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Objective: The aim of the study was to compare the effectiveness of a telerehabilitation exercise program versus "wait-and-see" on physical exertion, quality of life, dyspnea severity, heart rate, and oxygen saturation in patients with post-COVID fatigue and dyspnea., Design: Sixty-four patients were enrolled in this randomized clinical trial. A telerehabilitation program based on patient education, physical activity, airway clearing, and breathing exercise interventions was conducted. Self-perceived physical exertion during daily living activities, dyspnea severity, health-related quality of life and physiological outcomes, and the 6-min walking test were assessed at baseline, after the program and at 1- and 3-mo follow-up periods., Results: The experimental group experienced greater improvements in self-perceived physical exertion during daily living activities, dyspnea severity, health-related quality of life, and 6-min walking test (all, P < 0.001). In addition, patients undergoing the telerehabilitation program reported lower exertion scores at rest and after the 6-min walking test (both, P < 0.001). Between-group oxygen saturation differences were found at rest ( P < 0.001), but not after the 6-min walking test ( P = 0.024). Finally, significant between-group differences were found for heart rate after the 6-min walking test ( P < 0.001)., Conclusions: Although both groups showed a significant improvement after 3 mos of follow-up, the group receiving the telerehabilitation program described a greater improvement compared with the group receiving no intervention., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: SZ, SD, TL, MM, DL, JS, NZ No competing interests declared, (© 2024, Zhang, Deng et al.)

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.

Objective: The aim of the study is to compare the surface electromyographic amplitude, activation ratio, and onset latency of the main scapular stabilizing muscles between five typical rehabilitative exercises., Design: Twenty-seven healthy participants performed five scapular exercises (wall slide, wall push-up plus, prone horizontal abduction with external rotation, external rotation in side lying, and low row) while simultaneously recording surface electromyographic of serratus anterior, middle trapezius, lower trapezius, and upper trapezius. Surface electromyographic amplitudes, onset latencies, and activation ratios were calculated., Results: Prone horizontal abduction with external rotation showed an excellent upper trapezius/middle trapezius (0.43) and upper trapezius/lower trapezius (0.30) muscle balance with high (>50% maximum voluntary isometric contraction) middle trapezius and lower trapezius amplitudes, a low (<20% maximum voluntary isometric contraction) upper trapezius amplitude, and an early activation of the scapular stabilizing muscles (-474.7 to 89.9 ms) relative to upper trapezius. External rotation in side lying showed excellent upper trapezius/serratus anterior (0.26), upper trapezius/middle trapezius (0.32), and upper trapezius/lower trapezius (0.21) activation ratios and, along with low row and wall slide, showed early activation of the scapular stabilizing muscles (-378.1 to -26.6 ms)., Conclusions: Prone horizontal abduction with external rotation presented optimal scapular neuromuscular control. Although external rotation in side lying, low row, and wall slide did not meet all the criteria associated with optimal scapular neuromuscular control, these exercises could be used in early stages of shoulder rehabilitation because they favor early activation of the scapular stabilizing muscles., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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., (© 2022 Wiley Periodicals LLC.)

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.

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 K cat / K m 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.

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

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 PnpA SJ98 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.

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 CO 2 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.

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 β-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., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

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-NO 3 - g -1 dry soil d -1 ) and accumulation of N 2 O after 192 hours of incubation. Nitrification activity (NH 4 +  → NH 2 OH → NO → NO 2 -  → NO 3 - ) 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.

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 CO 2 , N 2 , and NH 4 + , 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.

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 (1.6 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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 (C 24 H 31 O 6 ). 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., (Copyright © 2019 American Society for Microbiology.)

