Regulation of manganese homeostasis in Bradyrhizobium japonicum
Hohle, Thomas H.
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Manganese is an essential element for various cellular processes, however it is toxic at high concentrations. Thus, transport of the metal into cells need to be regulated. This thesis explores manganese transport and regulation in Bradyrhizobium japonicum . We identified the mntH homolog of B. japonicum , and demonstrate that it is essential for Mn 2+ transport and maintenance of cellular manganese homeostasis. Transport activity was induced under manganese deficiency. The mntH mRNA was negatively regulated by manganese via Fur. Fur protected the mntH promoter in DNase I footprinting analysis in the presence of manganese. The findings identify mntH as the major Mn 2+ transporter in B. japonicum , and show that Fur is a manganese-responsive regulator in that organism. B. japonicum Fur has also been implicated in iron-dependent regulation of the irr gene. Thus, we sought to address the apparent discrepancy in Fur responsiveness to metals. irr is a transcriptional regulator found in iron-limited cells. Here, we show that irr gene mRNA was regulated by both iron and manganese, in an IRR and Fur dependent manner, respectively. Fur occupied the irr promoter in vivo in the presence of manganese and the absence of IRR protein. The IRR binding site within the irr promoter partially overlaps the Fur binding site, and IRR competes with Fur for binding to the promoter. We conclude that Fur mediates manganese-dependent repression of irr transcription and that IRR acts as an anti-repressor under iron limitation by preventing Fur binding to the promoter. Whole genome transcript analysis identified mnoP as a gene encoding a putative outer membrane protein that is co-regulated with the inner membrane Mn 2+ transporter gene mntH . The mnoP gene is required for growth in manganese-limited media and MnoP significantly increases the affinity of cells for Mn 2+ . MnoP allowed the transport of Mn 2+ and Fe 2+ , but not other divalent metals, into proteoliposomes, in the absence of an energy source. The data show that MnoP is an outer membrane channel required for manganese acquisition and utilization by cells. Moreover, the findings suggest that the outer membrane is a selective barrier conferred by the regulated expression of a solute-selective channel.