Evaluation of BACTEC™ MGIT™ 960 and the nitrate reductase assay in the National Laboratory Network of Colombia.

SETTING: The Mycobacteria Group of the National Reference Laboratory, National Laboratory Network, National Institute of Health and the Public Health Laboratories of Antioquia, Atlántico and Valle del Cauca, Colombia. OBJECTIVE: To compare BACTEC™ MGIT™ (Mycobacteria Growth Indicator Tube) 960 and the nitrate reductase assay (NRA) with the proportion method on Löwenstein-Jensen medium and to determine resistance to first-line anti-tuberculosis drugs. METHODS: Evaluation of diagnostic assays using time-adjusted convenience sampling. We analysed 183 isolates of Mycobacterium tuberculosis to compare MGIT 960 and NRA with the proportion method. RESULTS: MGIT 960 had a sensitivity and specificity of 90% for isoniazid (INH), and respectively 100% and 99.4% for rifampicin (RMP). NRA had a sensitivity and a specificity of respectively 86% and 94.7% for INH and 100% and 99% for RMP. CONCLUSION: Given its high sensitivity and specificity, and easy accessibility and interpretation of results, the NRA should be implemented in the National Laboratory Network for the detection of resistance to INH and RMP.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

Evaluation of bacterial nitrate reduction in the human oral cavity.

It is hypothesized that the enterosalivary nitrate circulation encourages nitrate reducing bacteria to reside within the oral cavity. Nitrite production may then limit the growth of acidogenic bacteria as a result of the production of antimicrobial oxides of nitrogen, including nitric oxide. This study was carried out with 10 subjects to characterize oral nitrate reduction and identify the bacteria responsible. Nitrate reduction varied between individuals (mean 85.4 +/- 15.9 nmol nitrite min(-1) with 10 ml 1 mm KNO(3) mouth wash) and was found to be concentrated at the rear of the tongue dorsal surface. Nitrate reductase positive isolates identified, using 16S rDNA sequencing, from the tongue comprised Veillonella atypica (34%), Veillonella dispar (24%), Actinomyces odontolyticus (21%), Actinomyces naeslundii (2%), Rothia mucilaginosa (10%), Rothia dentocariosa (3%) and Staphylococcus epidermidis (5%). Nitrite production rates, using intact and permeabilized cells, of the major tongue nitrate reducers were determined in the presence of methyl and benzyl viologen. Under anaerobic conditions in the presence of nitrate, rates in decreasing order were: A. odontolyticus > R. mucilaginosa > R. dentocariosa > V. dispar > V. atypica. In conclusion, Veillonella spp. were found to be the most prevalent taxa isolated and thus may make a major contribution to nitrate reduction in the oral cavity.

Proteome-level investigation of Brassica carinata-derived resistance to Leptosphaeria maculans.

Plants resistant to the fungal pathogen Leptosphaeria maculans were generated by an interspecific cross between the highly susceptible Brassica napus (canola) and the highly resistant Brassica carinata. Changes in the leaf protein profiles of these lines were investigated in order to understand the biochemical basis for the observed resistance. Two-dimensional electrophoresis followed by tandem mass spectrometry led to the identification of proteins unique to the susceptible (5 proteins) and resistant genotypes (7 proteins) as well those that were differentially expressed in the resistant genotype 48 h after challenge with the pathogen (28 proteins). Proteins identified as being unique in the resistant plant material included superoxide dismutase, nitrate reductase, and carbonic anhydrase. Photosynthetic enzymes (fructose bisphosphate aldolase, triose phosphate isomerase, sedoheptulose bisphosphatase), dehydroascorbate reductase, peroxiredoxin, malate dehydrogenase, glutamine synthetase, N-glyceraldehyde-2-phosphotransferase, and peptidyl-prolyl cis-trans isomerase were observed to be elevated in the resistant genotype upon pathogen challenge. Increased levels of the antioxidant enzyme superoxide dismutase were further validated and supported by spectrophotometric and in-gel activity assays. Other proteins identified in this study such as nitrate reductase and peptidylprolyl isomerase have not been previously described in this plant-pathogen system, and their potential involvement in an incompatible interaction is discussed.

Agromyces ulmi sp. nov., a xylanolytic bacterium isolated from Ulmus nigra in Spain.

Two xylan-degrading bacterial strains were isolated from a decayed Ulmus nigra tree in Spain. The isolates were Gram-positive, non-motile, aerobic and formed substrate mycelium which fragmented into irregular rods. 16S rRNA gene sequence analysis indicated that the isolates form a separate branch within the genus Agromyces phylogenetic cluster, with Agromyces mediolanus DSM 20152(T) being their closest relative (97.7 and 97.6 % sequence similarity). Catalase, nitrate reduction and urease tests differentiated these strains from A. mediolanus. Cell-wall peptidoglycan composition, major menaquinone, predominant fatty acids and phospholipid pattern were typical of the genus Agromyces. The DNA G+C content determined for the type strain XIL01(T) was 72 mol%. Based on the data presented, a novel species Agromyces ulmi sp. nov. is proposed. The type strain is XIL01(T) (=LMG 21954(T)=DSM 15747(T)).