Background: Postoperative pneumonia is associated with increased morbidity, mortality and costs. Prediction models of pneumonia that are currently available are based on retrospectively collected data and administrative coding systems., Objective: To identify independent variables associated with the occurrence of postoperative pneumonia., Design: A prospective observational study of a multicentre cohort (Prospective Evaluation of a RIsk Score for postoperative pulmonary COmPlications in Europe database)., Setting: Sixty-three hospitals in Europe., Patients: Patients undergoing surgery under general and/or regional anaesthesia during a 7-day recruitment period., Main Outcome Measure: The primary outcome was postoperative pneumonia., Definition: the need for treatment with antibiotics for a respiratory infection and at least one of the following criteria: new or changed sputum; new or changed lung opacities on a clinically indicated chest radiograph; temperature more than 38.3 °C; leucocyte count more than 12 000 μl., Results: Postoperative pneumonia occurred in 120 out of 5094 patients (2.4%). Eighty-two of the 120 (68.3%) patients with pneumonia required ICU admission, compared with 399 of the 4974 (8.0%) without pneumonia (P < 0.001). We identified five variables independently associated with postoperative pneumonia: functional status [odds ratio (OR) 2.28, 95% confidence interval (CI) 1.58 to 3.12], pre-operative SpO2 values while breathing room air (OR 0.83, 95% CI 0.78 to 0.84), intra-operative colloid administration (OR 2.97, 95% CI 1.94 to 3.99), intra-operative blood transfusion (OR 2.19, 95% CI 1.41 to 4.71) and surgical site (open upper abdominal surgery OR 3.98, 95% CI 2.19 to 7.59). The model had good discrimination (c-statistic 0.89) and calibration (Hosmer-Lemeshow P = 0.572)., Conclusion: We identified five variables independently associated with postoperative pneumonia. The model performed well and after external validation may be used for risk stratification and management of patients at risk of postoperative pneumonia., Trial Registration: NCT 01346709 (ClinicalTrials.gov).

Surface-enhanced Raman scattering (SERS) spectroscopy of pyridazine shows the selective enhancement of the bands recorded at about 1570, 1450 and 380 cm-1, which are assigned to two different types of vibrations. The first two correspond to in-plane 8a;νring and 19b;δ(CH) totally symmetric A1 modes, respectively, while the last band is assigned to the out-of-plane 16b;τring,B1 vibration. The selective enhancement has been analyzed on the basis of a resonant Raman process involving photoexcited metal (M)-to-molecule (A) charge transfer (CT: M-A + hν → M+-A-) states of the metal-adsorbate surface complex, which have also been related to the doublet electronic states of the corresponding radical anion of the adsorbate (A-). According to the selection rules of the electromagnetic/plasmonic SERS enhancement mechanism, the simultaneous enhancement of in-plane and out-of-plane modes could be attributed to different orientations of the adsorbate (perpendicular and parallel, respectively, or tilted) with respect to the metallic surface. The calculated resonance Raman-CT spectra (SERS-CT) and the vibrational wavenumbers of isolated pyridazine and of the pyridazine-Ag2 complex obtained from electronic structure calculations suggest a single type of molecule adsorbed with perpendicular orientation. The relative SERS enhancements of both in-plane and out-of-plane modes are due to Franck-Condon factors related to differences between the equilibrium geometries (A1 vibrations, ΔQ ≠ 0) and gradients (B1 vibrations, Δν ≠ 0 and ΔQ ≠ 0), respectively, of the potential energy surfaces of the involved ground and photoinduced CT electronic states. Therefore, the selective enhancement of the SERS bands of pyridazine is controlled by a general metal-to-molecule resonant CT mechanism. This conclusion supports the usefulness of SERS in studying the subtle electronic structure of charged interfaces as well as key processes such as electron transfer at the nanoscale.

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., (© 2018 John Wiley & Sons Ltd.)

Background: Currently used pre-operative prediction scores for postoperative pulmonary complications (PPCs) use patient data and expected surgery characteristics exclusively. However, intra-operative events are also associated with the development of PPCs., Objective: We aimed to develop a new prediction score for PPCs that uses both pre-operative and intra-operative data., Design: This is a secondary analysis of the LAS VEGAS study, a large international, multicentre, prospective study., Settings: A total of 146 hospitals across 29 countries., Patients: Adult patients requiring intra-operative ventilation during general anaesthesia for surgery., Interventions: The cohort was randomly divided into a development subsample to construct a predictive model, and a subsample for validation., Main Outcome Measures: Prediction performance of developed models for PPCs., Results: Of the 6063 patients analysed, 10.9% developed at least one PPC. Regression modelling identified 13 independent risk factors for PPCs: six patient characteristics [higher age, higher American Society of Anesthesiology (ASA) physical score, pre-operative anaemia, pre-operative lower SpO2 and a history of active cancer or obstructive sleep apnoea], two procedure-related features (urgent or emergency surgery and surgery lasting ≥ 1 h), and five intra-operative events [use of an airway other than a supraglottic device, the use of intravenous anaesthetic agents along with volatile agents (balanced anaesthesia), intra-operative desaturation, higher levels of positive end-expiratory pressures > 3 cmH2O and use of vasopressors]. The area under the receiver operating characteristic curve of the LAS VEGAS risk score for prediction of PPCs was 0.78 [95% confidence interval (95% CI), 0.76 to 0.80] for the development subsample and 0.72 (95% CI, 0.69 to 0.76) for the validation subsample., Conclusion: The LAS VEGAS risk score including 13 peri-operative characteristics has a moderate discriminative ability for prediction of PPCs. External validation is needed before use in clinical practice., Trial Registration: The study was registered at Clinicaltrials.gov, number NCT01601223.

