Discovery and analysis of iron export and iron import mechanisms of Bradyrhizobium japonicum and their roles in managing stress responses.

摘要

Nutritional iron acquisition by bacteria is well described, but almost nothing is known about bacterial iron export. Here, we show that Bradyrhizobium japonicum MbfA (Blr7895) is an inner membrane protein expressed in cells specifically under high iron conditions. An mbfA deletion mutant is severely defective in iron export activity, contains >2-fold more intracellular iron than the parent strain, and displays an aberrant iron-dependent gene expression phenotype. The findings suggest that iron export plays an important role in bacterial iron homeostasis, and MbfA is responsible for the iron export activity of B. japonicum. The N-terminal Ferritin like domain (FLD) of MbfA is localized to the cytoplasmic side of the inner membrane and is required for export activity. Purified FLD is a dimer in solution implying that MbfA functions as a dimer.;An mbfA mutant is sensitive to short term exposure to high levels iron or H2O2 but not when grown in elevated iron media, suggesting a stress response adaptation. The bfr gene encodes the iron storage protein bacterioferritin. An mbfA bfr double mutant showed a loss of stress adaptation, and had a severe growth phenotype in high iron media. The double mutant exhibits elevated intracellular iron content than the wild type, and displays aberrant gene expression even when grown in relatively low iron media. These results suggest that MbfA and Bfr work in concert to manage iron and oxidative stresses. In addition, the need for iron detoxification is not limited to extreme environments, but is also required for normal cellular function.;B. japonicum cannot make siderophores for acquisition of iron in aerobic environments. The mechanism of iron uptake in the absence of xenosiderophores is unknown. Exploiting the synthetic lethal phenotype of the mbfA bfr double mutant, we identified suppressor strains that can grow in high iron concentrations. The suppressor strains harbor loss of function mutations in the feoAB operon, which is a ferrous iron transport system. Interestingly, FeoAB system is required for ferric iron utilization and is required for high affinity uptake of both ferric and ferrous iron by B. japonicum. feoB and feoA incited small, poorly developed, non-nitrogen fixing nodules on soybean plants suggesting the requirement of FeoAB system for establishment of symbiosis. A suppressor strain harboring a Glu-40 to Lys mutation in FeoA (feoAE40K ) has diminished but measurable iron uptake activity in free living cells. It elicited nitrogen fixing nodules on soybean but the bacteroids in the nodules displayed lower iron uptake activity compared to wildtype bacteroids. This strongly suggests that the FeoAB transport system is involved in iron uptake within symbiotic bacteroids. Thus our results indicate that B. japonicum employs a single iron transporter to adapt to diverse environmental conditions.