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Cited by 311Gut microbiota and healthWastewater Treatment and Nitrogen RemovalProbiotics and Fermented Foods

Nitrate reduction to nitrite, nitric oxide and ammonia by gut bacteria under physiological conditions

Open access
Tiso, Mauro, Schechter, Alan N.·2015·PLOS ONE
DOI 10.1371/journal.pone.0119712PMC PMC4372352
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Abstract

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.

AI snapshot

Written from the paper’s full text.

TL;DR

In vitro cultures of common gut bacteria show that nitrate can be reduced first to nitrite and then to ammonia under oxygen and nitrate conditions relevant to the gastrointestinal tract. E. coli and L. plantarum were the main nitrate reducers, while lactic acid bacteria also generated NO from nitrite largely through acid-driven non-enzymatic reduction.

Key findings

  • E. coli and L. plantarum grown with 5 mM nitrate at low oxygen showed a growth benefit and produced nitrite and ammonia; nitrite and ammonia accumulated when nitrate was at least 2.5 mM, reaching about 260 μM NO2− and 180 μM NH4+ in E. coli media at 5–20 mM nitrate.
  • Time-course experiments at 2% O2 with 5 mM nitrate suggested nitrate is first converted to nitrite and subsequently to ammonia; nitrite peaked at 30–36 h at about 0.4 mM for E. coli and 0.1 mM for L. plantarum.
  • L. rhamnosus, L. acidophilus and B. longum infantis showed minor changes in nitrite or ammonia with nitrate, but all LAB cultures produced NO from exogenous nitrite; LAB lactic acid acidified the medium and drove non-enzymatic nitrite reduction to NO, whereas E. coli converted nitrite to NO even at neutral pH.
  • E. coli NO production from nitrite was low at 2% O2 (<40 ppb/109 CFU) and much larger after anaerobic growth, roughly correlating with increasing nitrate; arginine and L-NAME did not change NO production, arguing against bacterial NOS activity.

Methods

  • In vitro cultures of E. coli (MG1655 in Results; Table 1 lists MG1566), L. acidophilus ATCC 4356, L. plantarum ATCC BAA-793 (WCFS1), L. rhamnosus ATCC 7469, and B. longum infantis ATCC 15697 were grown at 37°C in modified LMRS broth without ammonium citrate, with controlled O2 levels from 0% to 21% and nitrate from 0 to 20 mM; LAB were supplemented with heme and vitamin K2.
  • Nitrite was measured by acidic tri-iodide-based gas-phase chemiluminescence, ammonia by colorimetric kits, NO gas by ozone-based chemiluminescence, and lactic acid enzymatically.
  • Experiments were performed in triplicate, values reported as mean ± SD, nitrite and ammonia normalized to OD600 after 24 h growth, and one-way ANOVA used where applicable.

Limitations

  • The paper states that the importance of this pathway in vivo demands further studies, that how much ammonia comes from nitrate-nitrite reduction versus deamination or urease activity requires detailed animal and/or human metabolic studies, and that the biological significance of dietary nitrate conversion in the intestinal lumen remains to be established.
  • The paper notes that very little information exists on in vivo O2 concentrations in intestinal fluids, so the oxygen conditions used are approximations of gastrointestinal levels.
  • Evident from the text: findings are from in vitro single-strain cultures, not complex gut communities or host tissue; ammonia measurements in cell pellets had large errors and possible ammonia evaporation, leading the authors to measure nitrate metabolites mainly in cell-free supernatant.

Model-written summary. Check it against the paper before relying on it.

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Crossref · 10.1371/journal.pone.0119712

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 35 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The presence of 5 mM nitrate induced nitrite generation in E. coli and L. plantarum bacteria grown at oxygen concentrations compatible with the gastrointestinal tract.

    “We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The O2 level of 2% used in some experiments is similar to the partial pressure found on the luminal surface of the intestinal mucosa.
  • Findingown data

    Nitrite was converted to NO by E. coli cultures even at neutral pH.

    “In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH.”
    Quote in the paperSubstantial claim
  • Findingown data

    At 2% O2, nitrate concentrations equal to or above 2.5 mM had a significant effect on nitrite and ammonia production only in L. plantarum cultures.

    “Nitrate concentrations equal to or above 2.5 mM had a significant effect on nitrite and ammonia production only in the L.plantarum cultures.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The O2 level of 2% used in some experiments is similar to the partial pressure found on the luminal surface of the intestinal mucosa.
  • Findingreasoning

    When supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate.

