Insight into the metabolic pathways of Paracoccus sp. strain DMF: a non-marine halotolerant methylotroph capable of degrading aliphatic amines/amides.

Paracoccus sp. strain DMF (P. DMF from henceforth) is a gram-negative heterotroph known to tolerate and utilize high concentrations of N,N-dimethylformamide (DMF). The work presented here elaborates on the metabolic pathways involved in the degradation of C1 compounds, many of which are well-known pollutants and toxic to the environment. Investigations on microbial growth and detection of metabolic intermediates corroborate the outcome of the functional genome analysis. Several classes of C1 compounds, such as methanol, methylated amines, aliphatic amides, and naturally occurring quaternary amines like glycine betaine, were tested as growth substrates. The detailed growth and kinetic parameter analyses reveal that P. DMF can efficiently aerobically degrade trimethylamine (TMA) and grow on quaternary amines such as glycine betaine. The results show that the mechanism for halotolerant adaptation in the presence of glycine betaine is dissimilar from those observed for conventional trehalose-mediated halotolerance in heterotrophic bacteria. In addition, a close genomic survey revealed the presence of a Co(I)-based substrate-specific corrinoid methyltransferase operon, referred to as mtgBC. This demethylation system has been associated with glycine betaine catabolism in anaerobic methanogens and is unknown in denitrifying aerobic heterotrophs. This report on an anoxic-specific demethylation system in an aerobic heterotroph is unique. Our finding exposes the metabolic potential for the degradation of a variety of C1 compounds by P. DMF, making it a novel organism of choice for remediating a wide range of possible environmental contaminants.

Biotransformation of 2,4,6-trinitrotoluene by Diaphorobacter sp. strain DS2.

Diaphorobacter strain DS2 degrades 3-nitrotoluene and 2-nitrotoluene via ring oxidation with 3-nitrotoluene dioxygenase (3NTDO). In the current study, we hypothesized that 3NTDO might also be involved in the degradation of 2,4,6-trinitrotoluene (TNT), a major nitroaromatic explosive contaminant in soil and groundwater. Strain DS2 transforms TNT as a sole carbon and nitrogen source when grown on it. Ammonium chloride and succinate in the medium accelerated the TNT degradation rate. A resting cell experiment suggested that TNT does not compete with 3NT degradation (no negative impact of TNT on the reaction velocity for 3NT). Enzyme assay with 3NTDO did not exhibit TNT transformation activity. The above results confirmed that 3NTDO of DS2 is not responsible for TNT degradation. In the resting cell experiment, within 10 h, 4ADNT completely degraded. The degradation of 2ADNT was 97% at the same time. We hypothesized that 3NTDO involve in this reaction. Based on the DS2 genome, we proposed that the N-ethylmaleimide reductases (nemA) were involved in the initial reduction of the nitro group and aromatic ring of TNT. Our findings suggest that strain DS2 could be helpful for the removal of TNT from contaminated sites with or without any additional carbon and nitrogen source and with minimal accumulation of undesirable intermediates.

Genomic profiling and characteristics of a C1 degrading heterotrophic fresh-water bacterium Paracoccus sp. strain DMF.

Paracoccus species are metabolically versatile gram-negative, aerobic facultative methylotrophic bacteria showing enormous promise for environmental and bioremediation studies. Here we report, the complete genome analysis of Paracoccus sp. strain DMF (P. DMF) that was isolated from a domestic wastewater treatment plant in Kanpur, India (26.4287 °N, 80.3891 °E) based on its ability to degrade a recalcitrant organic solvent N, N-dimethylformamide (DMF). The results reveal a genome size of 4,202,269 base pairs (bp) with a G + C content of 67.9%. The assembled genome comprises 4141 coding sequences (CDS), 46 RNA sequences, and 2 CRISPRs. Interestingly, catabolic operons related to the conventional marine-based methylated amines (MAs) degradation pathway were functionally annotated within the genome of an obligated aerobic heterotroph that is P. DMF. The genomic data-based characterization presented here for the novel heterotroph P. DMF aims to improve the understanding of the phenotypic gene products, enzymes, and pathways involved with greater emphasis on facultative methylotrophic motility-based latent pathogenicity.

