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.

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 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.

Structures and conformation of a benzo-12-crown-4 containing dipeptide.

Crystal structures of the dipeptide Boc-12-Crown-4-L-DOPA-Gly-OMe (chi) and Boc-12-Crown-4-D/L-DOPA-Gly-OMe (rac) were solved by single crystal X-ray diffraction. Analysis of the packing differences in the crystal reveals that the presence of a water molecule in chi enables intermolecular contacts with the solvent that result in a different conformation of the dipeptide backbone itself. An uncommon N-H…N interaction stabilizes the peptide in its solid state.

Effect of methylene group insertions on the structural rigidity of Aib containing helices.

Nonprotein amino acids are being extensively used in the design of synthetic peptides to create new structure mimics. In this study we report the effect of methylene group insertions in a heptapeptide Boc-Ala1-Leu2-Aib3-Xxx4-Ala5-Leu6-Aib7-OMe which nicely folds into a mixed 310 -/α-helical structure when Xxx= Ala. Analogs of this peptide have been made and studied by replacing central Xxx4 residue with Glycine (α-residue), ß-Alanine (ß-Αla), γ-aminobutyric acid (Gaba), and ε-aminocaproic acid (ε-Aca). NMR and circular dichroism were used to study the solution structure of these peptides. Crystals of the peptides containing alanine, ß-Αla, and Gaba reveal that increasing the number of central methylene (-CH2 -) groups introduces local perturbations even as the helical structure is retained.

Expression, purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.

3-Nitrotoluene dioxygenase from Diaphorobacter sp. strains: cloning, sequencing and evolutionary studies.

The first step in the degradation of 3-nitrotoluene by Diaphorobacter sp. strain DS2 is the dihydroxylation of the benzene ring with the concomitant removal of nitro group. This is catalyzed by a dioxygenase enzyme system. We report here the cloning and sequencing of the complete dioxygenase gene with its putative regulatory sequence from the genomic DNA of Diaphorobacter sp. strains DS1, DS2 and DS3. Analysis of the 5 kb DNA stretch that was cloned, revealed five complete open reading frames (ORFs) encoding for a reductase, a ferredoxin and two dioxygenase subunits with predicted molecular weights (MW) of 35, 12, 50 and 23 kDa respectively. A regulatory protein was also divergently transcribed from the reductase subunit and has a predicated MW of 34 kDa. Presence of parts of two functional ORFs in between the reductase and the ferredoxin subunits reveals an evolutionary route from a naphthalene dioxygenase like system of Ralstonia sp. strain U2. Further a 100 % identity of its ferredoxin subunit reveals its evolution via dinitrotoluene dioxygenase like system present in Burkholderia cepacia strain R34. A modeled structure of oxygenase3NT from strain DS2 was generated using nitrobenzene dioxygenase as a template. The modeled structure only showed minor changes at its active site. Comparison of growth patterns of strains DS1, DS2 and DS3 revealed that Diaphorobacter sp. strain DS1 has been evolved to degrade 4-nitrotoluene better by an oxidative route amongst all three strains.

Biomineralization of 3-nitrotoluene by Diaphorobacter species.

Three bacterial strains utilizing 3-nitrotoluene (3-NT) as a sole source of carbon, nitrogen and energy were isolated from an industrial wastewater treatment plant. Biochemical tests and 16S rDNA sequence analysis revealed that the isolated strains belonged to Diaphorobacter sp. Detailed studies were carried out with Diaphorobacter sp. strain DS2. Degradation of 3-NT by Diaphorobacter sp. strain DS2 was accompanied by the release of nitrite in the culture broth with increase in biomass. Total organic carbon analysis confirmed the extensive mineralization of 3-NT. The strain could degrade 3-methylcatechol, 4-methylcatechol and catechol easily suggesting that the degradation pathway could involve these as possible intermediates. Successful PCR amplification of the oxygenase large subunit and the presence of high activity for catechol 2,3-dioxygenase in the crude cell lysate further confirmed that the degradation of 3-NT occurred through (methyl)catechol intermediates in strain DS2. The strain DS2 was found to degrade other isomers of mononitrotoluene (2-NT and 4-NT) and nitrobenzene as well.

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.

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.

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.

Excited state relaxation dynamics of model green fluorescent protein chromophore analogs: evidence for cis-trans isomerism.

