Lupinus on 2 mM nitrate and 2 mM nitrite under anaerobiosis. Protein content was measured when the culture entered the stationary growth phase. Biomass production at 0 mM nitrate or nitrite resulted from fermentative metabolism.
Each value was calculated by substraction of initial protein content from total protein produced per liter. Anaerobic growth of Bradyrhizobium sp.
Lupinus in non-fermentable minimal medium. In each variant, yeast extract and mannitol were substituted with glycerol as non-fermentable carbon source.
The control variant contained no N oxyanions. Anaerobiosis induced NRA to a level six times higher than the activity in air Table 1. Nitrate addition 2 mM enhanced induction to a factor 2. Nitrite 2 mM was shown to be an equally effective inducer of the enzyme.
NRA of anaerobically grown bacteria was not affected by the presence of 10 mM ammonium Table 1. As a consequence, ammonium addition to anaerobic culture had no influence on nitrate utilization data not shown.
The data presented indicate that the enzyme is of respiratory type [4]. Simultaneously given 2 mM nitrate and 2 mM nitrite caused a twice as high induction of the enzyme than when given separately Table 1. This implies that nitrate and nitrite cause induction or depression of dissimilatory NR through supplementary regulatory pathways see discussion below.
The lack of reduction of higher nitrate levels was apparently due to intracellular accumulated nitrite, which could cause inhibition of nitrate uptake [16]. As a result, nitrate transport came to a halt before completion of its conversion. Anaerobic reduction of 2 mM nitrate panel A or 2 mM nitrite panel B to ammonium in cells of Bradyrhizobium sp.
Lupinus in the presence of MSX — an inhibitor of glutamine synthetase. In the absence of MSX, nitrite excretion and N oxyanion reduction proceeded in the same way not shown. The data are representative of five replicate experiments. MSX, l -methionine- d,l -sulfoximine. Levels of nitrate remaining and nitrite accumulated during anaerobic growth of Bradyrhizobium sp.
Lupinus with different concentrations of nitrate added. The concentrations of ions were measured after a cultivation time prolonged up to 25 h. Due to nitrite accumulation, the optimal initial concentration of nitrate was 2 mM. Anaerobiosis induced NiRA to a level two times higher than the activity in air Table 1. Nitrate addition 2 mM enhanced anaerobic induction two-fold through de novo enzyme synthesis. Nitrite 2 mM was shown to be a much better inducer, enhancing anaerobic enzyme induction five-fold.
Addition of 10 mM NH 4 Cl did not inhibit nitrate induction and only slightly lowered nitrite induction Table 1. As a result, ammonium had no influence on in vivo anaerobic nitrite reduction Fig. The data presented indicate that the enzyme is of dissimilatory type [4]. Simultaneously given 2 mM nitrate and 2 mM nitrite caused a much higher induction of the enzyme than when given separately, which is similar to the effect of these ions on NR induction Table 1. This demonstrates that in cells of B.
Lupinus such a synergy of nitrate and nitrite is a common phenomenon for anaerobic induction of both NR and NiR activities. These regulators are not supplementary since NarL and NarP bind competitively at the activation sites of the promoter region of the narG gene of NR [17] as well as of the nirB gene of NiR [18].
Considering that model, it is not clear how in the cells of B. Lupinus the induction of the enzymes in response to one N oxyanion can be stimulated by the presence of another. We propose that such a synergistic effect of N oxyanions on NR and NiR activities is a consequence of stimulation of nitrate transport by nitrite.
Accumulation of intracellular nitrate could in turn enhance induction of both membrane-bound NR and cytoplasmic NiR activities. Nevertheless, it should be noted that a substantial part of anaerobic nitrite reduction in B. Lupinus USDA could be associated also with the periplasmic form of the enzyme see below.
As a result, nitrite still remained in the medium after growth ceased data not shown. Anaerobic nitrate reduction led to external nitrite accumulation. These results indicate that the presence of nitrate at a concentration higher than 0. Several mechanisms of such an inhibition are conceivable [19]. Higher nitrate concentrations also had no inhibitory effect on NiRA induction manuscript in preparation.
