The herbicide-resistant species of the cyanobacterial D1 protein obtained by thorough and random in vitro mutagenesis

Abstract

Random mutations were introduced into the DNA fragment of the psbA2 gene of Synechocystis sp. PCC 6803, which encodes the carboxyl-terminal 178 amino acid region of the D1 protein of the PSII reaction center, by in vitro random mutagenesis to obtain D1 species resistant to herbicides and to understand the protein-herbicide interactions. The mutants were screened on the criterion of resistance to either 1 μM DCMU or 10 μM atrazine. In these mutants, amino acid substitutions were distributed throughout the entire area of the targeted region in the D1 protein. However, in every mutant, except for one case, the substitution was present in the region described as the 'herbicide-binding niche', i.e., between Phe211 and Leu275, although some amino acid substitutions which were not previously described were found at residues known to be involved with herbicide affinity. Thus, the result of random mutagenesis basically supports the validity of the proposed structural model for the D1 protein, as well as of the herbicide-binding niche. Preliminary characterization of the herbicide-resistant mutants obtained in this study has also been conducted.