A spectrophotometric trimethylamine monooxygenase assay.

Trimethylamine monooxygenase (Tmm, EC-1.14.13.148) belongs to the family of flavin-containing monooxygenases that oxidize trimethylamine into trimethylamine-N-oxide (TMAO). Conventional methods for assaying Tmm are accurate over a narrow range of substrate/product concentrations. Here we report a TMAO-specific enzymatic assay for Tmm using polyallylamine hydrochloride (PAHCl)-capped MnO2 nanoparticles (PAHCl@MnO2 ). We achieved TMAO specificity using iodoacetonitrile to remove interfering trimethylamine. The change in the concentration of TMAO is measured by observing the difference in the absorbance of 3,3',5,5'-tetramethylbenzidine (TMB) at 650 nm. The assay is tolerant to several interfering metal ions and other compounds. This method is more accessible and reliable than currently known methods. The limit of detection (LOD) and limit of quantitation (LOQ) are 1 µM and 10 µM, respectively, for direct TMAO measurement.

Effect of the Standardized ZnO/ZnO-GO Filter Element Substrate driven Advanced Oxidation Process on Textile Industry Effluent Stream: Detailed Analysis of Photocatalytic Degradation Kinetics.

A simple process of synthesizing coated filter element substrates (FES) containing zinc oxide (ZnO) nanorods and ZnO graphene-oxide nanocomposite for a pilot-scale industrial dye-effluent treatment plant is proposed. This work reports a detailed analysis of the photocatalysis mechanism on real industrial effluent streams containing a mixture of dyes. The analysis is very relevant for conducting advanced oxidation process-assisted effluent remediation at a field-level treatment operation. Estimation of the dye concentration shows nearly complete (≥98%) degradation from an initial dye sample concentration. A detailed study for the analysis of the initial reactive dyes and their degradation products was performed for quantification and identification of the degradation products through various spectral techniques. A design of the remediation mechanism through degradation pathways is proposed for characterizing the organic compounds in the degraded dye products. A regeneration and reusability study was performed on the FES presenting the durability of the FES-designed synthesis process originally for 11 cycles and regenerated FES for six cycles for achieving a threshold of 60% degradation efficiency. The experimental results demonstrate the efficacy of FES through the designed immobilized approach for the complete remediation of textile dye effluents for a 4 h treatment plant process and the consistent operability of the FES for the combined dye wastewater treatment operations.

Transformation of 2, 4, 6-trinitrotoluene by Stenotrophomonas strain SG1 under aerobic and anaerobic conditions.

TNT, or 2,4,6-trinitrotoluene, is a common explosive that can contaminate soil and groundwater in production sites, military training areas, and disposal locations. The compound is highly toxic; therefore, there is an urgent need to rehabilitate the impacted environments. Harnessing the microbial ability to biodegrade TNT into environmentally harmless compound(s) is one approach to remediating contaminated sites. In our study, we report on the genomic and metabolic ability of Stenotrophomonas strain SG1 to degrade TNT under aerobic and anaerobic conditions. The bacterial strain SG1 was first isolated as a contaminant from a culture of Diaphorobacter sp. strain DS2 over minimal media supplemented with TNT. The draft genome assembly of strain SG1 is ∼4.7 Mb and is distributed among 358 contigs. The homology search against the custom database of enzymes responsible for TNT biodegradation revealed the presence of three N-ethylmaleimide reductases (NemA) with a defined KEGG ortholog and KEGG pathway of TNT degradation. The presence of respiratory nitrate reductases has also been mapped, which supports denitrification under anaerobic conditions. Experimentally, the TNT transformation rate accelerated when carbon sources, such as sodium acetate, sodium citrate, sodium succinate, sucrose, and glucose (final concentration of 5 mM), were added. Citrate promoted the highest growth and TNT transformation ratio (88.35%) in 120 h. With the addition of 5 mM ammonium chloride, TNT completely disappeared in the citrate and sucrose-containing treatments in 120 h. However, higher biomass was obtained in the sucrose and glucose-containing treatments in 120 h. During incubation, the formation of amino dinitrotoluene isomers, dinitrotoluene isomers, trinitrobenzene, azoxy isomers, diaryl hydroxylamines, and corresponding secondary amines was confirmed by GC/MS and UPLC/MS. 2-Amino-4-nitrotoluene, 4-amino-2-nitrotoluene, and 2-amino-6-nitrotoluene were also identified in the culture supernatant by GC/MS. Under anaerobic conditions, TNT completely disappeared in the citrate and citrate plus nitrate treatments. Since the strain shows the ability to remove TNT, this research should be useful in basic research and practical applications for removing TNT from wastewater.