Moreover, in vivo nitrite reduction could start after a lag phase no longer than for nitrate reduction compare Fig. According to this explanation, nitrite accumulates due to a lack of balance between nitrate and nitrite reduction rate.
Since ATP is produced from the nitrate-to-nitrite step but not from nitrite reduction, it is more advantageous for the organism to divert its limited electron flow to the energy-producing step. This could result in nitrite accumulation. However, after nitrate depletion the need for a high-capacity electron sink became important, so nitrite reduction is expected from that moment [19]. Transient nitrite accumulation, depicted in Fig.
Under such conditions NiR should be more efficient in competition for electron donors with NR due to a decreased nitrate transport rate. As expected, nitrite accumulation did not occur even at an initial concentration of nitrate as high as 10 mM Fig. Moreover, addition of 10 mM nitrate doubled net protein production data not shown.
These results indicate that nitrite reduction in the presence of higher nitrate concentrations is possible but at temperatures more typical of the natural habitat of rhizobia. Higher temperatures of growth led to a lowered rate of nitrite reduction, most probably due to the unbalanced kinetic parameters of reduction of nitrate, which oxidizes the bulk of NADH [19].
Anaerobic reduction of 10 mM nitrate in cells of Bradyrhizobium sp. A: In the absence of MSX. B: In the presence of MSX. This indicates that the investigated strain has the capacity not only for respiration but also for efficient anaerobic assimilation of N oxyanions. An essential stage of such a process is ammonia production. These findings prompted the intriguing question if the investigated strain could activate ammonia-producing NiR under anaerobiosis. Lupinus were found to be located within the cytoplasm manuscript in preparation , which is in general agreement with the location of ammonia-producing NiR of E.
Nevertheless, a search for this product in anaerobic culture of B. Lupinus showed only a trace level of ammonium accumulation. Ammonium was eventually found as the end product of anaerobic reduction of both nitrate and nitrite when MSX l -methionine- d,l -sulfoximine , a potent inhibitor of glutamine synthetase, was used Fig.
Anaerobic ammonium excretion during nitrate reduction attests to a dissimilatory ammonification process [4 , 19]. There was no evidence of the presence of such a pathway in rhizobia so far. Nitrate respiration is the first reaction in this pathway and provides ATP through electron transport phosphorylation. The capacity for accepting six electrons per one reduced nitrite ion allows a very efficient reoxidation of NADH. Therefore, an electron-sink function is the most postulated role for the dissimilatory nitrite ammonification process [4].
Two explanations of the deficit in nitrate conversion to ammonium are conceivable. One of them is partial ammonium incorporation into amino acids even in the presence of MSX.
The investigated strain was capable of efficient ammonium assimilation under anaerobiosis data not shown and Fig. The second possibility is that nitrate is converted into gaseous N due to the activity of an additional — denitrifying — form of NiR.
Detection of denitrification products has not been carried out, but in the periplasmic fraction of USDA cells we detected methylviologen-dependent NiRA, which confirms such a supposition manuscript in preparation.
O'Hara and coworkers [21] reported that N 2 O is the end product of nitrate reduction in free-living cells and in bacteroids of B. Lupinus , which indicates a complete denitrification system.
The recently completed identification of the nucleotide sequence of the Sinorhizobium meliloti genome uncovered genes coding for two nitrite reductases [22]. One of them is strictly denitrifying, encoded by the nirK gene, and the second is soluble, NADH-dependent and ammonia-producing NiR, encoded by the nirB gene.
Recently, in B. In cells of B. Lupinus , the effect of ammonium on aerobic NiRA reflected the effect on anaerobic nitrite reduction. Under both aerobic and anaerobic conditions, addition of 10 mM NH 4 Cl did not inhibit induction of NiRA by nitrate and only slightly lowered nitrite induction Table 1. This indicates that NiR induction was not repressed by conditions of nitrogen excess regardless of the oxygen state of cells. Such results prompt the question if both activities are driven by a single enzyme form of dissimilatory type.
