Metal-regulated gene expression waves in Chlamydomonas

Paola Ferrante1, Claudia Catalanotti1, Giulia Bonente2, and Giovanni Giuliano1

1ENEA (Italian National Agency for New technologies, Energy and the Environment), Casaccia Research Center, Via Anguillarese 301, 00123 S.M. di Galeria, Rome, Italy 2Dipartimento Scientifico e Tecnologico, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy

Chlamydomonas reinhardtii is a model system for algal biology. The Chlamydomonas metal-responsive Cyc6 promoter is repressed by copper and induced by nickel ions, acting, respectively, at sub-micromolar and micromolar concentrations. However, in canonical TAP medium, induction of this promoter by nickel is weak (1/6 of the strong PsaD promoter) and poorly reversible by chelators. In addition, nickel has moderate toxicity effects. Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to increase the strength and reversibility of Cyc6 induction, and to alleviate toxicity effects. The first intron of the RbcS2 gene acts as a weak constitutive enhancer downstream of the Cyc6 promoter, increasing its expression both in inductive and non-inductive conditions. Use of an optimized TAP medium results in vastly increased Cyc6 induction by micromolar concentrations of nickel or of one copper specific chelator. Using different nickel and copper chelator concentrations, Cyc6 expression can be rapidly modulated from <0.01x to >2x of the PsaD promoter, with no (copper chelator) or moderate (nickel) toxicity effects. Copper chelator induction is rapidly reversible by addition of micromolar copper concentrations, thus resulting in a transient gene expression "wave". We conclude that the use of an expression cassette containing the Cyc6 promoter, and of growth media with optimized composition, is a reliable and cheap system for the temporal fine tuning of the expression of foreign genes in Chlamydomonas.

e-mail address of presenting author: paola.ferrante@casaccia.enea.it