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RESEARCH ARTICLE
Price et al., Access Microbiology 2025;7:001078.v3
DOI 10.1099/acmi.0.001078.v3
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Iron oxidation by Salmonella enterica serovar Typhimurium strain SL1344 is an abiotic byproduct of nitrate respiration
Alex Price, Ryan Kingston, Gary Rowley, Karen Olsson-Francis1 and Michael C. Macey1,*
Abstract
Background. Nitrate-dependent iron (Fe2+) oxidation (NDFO) is an important biogeochemical process, but whether NDFO provides a direct metabolic benefit to facultative anaerobes with diverse lifestyles, such as Salmonella enterica, and what role the respiratory nitrate reductases play in this process is unknown.
Methods. This study investigated NDFO in S. enterica serovar Typhimurium strain SL1344. We compared the WT strain with a ∆narGHIJ ∆narZYW mutant (∆nar), which lacks the primary respiratory nitrate reductase (Nar). Cultures were grown anaerobically in lysogeny broth medium amended with 4 mM nitrate and 10 mM FeSO4 as defined nitrate and iron sources. Growth, nitrate depletion, nitrite accumulation, and Fe2+ oxidation were monitored over 14 days. Abiotic controls amended with either nitrate or nitrite were included to control for abiotic iron oxidation.
Results. There was no significant difference in the growth rate and biomass production between the WT and ∆nar strains. However, there was significant variation in nitrite reduction and iron oxidation. 56.9% of the nitrate was depleted from the growth medium (initially 4 mM) with the WT cultures compared to 19.7% with the ∆nar strain. Fe2+ oxidation in the WT cultures reached a Fe²+/Fe total ratio of ~0.52–0.61 during exponential growth and was consistent during the stationary phase, whereas for the mutant, the maximum Fe²+/Fe total ratio was ~0.78, which returned to a more reduced state in stationary phase (Fe²+/ Fe total ~0.95). Abiotic controls amended with nitrite showed rapid Fe2+ oxidation, highlighting the role of nitrite as an oxidant.
Conclusion. The respiratory nitrate reductases are the primary drivers of NDFO in S. enterica SL1344 and are required for the generation of nitrite, which then abiotically oxidizes Fe2+ to Fe3+. This did not confer a growth advantage, suggesting NDFO is an indirect consequence of nitrate respiration rather than a direct energy-conserving pathway in this organism.
DATA SUMMARY
The data supporting the conclusions of this study are provided within the manuscript and the thesis of Dr. Alex Price (2020), titled ‘Microbial Nitrate Dependent Fe Oxidation: A Potential Early Mars Metabolism’, available on the Open University Repository Online at https://oro.open.ac.uk/72495/. This includes the results of the Bradford assay, nitrate, nitrite and iron concentration and speciation data and photographic logs of the cultures over the experiment.
INTRODUCTION
The geobiological process of nitrate- dependent Fe2+ oxidation (NDFO) metabolism was identified 30 years ago in environmental studies of anoxic soils, waters and sediments [1–3]. NDFO has been reported in various growth conditions, including apparent autotrophy and mixotrophy [2, 4–7]. However, the exact biochemical mechanisms underpinning NDFO are yet to be fully resolved. Several pathways have been proposed, including the direct enzymatic oxidation of Fe²+ by specialized cell surface- associated oxidoreductases and indirect oxidation via biologically produced reactive intermediates, most notably nitrite (NO₂⁻), which subsequently oxidizes Fe²+ abiotically [8–10]. Recent isotopic analysis confirmed that NDFO can be biologically induced, but that it is likely not directly coupled to the enzymatic mechanism of nitrate reduction [11] and that it plays a minor role relative to abiotic chemodenitrification, where nitrite reacts with Fe2+ in the absence of biological activity or enzymatic systems [12].
