Insights into nitrate-reducing Fe(II) oxidation by Diaphorobacter caeni LI3T through kinetic, nitrogen isotope fractionation, and genome analyses.

Nitrate (NO3-)-reducing Fe(II) oxidation (NRFO) is prevalent in anoxic environments. However, it is uncertain in which step(s) the biological Fe(II) oxidation is coupled with denitrification during NRFO. In this study, a heterotrophic NRFO bacterium, Diaphorobacter caeni LI3T, was isolated from paddy soil and used to investigate the transformation of Fe(II) and nitrogen as well as nitrogen isotopic fractionation (δ15N-N2O) during NRFO. Fe(II) oxidation was observed in the Cell+NO3- +Fe(II), Cell+NO2- + Fe(II), and NO2- + Fe(II) treatments, resulting in precipitation of amorphous Fe(III) minerals and lepidocrocite on the surface and in the periplasm of cells. The presence of Fe(II) slightly accelerated microbial NO3- reduction in the Cell+NO3- + Fe(II) treatment relative to the Cell+NO3- treatment, but slowed down the NO2- reduction in the Cell+NO2- + Fe(II) treatment relative to the Cell+NO2- treatment likely due to cell encrustation that blocking microbial NO2- reduction in the periplasm. The δ15N-N2O results in the Cell+NO3- + Fe(II) treatment were close to those in the Cell+NO3- and Cell+NO2- treatments, indicating that the accumulative N2O is primarily of biological origin during NRFO. The genome analysis found a complete set of denitrification and oxidative phosphorylation genes in strain LI3T, the metabolic pathways of which were closely related with cyc2 and cytc as indicated by protein-protein interactions network analysis. It is proposed that Fe(II) oxidation is catalyzed by the outer membrane protein Cyc2, with the resulting electrons being transferred to the nitrite reductase NirS via CytC in the periplasm, and the CytC can also accept electrons from the oxidative phosphorylation in the cytoplasmic membrane. Overall, our findings provide new insights into the potential pathways of biological Fe(II) oxidation coupled with nitrate reduction in heterotrophic NRFO bacteria.