Over Expression of the Cyanobacterial Pgr5-Homologue Leads to Pseudoreversion in a Gene Coding for a Putative Esterase in Synechocystis 6803

Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network.

Unconventional biochemical regulation of the oxidative pentose phosphate pathway in the model cyanobacterium Synechocystis sp. PCC 6803

Metabolite production from carbon dioxide using sugar catabolism in cyanobacteria has been in the spotlight recently. Synechocystis sp. PCC 6803 (Synechocystis 6803) is the most studied cyanobacterium for metabolite production. Previous in vivo analyses revealed that the oxidative pentose phosphate (OPP) pathway is at the core of sugar catabolism in Synechocystis 6803. However, the biochemical regulation of the OPP pathway enzymes in Synechocystis 6803 remains unknown. Therefore, we characterized a key enzyme of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PDH), and related enzymes from Synechocystis 6803. Synechocystis 6803 G6PDH was inhibited by citrate in the oxidative tricarboxylic acid (TCA) cycle. Citrate has not been reported as an inhibitor of G6PDH before. Similarly, 6-phosphogluconate dehydrogenase, the other enzyme from Synechocystis 6803 that catalyzes the NADPH-generating reaction in the OPP pathway, was inhibited by citrate. To understand the physiological significance of this inhibition, we characterized succinic semialdehyde dehydrogenase (SSADH) from Synechocystis 6803 (SySSADH), which catalyzes one of the NAD(P)H generating reactions in the oxidative TCA cycle. Similar to isocitrate dehydrogenase from Synechocystis 6803, SySSADH specifically catalyzed the NADPH-generating reaction and was not inhibited by citrate. The activity of SySSADH was lower than that of other bacterial SSADHs. Previous and this studies revealed that unlike the OPP pathway, the oxidative TCA cycle is a pathway with low efficiency in NADPH generation in Synechocystis 6803. It has, thus, been suggested that to avoid NADPH overproduction, the OPP pathway dehydrogenase activity is repressed when the flow of the oxidative TCA cycle increases in Synechocystis 6803.