The specific enzymatic systems responsible for nitrate reduction are central to understanding NDFO. Many facultative anaerobes possess two main nitrate reductase systems: the membrane- bound respiratory nitrate reductase (Nar), which contributes to energy conservation via proton motive force generation, and the periplasmic nitrate reductase (Nap) [13]. The Nar system, due to its role in energy transduction, has been considered a candidate for direct involvement in energy- yielding NDFO processes [5]. Conversely, the Nap respiratory nitrate reductase is proposed to have no role in promoting NDFO, as it is located within the periplasm, meaning Nap does not generate a proton motive force. Therefore, Nap does not contribute to ATP production [12] and the yield of energy, except when coupled to formate oxidation [14, 15].
This study aimed to identify the biochemical mechanisms of NDFO using a well- characterised and genetically tractable model: Salmonella enterica serovar Typhimurium strain SL1344 [16]. This study sought to (i) quantify the relative contribution of the respiratory Nar enzyme system to Fe²+ oxidation during nitrate reduction, (ii) investigate the role of nitrite accumulation and (iii) determine whether NDFO confers any growth advantage to this organism under the conditions tested.
METHODOLOGY
Cultivation of S. enterica serovar Typhimurium strain SL1344
Experiments were performed with cultures of S. enterica serovar Typhimurium strain SL1344 WT and the ΔnarGHIJ ΔnarZYW mutant, in which the nar genes coding for the Nar respiratory nitrate reductase enzymes were deleted [17]. The complete genome of the SL1344 strain has been sequenced previously [18] and the WT genome contains two copies of the Nar respiratory nitrate reductase (narGHIJ and narZYW), which were absent in the mutant. The mutant was created using the lambda red recombination method [19] targeting the nar genes that code for subunits of the Nar nitrate reductase enzyme, which were replaced with a kanamycin resistance gene [17]. The SL1344 genome also contains genes for the Nap periplasmic nitrate reductase, which remained unaffected in both the WT and the mutant strains.
Anaerobic cultures were prepared by growing the Salmonella strains (WT and ΔnarGHIJ ΔnarZYW mutant) in anoxic lysogeny broth (LB) medium [20], as this is a medium confirmed to support robust anaerobic growth by Salmonella [21, 22]. The medium was prepared by adding 10 g l-1 tryptone, 5 g l-1 yeast extract and 10 g l-1 NaCl to boiled distilled water, which was cooled under a continuous flow of N2 gas. The medium was then autoclaved (121 °C for 15 min at a pressure of 15 psi). A series of 10 ml aliquots was pipetted into Wheaton bottles under a continuous flow of N2 and then stoppered and crimped with aluminium seals [23]. Using a needle attached to the gas mixture with a 0.22-µm filter, the vials were flushed with a 90 : 10 N2:CO2 gas mixture. For the SL1344 WT or ΔnarGHIJ ΔnarZYW strains, the medium was inoculated to an initial concentration of 105 cells ml-1 using sterile syringes and needles, and the vials were incubated at 37 °C.
To compare the mutant to the WT strains, triplicate cultures of SL1344 WT and SL1344 ΔnarGHIJ ΔnarZYW were established. In parallel to the biotic test groups, two abiotic control test groups were established to control for abiotic iron oxidation – these were uninoculated media amended with nitrate and uninoculated media that were amended with nitrite. For all test groups, anoxic LB medium (19.3 ml) was dispensed into serum Wheaton bottles under an 80 : 15 : 5 N2:CO2:H2 atmosphere in a COY anaerobic glove box. Nitrate was added to the SL1344 WT and ΔnarGHIJ ΔnarZYW cultures and to the nitrate- amended abiotic control series, whilst nitrite was added to the nitrite- amended abiotic control. The final concentration of nitrate (or nitrite for the nitrite- amended series) in the medium was 4 mM. The biotic test groups were inoculated to an initial concentration of 105 cells ml-1 of either the SL1344 WT or ΔnarGHIJ ΔnarZYW. The final Fe2+ concentration in the medium was 10 mM, with Fe2+ supplied to the medium from a 1M anoxic stock solution of FeSO4. The Wheaton bottles were sealed with butyl rubber stoppers and aluminium crimping, before flushing with 90 : 10 N2:CO2 and incubation at 37 °C (Fig. 1).
