Version 2: Iron Oxidation by Salmonella enterica Serovar Typhimurium strain SL1344 is an Abiotic Byproduct of Nitrate Respiration

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 Salmonella enterica serovar Typhimurium strain SL1344. We compared the wild-type (WT) strain with a ∆narGHIJ ∆narZYW mutant (∆nar), which lacks the primary respiratory nitrate reductase (Nar). Cultures were grown anaerobically in Lysogeny Broth (LB) 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 4mM) 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 oxidises 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.