The work of Ka et al. Considering the fact that NiRA was detected in the periplasmic fraction of anaerobically grown USDA cells manuscript in preparation , it is conceivable that such a constitutive dissimilatory NiR is also present in this strain.
Additionally, since under aerobic conditions low levels of ammonium extraction were detected data not shown , a second, ammonia-producing form of aerobic NiR seems to occur apart from the periplasmic one. Since nitrite ammonification was proved in USDA cells also under anaerobiosis, the question remains to be elucidated if this process could be driven by a single enzyme form regardless of oxygen conditions.
In Bacillus subtilis the soluble, ammonia-producing nitrite reductase, encoded by homologs of E. Nevertheless, aerobic nitrite reduction in B. This is significantly different from the regulation observed in B. Lupinus cells, where periplasmic NiRA could mask the response of the cytoplasmic enzyme. O'Hara G. Daniel R. Soil Biol. Google Scholar. Sprent J. Raven J. Burris R. Evans H. Chapman and Hall , New York. Google Preview. Philipott L.
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Transport and phosphorylation of glucose, fructose and mannitol by Pseudomonas aeruginosa. Archives of Biochemistry and Biophysics : — Rigand, J. Effect of nitrite upon leghemoglobin and interaction with nitrogen fixation. Biochimica et Biophysica Acta : — Rowe, J. Nitrite inhibition of active transport and of respiration in Pseudomonas aeruginosa. Strahler, B. Firefly luminescence in the study of energy transfer mechanisms. In contrast, nitrite was converted to NO by E.
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Competing interests: Gladwin, M. United States Patents 20,,,; 20,,,; 20,,, Nitric oxide NO is a highly diffusible short-lived free radical gas, permeating biomembranes with a wide range of physiological functions [ 1 ].
Nitrate in the human intestine originates both from endogenous synthesis [ 7 ] and dietary products rich in nitrate [ 8 ]. Recent and past data have demonstrated that nitrate-rich diets increase plasma and tissue levels of nitrite [ 9 , 10 ], but this cannot be accounted solely by nitrate reduction from oral bacteria and other mechanisms have been implicated and are under investigation [ 11 ].
The metabolic fate of this unaccounted nitrate is still poorly understood. More recently, the normal bacterial flora has been shown to generate NO and gut luminal NO levels have been measured in vivo in rats [ 15 , 16 ]. Many enteric bacteria are also capable of catalytic reduction of nitrate to N 2 gas denitrification under anaerobic conditions, or to ammonia via two-steps dissimilatory or assimilatory pathways [ 17 , 18 ].
We hypothesize that the nitrogen imbalance detected in the early metabolic studies cited above could be at least in part attributed to the gut microbiota conversion of nitrate to ammonia via nitrite reduction. The ammonia thus generated would likely be carried to the liver via the portal vein, where it can enter the urea cycle and be converted into urea and amino acids.
However, as oxygen diffuses from the tissues underlying the mucosa, microbial activity will reduce its content, and the lumen of the colon has been considered for many aspects an anaerobic region.
In this study we investigated the formation of nitrite, NO and ammonia in cultures of representative species of gut bacteria grown with added nitrate under controlled oxygen concentrations existing in the human gastro-intestinal tract.
In particular we selected Escherichia coli , the best understood enteric bacteria, and four different species of lactic acid bacteria listed in Table 1 that have been previously shown to generate a substantial amount of NO when supplemented with 0.
Our findings suggest that, in the presence of relative high physiological nitrate concentrations, Escherichia coli and Lactobacillus plantarum , two common bacterial species colonizing the human intestine, generate nitrite and subsequently ammonia in an oxygen-dependent fashion. The importance of this pathway in vivo demands further studies. However, we found that different batches of LB broth from different vendors contained a considerable, but variable, amount of ammonia and therefore it was not considered suitable for this study and used exclusively for the preparation of E.