Resveratrol is among the best-known secondary plant metabolites because of its antioxidant, anti-inflammatory, and anticancer properties. It also is an important allelopathic chemical widely credited with the protection of plants from pathogens. The ecological role of resveratrol in natural habitats is difficult to establish rigorously, because it does not seem to accumulate outside plant tissue. It is likely that bacterial degradation plays a key role in determining the persistence, and thus the ecological role, of resveratrol in soil. Here, we report the isolation of an Acinetobacter species that can use resveratrol as a sole carbon source from the rhizosphere of peanut plants. Both molecular and biochemical techniques indicate that the pathway starts with the conversion of resveratrol to 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde. The aldehydes are oxidized to substituted benzoates that subsequently enter central metabolism. The gene that encodes the enzyme responsible for the oxidative cleavage of resveratrol was cloned and expressed in Escherichia coli to establish its function. Its physiological role in the resveratrol catabolic pathway was established by knockouts and by the reverse transcription-quantitative PCR (RT-qPCR) demonstration of expression during growth on resveratrol. The results establish the presence and capabilities of resveratrol-degrading bacteria in the rhizosphere of the peanut plants and set the stage for studies to evaluate the role of the bacteria in plant allelopathy. IMPORTANCE In addition to its antioxidant properties, resveratrol is representative of a broad array of allelopathic chemicals produced by plants to inhibit competitors, herbivores, and pathogens. The bacterial degradation of such chemicals in the rhizosphere would reduce the effects of the chemicals. Therefore, it is important to understand the activity and ecological role of bacteria that biodegrade resveratrol near the plants that produce it. This study describes the isolation from the peanut rhizosphere of bacteria that can grow on resveratrol. The characterization of the initial steps in the biodegradation process sets the stage for the investigation of the evolution of the catabolic pathways responsible for the biodegradation of resveratrol and its homologs., (Copyright © 2018 American Society for Microbiology.)

The photochemical decomposition of 4-methoxyphenyl azide (CH 3 O-Ph-N 3 ) is investigated using multiconfigurational second-order perturbation theory (MS-CASPT2). In addition, the multi-state resonance Raman spectra of the reactant, intermediates, and product are computed with a multi-state version of the vibronic theory of Albrecht. The results support that the key step of the photolysis of the parent azide is a 2 1 A'/2 3 A'' intersystem crossing which in a second step decays through a 2 3 A''/1 3 A'' conical intersection to give directly the formation of triplet 4-methoxyphenyl nitrene (CH 3 O-Ph-N) in its lowest electronic state, 1 3 A''. It is found that the efficiency of the cited intersystem crossing is enhanced by the close presence of a 2 1 A'/2 1 A'' conical intersection. On the other hand, the calculated spectra suggest that the only two species which would be observed in the gas phase experiments are the triplet nitrene plus 4,4'-dimethoxyazobenzene.

