Characterization of a heterotrophic mutant of Synechocystis sp. PCC 6803 created by random mutagenesis targeted to the psbAII gene

Abstract

In the attempt to identify specific molecular interactions responsible for the unique function of PSII, we have generated more than 100 random mutants impaired in the capacity of photoautotrophic growth due to amino acid substitution(s) in the targeted Ser148-Ala357 region on the D1 protein in Synechocystis sp. PCC6803. Out of these heterotrophic mutants, 11 strains were subjected to the preliminary characterization, which include 6 single-point mutants (N298I, W278R, A294E, Q187P, Q165L, F186Y) and 5 multi-point mutants (H195R/G276D, Q165H/Q241L, I192F/N267I, I238S/W317R, G171D/I192F/N322S). The 5 strains of those mutants, N298I, W278R, Q165L, F186Y and Q165H/Q241L, accumulated relatively large amounts of D1 protein, and it was confirmed that their PSII complexes preserved the activity of DCIP photoreduction with artificial electron donor almost comparable with that of the control strain on D1 protein basis. On the other hand, the activity with water as the electron donor was almost undetectable in these mutants, and modified thermoluminescence bands were observed in the W278R, Q165L and Q165H/Q241L mutants. An interesting general feature is that the mutation often affects the function on the other side of the complex. For example, H272R mutation on the acceptor side led to the impairment of oxygen-evolving activity; in this case no Mn-cluster can be formed. Detailed characteristics of each mutant will be presented in the poster.