Functional Characterization of the Co Transporter AitP in Sinorhizobium meliloti: A New Player in Fe Homeostasis.

Co induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe-export systems. An insertional mutant of AitP showed Co sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe sensitivity, despite AitP being a bona fide low affinity Fe exporter as demonstrated by the kinetic analyses of Fe uptake into everted membrane vesicles. Suggesting concomitant Fe-dependent induced stress, Co sensitivity was increased in strains carrying mutations in AitP and Fe exporters which did not correlate with the Co accumulation. Growth in the presence of sublethal Fe and Co concentrations suggested that free Fe-import might contribute to Co toxicity. Supporting this, Co induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe-export is likely counterbalanced via a nonfutile Fe-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe, Co induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co content may be encountered.