Nitrate was added as a filter-sterilized solution. Lactic acid bacteria cultures were supplemented, when indicated, with hemin stock solution: 0. All reagents were purchased from Sigma-Aldrich unless otherwise specified.
A full list of bacteria strains used in this study is in Table 1. Bacterial cell concentration was monitored by measuring the optical density OD at nm using a 1 cm pathlength cuvette. Typically, one hundred microliters of a 4 to 6 hours old inoculum of each strain with OD at nm between 0.
The bacteria cultures were agitated using either a magnetic stirrer or a Micromixer Mxi4t. The resulting suspension was sealed to prevent ammonia evaporation and used immediately to estimate ammonia and nitrite. The supernatant was used for nitrite and ammonia determination within 14 days.
This prevented the loss of ammonia content in the samples as determined by comparison with standards prepared from 10 mM NH 4 Cl.
To accurately measure nitrite concentration in cultures media and pellets after bacterial growth we used an acidic tri-iodide-based gas phase chemiluminescence method with a Sievers NO analyzer instrument NOA, model i, GE Analytical Instruments, Boulder, CO, USA as described previously [ 22 ]. Ammonia concentrations in all culture samples were determined using two commercially available colorimetric assay kits optimized for 96 well plate reader BioVision Inc. For the non-enzymatic reaction, samples were deproteinized prior to testing using a 10kDa cutoff spin column filter.
Experiments were carried out as following: 10 to mL of bacteria with their growth media at OD approximately 1. Once a stable baseline was established the indicated amount of nitrite was injected in the mixture as previously described [ 23 ].
We verify that the release of NO into the gas phase from the solutions can be used as a continuous measurement for the NO production building a calibration curve with amounts of NO produced by the injection of sodium nitrite standards into an 0. The assay was performed on the supernatant of cultures obtained after centrifugation at rpm for 10 min and diluted appropriately.
Data were analyzed using Origin 8. To account for the small growth differences between each bacterial batch the values for nitrite and ammonia determined in the cell free supernatant were normalized using the OD at nm measured after 24 h growth. Analysis for statistically significant differences among mean values was done, when applicable, using the one-way analysis of variance.
Error bars represent the SD of the measurement. We first compared E. In Fig. The presence of 5 mM nitrate provided a clear growth benefit to E. A Growth curves for E. B Concentration of nitrite and ammonia blue and red solid lines in E. The ammonia content of LMRS alone is indicated by the dashed lines. The average SD resulted smaller than the symbols dimensions 0. The E. These enzymes use nitrate as electron acceptor and produce nitrite which become toxic to the cell upon reaching high intracellular concentrations and is therefore transported outside the cell wall [ 25 ].
Alongside with transport E. However, it is unknown how O 2 levels affect these processes. We therefore measured both nitrite and ammonia in cell pellets Fig. The amount of ammonia detected followed the same trend but was at least 5 to 6 fold lower respect to the nitrite concentrations.
We then determined nitrite and ammonia in the media of E. Nitrite and ammonia concentrations remained steady when nitrate concentrations were lower or equal to 1. However, when nitrate reached 2. Lactic acid bacteria LAB are facultative anaerobe organisms that grow in abundance in the digestive tract of vertebrate animals. LAB also represent some of the most commonly used probiotic bacteria and are extensively used for the production of fermented foods yogurts, cheeses, sausages, pickles, etc.
It was believed that LAB depend strictly on a fermentative mode of metabolism since they do not possess heme containing enzymes essential for the respiratory chain. However, over the past 30 years it has been shown that many Lactobacilli species can incorporate heme from the environment and utilize menaquinones, also known as vitamins K , to eventually perform respiration [ 26 ]. In this regards, it is important to note that E. Brooijmans et al. We then grow single cultures of L.
Nitrate concentrations equal to or above 2. A smaller, but still considerable, effect on nitrite generation was observed in L. We then fixed the nitrate concentration in the bacterial cultures to 5 mM, a level sufficient to show a clear effect on nitrite and ammonia generation both in E.