    “However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate.”
    Quote in the paperSubstantial claim
  • Findingown data

    The presence of 5 mM nitrate induced ammonia generation in E. coli and L. plantarum bacteria grown at oxygen concentrations compatible with the gastrointestinal tract.

    “We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The O2 level of 2% used in some experiments is similar to the partial pressure found on the luminal surface of the intestinal mucosa.
  • May combine two claims. 32
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 6

  • May combine two claims.

    Findingown data

    In LAB cultures with 5 mM nitrate, when O2 tension was 4% or lower, nitrite and ammonia were both generated and excreted in the media and reached the highest concentrations under anaerobic conditions.

    “However when the O2 tension was 4% or lower nitrite and ammonia were both generated and excreted in the media and reached the highest concentrations in cultures grown under anaerobic conditions.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Under 2% O2, small amounts of NO (<40 ppb/10^9 CFU) were detected after injection of 100 μM exogenous nitrite in E. coli cultures almost independently of the nitrate concentration supplied.

    “Under 2% O2 small amounts of NO (< 40 ppb/109 CFU) were detected almost independently from the concentration of nitrate supplied.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In E. coli and L. plantarum cultures grown at 2% O2 with 5 mM nitrate, nitrite concentrations began to increase within 3 to 6 hours after nitrate addition and reached maxima at 30-36 h of about 0.4 mM for E. coli and 0.1 mM for L. plantarum.

    “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 0.4 mM for E.coli and 0.1 mM for L.plantarum (Fig. 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In E. coli and L. plantarum cultures grown at 2% O2 with 5 mM nitrate, ammonia concentrations increased almost linearly and continuously between 9 and 30 h, with different rates between species.

    “Similarly the ammonia concentrations plotted in Fig. 3B show well the almost linear and continuous increase measured between 9 and 30 h in both species but with different rates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 2% O2, a smaller but still considerable effect on nitrite generation was observed in L. rhamnosus and L. acidophilus cultures, and no significant changes were measured in B. longum infantis.

    “A smaller, but still considerable, effect on nitrite generation was observed in L.rhamnosus and L.acidophilus cultures and no significant changes were measured in B.longum infantis.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The O2 level of 2% used in some experiments is similar to the partial pressure found on the luminal surface of the intestinal mucosa.

  • May combine two claims.

    Findingown data

    After anaerobic growth, much larger quantities of NO were measured in E. coli cultures and the response roughly correlated with increasing nitrate concentrations.

    “Inversely, much larger quantities were measured in analogous experiments after anaerobic growth and the response roughly correlated with the increasing nitrate concentrations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In L. plantarum, nitrite concentration increased until around 30 h and afterwards began to decrease while ammonia kept increasing further.

    “Of particular note, nitrite concentration in L.plantarum increased until around 30 h and afterwards begun to decrease while ammonia kept increasing further.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LAB cultures produce substantial acidification of the media due to fermentation of glucose primarily to lactic acid, and lactic acid was confirmed in large amounts (mM) by direct detection.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The initial pH (6.5) decreased variably to values between 3.9 and 5.0 depending on the LAB species, with the least difference for L. plantarum.

    “The initial pH (6.5) decreased variably to values between 3.9 and 5.0 depending on the LAB species and the difference was the least for L. plantarum.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In E. coli cultures grown anaerobically for 24 h, when nitrate reached 2.5 mM, nitrite and ammonia accumulated in the medium and reached maxima around 260 μM nitrite and 180 μM ammonia at 5-20 mM nitrate.

    “However, when nitrate reached 2.5 mM both metabolites accumulated in the media and their concentrations were greatly increased and reached a maximum in the range 5–20 mM nitrate (to around 260 μM for NO2 − and 180 μM for NH4 +).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Replacement of growth media with fresh LMRS resulted in almost complete halt of NO generation in all LAB but only partially in E. coli cultures.

    “This procedure resulted in the almost complete halt of NO generation in all LAB (solid bars in Fig. 4B) but only partially in E.coli cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cultures of B. longum infantis did not grow at or above 6% O2 and showed negligible nitrite content and low ammonia independent of the oxygen level.

    “Of note, cultures of B. longum infantis did not grow at, or above, 6% O2 and showed negligible content of nitrite and low ammonia independent of the oxygen level.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    All LAB cultures grown with 5 mM nitrate at 2% O2 produced a considerable but variable amount of NO after 100 μM nitrite injection, while the same addition to fresh LMRS, with or without nitrate, had no effect.

