Cloning of the gene for 6-phosphogluconate dehydrogenase from the cyanobacterium Synechococcus sp. PCC 7942 and its regulation in response to stress

Duane David Culler, Purdue University

Abstract

The principal mode of energy metabolism in cyanobacteria is oxygenic photosynthesis. However, in the dark and under stressed conditions, cyanobacteria derive energy from degradation of glycogen granules accumulated during active photosynthesis. Previous work has shown that glycogen degradation occurs primarily, if not solely, via the oxidative pentose phosphate pathway. The gene for 6-phosphogluconate dehydrogenase, the second enzyme in this pathway, was cloned from a Synechococcus sp. PCC 7942 expression library. The deduced amino acid sequence has strong sequence identity to the corresponding sequences from E. coli, S. typhimurium B. subtilis, pig and sheep. Alignment of these sequences has permitted an evaluation of residues previously implicated in the structure and catalytic activity of the nucleotide and substrate binding sites of 6-phosphogluconate dehydrogenase. When 6-phosphogluconate dehydrogenase activity is measured in growing cultures of Synechococcus 7942, activity is six- to ten-fold higher in dense cultures than in dilute cultures. Relative transcript levels for this gene are about five-fold lower in dense cultures than in dilute cultures. When cultures of Synechococcus 7942 experience dark incubation, viability of mid-log phase cultures declines to less than 50% by three hours. There is a commensurate decrease in 6-phosphogluconate dehydrogenase activity. When late-log phase cultures are subjected to dark incubation, viability does not decline and 6-phosphogluconate dehydrogenase activity does not change. When cultures of Synechococcus 7942 experience nitrate starvation and recovery, 6-phosphogluconate dehydrogenase activity and relative transcripts begin increasing, usually within 30 minutes of nitrate starvation, and remain elevated for 24 hours. After addition of solid NaNO$\sb3$ 6-phosphogluconate dehydrogenase activity remains elevated while relative transcript levels decline. When Synechococcus 7942 experiences osmotic shock (0.2 M NaCl) 6-phosphogluconate dehydrogenase activity does not change until approx. 48 hours after treatment. Activity levels are three- to seven-fold higher by 72 hours after treatment. Relative levels of 6-phosphogluconate dehydrogenase transcripts decrease to about one-fifth of the initial value by three hours, and slowly increase during the next 21 hours. The data suggest that regulation of 6-phosphogluconate dehydrogenase activity is under both transcriptional and post-transcriptional control. Changes in 6-phosphogluconate dehydrogenase activity appear to be correlated with cyanobacterial requirement for additional fixed carbon.

Degree

Ph.D.

Advisors

Krogmann, Purdue University.

Subject Area

Biochemistry

COinS