DOI: 10.5061/dryad.stqjq2cf4
title: Distribution and activity of nitrate and nitrite reductases in the microbiota of the human intestinal tract

description: The human intestinal microbiota plays a vital role in health. One of the
 most protective benefits is the bacterial nitrogen metabolism of gut
 bacteria, which reduces nitrate (NO3–) and nitrite (NO2–) to ammonia or
 nitric oxide, preventing the formation of carcinogenic nitrosamines. In
 this study, we shed light on the gut bacterial NO2–/NO3– degradation, its
 efficacy, and the effects on the steady-state NO2– concentration in the
 human colon. Highly abundant gut bacteria that represent the most
 prominent phyla were analyzed for their potential to reduce NO2– or NO3–.
 Escherichia coli showed the greatest efficiency, which indicates a key
 role in the detoxification and the prevention of nitrosamine formation.
 Species of the genera Bacteroides and Phocaeicola also contributed to NO2–
 reduction due to their high abundance. The total activity of stool samples
 was about 620 µmol NO2– h-1, indicating that NO2– concentration in the
 human stool should be very low. We also show that bacterial NO2– reduction
 is necessary to allow NO2–-sensitive microorganisms to colonize the
 intestine, preventing a pathological shift in the composition of the
 intestinal microbiota. The results illustrate that the gut microbiota
 plays a central role in NO2– detoxification, ensuring microbiota integrity
 and potentially preventing nitrosamine formation and gut-associated
 cancers.
description: # Distribution and activity of nitrate and nitrite reductases in the
 microbiota of the human intestinal tract Dataset DOI:
 [10.5061/dryad.stqjq2cf4](10.5061/dryad.stqjq2cf4) ## Description of the
 data and file structure General experimental overview: This dataset
 contains the raw data and derived kinetic parameters used to assess
 nitrite and nitrate reduction by intestinal bacteria under anaerobic
 conditions. Experiments were performed in buffered brain heart infusion
 (BHI) or modified SHIME medium at 37°C under an N~2~/CO~2~ atmosphere.
 Optical density (OD~600~), pH, and nitrite/nitrate concentrations were
 measured at regular time intervals to determine growth and reduction
 kinetics. Nitrite was quantified colorimetrically using the Lunge reagent
 method (absorbance at 540 nm), and nitrate was determined by HPLC. Data
 were obtained from three biological replicates with three technical
 replicates each, unless otherwise indicated.
 \------------------------------------------------------------ **Figure 1
 and Figure 2 – Nitrite degradation by individual strains** Figure 1A:
 *Escherichia coli* (0.1 mM and 1 mM nitrite) and medium control Figure 1B:
 *Bacteroides xylanisolvens* (0.1 mM nitrite) Figure 2A: *Phocaeicola
 dorei* (0.1 mM nitrite) Figure 2B: *Bifidobacterium longum* (0.1 mM
 nitrite) Cells were grown in buffered BHI medium under anaerobic
 N~2~/CO~2~ at 37°C. Samples were taken over 24 h to determine OD~600~, pH,
 and nitrite concentration. The data were used to calculate nitrite
 consumption kinetics and to derive specific nitrite-reducing activities
 (µmol NO~2~^-^ gDW^-1^ h^-1^). Calculation of specific activities: Mean
 values of OD~600~, nitrite concentration, and pH were used for the
 construction of Figure 1 and 2. From these raw data, nitrite degradation
 kinetics were calculated. The functional equations describing NO₂⁻
 concentration versus time were determined using SciDAVis
 ([https://scidavis.sourceforge.net/),and](https://scidavis.sourceforge.net/\),and) their first derivatives were computed using the web tool Ableitungsrechner ([https://www.ableitungsrechner.net/](https://www.ableitungsrechner.net/)). These derivations provided the rate of NO₂⁻ consumption was plotted against the optical density. The slope definied the activity per unit of optical density (µmol OD^-1^ h^-1^). Specific enzymatic activities were normalized to dry weight (g DW) using the conversion factor 1 OD₆₀₀ = 0.735 g DW L⁻¹, which is based on 1.43 × 10¹² cells L⁻¹ and an average cell mass of 2.52 × 10⁻¹³ g cell⁻¹. The resulting values are expressed as µmol NO₂⁻ gDW⁻¹ h⁻¹. Each Excel file (Rawdata_Fig1A.xlsx, Rawdata_Fig1B.xlsx, etc.) contains the complete raw data with the following columns: Sample, Experiment, Nitrite_used_µM, Time_h, OD_600, Av_A540_Blank, A540_0_Nitrite, A540_100_Nitrite_1–3, A540_100_Nitrite_minus_Blank_1–3, Av_A540_100_Nitrite, Slope, A540_Sample_1–3, NO2-_µM_Sample_1–3, pH-value. \------------------------------------------------------------ **Figure 3 and Figure 4 – Synthetic gut mix and fecal microbiota** Figure 3: Synthetic gut mix with *Escherichia coli* Figure 4: Resting fecal microbiota from five human donors (pool of two donors per replicate). The synthetic gut mix consisted of 11 highly abundant intestinal bacterial species selected according to King *et al*. (2022), representing the dominant genera of the