. 4-Hydroxybenzaldehyde was subsequently used for the growth of the isolate, while 3,5-dimethoxybenzaldehyde was further converted to a dead-end metabolite with a molecular weight of 414 (C24H31O6). The gene that encodes the initial oxygenase was identified in the genome of strain JS1018, and its function was confirmed by heterologous expression in Escherichia coli This study reveals the biodegradation pathway of pterostilbene by plant-associated bacteria. The prevalence of such bacteria in the rhizosphere and plant tissues suggests a potential role of bacterial interference in plant allelopathy.IMPORTANCE Pterostilbene, an analog of resveratrol, is a stilbene allelochemical produced by plants to inhibit microbial infection. As a potent antioxidant, pterostilbene acts more effectively than resveratrol as an antifungal agent. Bacterial degradation of this plant natural product would affect the allelopathic efficacy and fate of pterostilbene and thus its ecological role. This study explores the isolation and abundance of bacteria that degrade resveratrol and pterostilbene in peanut tissues and rhizosphere, the catabolic pathway for pterostilbene, and the molecular basis for the initial cleavage of pterostilbene. If plant allelopathy is an important process in agriculture and management of invasive plants, the ecological role of bacteria that degrade the allelopathic chemicals must be equally important.
Subject(s)
Allelopathy , Arachis/microbiology , Sphingomonadaceae/metabolism , Stilbenes/metabolism , Antibiosis , Antioxidants , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biodegradation, Environmental , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Rhizosphere , Soil MicrobiologyABSTRACT
The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.