Zinc is a central player in the metalloproteomes of prokaryotes and eukaryotes. We used a bottom-up quantitative proteomic approach to reveal the repository of the zinc pools in the proteobacterium Cupriavidus metallidurans. About 60% of the theoretical proteome of C. metallidurans was identified, quantified, and the defect in zinc allocation was compared between a ΔzupT mutant and its parent strain. In both strains, the number of zinc-binding proteins and their binding sites exceeded that of the zinc ions per cell, indicating that the totality of the zinc proteome provides empty binding sites for the incoming zinc ions. This zinc repository plays a central role in zinc homeostasis in C. metallidurans and probably also in other organisms.

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An important application field of secondary ion mass spectrometry at the nanometer scale (NanoSIMS) is the detection of chemical elements and, in particular, metals at the subcellular level in biological samples. The detection of many trace metals requires an oxygen primary ion source to allow the generation of positive secondary ions with high yield in the NanoSIMS. The duoplasmatron oxygen source is commonly used in this ion microprobe but cannot achieve the same quality of images as the cesium primary ion source used to produce negative secondary ions (C(-), CN(-), S(-), P(-)) due to a larger primary ion beam size. In this paper, a new type of an oxygen ion source using a rf plasma is fitted and characterized on a NanoSIMS50L. The performances of this primary ion source in terms of current density and achievable lateral resolution have been characterized and compared to the conventional duoplasmatron and cesium sources. The new rf plasma oxygen source offered a net improvement in terms of primary beam current density compared to the commonly used duoplasmatron source, which resulted in higher ultimate lateral resolutions down to 37 nm and which provided a 5-45 times higher apparent sensitivity for electropositive elements. Other advantages include a better long-term stability and reduced maintenance. This new rf plasma oxygen primary ion source has been applied to the localization of essential macroelements and trace metals at basal levels in two biological models, cells of Chlamydomonas reinhardtii and Arabidopsis thaliana.

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

The bacterium is capable of reducing toxic gold(I/III)-complexes and biomineralizing them into metallic gold (Au) nanoparticles, thereby mediating the (trans)formation of Au nuggets. In Au-rich soils, most transition metals do not interfere with the resistance of this bacterium to toxic mobile Au-complexes and can be removed from the cell by plasmid-encoded metal efflux systems. Copper is a noticeable exception: the presence of Au-complexes and Cu-ions results in synergistic toxicity, which is accompanied by an increased cytoplasmic Cu content and formation of Au nanoparticles in the periplasm. The periplasmic Cu-oxidase CopA was not essential for formation of the periplasmic Au nanoparticles. As shown with the purified and reconstituted Cu efflux system CupA, Au-complexes block Cu-dependent release of phosphate from ATP by CupA, indicating inhibition of Cu transport. Moreover, Cu resistance of Au-inhibited cells was similar to that of mutants carrying deletions in the genes for the Cu-exporting P-type ATPases. Consequently, Au-complexes inhibit export of cytoplasmic Cu-ions, leading to an increased cellular Cu-content and decreased Cu/Au resistance. Uncovering the biochemical mechanisms of synergistic Au/Cu-toxicity in explains the issues this bacterium has to face in auriferous environments, where it is as an important contributor to the environmental Au cycle. lives in metal-rich environments, including auriferous soils that contain a mixture of toxic transition metal cations. We demonstrate here that copper ions and gold complexes exert synergistic toxicity because gold ions inhibit the copper-exporting P-type ATPase CupA, which is central to copper resistance in this bacterium. Such a situation should occur in soils overlying Au deposits, in which Cu:Au ratios usually are >> 1. Appreciating how solves the problem of living in environments that contain both Au and Cu is a pre-requisite to understand the molecular mechanisms underlying gold cycling in the environment, and the significance and opportunities of microbiota for specific targeting to Au in mineral exploration and ore processing.