Compound specific isotope analysis (CSIA) is widely used to monitor contaminant remediation in groundwater. CSIA-based approaches that use enrichment (ε) values to assess degradative processes rely on the assumption that the contaminant being investigated will have an ε value that is constant and specific to a catalytic pathway of a microorganism. Distinct ε values have been reported for aerobic degradation of cis-dichloroethene (cDCE), which has led to a number of proposed degradation mechanisms; however, cytochrome P450 catalyzed oxidation is the only biochemical mechanism that has been established in Polaromonas sp. JS666. Using CSIA we measured the ε values for microbial oxidation of cDCE (-18.8‰±1.5‰) and 1,2-dichloroethane (1,2-DCA) (-16.6‰±0.9‰) in wild-type JS666 and the oxidation of cDCE (-13.5‰±2.3‰) from a recombinant E. coli strain expressing the cytochrome P450 enzyme from JS666. This study supports the hypothesis that cytochrome P450 catalyzes the initial step in the degradation pathway of both cDCE and 1,2-DCA and provides evidence that a single enzyme can catalyze multiple pathways with different products and distinct ε values for a single substrate. Therefore, in cases where the products of the reaction cannot, or have not been characterized, caution must be used when employing ε values to interpret mechanisms, pathways, and their applications to environmental contaminant remediation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium, Variovorax sp. strain JS1663, that degrades N -nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene ( nnlA ) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed β-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives. IMPORTANCE The production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N -Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei This study reports the isolation of a Gram-negative soil bacterium, Variovorax sp. strain JS1663, that is able to use NNG as a sole growth substrate. The proposed degradation pathway occurs via a β-elimination reaction that releases nitrite from NNG. The novel NNG lyase requires iron(II) for activity. The identification of a novel enzyme and catabolic pathway provides evidence of a substantial and underappreciated flux of the antibiotic in natural ecosystems. Understanding the NNG biodegradation pathway will help identify other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in synthetic nitramine explosives., (Copyright © 2017 American Society for Microbiology.)

A single liter of water contains hundreds, if not thousands, of bacterial and archaeal species, each of which typically makes up a very small fraction of the total microbial community (<0.1%), the so-called "rare biosphere." How often, and via what mechanisms, e.g., clonal amplification versus horizontal gene transfer, the rare taxa and genes contribute to microbial community response to environmental perturbations represent important unanswered questions toward better understanding the value and modeling of microbial diversity. We tested whether rare species frequently responded to changing environmental conditions by establishing 20-liter planktonic mesocosms with water from Lake Lanier (Georgia, USA) and perturbing them with organic compounds that are rarely detected in the lake, including 2,4-dichlorophenoxyacetic acid (2,4-D), 4-nitrophenol (4-NP), and caffeine. The populations of the degraders of these compounds were initially below the detection limit of quantitative PCR (qPCR) or metagenomic sequencing methods, but they increased substantially in abundance after perturbation. Sequencing of several degraders (isolates) and time-series metagenomic data sets revealed distinct cooccurring alleles of degradation genes, frequently carried on transmissible plasmids, especially for the 2,4-D mesocosms, and distinct species dominating the post-enrichment microbial communities from each replicated mesocosm. This diversity of species and genes also underlies distinct degradation profiles among replicated mesocosms. Collectively, these results supported the hypothesis that the rare biosphere can serve as a genetic reservoir, which can be frequently missed by metagenomics but enables community response to changing environmental conditions caused by organic pollutants, and they provided insights into the size of the pool of rare genes and species. IMPORTANCE A single liter of water or gram of soil contains hundreds of low-abundance bacterial and archaeal species, the so called rare biosphere. The value of this astonishing biodiversity for ecosystem functioning remains poorly understood, primarily due to the fact that microbial community analysis frequently focuses on abundant organisms. Using a combination of culture-dependent and culture-independent (metagenomics) techniques, we showed that rare taxa and genes commonly contribute to the microbial community response to organic pollutants. Our findings should have implications for future studies that aim to study the role of rare species in environmental processes, including environmental bioremediation efforts of oil spills or other contaminants., (Copyright © 2017 American Society for Microbiology.)

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., (© 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Nitroaromatic compounds are typically toxic and resistant to degradation. Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), which is a molecule secreted by Streptomyces scabies, the plant pathogen responsible for potato scab. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid. We characterized 5NAA-A biochemically and obtained snapshots of its mechanism. 5NAA-A, an octamer that can use several divalent transition metals for catalysis in vitro, employs a nucleophilic aromatic substitution mechanism. Unexpectedly, the metal in 5NAA-A is labile but is readily loaded in the presence of substrate. 5NAA-A is specific for 5NAA and cannot hydrolyze other tested derivatives, which are likewise poor inhibitors. The 5NAA-A structure and mechanism expand our understanding of the chemical ecology of an agriculturally important plant and pathogen, and will inform bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroanilines and other challenging substrates.