Comparative effectiveness of cattle manure, poultry manure, phosphocompost and fertilizer-NPK on three cropping systems in vertisols of semi-arid tropics. II. Dry matter yield, nodulation, chlorophyll content and enzyme activity.

A field experiment was conducted on a deep Vertisol of Bhopal, India to compare root and shoot biomass, chlorophyll content, enzyme activity and nodulation in three cropping systems at three combinations of organic manure and inorganic-fertilizer: 75%NPK + 5 t farmyard manure (FYM), 75%NPK + 1.5 t poultry manure (PM), and 75%NPK + 5 t phosphocompost (PC) vis-a-vis 0%, 75% and 100% of fertilizer-NPK. In general, nodule number and its mass were lower in intercrop soybean than sole soybean. Also there was decrease in the nodule number with higher NPK dose. The FYM treated plots recorded 22.0% and 7.6% higher nodule mass than poultry manure and phosphocompost plots, respectively. Also, the total chlorophyll content was higher in organically treated plots than that in 100% NPK particularly at 30 days after sowing (DAS, pre-flowering). In sorghum the peak nitrate reductase (NR) activity was recorded at 60 DAS while in soybean it was at 30 DAS. The NR activity was higher in intercrop sorghum than that in sole sorghum. Maximum NR activity was observed in 100% NPK. Soybean/sorghum intercropping system recorded significantly higher root and shoot biomass than sole soybean and sorghum. The crop growth rates were relatively rapid during 30-60 DAS and followed the order; intercropping > sole sorghum > sole soybean. With the increase in NPK dose from 0% to 100% there was significant improvement in the dry matter (DM) production in sole sorghum and soybean/sorghum intercropping system. Soybean as preceding crop recorded the highest DM, chlorophyll content, NR activity in wheat while these values were the lowest in sorghum-wheat system.

Redox-triggered events in cytochrome c nitrite reductase.

Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.

Influence of maize mucilage on the diversity and activity of the denitrifying community.

In order to understand the effect of the maize rhizosphere on denitrification, the diversity and the activity of the denitrifying community were studied in soil amended with maize mucilage. Diversity of the denitrifying community was investigated by polymerase chain reaction (PCR) amplification of total community DNA extracted from soils using gene fragments, encoding the nitrate reductase (narG) and the nitrous oxide reductase (nosZ), as molecular markers. To assess the underlying diversity, PCR products were cloned and 10 gene libraries were obtained for each targeted gene. Libraries containing 738 and 713 narG and nosZ clones, respectively, were screened by restriction fragment analysis, and grouped based on their RFLP (restriction fragment length polymorphism) patterns. In all, 117 and 171 different clone families have been identified for narG and nosZ and representatives of RFLP families containing at least two clones were sequenced. Rarefaction curves of both genes did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity. Recovered NarG sequences were related to NarG from Actinomycetales and from Proteobacteria but most of them are not related to NarG from known bacteria. In contrast, most of the NosZ sequences were related to NosZ from alpha, beta, and gammaProteobacteria. Denitrifying activity was monitored by incubating the control and amended soils anaerobically in presence of acetylene. The N2O production rates revealed denitrifying activity to be greater in amended soil than in control soil. Altogether, our results revealed that mucilage addition to the soil results in a strong impact on the activity of the denitrifying community and minor changes on its diversity.

NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing.

Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA.

Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins.

Hydrophobic membrane proteins often have complex functions and are thus of great interest. However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation. We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis. CNBr selectively cleaves methionine residues. But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces. To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel. The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied. We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature. This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples. The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media.

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.

Quantitative measurement of the nitrate reductase activity in the human oral cavity.

To quantitatively characterise the nitrate reductase activity in the human oral cavity, a new assay based on holding 20 ml of 10 mg nitrate-N/L solution in the mouth was developed. The mouth assay appeared to relate primarily to the oral cavity surface rather than to the saliva. Nitrite formation in the assay was 50-100 times higher compared to in vitro incubation. In the proposed assay, the nitrite formation linearly increased over a period of 3 min. The average nitrate reductase activity in the oral cavity of 20 subjects was 2.39+/-1.52 microg nitrite-N formed/person x min. The nitrate reductase activity measured for two subjects at different hours varied about 15% for the same subject. The average nitrate reductase activity measured in June for 10 subjects (3.43+/-1.75 microg-N/person x min) was significantly higher than that measured in November for 10 other subjects (1.54+/-0.46 microg-N/person x min). Therefore, the nitrate reductase activity in the oral cavity appears to be influenced by the seasonal conditions. Although the amounts of nitrite formed in the mouth assay increased with increasing levels of nitrate, the rate of nitrate to nitrite reduction decreased with increasing levels of nitrate. The nitrite formation was also affected by the pH, with an optimal pH about 8. The nitrite formation was not influenced by uptake in the mouth of glucose, L-ascorbic acid and L-arginine.

Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis.

Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum.

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.

Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci.

