Adaptive response of Amphibacillus xylanus to normal aerobic and forced oxidative stress conditions.

Amphibacillus xylanus grows at the same rate and with the same cell yield under aerobic and anaerobic conditions. Under aerobic conditions, it exhibits vigorous oxygen consumption in spite of lacking a respiratory system and haem catalase. To understand the adaptive response of A. xylanus to oxidative stresses, a genomic analysis of A. xylanus was conducted. The analysis showed that A. xylanus has the genes of four metabolic systems: two pyruvate metabolic pathways, a glycolytic metabolic pathway and an NADH oxidase (Nox)-AhpC (Prx) system. A transcriptional study confirmed that A. xylanus has these metabolic systems. Moreover, genomic analysis revealed the presence of two genes for NADH oxidase (nox1 and nox2), both of which were identified in the transcriptional analysis. The nox1 gene in A. xylanus was highly expressed under normal aerobic conditions but that of nox2 was not. A purification study of NADH oxidases indicated that the gene product of nox1 is a primary metabolic enzyme responsible for metabolism of both oxygen and reactive oxygen species. A. xylanus was successfully grown under forced oxidative stress conditions such as 0.1 mM H2O2, 0.3 mM paraquat and 80 % oxygen. Proteomic analysis revealed that manganese SOD, Prx, pyruvate dehydrogenase complex E1 and E3 components, and riboflavin synthase ß-chain are induced under normal aerobic conditions, and the other proteins except the five aerobically induced proteins were not induced under forced oxidative stress conditions. Taken together, the present findings indicate that A. xylanus has a unique defence system against forced oxidative stress.

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

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 µm, 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 µm free FAD, the catalytic efficiency (kcat/Km) 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.

NADH oxidase and alkyl hydroperoxide reductase subunit C (peroxiredoxin) from Amphibacillus xylanus form an oligomeric assembly.

The NADH oxidase-peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from ß-NADH passed through the secondary disulfide, Cys128-Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.