    “All the cultures produced a considerable but variable amount of NO after 100 μM nitrite injection, while the same addition to fresh LMRS, with or without nitrate, had no effect (bars with oblique lines).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    LAB cultures grown with 5 mM nitrate that grew at O2 levels equivalent to or greater than 6% showed no significant changes in nitrite or ammonia.

    “LAB cultures that grew at O2 levels equivalent or greater than 6% showed no significant changes in nitrite or ammonia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Replacing the growth media with fresh LMRS (pH = 6.5) almost completely blocked the LAB cultures' ability to convert nitrite to NO but not E. coli's.

    “Replacing the growth media with fresh LMRS (pH = 6.5) almost completely blocked the LAB cultures ability to convert nitrite to NO but not in E.coli.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    E. coli NO generation is greatly reduced at 2% oxygen and becomes nitrate independent.

    “This NO generation, however is greatly reduced at 2% oxygen and become nitrate independent.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Active NOS enzymes are absent in E. coli and L. plantarum.

    “Our results also excluded the presence of active NOS enzymes in E.coli and L.plantarum”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    E. coli expresses two nitrite reductase enzymes (Nrf and Nir) that detoxify NO2− by rapidly converting it to ammonia through a six-electron reduction.

    “Alongside with transport E.coli expresses two nitrite reductase enzymes (Nrf and Nir) that detoxify NO2 - by rapidly converting it to ammonia through a six-electron reduction [18].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] J Simon, 2002, Enzymology and bioenergetics of respiratory nitrite ammonification, FEMS Microbiol Rev source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A 5 mM nitrate concentration is compatible with levels found in the upper intestinal tract of healthy volunteers and with values measured in the mouse intestinal mucus.

    “a concentration compatible with levels found in the upper intestinal tract of healthy volunteers and with values measured in the mouse intestinal mucus [24].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] SA Jones, 2007, Respiration of Escherichia coli in the mouse intestine, Infection and Immunity source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The E. coli genome encodes at least three distinct nitrate reductase enzymes that are known to be expressed during anaerobic respiration.

    “The E.coli genome encodes at least three distinct nitrate reductase enzymes [4] that are known to be expressed during anaerobic respiration [17].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] JM Lundberg, 2004, Nitrate, bacteria and human health, Nat Rev Micro source

      Not checked in the free run.

    • cites [17] C Sparacino-Watkins, 2013, Nitrate and periplasmic nitrate reductases, Chem Soc Rev source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of overall mammalian nitrate/nitrite/NO metabolism and is another way in which the microbiome links diet and health.

    “We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Ammonia generated by gut microbiota from nitrate would likely be carried to the liver via the portal vein and enter the urea cycle to be converted into urea and amino acids.

  • May combine two claims.

    Premisecitation

    Many Lactobacilli species can incorporate heme from the environment and utilize menaquinones (vitamin K) to perform respiration.

    “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].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] G Wolf, 1990, Heme-dependent and heme-independent nitrite reduction by lactic acid bacteria results in different N-containing products, Int J Food Microbiol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction.

    “Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    These results indicate that at least two processes producing NO are present in E. coli: one predominates at 2% O2 tension and the other in anaerobic atmosphere.

    “These results indicate that at least 2 processes producing NO are present in E.coli: one predominates at 2% O2 tension and the other in anaerobic atmosphere.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Exogenous addition of menaquinone 4 (vitamin K2) along with heme stimulates nitrate reduction in L. plantarum.

    “Brooijmans et al. [27] have also recently reported that exogenous addition of menaquinone 4 (vitamin K2) along with heme stimulate nitrate reduction in L.plantarum.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Brooijmans et al. [27] RJW Brooijmans, 2009, Lactobacillus plantarum WCFS1 electron transport chains, Applied and Environmental Microbiology source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Many enteric bacteria are capable of catalytic reduction of nitrate to N2 gas (denitrification) under anaerobic conditions, or to ammonia via two-step dissimilatory or assimilatory pathways.

    “Many enteric bacteria are also capable of catalytic reduction of nitrate to N2 gas (denitrification) under anaerobic conditions, or to ammonia via two-steps dissimilatory or assimilatory pathways [17,18].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [17] C Sparacino-Watkins, 2013, Nitrate and periplasmic nitrate reductases, Chem Soc Rev source

      Not checked in the free run.

    • cites [18] J Simon, 2002, Enzymology and bioenergetics of respiratory nitrite ammonification, FEMS Microbiol Rev source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several following studies have proposed different mechanisms of bacterial NO formation independent of respiratory denitrification, such as arginine-dependent bacterial NOS activity, DNRA, and non-enzymatic processes.