The objective of this work was to study the production of catalase and nitrate reductase by staphylococci in order to understand their role in lipid oxidation during sausage manufacturing. Catalase and nitrate reductase were measured in resting cells and supernatants of staphylococci grown in different conditions. All staphylococci (except S. warneri) synthetized nitrate reductase. In static condition, the synthesis was maximal during exponential growth phase, whereas in shaking condition, the synthesis was maximal at the beginning of stationary phase. The production of nitrate reductase was increased in presence of nitrate, this effect was particularly important for the two S. carnosus strains which exhibited the highest activity. For all staphylococci, the production of catalase was maximal at the end of the exponential growth phase. The lowest amount of catalase was produced by S. warneri and the highest by S. carnosus. Only S. xylosus 873 and S. saprophyticus 852 released high amounts of catalase in the supernatant growth. Staphylococci produced higher amounts of catalase in shaking conditions. Addition of nitrate in the growth media favoured the synthesis of catalase, with a pronounced effect for S. carnosus. Nitrate also favoured the release of catalase.

Disruption of narG, the gene encoding the catalytic subunit of respiratory nitrate reductase, also affects nitrite respiration in Pseudomonas fluorescens YT101.

The Pseudomonas fluorescens YT101 gene narG, which encodes the catalytic alpha subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar(-) mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N(2)O respiration was not affected, anaerobic growth with NO(2) as the sole electron acceptor was delayed for all of the Nar(-) mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.

Salivary nitrate, nitrite and nitrate reductase activity in relation to risk of oral cancer in Egypt.

It has been suggested that nitrate and nitrite may play a role in the etiology of human oral cancer. We investigated whether salivary nitrate and nitrite and the activity of nitrate reductase (NRase) may affect the risk of oral cancer in Egypt, an area with high levels of environmental nitrosating agents. Levels of salivary nitrite (8.3 +/- 1.0 micrograms/ml) and nitrate (44 +/- 3.7 micrograms/ml) and activity of NRase (74 +/- 10 nmol/ml/min) were significantly (P < 0.05) higher in oral cancer patients (n = 42) compared to control Egyptian healthy individuals (n = 40, nitrite = 5.3 +/- 0.3 micrograms/ml, nitrate = 27 +/- 1.2 micrograms/ml, and NRase activity = 46 +/- 4 nmol/ml/min). The adjusted odds ratio (OR) and the 95% confidence intervals (C.I.) for risk of oral cancer, categorized by the levels of salivary nitrate and nitrite and NRase activity, showed a higher cancer risk associated with nitrite > 7.5 micrograms/ml (OR: 3.0, C.I.: 1.0-9.3), nitrite > 40 micrograms/ml (OR: 4.3, C.I.: 1.4-13.3) and NRase activity > 50 nmol/ml/min (OR: 2.9, C.I.: 1.1-7.4). Our findings suggest that increased consumption of dietary nitrate and nitrite is associated with elevated levels of salivary nitrite. Together with the increased activity of salivary NRase, these observations may explain, at least in part, the role of nitrate and nitrite in the development of oral cancer in individuals from an area with a high burden of N-nitroso precursors.

Characterization of moeB--part of the molybdenum cofactor biosynthesis gene cluster in Staphylococcus carnosus.

Transposon mutagenesis of Staphylococcus carnosus led to the identification of a gene cluster comprising nine genes that are important for molybdenum cofactor biosynthesis. Two nitrate-reductase-negative Tn917-insertion mutants were defective in MoeB. In cell-free extracts of an moeB mutant, the molybdenum-cofactor-deficient nitrate reductase could be reconstituted with a low-molecular-mass component (most likely free molybdenum cofactor) from an S. carnosus mutant that is defective in the nitrate reductase structural genes. The expression of moeB was studied in response to oxygen and nitrate. Primer-extension studies indicated that anaerobiosis and nitrate each enhance transcription of moeB.

Deletion of the N-terminal region of the AREA protein is correlated with a derepressed phenotype with respect to nitrogen metabolite repression.

The entire areA gene and a truncated version lacking the sequence encoding the N-terminal 389 amino acids were expressed from the qutE promoter and terminator in an Aspergillus nidulans strain with the endogenous areA gene deleted. This expression system was used to decouple the effects of transcription regulation and mRNA stability mediated by the native promoter and terminator from any posttranslational modulation of AREA activity. Both the full-length AREA protein and the truncated form were able to function in the deletion strain, conferring the ability to use alternate nitrogen sources. Transformants containing the entire areA gene had a repressible phenotype with respect to nitrogen metabolite repression, whereas those containing the truncated form of the areA gene had a derepressed phenotype. The truncated areA gene was expressed in an A. nidulans strain containing a normally regulated wild-type areA gene, and transformants displayed a quinate-inducible nitrogen metabolite derepressed phenotype. Northern blot analysis of transformed strains showed that areA-specific mRNAs of the expected sizes were being produced. The truncated AREA protein was overproduced in Escherichia coli as a fusion protein and purified to homogeneity by a single-step immobilized metal affinity chromatography, and the purified protein was shown to bind specifically to the niaD promoter. Revised sequences of the 5' region of the areA gene and the entire meaB gene are reported.

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct.