Monitoring Cytosolic pH of Carboxysome-Deficient Cells of Synechocystis sp. PCC 6803 Using Fluorescence Analysis

Abstract

Disruption of the ccmM gene in the cyanobacterium Synechocystis sp. PCC 6803 causes a deficiency of carboxysomes and impairs growth in ambient CO₂. The effect of this gene defect on cellular metabolism was investigated using electron microscopy, biochemical and fluorescence analysis. Mutant cells were devoid of the characteristic dense polyhedral bodies called carboxysomes. The photosynthetic oxygen evolution was considerably lower in mutant cells compared to wild type, while Rubisco activity in cell extracts was similar. During photosynthetic CO₂-dependent oxygen evolution, Rubisco V _max dropped from 142 micromol mg^-1 chlorophyll h^-1 (WT) to 77 micromol mg^-1 chlorophyll h^-1 in the mutant cells, and the K _m for Ci (inorganic carbon) increased from 0.5 mM (WT) to 40 mM. The fluorescent indicator, acridine yellow, was used for non-invasive measurements of cytoplasmic pH changes in whole cells induced by addition of Ci, making use of the decrease in fluorescence yield that accompanies cytoplasmic acidification. The experimental results indicate that control of the cytoplasmic pH is linked to the internal carbon pool (Ci). Both wild-type and ccmM-deficient cells showed a linear response of acridine yellow fluorescence quenching and, thus, of internal acidification, with respect to externally added inorganic carbon. However, the fluorescence analysis of mutant (carboxysome-free) cells indicated slower kinetics of Ci accumulation.

Key words

Acridine dyes - carbon concentrating mechanism (ccm) - carboxysome - cyanobacteria - fluorescence analysis - Rubisco

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Editor: W. Martin