Toxic effects of vanadium (V) on a combined autotrophic denitrification system using sulfur and hydrogen as electron donors.

Vanadium (V) is a common heavy metal and often co-occurs with nitrate in effluents from mining and metal finishing industry. In the present study, the toxic effects of V(V) were examined in a sulfur and hydrogen based autotrophic denitrification system. This combined system achieved simultaneously microbial denitrification and V(V) reduction. High concentration of V(V) (60 and 100 mg/L) inhibited the denitrification activities, while 30 mg/L V(V) had a very slight effect. V(V) induced increases of lactate dehydrogenase release and reactive oxygen species production, which may inhibit nitrate and nitrite reductases activities and abundances of denitrifying functional genes. Moreover, the extracellular polymeric substance production was also suppressed under V(V) stress, thereby decreasing the amount of biofilm biomass. Microbial community analyses suggesting the genus Bacillus may have higher tolerance to V(V). These findings can provide scientific basis for the optimized design of treatment system to remove nitrate and V(V) simultaneously.

Measuring nitrate reductase activity from human and rodent tongues.

Reduction of salivary nitrate to nitrite by oral microbes expressing nitrate-reductase has emerged as a crucial pathway in systemic NO homeostasis in humans and other mammals. Selective depletion of oral microbes prevents dietary nitrate-dependent lowering of blood pressure, inhibition of platelet aggregation and ischemic injury. To date, most studies interrogate enterosalivary nitrate reduction by following changes in saliva or plasma nitrite and NO-signaling (functional) end points. Little is known about whether, and if so how, nitrate-reductase enzymatic activity per se (i.e. independent of nitrate levels) is a variable and may account for any individual to individual variation. Here, we describe a minimally invasive protocol that allows for NR activity determination from human, rat and mouse tongue scrapes/swabs. We validate this method using selective application of antiseptic agents to the distal tongue surface which decreased NR activity by >80% and show that bacterial number is a significant variable in measured NR activities between males and females. Also, we show that NR activity is >80% lower in smokers (humans) and after bromine gas exposure (mice), suggesting that exposure to inhaled reactive substances inhibit NR activity identifying a potentially new mechanism by which environmental toxicants promote dysfunction in NO-bioavailability. The described method will facilitate studies testing whether NR specific activity is a variable in different pathophysiologic settings, and in turn how this activity modulates enterosalivary nitrate-reduction.

Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801.

The assimilatory nitrate reductase (NarB) of N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (K(m)1, ∼1µM; K(m)2, âˆ¼270µM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe-4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site.

Verticillium dahliae toxins-induced nitric oxide production in Arabidopsis is major dependent on nitrate reductase.

The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.

Physiological responses of Ulva pertusa and U. armoricana to copper exposure.

A comparative study of copper (Cu) toxicity and tolerance in two species of Ulva from Korea, the native Ulva pertusa and alien Ulva armoricana, was conducted by examining the effects on growth, pigmentation, chlorophyll fluorescence, antioxidant capacity and nitrate reductase activity. Toxic effects of Cu were less expressed in U. armoricana than in U. pertusa. At lower concentrations (25-50 microgL(-1)), exposure to Cu did not affect thallus growth of U. armoricana, whilst growth was significantly reduced in U. pertusa. An increase in chlorophyll concentrations was observed in U. armoricana exposed up to 100 microgL(-1), whereas Cu caused a significant chlorophyll reduction in U. pertusa. Chlorophyll b was reduced to a lesser extent than chlorophyll a by higher Cu concentrations. In U. armoricana, the maximum efficiency of photosystem II, minimum fluorescence, maximum electron transport rate and non-photochemical quenching were unaffected by Cu except at the highest concentration tested. U. pertusa showed a significant decrease in those parameters at much lower Cu concentrations. It was notable that in this alga the maximum efficiency of photosystem II was reduced at higher Cu concentrations than relative electron transport rate. Elevated concentrations of Cu induced a strong activation of antioxidant activity in U. armoricana, whereas the generation of high levels of reactive oxygen species probably decreased the non-enzymatic antioxidant defense system in U. pertusa. An increase in the nitrate reductase activity of U. armoricana at 50-100 microgL(-1) Cu coincided with the increase in chlorophyll contents, whereas U. pertusa showed a significant decrease at the higher Cu concentration. Differences in the sensitivity of the two species of Ulva to Cu may influence their competitive interactions in Korean coastal waters experiencing temporal increases in the loading of heavy metals.

Probing the role of copper in the biosynthesis of the molybdenum cofactor in Escherichia coli and Rhodobacter sphaeroides.

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803-806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.