Of note, cultures of B. We measured relatively high concentrations of nitrite and ammonia in E. Previous works on different E. However this enzyme is subject to repression by oxygen and induction by high nitrite concentrations. In order to limit the effects of the different growth rates between species and batch cultures, in this experiment nitrate was added after the organisms exponential phase of growth upon cultures media reaching OD about 1. We found that nitrite concentrations begun to increase within 3 to 6 hours after nitrate addition and accumulated steadily to reach a maximum in 30—36 h at about 0.
Similarly the ammonia concentrations plotted in Fig. Of particular note, nitrite concentration in L. The formation of ammonia from nitrate indeed is proposed to occur via nitrite in two successive elementary steps, each with its rate law and characteristic kinetic parameters. We plotted this ratio in Fig. This result suggests that nitrate is first converted to nitrite and after some accumulation it is reduced to ammonia or other reduced nitrogen compounds.
Samples from E. Ratio between nitrite and ammonia concentrations measured at each time point. Black lines represent E. However, several following studies on bacterial NO formation have proposed different mechanisms independent of respiratory denitrification such as arginine dependent bacterial NOS enzymatic activity, DNRA and non-enzymatic processes [ 15 , 32 — 34 ]. Inversely, much larger quantities were measured in analogous experiments after anaerobic growth and the response roughly correlated with the increasing nitrate concentrations.
These results indicate that at least 2 processes producing NO are present in E. LAB cultures are well known to produce substantial acidification of the media due to the fermentation of glucose primarily to lactic acid and we confirmed its formation in large amounts mM by direct detection.
The concentrations measured at 24 h are reported in Table 1 together with the corresponding culture broth final pH. The initial pH 6. To elucidate if this acidification could be responsible for the generation of NO by the known non-enzymatic nitrite disproportionation, each bacterial preparation was split in equal volumes 10 ml and tested before and after the replacement of its growth media with fresh LMRS by short centrifugation and decantation. Lysis by brief sonication in fresh media of the LAB cells to extract the cytosolic enzymes did not restored the generation of NO after injection of nitrite.
We concluded that LAB production of lactic acid causes sufficient medium acidification to induce the chemical nitrite conversion to NO, instead this process in E. Increasing amounts of NO were produced by the non-enzymatic nitrite disproportionation as the media pH decreased. The logarithmic plot of the total amount of NO detected ppb versus the pH revealed a linear correlation, similar to previously reported results for acidified nitrite solution in MRS broth or phosphate buffer [ 32 ].
The human microbiota comprises more than a thousand distinct bacterial species [ 35 ] and plays a major role in human health by promoting nutrient supply, preventing pathogen colonization and shaping and maintaining normal mucosal immunity. Commensal gut bacteria have recently been appreciated as having a true symbiotic relationship with the host [ 36 , 37 ]; within this large pool of bacteria, probiotic supplements containing LAB i.
Lactobacilli and Bifidobacteria have been claimed to have a variety of beneficial effects on human health, such as prevention of diarrhea and inflammatory bowel disease or prophylaxis of urogenital infections [ 38 ].
However, our knowledge of the biochemical roles that specific species and strains play in human health and disease is severely limited. In this study we aimed to advance the understanding of the nitrate reduction pathways in selected common bacterial species colonizing the human intestine using in vitro conditions compatible with nitrate-rich diets and oxygen levels found on the mucosal surfaces of the GI tract.
The primary findings of our investigation indicate that: 1 E. Most eukaryotes derive their energy primarily through oxidative phosphorylation and must breathe O 2 for the formation of ATP, however many enteric bacteria, including E. It has also been recently shown that nitrate generated as a by-product of host inflammation can be used by E. This bacterium presents the typical facultative heterofermentative pathway of the LAB family but, unique to this species, genes that encode a putative nitrate-reductase system narGHJI were recently identified in the L.
Indeed a recently published genetic analysis of L. In our experiments significant nitrate reductase activity was detected both in E.
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