Two green fluorescent protein (GFP) chromophore analogs (4Z)-4-(N,N-dimethylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DMPI) and (4Z)-4-(N,N-diphenylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DPMPI) were investigated using femtosecond fluorescence up-conversion spectroscopy and quantum chemical calculations with the results being substantiated by HPLC and NMR measurements. The femtosecond fluorescence transients are found to be biexponential in nature and the time constants exhibit a significant dependence on solvent viscosity and polarity. A multicoordinate relaxation mechanism is proposed for the excited state relaxation behavior of the model GFP analogs. The first time component (τ(1)) was assigned to the formation of twisted intramolecular charge transfer (TICT) state along the rotational coordinate of N-substituted amine group. Time resolved intensity normalized and area normalized emission spectra (TRES and TRANES) were constructed to authenticate the occurrence of TICT state in subpicosecond time scale. Another picosecond time component (τ(2)) was attributed to internal conversion via large amplitude motion along the exomethylenic double bond which has been enunciated by quantum chemical calculations. Quantum chemical calculation also forbids the involvement of hula-twist because of high activation barrier of twisting. HPLC profiles and proton-NMR measurements of the irradiated analogs confirm the presence of Z and E isomers, whose possibility of formation can be accomplished only by the rotation along the exomethylenic double bond. The present observations can be extended to p-HBDI in order to understand the role of protein scaffold in reducing the nonradiative pathways, leading to highly luminescent nature of GFP.

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.

Synchronized Electromechanical Shock Wave-Induced Bacterial Transformation.

We report a simple device that generates synchronized mechanical and electrical pressure waves for carrying out bacterial transformation. The mechanical pressure waves are produced by igniting a confined nanoenergetic composite material that provides ultrahigh pressure. Further, this device has an arrangement through which a synchronized electric field (of a time-varying nature) is initiated at a delay of ≈85 µs at the full width half-maxima point of the pressure pulse. The pressure waves so generated are incident to a thin aluminum-polydimethylsiloxane membrane that partitions the ignition chamber from the column of the mixture containing bacterial cells (Escherichia coli BL21) and 4 kb transforming DNA. A combination of mechanical and electrical pressure pulse created through the above arrangement ensures that the transforming DNA transports across the cell membrane into the cell, leading to a transformation event. This unique device has been successfully operated for efficient gene (∼4 kb) transfer into cells. The transformation efficacy of this device is found comparable to the other standard methods and protocols for carrying out the transformation.

Crosslinking of agar by diisocyanates.

In the present study, crosslinking of agar using diisocyanate (DI) was demonstrated to limit the high water absorption property of agar. In addition, the efficacy of aromatic diisocyanate, DDI (4, 4 diphenyl diisocyanate) and aliphatic diisocyanate, HDI (1, 6 hexamethylene diisocyanate) on crosslinked agar properties was compared. The water uptake was successfully reduced by crosslinking and its minimum values observed for DDI and HDI crosslinked agar was 33.6% and 43.6%, respectively in comparison to agar (206%). The maximum tensile strength was observed for DDI crosslinked agar (45.3 MPa) which was higher than HDI crosslinked agar (30.6 MPa) and agar (31.7 MPa). The aromatic diisocyanates crosslinked agar showed better thermal resistance at higher temperature. It was observed that aromatic diisocyanate crosslinked agar more effectively than the aliphatic diisocyanate due to the higher reactivity. The crosslinked agar samples were hemocompatible and show non-toxic nature for cell proliferation.

Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.

3-nitrotoluene dioxygenase (3NTDO) from Diaphorobacter sp. strain DS2 catalyses the conversion of 3-nitrotoluene (3NT) into a mixture of 3- and 4-methylcatechols with release of nitrite. We report here, X-ray crystal structures of oxygenase and ferredoxin components of 3NTDO at 2.9 Å and 2.4 Å, respectively. The residues responsible for nitrite release in 3NTDO were further probed by four single and two double mutations in the catalytic site of α-subunit of the dioxygenase. Modification of Val 350 to Phe, Ile 204 to Ala, and Asn258 to Val by site directed mutagenesis resulted in inactive enzymes revealing the importance of these residues in catalysis. Docking studies of meta nitrotoluene to the active site of 3NTDO suggested possible orientations of binding that favor the formation of 3-methylcatechol (3MC) over 4-methylcatechol energetically. The electron transfer pathway from ferredoxin subunit to the active site of the oxygenase subunit is also proposed.

Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms.

The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples.