Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Modeling the Biodegradation Kinetics of Perchlorate in the Presence of Oxygen and Nitrate as Competing Electron Acceptors

A mathematical model was developed to describe the biodegradation kinetics of perchlorate in the presence of nitrate and oxygen as competing electron acceptors. The rate of perchlorate degradation is described as a function of the electron donor (acetate) degradation rate, the concentration of the alternate electron acceptors, and rates of biomass growth and decay. The kinetics of biomass growth are described using a modified Monod model, and inhibition factors are incorporated to describe the influence of oxygen and nitrate on perchlorate degradation. In order to develop input parameters for the model, a series of batch biodegradation studies were performed using Azospira suillum JPLRND, a perchlorate-degrading strain isolated from groundwater. This strain is capable of utilizing oxygen, nitrate, or perchlorate as terminal electron acceptors. The maximum specific growth rate (μ_max) and half-saturation constant (K _S ^don) for the bacterium when utilizing either perchlorate or nitrate were similar; 0.16 per h and 158 mg acetate/L, respectively. However, these parameters were different when the strain was growing on oxygen. In this case, μ_max and K _S ^don were 0.22 per h and 119 mg acetate/L, respectively. The batch experiments also revealed that nitrate inhibits perchlorate biodegradation by this strain. This finding was incorporated into the model by applying an inhibition coefficient (K _i ^nit) value of 25 mg nitrate/L. Combined with appropriate groundwater transport models, this model can be used to predict perchlorate biodegradation during in situ remediation efforts.     

Biodegradation of Methyl tert-Butyl Ether by a Pure Bacterial Culture

Biodegradation of methyl tert-butyl ether (MTBE) by the hydrogen-oxidizing bacterium Hydrogenophaga flava ENV735 was evaluated. ENV735 grew slowly on MTBE or tert-butyl alcohol (TBA) as sole sources of carbon and energy, but growth on these substrates was greatly enhanced by the addition of a small amount of yeast extract. The addition of H₂ did not enhance or diminish MTBE degradation by the strain, and MTBE was only poorly degraded or not degraded by type strains of Hydrogenophaga or hydrogen-oxidizing enrichment cultures, respectively. MTBE degradation activity was constitutively expressed in ENV735 and was not greatly affected by formaldehyde, carbon monoxide, allyl thiourea, or acetylene. MTBE degradation was inhibited by 1-amino benzotriazole and butadiene monoepoxide. TBA degradation was inducible by TBA and was inhibited by formaldehyde at concentrations of >0.24 mM and by acetylene but not by the other inhibitors tested. These results demonstrate that separate, independently regulated genes encode MTBE and TBA metabolism in ENV735.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Bioremediation in Groundwater: Are Known RDX-Degrading Bacteria the Dominant Players?

Biodegradation of explosives in groundwater represents a promising remedial approach for these compounds. Although a range of bacteria capable of degrading the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in pure culture have been described, the role of these known strains (and the genera they represent) during RDX degradation in groundwater has not been established. RDX-contaminated groundwater was collected from the Pueblo Chemical Depot (CO, USA) and the Picatinny Arsenal (NJ, USA) where bioremediation technologies are being tested. Soil columns and enrichment cultures were derived from Picatinny Arsenal groundwater. Bacteria-specific primers were used to amplify the 16S rRNA genes that were used for phylogenetic analysis. The species detected ranged across multiple genera, many of which have not been previously associated with RDX biodegradation. None of the retrieved sequences were exact matches to previously described RDX-degrading strains, although multiple sequences that grouped with known explosive-degrading strains of Clostridium and Pseudomonas were recovered. Genes previously reported to be associated with RDX degradation, including xplA, hydA, onr, xenA, and xenB, were not detected in any of the groundwater samples. These preliminary results indicate that the previously described RDX-degrading bacteria likely do not capture the microbial diversity associated with RDX bioremediation in groundwater, especially under the general biostimulation approaches used during most remediation efforts.     

