Behind the Shield of Czc: ZntR Controls Expression of the Gene for the Zinc-Exporting P-Type ATPase ZntA in Cupriavidus metallidurans

Abstract

In the metallophilic beta-proteobacterium Cupriavidus metallidurans, the plasmid-encoded Czc metal homeostasis system adjusts the periplasmic zinc, cobalt and cadmium concentration, which influences subsequent uptake of these metals into the cytoplasm. Behind this shield, the PIB2-type APTase ZntA is responsible for removal of surplus cytoplasmic zinc ions, thereby providing a second level of defense against toxic zinc concentrations. ZntA is the counterpart to the Zur-regulated zinc uptake system ZupT and other import systems; however, the regulator of zntA expression was unknown. The chromid-encoded zntA gene is adjacent to the genes czcI2C2B2’, which are located on the complementary DNA strand and transcribed from a common promoter region. These genes encode homologs of plasmid pMOL30-encoded Czc components. Candidates for possible regulators of zntA were identified and subsequently tested: CzcI, CzcI2, and the MerR-type gene products of the locus tags Rmet_2302, Rmet_0102, Rmet_3456. This led to the identification of Rmet_3456 as ZntR, the main regulator of zntA expression. Moreover, both CzcIs decreased Czc-mediated metal resistance, possibly to avoid “over-excretion” of periplasmic zinc ions, which could result in zinc starvation due to diminished zinc uptake into the cytoplasm. Rmet_2302 was identified as CadR, the regulator of the cadA gene for an important cadmium-exporting PIB2-type ATPase, which provides another system for removal of cytoplasmic zinc and cadmium. Rmet_0102 was not involved in regulation of the metal resistance systems examined here. Thus, ZntR forms a complex regulatory network with CadR, Zur and the CzcIs. Moreover, these discriminating regulatory proteins assign the efflux systems to their particular function. Importance Zinc is an essential metal for numerous organisms from humans to bacteria. The transportome of zinc uptake and efflux systems controls the overall cellular composition and zinc content in a double feed-back loop. Zinc starvation mediates, via the Zur regulator, an up-regulation of the zinc import capacity via the ZIP-type zinc importer ZupT and an amplification of zinc storage capacity, which together raise the cellular zinc content again. On the other hand, an increasing zinc content leads to ZntR-mediated up-regulation of the zinc efflux system ZntA, which decreases the zinc content. Together, the Zur regulon components and ZntR/ZntA balance the cellular zinc content under both high external zinc concentrations and zinc starvation conditions.

A Novel Zinc Exporter CtpG Enhances Resistance to Zinc Toxicity and Survival in Mycobacterium bovis

“…For example, in B. abortus , the expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by Zur ( 44 ). And ZntR controls the expression of the gene encoding the zinc exporter ZntA by binding directly to its promoter to adaptation to zinc toxicity environment ( 45 – 47 ). However, only the zinc uptake repressor Zur have been characterized in mycobacterial species until now.…”

Importance of RpoD- and Non-RpoD-Dependent Expression of Horizontally Acquired Genes in Cupriavidus metallidurans

“…CzcCBA is the RND-driven transenvelope efflux system for cobalt, zinc, and cadmium ions, CzcRS, a two-component regulatory system, CzcD, an inner membrane efflux pump and one of the founders of the CDF protein family, CzcE, a periplasmic protein involved in regulation of czc , CzcP, a P IB4 -type exporter of loosely bound cytoplasmic zinc ions, CzcJ and CzcI, additional periplasmic proteins, and CzcN and FlgB, which are of unknown functions ( 16 , 50 ). CzcI functions as a “quencher” of Czc activity to avoid overpumping of essential transition metal cations, mainly cobalt ( 77 ). MgtC-like proteins such as CzcM inhibit phosphate uptake under conditions of magnesium starvation ( 78 ).…”

“…S5B and C). Expression of zntA for the main zinc-exporting P IB2 -type ATPase of C. metallidurans ( 50 , 79 ) was under the control of RpoD, the ZntR regulatory protein ( 77 ). RpoD was clearly responsible for expression of most genes of the czc2 determinant, including czcI2C2B2 ′.…”

“…S5N to Z). The chromosomal cad determinant, composed of the cadR gene for the MerR-type regulator ( 77 ) in one direction of transcription and cadAC for the CadA P IB2 -type ATPase and protein CadC, started from two RpoD-dependent TSSs in a common promoter region (Fig. S5N).…”

Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes

“…Expression of the paralogous genes may be controlled by different regulatory circuits. For instance, the three genes zntA , cadA , and pbrA , encoding P IB2 -type ATPases that export Zn(II), Cd(II), and Pb(II), respectively, are under the control of their own specific MerR-type regulators zntR , cadR , and pbrR , respectively ( 19 – 22 ). Of the 10 determinants encoding transenvelope efflux systems for divalent transition metal cations, which are composed of an inner membrane component of the RND (resistance nodulation protein family), an outer membrane factor, and a connecting membrane fusion (MFP) or adapter protein, many are inactivated by deleterious mutations of the central RND- or MFP-encoding genes, which identifies them as recessive determinants ( 15 ).…”