Bacterial copper (Cu) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of to external 0.5 mm CuSO, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu efflux systems (a P-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu with high affinity. Importantly, novel transmembrane transporters probably mediating Cu influx were among those largely repressed upon Cu stress. Compartmental Cu levels appear independently controlled; the cytoplasmic Cu sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu Analysis of Δ and Δ mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu levels in compartments.

The early discovery of the human Cu+-ATPases and their link to Menkes and Wilson's diseases brought attention to the unique role of these transporters in copper homeostasis. The characterization of bacterial Cu+-ATPases has significantly furthered our understanding on the structure, selectivity and transport mechanism of these enzymes, as well as their interplay with other elements of Cu+ distribution networks. This review focuses on the structural-functional insights that have emerged from studies of bacterial Cu+-ATPase at the molecular level and how these observations have contributed to draw up a comprehensive picture of cellular copper homeostasis.

Summary Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen‐fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron‐based X‐ray fluorescence assays were carried out in the nodule‐specific M. truncatula ferroportin Medicago truncatula nodule‐specific gene Ferroportin2 (MtFPN2) is an iron‐efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early‐fixation zone of the nodules. Loss‐of‐function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen‐fixing bacteroids in M. truncatula nodules.

Zinc transporters facilitate metal mobilization and compartmentalization, playing a key role in cellular development. Little is known about the mechanisms and pathways of Zn movement between Zn transporters and metalloproteins during myoblast differentiation. We analyzed the differential expression of ZIP and ZnT transporters during C2C12 myoblast differentiation. Zn transporters account for a transient decrease of intracellular Zn upon myogenesis induction followed by a gradual increase of Zn in myotubes. Considering the subcellular localization and function of each of the Zn transporters, our findings indicate that a fine regulation is necessary to maintain correct metal concentrations in the cytosol and subcellular compartments to avoid toxicity, maintain homeostasis, and for loading metalloproteins needed during myogenesis. This study advances our basic understanding of the complex Zn transport network during muscle differentiation.

The genome of Arabidopsis thaliana encodes approximately 260 copper (Cu)‐dependent proteins, which includes enzymes in central pathways of photosynthesis, respiration and responses to environmental stress. Under Cu‐deficient growth conditions, Squamosa promoter binding Protein‐Like 7 ( SPL 7) activates the transcription of genes encoding Cu acquisition systems, and it mediates a metabolic reorganization to economize on Cu. The transcription factor SPL 7 groups among comparably large proteins in the SPL family, which additionally comprises a second group of small SPL proteins targeted by mi RNA 156 with roles in plant development. SPL 7 shares extended regions of sequence homology with SPL 1 and SPL 12. Therefore, we investigated the possibility of a functional overlap between these three members of the group of large SPL family proteins. We compared the spl1 spl12 double mutant and the spl1 spl7 spl12 triple mutant with both the wild type and the spl7 single mutant under normal and Cu‐deficient growth conditions. Biomass production, chlorophyll content and tissue elemental composition at the seedling stage, as well as plant and flower morphology during reproductive stages, confirmed the involvement of SPL 7, but provided no indication for important roles of SPL 1 or SPL 12 in the acclimation of Arabidopsis to Cu deficiency. Furthermore, we analyzed the effects of zinc (Zn) deficiency on the same set of mutants. Different from what is known in the green alga Chlamydomonas reinhardtii , Arabidopsis did not activate Cu deficiency responses under Zn deficiency, and there was no Cu overaccumulation in either shoot or root tissues of Zn‐deficient wild type plants. Known Zn deficiency responses were unaltered in spl7 , spl1 spl12 and spl1 spl7 spl12 mutants. We observed that CuZn SOD activity is strongly downregulated in Zn‐deficient A. thaliana , in association with an about 94% reduction in the abundance of the CSD 2 transcript, a known target of miR398. However, different from the known Cu deficiency responses of Arabidopsis, this Zn deficiency response was independent of SPL 7 and not associated with an upregulation of MIR 398b primary transcript levels. Our data suggest that there is no conservation in A. thaliana of the crosstalk between Zn and Cu homeostasis mediated by the single SPL family protein CRR 1 of Chl...