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bioRxiv · 10.1101/2022.09.08.507232

Functional characterization of the Co2+ transporter AitP in Sinorhizobium meliloti: a new player in Fe2+ homeostasis

Mihelj, P.·Abreu, I.·Moreyra, T. E.·Gonzalez-Guerrero, M.·Raimunda, D. C.
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Abstract

Co2+ 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 N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting Cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this sub-family, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sub-lethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ 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 Fe2+-export is likely counterbalanced via a non-futile Fe2+-import pathway. Two lines of evidence support this: i) an increased hemin uptake in presence of Co2+ was observed in WT vs. AitP mutant, and ii) hemin reversed the Co2+ 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. ImportanceCross-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 Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the N2-fixating rhizobial cells solve this perturbation by clearing Fe through a Co2+-exporter, in order to reestablish intracellular Fe-levels by importing non-free Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

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Mihelj, P., Abreu, I., Moreyra, T. E., Gonzalez-Guerrero, M., Raimunda, D. C.. 2022-09-11. Functional characterization of the Co2+ transporter AitP in Sinorhizobium meliloti: a new player in Fe2+ homeostasis. https://doi.org/10.1101/2022.09.08.507232

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