Accurate and convenient detection of explosive components is vital for a wide spectrum of applications ranging from national security and demilitarization to environmental monitoring and restoration. With the increasing use of DNAN as a replacement for 2,4,6-trinitrotoluene (TNT) in insensitive explosive formulations, there has been a growing interest in strategies to minimize its release and to understand and predict its behavior in the environment. Consequently, a convenient tool for its detection and destruction could enable development of more effective decontamination and demilitarization strategies. Biosensors and biocatalysts have limited applicability to the more traditional explosives because of the inherent limitations of the relevant enzymes. Here, we report a highly specific, convenient and robust biocatalyst based on a novel ether hydrolase enzyme, DNAN demethylase (that requires no cofactors), from a Nocardioides strain that can mineralize DNAN. Biogenic silica encapsulation was used to stabilize the enzyme and enable it to be packed into a model microcolumn for application as a biosensor or as a bioreactor for continuous destruction of DNAN. The immobilized enzyme was stable and not inhibited by other insensitive munitions constituents. An alternative method for DNAN detection involved coating the encapsulated enzyme on cellulose filter paper. The hydrolase based biocatalyst could provide the basis for a wide spectrum of applications including detection, identification, destruction or inertion of explosives containing DNAN (demilitarization operations), and for environmental restorations.

TD-DFT calculations predict a linear dependence of the energies of charge transfer states of Ag n -pyrazine-Ag n molecular junctions on the inverse of the size (1/n) of the linear metal chains. The density of charge (q eff = q/n) in the metal-to-metal charge transfer excited states (CT MM : Ag n q -pyrazine-Ag n -q ) smoothly tunes the electronic structure of the junction, especially the metal-to-molecule charge transfer states (CT 0 and CT 1 ) and the first excited singlet of pyrazine (S 1,Pz ). In enlarged junctions, pyrazine bonds preferably to one of the Ag n clusters and this weak adsorption produces a significant unexpected asymmetry for forward and reverse charge transfer processes.

When anoxic polluted groundwater encounters the overlying vadose zone an oxic/anoxic interface is created, often near the capillary fringe. Biodegradation of volatile contaminants in the capillary fringe can prevent vapor migration. In contrast, the biodegradation of nonvolatile contaminants in the vadose zone has received comparatively little attention. Nonvolatile compounds do not cause vapor intrusion, but they still move with the groundwater and are major contaminants. Aniline (AN) and diphenylamine (DPA) are examples of toxic nonvolatile contaminants found often at dye and munitions manufacturing sites. In this study, we tested the hypothesis that bacteria can aerobically biodegrade AN and DPA in the capillary fringe and decrease the contaminant concentrations in the anoxic plume beneath the vadose zone. Laboratory multiport columns that represented the unsaturated zone were used to evaluate degradation of AN or DPA in contaminated water. The biodegradation fluxes of the contaminants were estimated to be 113 ± 26 mg AN·m(-2)·h(-1) and 76 ± 18 mg DPA·m(-2)·h(-1) in the presence of bacteria known to degrade AN and DPA. Oxygen and contaminant profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place within the lower part of the capillary fringe. The results indicate that bacteria capable of contaminant biodegradation in the capillary fringe can create a sink for nonvolatile contaminants.

Most of the models for simulating vapor intrusion accept the local equilibrium assumption for multiphase concentration distributions, that is, concentrations in solid, liquid and vapor phases are in equilibrium. For simulating vapor transport with aerobic biodegradation controlled by counter-diffusion processes, the local equilibrium assumption combined with dual-Monod kinetics and biomass decay may yield near-instantaneous behavior at steady state. The present research investigates how predicted concentration profiles and fluxes change as interphase mass transfer resistances are increased for vapor intrusion with aerobic biodegradation. Our modeling results indicate that the attenuation coefficients for cases with and without mass transfer limitations can be significantly different by orders of magnitude. Rate-limited mass transfer may lead to larger overlaps of contaminant vapor and oxygen concentrations, which cannot be simulated by instantaneous reaction models with local equilibrium mass transfer. In addition, the contaminant flux with rate-limited mass transfer is much smaller than that with local equilibrium mass transfer, indicating that local equilibrium mass transfer assumption may significantly overestimate the biodegradation rate and capacity for mitigating vapor intrusion through the unsaturated zone. Our results indicate a strong research need for field tests to examine the validity of local equilibrium mass transfer, a widely accepted assumption in modeling vapor intrusion.