    “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].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [15] T Sobko, 2005, Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite, Nitric Oxide source

      Not checked in the free run.

    • cites [32] J Xu, 2001, Evaluation of nitric oxide production by lactobacilli, Appl Microbiol Biotechnol source

      Not checked in the free run.

    • cites [33] H Corker, 2003, Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp, Journal of Biological Chemistry source

      Not checked in the free run.

    • cites [34] J Vermeiren, 2009, Nitric oxide production by the human intestinal microbiota by dissimilatory nitrate reduction to ammonium, Journal of Biomedicine and Biotechnology

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Ji and Hollocher first observed generation of NO from nitrite by E. coli in anaerobic conditions and later concluded that nitrite-dependent NO production was due to respiratory membrane-associated nitrate reductase enzymes.

    “In 1988, Ji and Hollocher first observed the generation of NO from nitrite by E.coli in anaerobic conditions [30] and later concluded that nitrite-dependent NO production was due to the activity of the respiratory membrane-associated nitrate reductase enzymes [31].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [30] XB Ji, 1988, Reduction of nitrite to nitric oxide by enteric bacteria, Biochem Biophys Res Commun source

      Not checked in the free run.

    • cites [31] XB Ji, 1989, Nitrate Reductase of Escherichia-Coli as a No-Producing Nitrite Reductase, Biochemical Archives

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    LAB cultures examined produce large amounts of lactic acid, causing sufficient acidification of culture media to drive non-enzymatic nitrite conversion to NO.

    “We concluded that LAB production of lactic acid causes sufficient medium acidification to induce the chemical nitrite conversion to NO”
    • Also stated in: § Discussion · plain
      “4) All LAB cultures examined generate large amounts of lactic acid causing sufficient acidification of culture media to drive nitrite disproportionation to NO.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Nitrate-rich diets increase plasma and tissue levels of nitrite, but this cannot be accounted solely by nitrate reduction from oral bacteria.

    “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].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [9] M Govoni, 2008, The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash, Nitric Oxide source

      Not checked in the free run.

    • cites [10] NJH Raat, 2009, Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response, Free Radical Biology and Medicine source

      Not checked in the free run.

    • cites [11] E Weitzberg, 2013, Novel aspects of dietary nitrate and human health, Annu Rev Nutr source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    In E. coli, the conversion of nitrite to NO is enzymatic and occurs around pH 6.5.

    “instead this process in E.coli is enzymatic and occurs around pH 6.5.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The normal bacterial flora generates NO and gut luminal NO levels have been measured in vivo in rats.

    “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].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] T Sobko, 2005, Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite, Nitric Oxide source

      Not checked in the free run.

    • cites [16] T Sobko, 2006, Generation of NO by probiotic bacteria in the gastrointestinal tract, Free Radical Biology and Medicine source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In healthy individuals, about one third of daily nitrate intake reaches the lower intestine while only 1% is recovered in the feces.

    “In healthy individuals dietary nitrate is usually well absorbed in the upper intestinal tract, however a considerable fraction of the daily nitrate intake (about 1/3) was found to reach the lower intestine while only 1% of it is recovered in the feces [12].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] B Bartholomew, 1984, The pharmacology of dietary nitrate and the origin of urinary nitrate, Food Chem Toxicol source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Respiratory denitrification can generate small but relevant amounts of NO as an intermediate product and has been implicated in bacterial NO production in the gut.

    “Respiratory denitrification can also generate small but relevant amounts of NO as an intermediate product and has been implicated in the bacterial NO production in the gut [32,33].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] J Xu, 2001, Evaluation of nitric oxide production by lactobacilli, Appl Microbiol Biotechnol source

      Not checked in the free run.

    • cites [33] H Corker, 2003, Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp, Journal of Biological Chemistry source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In contrast to conventional rats, NO levels in the intestine of germ-free rats are extremely low, and inoculation with normal bacteria flora increased observed NO production 10-fold.

    “Sobko et al. [15] showed that in contrast to conventional rats, NO levels in the intestine of germ-free rats are extremely low and when inoculated with normal bacteria flora the observed NO production increased 10 fold.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] T Sobko, 2005, Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite, Nitric Oxide source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    E. coli K12 strains express three molybdenum-containing nitrate reductases.

