The random mutational analysis of the structure and function of photosystem II reaction center targeting to the psbAII gene of Synechocystis sp. PCC 6803

Abstract

In order to analyze the structure¿function relationships in the photosystem II (PSII), mutants of Synechocystis sp. PCC 6803 having random amino acid substitution(s) in the D1 subunit were created in this study. The targeted random mutagenesis covered 210 amino acid segment of the D1 protein (S148-A357). The kanamycin-resistant transformants carrying the mutated fragment of psbAII gene, created by in vitro random PCR, were screened based on the loss of photoautotrophy, either by direct inspection of growth on agar plates or systematic nitrofurantoin selection under illumination diminishing photosynthetically active cells (Yamasato and Satoh, 2001). By this method, we have succeeded in isolating approximately 150 photosynthesis-deficient mutants, moreover, 73 of them were heterotrophic mutants and did not contain any stop codon or codon shift. In the 73 mutants, the amino acid substitutions were distributed almost evenly in the targeted region. However, the frequency of mutation was high at specific sites, which have established to be important in the PSII, such as Y161, H198 and H272, proving the effectiveness of this method in identifying amino acid residues of functional importance in the PSII. On the other hand, some novel sites, such as Q165, Q187, N298 and W278, were identified to be important to the manifestation of photoautotrophy of this organism. The analysis of these random mutants provides entirely new aspects in understanding the structure¿function relationships in the PSII, which could not be elucidated by the crystallographic analysis. The preliminary characterization of these mutants will be presented in the accompanying poster.