2,4-Dinitroanisole (DNAN), a low sensitivity replacement for TNT, is a key component of a new class of melt cast formulations designed for use in insensitive munitions. It is therefore essential that its fate and transport in the environment be assessed before its large scale implementation. Several recent studies have described reductive biotransformation pathways leading to dead-end products. Recently a Nocardioides strain, JS1661 was isolated based on its ability to mineralize DNAN via the 2,4-dinitrophenol (DNP) pathway. However, its potential for degrading DNAN under environmentally relevant conditions was not examined. Therefore we evaluated the aerobic biodegradation of DNAN by JS1661 in non-sterile soil, aqueous media and in a fluidized bed bioreactor over a wide range of DNAN concentrations. DNAN was completely degraded under all tested conditions with little or no accumulation of DNP and almost stoichiometric release of nitrite. Furthermore, when DNAN was used as the sole carbon and nitrogen source, the accumulation of nitrite was dramatically reduced. The results of the study revealed the robustness of the strain over a range of loading rates in various physical environments suggesting that it could provide the basis for waste treatment, bioremediation and bioaugmentation applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Vapor intrusion from volatile subsurface contaminants can be mitigated by aerobic biodegradation. Laboratory column studies with contaminant sources of chlorobenzene and a mixture of chlorobenzene, 1,2-dichlorobenzene, and 1,4-dichlorobenzene showed that contaminants were rapidly degraded in thin reactive zones with high biomass and low substrate concentrations in the vicinity of the capillary fringe. Such behavior was well characterized by a model that includes oxygen-, substrate-, and biomass-dependent biodegradation kinetics along with diffusive transport processes. An analytical solution was derived to provide theoretical support for the simplification of reaction kinetics and the approximation of reactive zone location and mass flux relationships at steady state. Results demonstrate the potential of aerobic natural attenuation in the capillary fringe for preventing contaminant migration in the unsaturated zone. The solution indicates that increasing contaminant mass flux into the column creates a thinner reactive zone and pushes it toward the oxygen boundary, resulting in a shorter distance to the oxygen source and a larger oxygen mass flux that balances the contaminant mass flux. As a consequence, the aerobic biodegradation can reduce high contaminant concentrations to low levels within the capillary fringe and unsaturated zone. The results are consistent with the observations of thin reactive layers at the interface in unsaturated zones. The model considers biomass while including biodegradation in the capillary fringe and unsaturated zone and clearly demonstrates that microbial communities capable of using the contaminants as electron donors may lead to instantaneous degradation kinetics in the capillary fringe and unsaturated zone.

2,4-Dinitroanisole (DNAN) is an insensitive munition ingredient used in explosive formulations as a replacement for 2,4,6-trinitrotoluene (TNT). Little is known about the environmental behavior of DNAN. There are reports of microbial transformation to dead-end products, but no bacteria with complete biodegradation capability have been reported. Nocardioides sp. strain JS1661 was isolated from activated sludge based on its ability to grow on DNAN as the sole source of carbon and energy. Enzyme assays indicated that the first reaction involves hydrolytic release of methanol to form 2,4-dinitrophenol (2,4-DNP). Growth yield and enzyme assays indicated that 2,4-DNP underwent subsequent degradation by a previously established pathway involving formation of a hydride-Meisenheimer complex and release of nitrite. Identification of the genes encoding the key enzymes suggested recent evolution of the pathway by recruitment of a novel hydrolase to extend the well-characterized 2,4-DNP pathway., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Volatile chlorinated compounds are major pollutants in groundwater, and they pose a risk of vapor intrusion into buildings. Vapor intrusion can be prevented by natural attenuation in the vadose zone if biodegradation mechanisms can be established. In this study, we tested the hypothesis that bacteria can use cis-dichloroethene (cis-DCE) or vinyl chloride (VC) as an electron donor in the vadose zone. Anoxic water containing cis-DCE or VC was pumped continuously beneath laboratory columns that represented the vadose zone. Columns were inoculated with Polaromonas sp. strain JS666, which grows aerobically on cis-DCE, or with Mycobacterium sp. JS60 and Nocardiodes sp. JS614 that grow on VC. Complete biodegradation with fluxes of 84 ± 15 μmol of cis-DCE · m(-2) · hr(-1) and 218 ± 25 μmole VC·m(-2) · h(-1) within the 23 cm column indicated that microbial activities can prevent the migration of cis-DCE and VC vapors. Oxygen and volatile compound profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place in the first 10 cm above the saturated zone within the capillary fringe. The results revealed that cis-DCE and VC can be biodegraded readily at the oxic/anoxic interfaces in the vadose zone if appropriate microbes are present.