    “it is well known that E.coli K12 strains express three molybdenum-containing nitrate reductases [17]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] C Sparacino-Watkins, 2013, Nitrate and periplasmic nitrate reductases, Chem Soc Rev source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The major product of nitrite reduction in E. coli is ammonia, with about only 1% being reduced to NO at neutral pH.

    “Cole and colleagues reported that the major product of nitrite reduction in E.coli is ammonia with about only 1% being reduced to NO at neutral pH [51].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [51] J Cole, 1982, Independent pathways for the anaerobic reduction of nitrite to ammonia by Escherichia coli, Biochem Soc Trans source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Unique to L. plantarum, genes encoding a putative nitrate-reductase system (narGHJI) were identified in the L. plantarum WCFS1 genome, suggesting it is capable of using nitrate as an electron acceptor.

    “unique to this species, genes that encode a putative nitrate-reductase system (narGHJI) were recently identified in the L. plantarum WCFS1 genome, which suggests that it is capable of using nitrate as an electron acceptor [27].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] RJW Brooijmans, 2009, Lactobacillus plantarum WCFS1 electron transport chains, Applied and Environmental Microbiology source

      Not checked in the free run.

38 of 38

  • The paper relies on this without support.

    The O2 level of 2% used in some experiments is similar to the partial pressure found on the luminal surface of the intestinal mucosa.

    “with the O2 level fixed at 2%, a partial pressure similar to the one found on the luminal surface of the intestinal mucosa”

  • The paper relies on this without support.

    Ammonia generated by gut microbiota from nitrate would likely be carried to the liver via the portal vein and enter the urea cycle to be converted into urea and amino acids.

    “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.”

  • The paper relies on this without support.

    The in vitro conditions used are compatible with nitrate-rich diets and oxygen levels found on the mucosal surfaces of the GI tract.

    “using in vitro conditions compatible with nitrate-rich diets and oxygen levels found on the mucosal surfaces of the GI tract”

  • The paper relies on this without support.

    The intestinal pH ranges between 5.7 and 7.5.

    “Measurements of the intestine pH ranges between 5.7 and 7.5”

  • The paper relies on this without support.

    Approximately 60% of an ingested nitrate dose in humans is not accounted for in excreted nitrogen-containing compounds.

    “the biological fate of exogenous nitrate could not be accounted for in the excreted nitrogen-containing compounds which amount to approximately 60% of an ingested nitrate dose in humans”

  • The paper relies on this without support.

    The bacterial species and strains studied in vitro are representative of those performing nitrate reduction in the human gut.

    “selected common bacterial species colonizing the human intestine”
6 of 6

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Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • E. coli strain identifier differs between Table 1 and the results

    Minor

    Table 1 identifies the E. coli strain used in the study as MG1566, whereas the results identify the strain in the growth experiments as MG1655. The text does not establish whether these are different strains or a mislabeled strain.

1 of 1

Cites

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Research works printed but not linked: 1

  1. XB Ji, 1989, Nitrate Reductase of Escherichia-Coli as a No-Producing Nitrite Reductase, Biochemical Archives, 5, 61

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Authors
Tiso, M., & Schechter, A. N.
Affiliations
National Institutes of Health (US)
Corresponding author
Schechter, Alan N.
ORCID
Schechter, Alan N.
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-03-24
Volume
10
Issue
3
Pages
e0119712
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0119712
PMC
PMC4372352
OpenAlex
W2016792399
Semantic Scholar
85a9ecdb44af363910ac2a1b792ff39fd2cee804
Cited by
311
References
55
Citations by year
2026: 8 · 2025: 30 · 2024: 41 · 2023: 39 · 2022: 27 · 2021: 32 · 2020: 43 · 2019: 22 · 2018: 32 · 2017: 21 · 2016: 11 · 2015: 4
FWCI
9.85
Type
journal article
Language
en
Update notices
correction (2015-05-06)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, public-domain)
  • Europe PMC (PubMed Central) (publishedVersion, public-domain)
  • PubMed Central (publishedVersion, public-domain)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institutes of Health
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Physical Sciences › Environmental Science › Pollution › Wastewater Treatment and Nitrogen Removal
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
Keywords
nitrate reduction, nitrite, nitric oxide, ammonia synthesis, gut microbiota, Escherichia coli, Lactobacillus, Bifidobacterium, lactic acid
MeSH terms
Gastrointestinal Microbiome, Ammonia, Analysis of Variance, Bifidobacterium, Escherichia coli, Humans, Lactobacillus, Nitrates, Nitric Oxide, Nitrites, Oxygen, Lactic Acid, Nitrogen Cycle

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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