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The role of extracytoplasmic function (ECF) sigma factors in multiple metal homeostasis of the metallophilic bacterium Cupriavidus metallidurans was studied. RNA sequencing was used to predict 3084 operons in the genome of this bacterium, including 11 for ECF sigma factors, and to measure transcript abundances. Mutants carrying multiple deletions in genes for ECF sigma factors were constructed and characterized. Mutants and parent were challenged with a metal mix, changes in the global gene expression profile and the overall metal content determined. All 11 ECF sigma factors were involved in metal homeostasis. The three ECF sigma factors RpoI, RpoJ and RpoK synchronized iron homeostasis with that of other divalent metal cations, RpoO, RpoL and RpoM magnesium and phosphorous homeostasis with that of zinc and with cadmium resistance. Factors RpoE, CnrH and RpoP controlled the response to nickel and cobalt, RpoQ and RpoR may be assigned to the thiol and sulfide metabolism. All 11 ECF sigma factors overlap in their function and control gene expression involved in metal homeostasis, however, except CnrH, no other ECF sigma factor was needed for up-regulation of 63 predicted operons responding to metal shock, 48 of these encoding metal efflux pumps. Moreover, disturbance of the cellular metal content resulting from missing sigma factors also affected silencing and un-silencing of genomic islands. Together, these data demonstrate on a global and systemic level how a robust network of ECF sigma factors and other regulators allow C. metallidurans to handle a mixture of toxic transition metal cations, which are conditions the bacterium faces in its natural environment. Iron homeostasis is to be maintained at any cost, followed by the necessity for magnesium, phosphorous and zinc homeostasis on the second level, and cobalt plus nickel coming last.

The zinc importer ZupT is required for the efficient allocation of zinc to zinc-dependent proteins in the metal-resistant bacterium Cupriavidus metallidurans but not for zinc import per se . The expression of zupT is upregulated under conditions of zinc starvation. C. metallidurans contains three members of the Fur family of regulators that qualify as candidates for the zupT regulator. The expression of a zupT-lacZ reporter gene fusion was strongly upregulated in a Δ furC mutant but not in a Δ furA or Δ furB mutant. Expression of the genes for transition-metal importers ( pitA , corA1 , corA2 , and corA3 ) was not changed in this pattern in all three Δ fur mutants, but they were still downregulated under conditions of elevated zinc concentrations, indicating the presence of another zinc-dependent regulator. FurA was a central regulator of the iron metabolism in C. metallidurans , and furA was constitutively expressed under the conditions tested. Expression of furB was upregulated under conditions of iron starvation, and FurB could be an iron starvation Fur connecting general metal and iron homeostasis, as indicated by the phenotype of a Δ furB Δ furC double mutant. FurC was purified as a Strep-tagged protein and retarded the electrophoretic mobility of a DNA fragment upstream of zupT . Binding of FurC to this operator region was influenced by the presence of zinc ions and EDTA. Thus, FurC is the main zinc uptake regulator (Zur) of C. metallidurans and represses synthesis of the central zinc importer ZupT when sufficient zinc is present.

Primary and/or secondary injury of the renal microvascular endothelium is a common finding in various renal diseases. Besides well-known endothelial repair mechanisms, including endothelial cell (EC) proliferation and migration, homing of extrinsic cells such as endothelial progenitor cells (EPC) and hematopoietic stem cells (HSC) has been shown in various organs and may contribute to microvascular repair. However, these mechanisms have so far not been studied after selective microvascular injury in the kidney. The present study investigated the time course of EPC and HSC stimulation and homing following induction of selective EC injury in the mouse kidney along with various angiogenic factors potentially involved in EC repair and progenitor cell stimulation. Erythropoietin was used to stimulate progenitor cells in a therapeutic approach. We found that selective EC injury leads to a marked stimulation of EPCs, HSCs, and various angiogenic factors to orchestrate microvascular repair. Angiogenic factors started to increase as early as 30 min after disease induction. Progenitor cells could be first detected in the circulation and the spleen before they selectively homed to the diseased kidney. Injection of a high dose of erythropoietin 2 h after disease induction markedly attenuated vascular injury through nonhemodynamic mechanisms, possibly involving vascular endothelial growth factor release.