Biotransformation of N-Nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of ¹⁸O₂ and H₂¹⁸O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O₂. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH₃OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial α-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Alterations in intermediate filament proteins in rat kidney proximal tubule epithelial cells

Changes in the intermediate filament composition of rat kidney proximal tubule cells in culture have been investigated. The data suggest that differentiated tubular epithelial cells do not express vimentin, but vimentin expression is induced when the cells begin to proliferate in culture. The cultured cells are positive for both cytokeratins and vimentin by immunofluorescence microscopy. The data support the concept that the intermediate filament composition of proximal tubule epithelial cells can be altered during proliferation induced by nephrotoxic chemicals or by neoplastic transformation.     

Neuroendocrine Regulation and Metabolism of Glucose and Lipids in Primary Chronic Insomnia: A Prospective Case-Control Study

Objectives

To investigate the relation between primary chronic insomnia and insulin sensitivity, visceral adiposity, non alcoholic fatty liver disease and neuroendocrine hormones.

Materials and Methods

In a case-controlled, prospective clinical trial 13 women with primary chronic insomnia according to DSM-IV criteria were compared to 12 healthy controls matched for age, sex, BMI, body composition and menopausal status. All participants had a sleep assessment including polysomnographic studies and neuropsychiatric evaluation. Insulin sensitivity was evaluated using the euglycaemic hyperinsulinemic clamp. Hepatic fat content, visceral adipose tissue and intramyocellular lipid accumulation were assessed using magnetic resonance imaging and spectroscopy. The hormonal stress axis was evaluated by measurements of midnight and early morning salivary cortisol, urinary catecholamines and plasma metanephrines. Body composition was determined using body impedance analysis and indirect calorimetry.

Results

Although the diagnosis of primary chronic insomnia was made by established clinical criteria, standard polysomongraphic studies failed to identify altered sleep continuity and architecture when compared to matched controls. However, women with primary chronic insomnia showed significantly higher midnight salivary cortisol concentrations (1.46 vs. 0.76 nmol/l, p = 0.02), indicating dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis. Plasma glucose and lipid concentrations, insulin sensitivity, hepatic and intramyocellular fat content, visceral adipose tissue mass and body composition did not differ between the two groups.

Conclusion

Healthy women with clinically diagnosed primary chronic insomnia demonstrate a dysregulation of circadian cortisol secretion despite normal sleep continuity and architecture. Increased midnight cortisol levels, however, were not associated with impaired metabolism of glucose and lipids.     

Enhancing transport of hydrogenophaga flava ENV735 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.     

Relationship between the number of bacteria added to soil or seeds and their abundance and distribution in the rhizosphere of alfalfa

A study was conducted of the relationship between the density of several bacterial strains introduced into soil or onto seeds and their abundance in the rhizosphere of alfalfa. The abundance of six species in the rhizosphere was directly correlated with the density of bacteria initially added to soil. The density of six species in the rhizosphere of 15-day-old plants also was directly correlated with the density of each strain in nonrhizosphere soil. Tests of seven species added to soil at four inoculum densities showed that bacteria that survived well in the soil attained the highest densities in the rhizosphere and those that survived poorly in the soil were present at the lowest densities in the rhizosphere. Sixteen of 19 bacterial strains added to alfalfa seeds at 10⁷ or 10⁸ cells per g colonized the rhizosphere of 15-day-old plants, but nearly all of the cells were localized in the upper third of the rhizosphere. A study of 12 bacterial strains that failed to colonize the lower part of the rhizosphere if inoculated onto seeds showed that the bacteria colonized the entire rhizosphere of 15-day-old alfalfa plants if initially inoculated throughout the soil. The data suggest that the density of individual bacterial strains in the rhizosphere is dependent on their density in the soil and that seed inoculation only has an effect on the population in the proximal portion of the alfalfa root system.