human colon microbiota. Each strain was cultivated individually under anaerobic conditions, harvested, and combined according to their relative abundances reported by King et al. to approximate the microbial composition of the large intestine.Cells were washed and resuspended in modified SHIME medium (29.8 mM NaHCO₃, 34.2 mM NaCl, 18.7 mM NH₄Cl, 49.5 mM K₂HPO₄, 49.8 mM KH₂PO₄, 0.07 mM CaCl₂·2H₂O, 0.03 mM MgSO₄·7H₂O, 1 mg L⁻¹ resazurin) containing 1.5 mM L-cysteine. The resulting resting cells were metabolically active but no longer dividing during incubation. Nitrite concentrations were monitored over time under an anaerobic N₂/CO₂ atmosphere at 37 °C, and the data were used to determine specific nitrite-reducing activities (µmol NO₂⁻ gDW⁻¹ h⁻¹). Fecal microbiota samples were prepared in parallel from fresh stool of five donors. Samples were diluted (1:10) in modified SHIME medium containing 5 mM dithiothreitol and immediately flushed with N₂/CO₂ to ensure anaerobic conditions. After removal of debris and washing steps, the bacterial pellets were resuspended to an optical density of 8–10 (OD₆₀₀) in SHIME buffer containing 1.5 mM L-cysteine. The preparations were incubated at 37 °C under anaerobic N₂/CO₂ conditions, and nitrite degradation kinetics were analyzed as described above. Each Excel file (Rawdata_Fig3.xlsx, Rawdata_Fig4.xlsx, etc.) contains the complete raw data with the following columns: Sample, Experiment, Nitrite_used_µM, Time_h, OD_600, Av_A540_Blank, A540_0_Nitrite, A540_100_Nitrite_1–3, A540_100_Nitrite_minus_Blank_1–3, Av_A540_100_Nitrite, Slope, A540_Sample_1–3, NO2-_µM_Sample_1–3, pH-value. \------------------------------------------------------------ **Figure 5 – Nitrite inhibition of bacterial growth** Strains: *Veillonella atypica*, *Agathobacter rectalis*, *Faecalibacterium prausnitzii*. Cells were cultivated in 48-well plates (Greiner CELLSTAR) and growth was monitored in a Tecan Infinite M200 plate reader at 600 nm for 24 h under anaerobic CO~2~/N~2~/H~2~ (49/49/2 %) atmosphere. Nitrite concentrations ranged from 0 to 1000 µM. Data include OD~600~ values for three biological replicates with three technical replicates each. Mean values and standard deviations were calculated, and growth rate (µ), doubling time, and lag phase were determined. The Excel file (Rawdata_Fig5.xlsx etc.) contains the complete raw data with the following columns: Sample, Experiment, Time_s, 0_µM_Nitrite_1–3, 10_µM_Nitrite_1–3, 20_µM_Nitrite_1–3, 50_µM_Nitrite_1–3, 100_µM_Nitrite_1–3, 200_µM_Nitrite_1–3, 500_µM_Nitrite_1–3, 1000_µM_Nitrite_1–3. \------------------------------------------------------------ **Figure 6 – Nitrate degradation kinetics** Nitrate degradation was analyzed for the synthetic gut mix and fecal microbiota samples to complement the nitrite degradation experiments. The gut mix consisted of 11 highly abundant intestinal bacterial species according to King et al. (2022), representing the dominant genera of the human colon. In parallel, fecal microbiota samples were prepared from five donors as described for Figure 4. Cells were resuspended in modified SHIME medium under anaerobic N₂/CO₂ conditions and incubated at 37 °C. Nitrate degradation was quantified by HPLC under anaerobic conditions in BHI or SHIME medium. The Excel file (Rawdata_Fig6.xlsx etc.) contains the complete raw data with the following columns: Sample, Experiment, Nitrate_used_µM, Time_h, OD_600, pH_value, Nitrate_µM_Blank, Nitrate_µM_Sample. \------------------------------------------------------------ **Figure 7 – Abiotic controls (cysteine and pH dependence)** Figure 7A: BHI medium with varying cysteine concentrations (0–4 mM) incubated without cells for 24 h under anaerobic conditions with 150 µM nitrite at 37°C. Figure 7B: BHI medium with varying pH values incubated anaerobically for 24 h with 100 µM nitrite at 37°C. Nitrite concentration was determined colorimetrically (Lunge reagent method). Each measurement was performed in triplicate. \------------------------------------------------------------ **Tables 2–4 – Kinetic parameters and specific activities** Tables summarize the specific nitrite- and nitrate-reducing activities of intestinal bacterial strains expressed as µmol gDW⁻¹ h⁻¹. Table 2 contains the specific nitrite-reducing activities of the following strains: *Agathobacter rectalis, Akkermansia muciniphila, Bacteroides cellulosilyticus, Bacteroides xylanisolvens, Bifidobacterium longum, Escherichia coli K-12, Faecalibacterium prausnitzii, Hominimerdicola aceti, Phocaeicola dorei, Phocaeicola vulgatus, and Roseburia intestinalis*. Table 3 lists nitrite-reducing activities of lactic acid bacteria and species of the genera *Streptococcus* and *Veillonella*: *Lacticaseibacillus casei, Limosilactobacillus balticus, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Streptococcus hyointestinalis, Streptococcus thermophilus, Streptococcus vestibularis, Veillonella atypica, Veillonella