Analysis of the cultures
At specified time points (0, 1, 2, 3, 4, 7 and 14 days), 1.5 ml samples were aseptically withdrawn from each series (n=3) using a sterile N₂-flushed syringe after gentle swirling to ensure homogeneity (Fig. 1). For immediate Fe2+/Fe3+ measurements, 20 µl aliquots were processed with the Ferrozine assay. Five hundred microlitres aliquots for protein analysis and 100 µl aliquots for dissolved nitrate/nitrite analysis were transferred to 2- ml microcentrifuge tubes and stored at -20 °C until analysed.
Ferrozine assay
Fe2+/Fe3+ was measured spectrophotometrically using the Ferrozine assay [21]. For this analysis, 40 µl of fluid was collected from each test group, and a 20 µl aliquot was mixed with 0.5M HCl to a total volume of 1 ml, to enable measurement of the oxidized iron concentration. The other 20 µl was mixed with 0.5M HCl and 0.3M hydroxylamine hydrochloride to a total volume of 1 ml, converting the Fe3+ to Fe2+ and enabling measurement of the total iron concentration. After 1 h of incubation at room temperature, the samples were refrigerated at 4 °C for later analysis. Twenty microlitres of these incubated fluids was then added to Ferrozine

Fig. 1. Experimental setup for Nar enzyme knockout experiment. Anaerobic LB batch cultures, amended with 4mM NO3⁻, were inoculated with (i) SL1344 wild- type (WT), (ii) SL1344 ∆narGHIJ ∆narZYW mutant (∆nar), (iii) sterile LB media with 4 mM NO2⁻ (nitrite control) or (iv) sterile LB media with 4 mM NO3⁻ (nitrate control). All cultures and controls were amended with 10 mM Fe²+ post-i noculation and incubated at 37 °C for 14 days. Aseptic samples were collected anaerobically for chemical analyses.
solution (1 g l-1 3- (2- pyridyl)- 5,6- diphenyl- 1,2,4- triazine- p,p′-disulfonic acid monosodium salt hydrate, 11.96 g l-1 HEPES) and mixed (total volume 2 ml). Uninoculated and unamended LB medium was measured alongside the abiotic test groups as an additional control. The absorbance measurements were made using a Camspec M107 visible spectrophotometer at a wavelength of 592 nm. The Fe2+ concentration was estimated by comparison with a standard absorbance curve with FeSO4 (Alfa Aesar, UK, 98% purity) [24] using the line equation from the standard curve.
Bradford protein assay
The amorphous Fe- bearing phases in 500 µl aliquots of the culture fluids were dissolved using Tamm’s reagent [25]. Protein concentration in these samples was then measured using the Bradford assay [26]. Protein concentration was used as an indirect measurement of microbial growth to prevent the impact of Fe3+ oxide precipitation on optical density measurements [22].
The protein was precipitated using trichloroacetic acid, as previously described [27]. The supernatant was removed via pipetting, and the pelleted protein was resuspended in 500 µl of 0.1M NaOH. The resuspended protein was heated to 60 °C in a dry bath for 6 min to disrupt the secondary and tertiary structure of the proteins, allowing the dye to bind more effectively. Two hundred fifty microlitres of the sample was then mixed with an equal volume of Bradford reagent, and the absorbance was measured at 595 nm after 5 min using a Camspec M107 visible spectrophotometer [26]. A standard curve was prepared using γ- globulin (0 to 25 µg ml-1 prepared in 0.1M NaOH solution), and the values of the samples were calculated using the line equation from the standard curve.
Nitrate and nitrite measurement
The nitrite concentration was measured using Griess reagent [25, 26]. For the assay, 100 µl of fluid from the test groups, and uninoculated and unamended medium used as a control, was centrifuged at 15,500 g for 10 min, and 50 µl of supernatant was transferred into the well of a 96- well flat- bottomed, optically clear ELISA microplate. One hundred microlitres of 1×Griess reagent solution (Sigma- Aldrich) was added to each well (150 µl total volume) and incubated at room temperature for 15 min. The absorbance was measured using a BioTek ELx808 microplate reader with a 540- nm filter. A standard curve was prepared (1 to 100 µM NaNO2), which was used to calculate the values of the samples. The concentration of nitrate ions was measured using an ELIT 0821 ion- selective NO3- electrode and an ELIT 003 lithium acetate reference electrode (Nico2000) connected to a conductivity metre (HANNA instruments). A standard curve was prepared (10 µM to 10 mM NaNO3), which plotted concentration against conductivity (mV). The nitrate concentration was determined using the line equation from the standard curve.
