Mechanismus und Kontrolle der Zink-Aufnahme im Modellorganismus Cupriavidus metallidurans CH34
Zusammenfassung der Projektergebnisse
1. Up to a nine-fold deletion mutant, the genes for seven secondary (zupT, pitA, corA1, corA2, corA3, zntB, hoxN) and two primary transport systems (mgtA, mgtB) that might be involved in zinc uptake in C. metallidurans were deleted in the plasmid-free strain AE104 and the mutants were studied. 2. PitA was an important uptake system for metal:phosphate. The gene pitA was upregulated by increasing phosphate concentrations indicating emphasis on the export of metals rather on that of phosphate. Deletion of pitA led to a three- to –four-fold higher cellular magnesium content and up-regulation of the phosphate starvation response. 3. While CorA was an important uptake system for magnesium, all CorAs cooperated to control import of cobalt and nickel ions. Deletion of zntB for a possible zinc-exporting CorA-like proteins in a ∆5 mutant strain (∆zupT ∆pitA ∆corA1 ∆corA2 ∆corA3) or of hoxN in ∆6 (∆5 ∆zntB) had no effect. 4. Deletion of ∆zupT for a member of the ZIP (= ZRT/IRT) protein family did not influence zinc import on the first glance. However, the ∆zupT mutant had problems to acquire zinc efficiently at low concentrations, e.g. in non-amended low-phosphate Tris-buffered mineral salts medium (TMM) or in the presence of more than 300 µM EDTA, and suffered from several pleitropic effects: (i) inefficient folding of the zinc-containing RpoC subunit of the RNA polymerase causing excretion of the not properly folded protein into inclusion bodies; (ii) decreased oxidative stress resistance connected to a lower number of periplasmic CuZn superoxide dismutase proteins; (iii) inability to harbor the CzcCBA transenvelope complex for cobalt, zinc and cadmium after recurrent transfer on non zinc-amended TMM stock plates, and (iv) un-silencing of a region encoding the nickeldependent NAD-reducing hydrogenase plus Calvin cycle enzymes. This region is part of a genomic island that is silenced in zinc-stressed C. metallidurans cells. 5. This defines a battery of redundant high-rate, low affinity uptake systems that supply a wide range of transition metal cations plus Mg2+ and phosphate to the cells and interact with specifically produced metal efflux systems that “worry later” should a particular metal be imported in surplus. This process results in a kinetical flow equilibrium of import and export reactions, which controls the cytoplasmic content of the individual trace element cations plus the overall composition of the cation “bouquet” to prevent toxic actions of these metals, e.g. by interference. 6. The counterbalance and buffer of this transport flow equilibrium in C. metallidurans is the zinc repository, a large number of empty zinc-binding sites in cytoplasmic proteins of C. metallidurans. This repository should be able to accommodate any incoming zinc ion, sequester it to prevent interference with other metal cation or binding to thiol groups, and stores is until needed as cofactor or exported again. The zinc repository may also be able to harbor other transition metal cations such as cobalt, copper or gold. 7. The main regulator of zupT, Zur (= FurC), was identified. Biggest surprises were the existence of the zinc repository and the silencing/un-silencing of the autotrophic region of the genomic island CMGI-3.
Projektbezogene Publikationen (Auswahl)
- 2009. CzcP is a novel efflux system contributing to transition metal resistance in Cupriavidus metallidurans CH34. Mol Microbiol 73:601-621
Scherer J, and Nies DH
- 2011. Contributions of five secondary metal uptake systems to metal homeostasis of Cupriavidus metallidurans CH34. J Bacteriol 193:4652-4663
Kirsten A, Herzberg M, Voigt A, Seravalli J, Grass G, Scherer J, and Nies DH
- 2012. Metal selectivity determinants in a family of transition metal transporters. J Biol Chem 287:3185-3196
Podar D, Scherer J, Noordally Z, Herzyk P, Nies D, and Sanders D
(Siehe online unter https://doi.org/10.1074/jbc.M111.305649) - 2013. Colonization resistance against genetically modified Escherichia coli K12 (W3110) strains is abrogated following broad-spectrum antibiotic treatment and acute ileitis. Eur J Microbiol Immunol 3:222-228
Heimesaat MM, Kupz A, Fischer A, Nies DH, Grass G, Göbel UB, and Bereswill S
- 2013. HypD is the scaffold protein for Fe–(CN)2CO cofactor assembly in {NiFe}–hydrogenase maturation. Biochemistry 52:3289-3296
. Stripp ST, Soboh B, Lindenstrauss U, Braussemann M, Herzberg M, Nies DH, Sawers G, and Heberle J
(Siehe online unter https://doi.org/10.1021/bi400302v) - 2013. Impact of metal ion homeostasis of genetically modified Escherichia coli Nissle 1917 and K12 (W3110) strains on murine intestinal colonization. Eur J Microbiol Immunol 3:229-335
Kupz A, Fischer A, Nies DH, Grass G, Göbel UB, Bereswill S, and Heimesaat MM
- 2013. The DNA uptake ATPase PilF of Thermus thermophilus: a reexamination of the zinc content. Extremophiles 17:697-698
Salzer R, Herzberg M, Nies D, Biukovic G, Gruber G, Müller V, and Averhoff B
- 2014. Deletion of the zupT gene for a zinc importer influences zinc pools in Cupriavidus metallidurans CH34. Metallomics 6:421- 436
Herzberg M, Bauer L, and Nies DH
(Siehe online unter https://doi.org/10.1039/c3mt00267e) - 2014. FurC regulates expression of zupT for the central zinc importer ZupT of Cupriavidus metallidurans. J Bacteriol 196:3461-3471
Schmidt C, Schwarzenberger C, Grosse C, and Nies DH
(Siehe online unter https://doi.org/10.1128/JB.01713-14) - 2014. The zinc repository of Cupriavidus metallidurans. Metallomics 6:2157-2165
Herzberg M, Dobritzsch D, Helm S, Baginski S, and Nies DH
(Siehe online unter https://doi.org/10.1039/c4mt00171k) - 2014. Zinc and ATP binding of the hexameric AAA-ATPase PilF from T. thermophilus: Role in complex stability, piliation, adhesion, twitching motility and natural transformation. J Biol Chem 289:30343-30354
Salzer R, Herzberg M, Nies DH, Joos F, Rathmann B, Thielmann Y, and Averhoff B
(Siehe online unter https://doi.org/10.1074/jbc.M114.598656) - 2015. Synthesis of nickel-iron hydrogenase in Cupriavidus metallidurans is controlled by metal-dependent silencing and un-silencing of genomic islands. Metallomics
Herzberg M, Schüttau M, Reimers M, Grosse C, Schlegel HG, and Nies DH
(Siehe online unter https://doi.org/10.1039/c4mt00297k)
