Intracellular free flavin and its associated enzymes participate in oxygen and iron metabolism in Amphibacillus xylanus lacking a respiratory chain

Abstract

Amphibacillus xylanus is a recently identified bacterium which grows well under both aerobic and anaerobic conditions and may prove useful for biomass utilization. Amphibacillus xylanus, despite lacking a respiratory chain, consumes oxygen at a similar rate to Escherichia coli (130–140 μmol oxygen·min −1 ·g −1 dry cells at 37 °C), suggesting that it has an alternative system that uses a large amount of oxygen. Amphibacillus xylanus NADH oxidase (Nox) was previously reported to rapidly reduce molecular oxygen content in the presence of exogenously added free flavin. Here, we established a quantitative method for determining the intracellular concentrations of free flavins in A. xylanus , involving French pressure and ultrafiltration membranes. The intracellular concentrations of flavin adenine dinucleotide ( FAD ), flavin mononucleotide ( FMN ), and riboflavin were estimated to be approximately 8, 3, and 1 μ m , respectively. In the presence of FAD , the predominant free flavin species, two flavoproteins Nox (which binds FAD ) and NAD (P)H oxidoreductase (Npo, which binds FMN ), were identified as central free flavin‐associated enzymes in the oxygen metabolic pathway. Under 8 μ m free FAD , the catalytic efficiency ( k cat / K m ) of recombinant Nox and Npo for oxygen increased by approximately fivefold and ninefold, respectively. Nox and Npo levels were increased, and intracellular FAD formation was stimulated following exposure of A. xylanus to oxygen. This suggests that these two enzymes and free FAD contribute to effective oxygen detoxification and NAD (P) + regeneration to maintain redox balance during aerobic growth. Furthermore, A. xylanus required iron to grow aerobically. We also discuss the contribution of the free flavin‐associated system to the process of iron utilization.

Simultaneous Two-Photon Fluorescence Microscopy of NADH and FAD Using Pixel-to-Pixel Wavelength-Switching

“…In our experiment, NADH has a smaller cross-section than FAD, but the fluorescence is stronger in NADH images (Figure 4). We consider that FAD in mouse colonic mucosa is relatively sparse comparing with NADH, which is consistent with previously published data [17,18]. The experimental results show that the system can effectively perform simultaneous imaging of NADH and FAD, and metabolism-related quantitative analysis may be carried out with sufficient prior knowledge of tissues.…”

High vitamin B2 production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon

“…2A and C). The estimated intracellular concentrations of RF+FMN and FAD in Lp UFG9 are ~3.5 μM and ~20 μM, respectively, which do not greatly differ from the values reported for the Firmicute bacterium Amphibacillus xylanus (Kimata et al, 2018). In contrast, the intracellular concentration of RF+FMN in Lp B2 was estimated to be ~20 μM.…”

Cannabidiol Modulates Mitochondrial Redox and Dynamics in MCF7 Cancer Cells: A Study Using Fluorescence Lifetime Imaging Microscopy of NAD(P)H

“…Solution concentrations as high as 0.5 mM are observed in the flavin-detecting channel only. The intracellular FAD concentration has been estimated to be around 8 μM ( Kimata et al, 2018 ). For each of the five experimental groups (Control, 1, 5, 10, and 20 μm CBD), five spatially distinct 2P-FLIM images were acquired.…”