RESULTS
Microbial growth
Fig. 2 shows protein concentration (normalized to the uninoculated and unamended medium) for the WT, ∆narGHIJ ∆narZYW mutant and the abiotic control that was amended with nitrite. Exponential growth for both the WT and ∆nar strains was achieved within the first 2 days of inoculation, after which growth plateaued. No growth was detected in the abiotic controls.
Regarding visual observations of the cultures (Fig. 3), at day 1, the nitrite-a mended controls had become orange in colouration in comparison with the yellow of the other series, but this faded marginally at day 2, whereas an orange- brown colouration developed in the SL1344 WT cultures at day 2 and persisted throughout the experiment. Weak orange- yellow colouration was observed in the liquid phase of the SL1344 ΔnarGHIJ cultures at day 2 and gradually became less pronounced over the course of the experiment. No colour change was observed in the uninoculated and unamended control series.
Nitrite and nitrate concentrations in the growth medium
The Griess assay was used to measure the production of nitrite through biological nitrate reduction and the depletion of nitrite due to abiotic reactions in the nitrite- amended control (Fig. 4). For the WT strain, the nitrite concentration increased rapidly to a maximum of 132.4 µM (±15.2) at day 2 (correlating with the exponential growth observed via the Bradford assay) (Fig. 2). This trend was also observed in the mutant strain, albeit with a lower concentration of nitrite (65.9 µM±3.0) measured at day 2. For each strain, the nitrite concentration declined during the stationary phase, staying below 41.1 µM (±3.8) for the WT and 7.7 µM (±0.3) for the mutant cultures. In the nitrite- amended controls, the concentration of nitrite decreased from 4 mM to 89.4 µM (±4.3) at day 1 and eventually to 15.6 µM (±1.2) by day 14 (Fig. 4), suggesting the rapid abiotic chemodenitrification of nitrite via reaction with Fe2+ [12]. The nitrite in the abiotic nitrate- amended control series remained around 1 µM throughout the course of the experiment, indicating that abiotic reduction of nitrate was minimal or absent.
Measurements of the concentration of nitrate after 14 days showed that the nitrate concentration in the SL1344 WT cultures depleted by 56.9% (±1.7%) relative to the 4 mM starting concentration. The depletion over the same period for the SL1344 ΔnarGHIJ ΔnarZYW test group was 19.7% (±3.7%) and 4.0% (±1.0%) in the controls. The nitrate depletion correlates

Fig. 2. Protein concentration over time for SL1344 WT (•, filled circles, dashed line), SL1344 ∆narGHIJ ∆narZYW (■, filled squares, dashed line) and the abiotic nitrite-amended control series (△, open triangles, dotted line). Cultures were incubated anaerobically at 37 °C. Error bars represent ±standard error of the mean (se) for triplicate cultures.
proportionally to the extent of nitrite production in the cultures, providing further evidence for both microbial nitrate reduction and the diminished capacity for this process in the ΔnarGHIJ ΔnarZYW mutant. Nitrate consumption and nitrite production, but not growth, also mirrored the Fe2+ oxidation observed in both the SL1344 WT and ΔnarGHIJ ΔnarZYW test groups. This indicates that microbial nitrate reduction and Fe2+ oxidation are intrinsically linked to one another in this instance, but not to increased biomass production. The nitrite- amended control series is not shown as no nitrate was present in the initial medium composition.
