This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Valverde, F. Articles by Serrano, A. Search for Related Content PubMed PubMed Citation Articles by Valverde, F. Articles by Serrano, A.

 Previous Article  |  Next Article 

J. Bacteriol., Jul 1997, 4513-4522, Vol 179, No. 14
Copyright © 1997, American Society for Microbiology

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

F Valverde, M Losada and A Serrano
Instituto de Bioquimica Vegetal y Fotosintesis, Centro de Investigacion Isla de la Cartuja, Universidad de Sevilla-CSIC, Seville, Spain.

The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3- phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia 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 photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 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 gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic 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-dependent 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-expressing 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, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.

This article has been cited by other articles:

• Singh, A. K., Sherman, L. A. (2005). Pleiotropic Effect of a Histidine Kinase on Carbohydrate Metabolism in Synechocystis sp. Strain PCC 6803 and Its Requirement for Heterotrophic Growth. J. Bacteriol. 187: 2368-2376 [Abstract] [Full Text]  
• Lopez-Marques, R. L., Perez-Castineira, J. R., Losada, M., Serrano, A. (2004). Differential Regulation of Soluble and Membrane-Bound Inorganic Pyrophosphatases in the Photosynthetic Bacterium Rhodospirillum rubrum Provides Insights into Pyrophosphate-Based Stress Bioenergetics. J. Bacteriol. 186: 5418-5426 [Abstract] [Full Text]  
• Knowles, V. L., Plaxton, W. C. (2003). From Genome to Enzyme: Analysis of Key Glycolytic and Oxidative Pentose-Phosphate Pathway Enzymes in the Cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 44: 758-763 [Abstract] [Full Text]  
• Perez-Castineira, J. R., Lopez-Marques, R. L., Villalba, J. M., Losada, M., Serrano, A. (2002). Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases. Proc. Natl. Acad. Sci. USA 99: 15914-15919 [Abstract] [Full Text]  
• Mrazek, J., Bhaya, D., Grossman, A. R., Karlin, S. (2001). Highly expressed and alien genes of the Synechocystis genome. Nucleic Acids Res 29: 1590-1601 [Abstract] [Full Text]