Bacterioferritin-driven oxidative stress resistance in anammox bacteria: Mechanistic insights into reactive oxygen species scavenging.

Bacterioferritin in anammox bacteria plays dual roles: storing iron and mitigating oxidative stress induced by heavy metals. We hypothesized that bacterioferritin mediates the antioxidant defense pathway in anammox bacteria. The potential clearance mechanism of bacterioferritin against typical reactive oxygen species, hydrogen peroxide (H₂O₂), was explored. The results indicated that bacterioferritin is widely distributed among the genera Jettenia, Brocadia, Kuenenia, and Scalindua. Using two bacterioferritins (K.S Bfr and Q1Q315) from Candidatus. Kuenenia stuttgartiensis, engineered E. coli strains expressing these proteins exhibited higher survival and preserved cellular integrity under 10 mM H₂O₂ stress compared with the wild-type bacteria (WB). In vitro experiments confirmed that bacterioferritin acts as a reactive oxygen species scavenger by eliminating H₂O₂ through iron oxidation at its ferroxidase center. Furthermore, it was found that the gene abundances of K.S Bfr and Q1Q315 increased.in anammox granules exposed to H₂O₂. Proteomic analysis revealed notable damage to membrane- and DNA-associated proteins in WB under H₂O₂ stress, accompanied by significant upregulation of conventional antioxidant proteins. However, this response was insufficient to protect WB from high concentrations of H₂O₂. In contrast, engineered bacteria showed a significant upregulation of ribosomal metabolic pathways, enhancing intracellular protein synthesis, particularly of K.S Bfr and Q1Q315. These proteins mediated reactions that consumed H₂O₂, thereby providing a crucial buffer period for cellular ribosomal activity. Overall, bacterioferritin is a key antioxidant protein widely present in anammox bacteria, playing a pivotal role in cellular oxidative stress mitigation.