Linkages between iron oxidation and nitrate reduction
In the WT cultures, iron oxidation progressed until day 3 (correlating with exponential growth) and then stabilized at an Fe2+/ Fe total value of 0.52 (±0.01) with no significant difference observed during the stationary phase. This is also reflected in the formation of nitrite (Fig. 4) during the exponential stage and the decrease in nitrite formation during the stationary phase. During the stationary phase, the reducing medium and oxidation from the continued Nar- driven supply of nitrite potentially reached an equilibrium, allowing Fe to remain partially oxidized even under reducing conditions. In contrast, in the SL1344 ΔnarGHIJ ΔnarZYW knockout mutant, the Fe2+/Fe total ratio decreased from 1 to 0.78 (±0.01) during exponential growth. The ratio then increased again to 0.91 (±0.03), suggesting that there was no biogenically produced nitrite. In the abiotic, nitrite- amended samples, there was a rapid decrease in the Fe2+/Fe total ratio from 1 to 0.38 (±0.02), which agrees with previous studies demonstrating the abiotic oxidation of iron by nitrite [8, 28, 29]. After this time, the Fe2+/Fe total ratio recovered to 0.75 (±0.02) and with only small changes observed that were potentially caused by interactions between iron and the organic components in the LB medium [30–33]. The Fe2+/Fe total ratio in the abiotic, nitrate- amended control series remained between 1 (±0.02) and 0.95 (±0.05) throughout the experiment.
DISCUSSION
NDFO is a biogeochemical process occurring in diverse anoxic environments, yet the specific microbial mechanisms and direct energetic benefits, particularly for heterotrophic organisms, remain subjects of active research [2, 4–6]. Several pathways have been proposed, including direct enzymatic Fe2+ oxidation and indirect oxidation via reactive nitrogen species [4, 5, 7]. This study aimed to elucidate the primary mechanism of NDFO in the facultative anaerobe S. enterica serovar Typhimurium SL1344 and to determine the role of the Nar system in this process, specifically investigating whether Nar- mediated NDFO contributes to growth.

Fig. 3. SL1344 WT, SL1344 ∆nar, nitrite- amended and an uninoculated and unamended control series at days 1–14. Orange colouration is seen throughout in the nitrite-amended series, developing from day 2 in the SL1344 WT series. Turbidity was evident from day 2 in SL1344 WT and ∆narGHIJ series.

Fig. 4. Fe2+/Fe total (open symbols, dotted lines) and nitrite (filled symbols, solid lines) ion concentration over time for SL1344 WT (circles: • NO2⁻, ○ Fe²+/Fe_total), SL1344 ∆narGHIJ ∆narZYW (squares: ■ NO2⁻, □ Fe²+/Fe_total), abiotic nitrite- amended control (triangles: ▲ NO2⁻, △ Fe²+/Fe_total) and abiotic nitrate control [diamonds: ♦ NO2⁻ (expected to be near zero and may not be distinctly visible if overlapping with other low values), ◊ Fe²+/ Fe_total]. The initial NO2⁻ concentration at day 0 for the abiotic nitrite-amended series was 4 mM (4,000 µM). Cultures were incubated anaerobically at 37 °C. Error bars represent ±standard error of the mean (se) for triplicate cultures.
Our findings suggest that whilst NDFO occurs in S. enterica SL1344, it is primarily an indirect process driven by nitrite produced via Nar- dependent nitrate respiration, and it does not confer a discernible growth advantage under the conditions tested in this study. If NDFO were linked by direct electron donation to Nar from Fe2+ as hypothesized, this would be reflected in microbial growth; however, the concentrations of biomass in SL1344 WT and SL1344 ΔnarGHIJ ΔnarZYW cultures are comparable throughout the growth experiment. The iron oxidation data support these findings. Fe oxidation progressed to a significantly greater extent with the WT than the ΔnarGHIJ ΔnarZYW mutant, suggesting that NDFO is not directly linked to the growth of SL1344. The ΔnarGHIJ ΔnarZYW mutant, which retains the Nap system, exhibited transient nitrite accumulation and minor Fe²+ oxidation. This observation confirms that the Nap system can reduce nitrate in the absence of Nar, but its contribution is significantly less extensive and is not sustained into the stationary phase under these conditions. As a result, nitrite was depleted through abiotic reactions with Fe²+ [34], and Fe³+ was subsequently reduced as a consequence of the reducing fluid chemistry, as was observed in the abiotic nitrite- amended controls.