Inhibition effect of 2,4,6-trinitrotoluene (TNT) on RDX degradation by rhodococcus strains isolated from contaminated soil and water.

2,4,6-trinitrotoluene (TNT) is a highly toxic explosive that contaminates soil and water and may interfere with the degradation of co-occurring compounds, such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We proposed that TNT may influence RDX-degrading bacteria via either general toxicity or a specific effect on the |RDX degradation mechanisms. Thus, we examined the impact of TNT on RDX degradation by Rhodococcus strains YH1, T7, and YY1, which were isolated from an explosives-polluted environment. Although partly degraded, TNT did not support the growth of any of the strains when used as either sole carbon or sole nitrogen sources, or as carbon and nitrogen sources. The incubation of a mixture of TNT (25 mg/l) and RDX (20 mg/l) completely inhibited RDX degradation. The effect of TNT on the cytochrome P450, catalyzing RDX degradation, was tested in a resting cell experiment, proving that TNT inhibits XplA protein activity. A dose-response experiment showed that the IC50/trans values for YH1, T7, and YY1 were 7.272, 5.098, and 9.140 (mg/l of TNT), respectively, illustrating variable sensitivity to TNT among the strains. The expression of xplA was also strongly suppressed by TNT. Cells that were pre-grown with RDX (allowing xplA expression) and incubated with ammonium chloride, glucose, and TNT, completely transformed into their amino dinitrotoluene isomers and formed azoxy toluene isomers. The presence of oxygen-insensitive nitroreductase that enable reduction of the nitro group in the presence of O2 in the genomes of these strains suggests that they are responsible for TNT transformation in the cultures. The experimental results concluded that TNT has an adverse effect on RDX degradation by the examined strains. It inhibits RDX degradation due to the direct impact on cytochrome P450, xplA, or its expression. The tested strains can transform TNT independently of RDX. Thus, degradation of both compounds is possible if TNT concentrations are below their IC50 values.

Integrated DEP Assisted Detection of PCR Products With Metallic Nanoparticle Labels Through Impedance Spectroscopy.

Electrochemical impedance spectroscopy (EIS) is gaining immense popularity in the current times due to the ease of integration with microelectronics. Keeping this aspect in mind, various detection schemes have been developed to make impedance detection of nucleic acids more specific. In this context, the current work makes a strong case for specific DNA detection through EIS using nanoparticle labeling approach and also an added selectivity step through the use of dielectrophoresis (DEP), which enhances the detection sensitivity and specificity to match the detection capability of quantitative polymerase chain reaction (qPCR) in real-time context as compared to the individually amplified DNA (Liu et al., 2008). The detection limit of the proposed biochip is observed to be 3-4 PCR cycles for 582 bp bacterial DNA, where the complete procedure of detection starts in less than 10 min. The process of integrated DEP capture of labeled products coming out of PCR and their impedance-assisted detection is carried out in an in-house micro-fabricated biochip. The gold nanoparticles, which possess excellent optical, chemical, electronic, and biocompatibility properties and are capable of generating lump-like DNA structure without modifying its basic impedance signature are introduced to the amplified DNA through the nanoparticle labeled primers.

Comparison of CryoEM and X-ray structures of dimethylformamidase.

Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54 °C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.

A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2 ß2 or (α2 ß2 )2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.