Benzalkonium chlorides (BACs) are disinfectants widely used in a variety of clinical and environmental settings to prevent microbial infections, and they are frequently detected in nontarget environments, such as aquatic and engineered biological systems, even at toxic levels. Therefore, microbial degradation of BACs has important ramifications for alleviating disinfectant toxicity in nontarget environments as well as compromising disinfectant efficacy in target environments. However, how natural microbial communities respond to BAC exposure and what genes underlie BAC biodegradation remain elusive. Our previous metagenomic analysis of a river sediment microbial community revealed that BAC exposure selected for a low-diversity community, dominated by several members of the Pseudomonas genus that quickly degraded BACs. To elucidate the genetic determinants of BAC degradation, we conducted time-series metatranscriptomic analysis of this microbial community during a complete feeding cycle with BACs as the sole carbon and energy source under aerobic conditions. Metatranscriptomic profiles revealed a candidate gene for BAC dealkylation, the first step in BAC biodegradation that results in a product 500 times less toxic. Subsequent biochemical assays and isolate characterization verified that the putative amine oxidase gene product was functionally capable of initiating BAC degradation. Our analysis also revealed cooperative interactions among community members to alleviate BAC toxicity, such as the further degradation of BAC dealkylation by-products by organisms not encoding amine oxidase. Collectively, our results advance the understanding of BAC aerobic biodegradation and provide genetic biomarkers to assess the critical first step of this process in nontarget environments., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBank(TM) as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with k(cat)(app)/K(m)(app) of 12 × 10(6) M(-1) s(-1), k(cat)(app) of 1300 s(-1), and K(m)(app) of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Monooxygenation is an important route of nitroaromatic compound (NAC) biodegradation and it is widely found for cometabolic transformations of NACs and other aromatic pollutants. We investigated the C and N isotope fractionation of nitrophenol monooxygenation to complement the characterization of NAC (bio)degradation pathways by compound-specific isotope analysis (CSIA). Because of the large diversity of enzymes catalyzing monooxygenations, we studied the combined C and N isotope fractionation and the corresponding (13)C- and (15)N-apparent kinetic isotope effects (AKIEs) of four nitrophenol-biodegrading microorganisms (Bacillus spharericus JS905, Pseudomonas sp. 1A, Arthrobacter sp. JS443, Pseudomonas putida B2) in the pH range 6.1-8.6 with resting cells and crude cell extracts. While the extent of C and N isotope fractionation and the AKIE-values varied considerably for the different organisms, the correlated C and N isotope signatures (δ(15)N vs δ(13)C) revealed trends, indicative of two distinct monooxygenation pathways involving hydroxy-1,4-benzoquinone or 1,2- and 1,4-benzoquinone intermediates, respectively. The distinction was possible based on larger secondary (15)N-AKIEs associated with the benzoquinone pathway. Isotope fractionation was neither masked substantially by nitrophenol speciation nor transport across cell membranes. Only when 4-nitrophenol was biodegraded by Pseudomonas sp. 1A did isotope fractionation become negligible, presumably due to rate-limiting substrate binding steps pertinent to the catalytic cycle of flavin-dependent monooxygenases.

Variovorax paradoxus is a ubiquitous betaproteobacterium involved in plant growth promotion, the degradation of xenobiotics, and quorum-quenching activity. The genome of V. paradoxus strain EPS consists of a single circular chromosome of 6,550,056 bp, with a 66.48% G+C content.

