Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii

Abstract

The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni(2+)-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.

FIG. 1.

Nickel and cobalt ions can activate Cyc6 and Cpx1 expression. (A) CC125 cells were sampled 5 h after the addition of the indicated concentrations of NiCl₂ or CoCl₂, and total RNA was isolated and analyzed by blot hybridization. (B) The RNA blots were exposed to a PhosphorImager screen, and the relative band intensities were quantified and normalized to the respective RbcS2 hybridization signals. Bars: □, Cpx1; ▪, Cyc6.

FIG. 2.

Comparison of Cu²⁺, Ni²⁺, and O₂ signals. Relative accumulation of Cpx1, Cyc6, and Crd1 transcripts in strain CC125 in response to each signal: Cu²⁺ deficiency, O₂ deficiency (0% air, 98% nitrogen, 2% CO₂), or Ni²⁺ (25 μM) addition as described in the legend to Fig. 1.

FIG. 3.

Ni²⁺ ions do not prevent the utilization of copper ions from the medium. CuCl₂ (+Cu), NiCl₂ (+Ni), or both together (Cu+Ni) were added as indicated to replicate copper-deficient cultures of CC125 at t = 0. Soluble protein was prepared from an aliquot of each culture at the indicated times and analyzed by native gel electrophoresis, followed by immune detection with anti-plastocyanin serum at a 1/1,000 dilution. The positions of migration of holoplastocyanin and cytochrome c₆ are indicated.

FIG. 4.

Analysis of Ni-responsive expression of Cyc6-Ars2 reporter gene constructs. Strains containing the indicated Cyc6 promoter sequences fused to the Ars2 reporter gene were grown to mid to late log phase (5 × 10⁶ to 10 × 10⁶) in copper-supplemented TAP medium. Nickel chloride was added to 25 μM, and samples of the cultures were harvested at the indicated times for preparation and analysis of total RNA. X, mutation of GTAC core of a CuRE; 0, mutation of GTAC that is not part of a CuRE. The series of panels labeled Ars2 were probed with the Ars2 cDNA to analyze expression of the reporter gene. Expression of the endogenous Cyc6 was probed as a positive control for nickel induction.

FIG. 5.

Analysis of Cpx1-Ars2 reporter gene constructs. An analysis of nickel-responsive expression was carried out as follows. Strains containing the indicated Cpx1 5′ upstream sequences fused to the Ars2 reporter gene were grown to 4 × 10⁶ to 9 × 10⁶ cells/ml in copper-supplemented TAP medium. NiCl₂ was added to 25 μM, and samples of the cultures harvested at the indicated times for preparation and analysis of total RNA. X, mutation of GTAC core of CuRE(s); 0, mutation of GTAC that is not part of a CuRE. Endogenous Cpx1 was probed as a positive control for the efficacy of the nickel treatment.

FIG. 6.

CRR1 is required for Ni-responsive expression of Cpx1 and Cyc6. Total RNA was isolated from the mutant crr1 and a wild-type strain (CRR1 = CC425) 5 h after addition of NiCl₂ (to 25 μM) and analyzed for Cpx1 and Cyc6 expression.

FIG. 7.

Reversibility of Ni-induced gene expression. Expression of target genes in strain CC125 at 3 × 10⁶ cells/ml is shown in lane 1 (t = 0). The culture was treated with either 25 μM NiCl₂ for 5 h (lane 2) or 50 μM EDTA for 5 or 19 h (lanes 7 and 8) The reversibility of the 5 h Ni²⁺ treatment was ascertained either by addition of excess EDTA (50 μM) to chelate the Ni²⁺ ions or by transfer to fresh medium and then sampled 5 or 19 h later to assess gene expression (lanes 5 and 6 and lanes 9 and 10, respectively). Ni-treated cultures were maintained in parallel for another 5 or 19 h as a control for the EDTA treatment (lanes 3 and 4, respectively), or in the case of the washed cells, NiCl₂ was added back, and the culture sampled 5 or 19 h later (lanes 11 and 12). The total time of exposure to Ni²⁺ was, therefore, 10 or 24 h. Total RNA was isolated and analyzed by RNA blot hybridization for Cyc6 and Cpx1 expression. Cβlp expression was used as a loading control. The circular arrow indicates that the cells were transferred to fresh TAP medium as described in Materials and Methods.

FIG. 8.

Strain- and culture density-dependent optimization of Ni-responsive Cyc6 and Cpx1 expression. (A) Wild-type strains (CC125, CC425, and 2137) were treated with nickel concentrations between 25 and 50 μM. Samples were taken at the indicated times over a 24-h period and analyzed by RNA blot hybridization for Cyc6 and Cpx1 gene expression. Cβlp expression was monitored as a loading control. (B) The indicated concentration of NiCl₂ was added to cultures of CC125 at various cell densities. The cultures were sampled 5 h later for isolation and analysis of total RNA.