Procedures for the Isolation of Cyanobacterial Random Mutants with a Digital Imaging Spectrophotometer

Abstract

We investigated the use of the Digital Imaging Spectrophotometer (Youvan et al., 1995) for the primary isolation of photosynthetic mutants in the cyanobacterium Synechocystis sp. PCC 6803. We tested the system with two previously characterized mutants of Synechocystis sp. PCC 6803: the Del-1 mutant, a partial deletion mutant of the psbB gene (Eaton-Rye and Vermaas, 1991), and the ΔpsbO mutant, a complete deletion of the psbO gene (Burnap and Sherman, 1991). We found that the considiration of colony sizes vs camera resolution is important for avoiding the isolation of false positive mutants. We modified the instrument by adding a magnifying lens for fluorescence imaging of plates inside the sphere. We proposed three ways in which the DIS can be used to isolate cyanobacterial random mutants: direct fluorescence intensity, fluorescence image ratios, and PC/Chl ratios calculated from absorbance. The reliabilty of each of those methods is excellent for differentiating existing PSII deletion mutants. We also proposed a statistical criterion for selecting significantly different mutants.