3-Nitropropionate (3-NPA) is a nitro aliphatic compound found in numerous plants and fungi. The nitro compound exists in equilibrium with its conjugate base, propionate 3-nitronate (P3N) and has a pKa approaching the physiological range of 9.1. Since 1920, more than 30 species of plant and fungi have been identified as producing 3-NPA as a means of defense from herbivores. Glycoside products containing moieties of 3-NPA found in parts of the plants most accessible to herbivores can be easily hydrolyzed to free 3-NPA by bacterial enzymes in the gut of animals. In addition to providing a defense mechanism, the nitro compound is an intermediate in the nitrification process of leguminous plants. The synthesis of 3-NPA in these plants and fungi is poorly understood. P3N, which readily forms from 3-NPA at physiological pH, is a potent inhibitor of the key enzyme succinate dehydrogenase in the Krebs cycle and electron transport chain. Inhibition of succinate dehydrogenase in humans and livestock causes neurotoxicity and in some cases death. Several enzymes catalyze the oxidation of 3-NPA or P3N; all contain a noncovalently bound flavin cofactor and are found in the organisms that produce 3-NPA. With k(cat)/K(m) values of >10(6) M(-1) s(-1), nitronate monooxygenases can quickly and efficiently oxidize P3N to malonic semialdehyde as a means of protecting the organism from killing itself. Although it was discovered almost a century ago, the biochemistry and physiological role of 3-NPA/P3N are just emerging., (© 2013 International Union of Biochemistry and Molecular Biology. © 2013 IUBMB Life.)

Assessing the fate of nitroaromatic explosives in the subsurface is challenging because contaminants are present in different phases (e.g., bound to soil or sediment matrix or as solid-phase residues) and transformation takes place via several potentially competing pathways over time-scales of decades. We developed a procedure for compound-specific analysis of stable C, N, and H isotopes in nitroaromatic compounds (NACs) and characterized biodegradation of 2,4,6-trinitrotoluene (TNT) and two dinitrotoluene isomers (2,4-DNT and 2,6-DNT) in subsurface material of a contaminated site. The type and relative contribution of reductive and oxidative pathways to the degradation of the three contaminants was inferred from the combined evaluation of C, N, and H isotope fractionation. Indicative trends of Δδ(15)N vs Δδ(13)C and Δδ(2)H vs Δδ(13)C were obtained from laboratory model systems for biodegradation pathways initiated via (i) dioxygenation, (ii) reduction, and (iii) CH3-group oxidation. The combined evaluation of NAC isotope fractionation in subsurface materials and in laboratory experiments suggests that in the field, 86-89% of 2,4-DNT transformation was due to dioxygenation while TNT was mostly reduced and 2,6-DNT reacted via a combination of reduction and CH3-group oxidation. Based on historic information on site operation, our data imply biodegradation of 2,4-DNT with half-lives of up to 9-17 years compared to 18-34 years for cometabolic transformation of TNT and 2,6-DNT.

Much of the microbial activity in nature takes place at interfaces, which are often associated with redox discontinuities. One example is the oxic/anoxic interface where polluted groundwater interacts with the overlying vadose zone. We tested whether microbes in the vadose zone can use synthetic chemicals as electron donors and thus protect the overlying air and buildings from groundwater pollutants. Samples from the vadose zone of a site contaminated with chlorobenzene (CB), 1,2-dichlorobenzene (12DCB), and 1,4-dichlorobenzene (14DCB) were packed in a multiport column to simulate the interface of the vadose zone with an underlying groundwater plume. A mixture of CB, 12DCB, and 14DCB in anoxic water was pumped continuously through the bottom of column to an outlet below the first sampling port to create an oxic/anoxic interface and a capillary fringe. Removal to below the detection limits by rapid biodegradation with rates of 21 ± 1 mg of CB • m(-2) • d(-1), 3.7 ± 0.5 mg of 12DCB • m(-2) • d(-1), and 7.4 ± 0.7 mg of 1.4 DCB • m(-2) • d(-1) indicated that natural attenuation in the capillary fringe can prevent the migration of CB, 12DCB, and 14DCB vapors. Enumeration of bacteria capable of degrading chlorobenzenes suggested that most of the biodegradation takes place within the first 10 cm above the saturated zone. Biodegradation also increased the upward flux of contaminants and thus enhanced their elimination from the underlying water. The results revealed a substantial biodegradation capacity for chlorinated aromatic compounds at the oxic/anoxic interface and illustrate the role of microbes in creating steep redox gradients.