In the WT SL1344 cultures, the expression and activity of the Nar respiratory nitrate reductase appeared to persist during the stationary phase, facilitating a sustained and low- level production of nitrite. This ongoing nitrite generation likely contributed to a dynamic equilibrium between abiotic Fe²+ oxidation and Fe³+ reduction under the chemically reducing conditions. The plateau in nitrite concentration after day 4 in SL1344 WT contrasts with the depletion of nitrite in the abiotic nitrite- amended series and necessitates a continually active formation mechanism in the stationary phase. In this case, that mechanism is likely to be respiratory nitrate reduction catalysed by NarGHIJ. The persistent partial oxidation of Fe in the presence of the SL1344 WT suggests that it is the presence and activity of Nar as a source of nitrite ions that drives the iron oxidation. In the absence of evidence for the other hypotheses in this organism, nitrite- mediated oxidation is likely to be the sole or dominant mechanism for the Fe2+ oxidation observed.
By utilizing the ΔnarGHIJ ΔnarZYW mutant, this study demonstrates that the activity of the Nar respiratory nitrate reductase, via the production of nitrite ions, contributes to Fe oxidation and maintenance of Fe3+ under chemically reducing conditions that favour the existence of Fe2+. This result has several implications, first among which is the confirmation that the interaction of biogenic nitrite with Fe2+ ions is likely to be a key contributor to NDFO. This supports previous findings that suggest that nitratereducing species are likely capable of NDFO via the abiotic reaction of nitrite with Fe2+ [8–10]. Taken together with the protein measurements, which showed parallel growth in SL1344 WT and ΔnarGHIJ ΔnarZYW, this study suggests that Fe oxidation via this mechanism does not metabolically benefit this species. Whilst not within the scope of this study, further research using a triple mutant (ΔnarGHIJ, ΔnarZYW, Δnap) would define the possible role of Nap in contributing to iron oxidation and the residual nitrate reduction observed in the double mutant.
CONCLUSION
This study demonstrates that nitrate- dependent Fe2+ oxidation in S. enterica serovar Typhimurium SL1344 is principally mediated by the activity of the Nar respiratory nitrate reductase. The primary mechanism involves the biological production of nitrite during nitrate respiration, which subsequently acts as an abiotic oxidant of Fe2+. Whilst the ∆narGHIJ ∆narZYW mutant, which utilizes the periplasmic Nap system, exhibited some initial nitrite production and Fe2+ oxidation, these were significantly less extensive and less sustained compared to the WT. Crucially, no discernible growth benefit, in terms of increased biomass, was associated with Fe2+ oxidation in either the WT or the mutant strain under the rich conditions of the media. These findings suggest that in S. enterica SL1344, NDFO is an indirect consequence of nitrate reduction via nitrite, rather than a dedicated metabolic strategy for energy generation through Fe2+ oxidation. This supports the broader understanding that nitrite- mediated Fe2+ oxidation is a key pathway in NDFO processes across diverse nitrate- reducing bacteria.
Funding information
This research was supported by the Science and Technology Facilities Council, the Open University and Research England via the 'Expanding Excellence in England' grant number 124.18.
Acknowledgements
This manuscript details research that was performed as part of the PhD of Dr Alex Price (2020), titled ‘Microbial Nitrate Dependent Fe Oxidation: A Potential Early Mars Metabolism’. This thesis is available at https://oro.open.ac.uk/72495/.
Author contributions
A.P.: Conceptualization, methodology, investigation, formal analysis, visualization and writing – original draft. R.K.: Writing – original draft and writing – review and editing. G.R.: Resources, methodology and writing – review and editing. K.O.-F.: Conceptualization, supervision, funding acquisition, validation, project administration and writing – review and editing. M.C.M.: Conceptualization, supervision, funding acquisition, project administration, methodology and writing – review and editing.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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