magna, and Veillonella ratti*. Table 4 summarizes the nitrate-reducing activities of the following strains, determined by HPLC under anaerobic conditions: *Agathobacter rectalis, Akkermansia muciniphila, Bacteroides cellulosilyticus, Bacteroides xylanisolvens, Bifidobacterium longum, Escherichia coli* *K-12, Faecalibacterium prausnitzii, Phocaeicola dorei, Phocaeicola vulgatus, Roseburia intestinalis, Limosilactobacillus reuteri, Streptococcus vestibularis, and Veillonella atypica*. Each Excel file (Rawdata_Tab2.xlsx, Rawdata_Tab3.xlsx, Rawdata_Tab4.xlsx etc.) contains the corresponding raw data with the following columns: Sample, Experiment, Substrate_used_µM (Nitrite or Nitrate), Time_h, OD_600, pH_value, Substrate_µM_Blank, Substrate_µM_Sample, Rate_µmol_gDW⁻¹_h⁻¹, and Remarks (if applicable). \------------------------------------------------------------ **Column descriptions (all raw data files):** Sample – bacterial strain or condition Experiment – replicate identifier Nitrite_used_µM / Nitrate_used_µM – initial substrate concentration Time_h/sek – sampling time (hours or seconds) OD_600 – optical density at 600 nm (unitless) Av_A540_Blank – mean absorbance of blank A540_0_Nitrite – absorbance of 0 µM nitrite standard A540_100_Nitrite_1–3 – 100 µM nitrite standard replicates A540_100_Nitrite_minus_Blank_1–3 – blank-corrected nitrite standards Av_A540_100_Nitrite – mean of corrected nitrite standards Slope – calibration slope (A540 per µM NO2-) A540_Sample_1–3 – sample absorbance values NO2-_µM_Sample_1–3 – calculated nitrite concentrations (µM) pH-value – pH at sampling time \------------------------------------------------------------ **Abbreviations and units:** OD600 – optical density at 600 nm (unitless) A540 – absorbance at 540 nm (unitless) µM – micromolar gDW – grams dry weight SD – standard deviation NO2- – nitrite NO3- – nitrate ### Files and variables #### File: Rawdata_Hager_et_al._2025_Revised.zip **Description:** All data are provided in `.xlsx` (Microsoft Excel) format. Each file contains raw or processed data that were used to generate the corresponding figure or table in the manuscript. Files in `.docx` format contain supplementary explanations. ## Code/software For data analysis and visualization, we used a combination of open-source and commercial software tools. Sequence similarity searches were performed using **BLASTp** (NCBI BLAST+), always in the latest available version at the time of analysis. These searches were conducted against the IMG database to identify genes encoding nitrate and nitrite reductases, using well-characterized reference sequences under default parameters (E-value threshold 1e-40). For scientific plotting, we used **Veusz** version **3.6** (open-source), which enabled detailed graphical representation of kinetic data such as nitrite degradation and bacterial growth curves. Raw data from Excel files were imported into Veusz, where curves were plotted, annotated, and arranged in multi-panel layouts. In addition, **GraphPad Prism** version **10.0.2** (GraphPad Software, San Diego, USA) was used for graphical visualization. Prism was employed to generate dose-response and bar plots, and to visualize standard deviations and biological replicates across experimental conditions. To calculate kinetic parameters from time-dependent concentration changes, we used **SciDAVis** (version 2.8) for curve fitting and function modeling. The resulting mathematical functions were then analyzed using the **online derivative calculator** ([https://www.ableitungsrechner.net](https://www.ableitungsrechner.net)) to determine reaction rates by computing first derivatives. ## Access information Other publicly accessible locations of the data: * This dataset is not available from any other publicly accessible repository. It is exclusively provided through Dryad in connection with the associated manuscript. Data was derived from the following sources: * All data were generated by the authors as part of the study. No external datasets were used. Gene and protein information used in BLASTp analyses were obtained from the Integrated Microbial Genomes (IMG) database ([https://img.jgi.doe.gov/](https://img.jgi.doe.gov/)). ## Human subjects data Human subjects de-identification statement: All human-derived fecal samples used in this study were obtained in an anonymized form from collaborating institutions. No personally identifiable information (PII) or metadata that could link the samples to individual donors was provided to, or collected by, our laboratory. The samples were used exclusively for microbial community and biochemical activity analyses, and no human genetic or clinical data were generated. Because donor identification was not possible at any stage of the study, no additional de-identification procedures were necessary. The use of anonymized fecal material complied with institutional ethical standards and applicable data protection regulations.