Sudden anaerobization in Amphibacillus xylanus increases intracellular labile ferrous iron and inhibits cell growth

Amphibacillus xylanus is an aerotolerant anaerobe that consumes large amounts of oxygen and requires iron as an essential micronutrient during its aerobic growth. However, this bacterium lacks a typical respiratory chain; therefore, intracellular free flavins and their reductases are thought to participate in the reduction of molecular oxygen and ferric iron (Fe³⁺). This system can potentially generate hydroxyl radicals through the Fenton reaction, highlighting the importance of regulating the production of redox-active ferrous iron (Fe²⁺). In this study, we employed a strategy of abruptly switching the cell culture system from aerobic to anaerobic conditions to examine Fe²⁺ production via free flavins. Sudden anaerobization induced growth inhibition and a significant increase in intracellular labile Fe²⁺. Whole-cell ⁵⁷Fe Mössbauer spectroscopy revealed that in aerobic cells, high-spin Fe³⁺ is the major chemical species, whereas in anaerobic conditions, the intracellular iron pool is completely converted into Fe²⁺ and consists of low- and high-spin Fe²⁺ species. Increased Fe²⁺ production was mimicked using cell-free extracts and the reductase activity promoted electron transfer from NADH to Fe³⁺ via physiological concentrations of free flavin adenine dinucleotide under anaerobic conditions. RNA sequencing showed that the electrons of NADH generated through glycolysis and the pyruvate metabolic pathway can flow into a flavoprotein with flavin reductase activity. These findings suggest that the production of labile Fe²⁺ via free flavin is regulated by molecular oxygen, allowing safe iron utilization during the aerobic growth of A. xylanus.