Two Glyceraldehyde-3-phosphate Dehydrogenases with Opposite Physiological Roles in a Nonphotosynthetic Bacterium

Abstract

Bacillus subtilis possesses two similar putative phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding genes, gap (renamed gapA) and gapB. A gapA mutant was unable to grow on glycolytic carbon sources, although it developed as well as the wild-type strain on gluconeogenic carbon sources. A gapB mutant showed the opposite phenotype. Purified GapB showed a 50-fold higher GAPDHase activity with NADP ؉ than with NAD ؉ , with K m values of 0.86 and 5.7 mM, respectively. lacZ reporter gene fusions revealed that the gapB gene is transcribed during gluconeogenesis and repressed during glycolysis. Conversely, gapA transcription is 5-fold higher under glycolytic conditions than during gluconeogenesis. GAPDH activity assays in crude extracts of wild-type and mutant strains confirmed this differential expression pattern at the enzymatic level. Genetic analyses demonstrated that gapA transcription is repressed by the yvbQ (renamed cggR) gene product and indirectly stimulated by CcpA. Thus, the same enzymatic step is catalyzed in B. subtilis by two enzymes specialized, through the regulation of their synthesis and their enzymatic characteristics, either in catabolism (GapA) or in anabolism (GapB). Such a dual enzymatic system for this step of the central carbon metabolism is described for the first time in a nonphotosynthetic eubacterium, but genomic analyses suggest that it could be a widespread feature.Glycolysis is the main pathway for degradation of carbohydrates and is found in nearly all groups of organisms. The formation of the final product of glycolysis, pyruvate, from glucose is achieved by nine enzymatic steps, most of which function in the reverse direction during gluconeogenesis. The phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1 occupies a pivotal role in the Embden-Meyerhoff pathway not only in glycolysis but also in gluconeogenesis because of the reversibility of the oxidation of glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (1,3dPG). In plants, two distinct types of phosphorylating GAPDH co-exist: (i) a strictly NAD-dependent cytoplasmic GAPDH involved in glycolysis and gluconeogenesis; and (ii) a chloroplastic GAPDH, which is involved in photosynthetic CO 2 assimilation and exhibits a dual coenzyme specificity with a preference for NADP (1, 2).Recently, two gap genes, named gap1 and gap2, have been characterized in the cyanobacterium Synechocystis sp. PPC 6803. The NAD-dependent enzyme Gap1 was reported to be essential for glycolytic glucose breakdown, whereas the enzyme Gap2, which exhibits dual coenzyme specificity, was shown to be operative in the photosynthetic Calvin cycle and in nonphotosynthetic gluconeogenesis (3). Thus, at least in some photoautotrophic bacterial species (in which the photosynthetic Calvin cycle and glycolysis/gluconeogenesis function in the same cellular compartment, in contrast to what happens in land plants and algae) two distinct GAPDHs, a strictly NAD-dependent one and the photosynthetic one, catalyze the two ...

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

“…Two GAPDH encoding genes were identified in the genomes of Methanosarcina species suggesting different metabolic functions. A similar situation is reported in Bacillus subtilis which contains two distinct GAPDHs, one acting in gluconeogenic direction and the other acting in glycolytic direction (Fillinger et al 2000). In order to address if this by-pass is generally found in (hyper)thermophiles the distribution of GAP converting enzymes in (hyper)thermophilic bacteria was studied.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…127) B. subtilis possesses NADH-and NADPH-dependent glyceraldehyde-3-P dehydrogenases, which are specific for glycolysis and gluconeogenesis and are encoded by gapA and gapB respectively. 131) This protein also represses sr1, 132) which encodes a small non-coding regulatory RNA that inhibits the translation of ahrC encoding a transcriptional regulator that activates the rocABC and rocDEF operons for arginine catabolism and represses the gene cluster for arginine biosynthesis. 108,[133][134][135] CcpN is active when cells are growing on a glycolytic substrate, even if the medium also contains a gluconeogenic substrate.…”

Glyceraldehyde-3-phosphate dehydrogenase of Xanthomonas campestris pv. campestris is required for extracellular polysaccharide production and full virulence

“…In the first phase of gluconeogenesis, pyruvates are converted after several reactions to 1,3-bisphosphoglycerates, which are converted further to GAPs by GAPDH. It has been demonstrated that, in B. subtilis and the cyanobacterium Synechocystis, one of the two GAPDH-encoding gap genes is involved in gluconeogenesis (Fillinger et al, 2000;Koksharova et al, 1998). Our previous work has shown that Xcc strain 8004 possesses a functional gluconeogenesis pathway that is indispensable for the bacterium to utilize pyruvate as the sole carbon source .…”

“…Data are the means±SD from three replicates. Fillinger et al, 2000;Koksharova et al, 1998). In many bacteria, glucose is catabolized through the glycolysis pathway, which functions primarily to provide energy in the form of ATP (Gottschalk, 1986).…”

“…For instance, Escherichia coli contains two GAPDHencoding genes, gapA and gapB; mutational studies revealed that gapA is essential for glycolysis, whilst gapB is dispensable for both glycolysis and the pyridoxal biosynthesis pathway (Seta et al, 1997). Bacillus subtilis and the cyanobacterium Synechocystis also possess two gap genes, of which one is essential for glycolysis whilst the other is operative in gluconeogenesis (Fillinger et al, 2000;Koksharova et al, 1998).…”