FUNCTIONAL COMPLEMENTATION OF AN ESCHERICHIA-COLI GAP MUTANT SUPPORTSAN AMPHIBOLIC ROLE FOR NAD(P)-DEPENDENT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE OF SYNECHOCYSTIS SP STRAIN PCC-6803

The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosp hate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. st rain PCC 6803. was cloned by functional complementation of an Escheric hia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in phot osynthetic carbon assimilation. The Synechocystis DNA region able to c omplement the E. coli gap mutant was narrowed down tee 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative g ap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of p utative promoter and terminator sites, and orientation of the truncate d flanking genes suggested the gap-2 transcript should be monocystroni c, a possibility further confirmed by Northern blot studies. Both natu ral and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. Th e recombinant GAPDH2 showed the dual pyridine nucleotide specificity c haracteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones . The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-depen dent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-exp ressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, althoug h at different levels, in Synechocystis cells grown in different metab olic regimens, even under chemoheterotrophic conditions. A possible am phibolic role of the cyanobacterial GAPDH2, namely, anabolic for photo synthetic carbon assimilation and catabolic for carbohydrate degradati ve pathways, is discussed.