The Incorporation and Metabolism of Glucose by Anabaena variabilis

Pearce, J.; Carr, N. G.

doi:10.1099/00221287-54-3-451

Cited by 101 publications

(34 citation statements)

References 28 publications

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“…strain PCC 6803 cells growing under different trophic conditions, even in strict chemoheterotrophy when no photosynthesis is performed at all, so that it seems able to accomplish by itself all potential metabolic functions, without the need for another gap product. Our data on GAPDH2 regulation shed new light on several contradictory reports in the early literature concerning specific activity measurements in cell extracts of cyanobacteria (3,13,24,32,33). The good correlation found between specific activity, GAPDH2 protein, and mRNA gap-2 transcript levels during the dramatic decrease of these parameters observed after glucose addition to Synechocystis sp.…”

mentioning

confidence: 53%

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

1997

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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 NADPdependent 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.Phosphorylating D-glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) are enzymes involved in the central pathways of carbon metabolism that have been found in all organisms studied so far (16). Due to their key metabolic roles, these proteins and their corresponding gap genes have been well characterized in many organisms (8,16). In the case of the widespread glycolytic GAPDH, by far the best-studied member of this enzyme family, the knowledge of its structure has allowed directed mutagenesis of essential amino acid residues of the active site and the eventual detailed description of the enzymatic mechanism (12,18,44).Three distinct GAPDH enzymes with different subcellular localizations and performing diverse roles are present in photosynthetic eukaryotic organisms: (i) a typical NAD-dependent...

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confidence: 53%

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

1997

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“…These activities are of the same order of magnitude as rates of photosynthesis measured for intact filaments (unpublished data). In other blue-green algae, the oxidative pentose phosphate pathway is the major route of sugar dissimilation (3,(10)(11)(12). Thus, heterocysts may possibly play a major role in the dissimilation of sugars in whole filaments, and may conceivably supplement the NADPH of adjacent vegetative cells.…”

Section: Resultsmentioning

confidence: 99%

“…Hexokinase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, and glyceraldehyde-3-P dehydrogenase were assayed under the conditions described by Pearce and Carr (10).…”

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confidence: 99%

Activities of Enzymes of the Oxidative and the Reductive Pentose Phosphate Pathways in Heterocysts of a Blue-Green Alga

Winkenbach¹,

Wölk²

1973

Plant Physiol.

138

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Preparations of heterocysts of Anabaena cylindrica Brunswick, N.J.) continuously illuminated with six fluorescent tubes (daylight, 15 w) and gassed with air to a density of 3.0 ,ug Chl/ml. The doubling time based on Chl was approximately 17 hr. Cultures were checked regularly for bacterial contamination by microscopic examination, and occasionally by subculture of 1 ml into 10 ml of culture medium supplemented with 1% glucose and 0.5% peptone. Every 24 hr 7 to 8 liters of the 12 liters of algal suspension were removed and replaced with fresh medium. Filaments concentrated by centrifugation (5 min, 1100g) were resuspended in a small volume of used growth medium and stored on ice for 1 to 3 hr. Harvest, concentration, and resuspension of filaments were accomplished within 30 min.Preparation of Cell-Free Extracts. Immediately prior to use, the concentrated suspension was centrifuged (2 min, 5000g) and resuspended either approximately 100 ug Chl/ml in ice-cold tris buffer (20 mm tris HCl, pH 7.6, containing 0.1 mm EDTA and 10 mm 2-mercaptoethanol), or approximately 250 ,ug Chl/ ml in ice-cold phosphate buffer (40 mm KH}P04-K,HPO4 pH 7.0). The dense suspension was cavitated at setting no. 3 of a Model S-125 Sonifier (Heat Systems, Inc., Melville, N.Y.), which was cooled with a water jacket (12 C). Cavitation for 5 to 11 sec/ml broke most of the vegetative cells and left the major portion of the heterocysts apparently intact. After 1 min/ml of cavitation, virtually all of the vegetative cells and heterocysts were broken. In certain experiments, the phosphate buffer was supplemented with 1.5 mM glucose-6-P and 3 mM MgCl,. Cell-free extracts were obtained by centrifugation for 4 or 5 min at 50OOg or 12,000g.For studies on the time course of solubilization of glucose-6-P dehydrogenase an algal suspension, initial volume 22 ml, was cavitated for 5 sec/ml and then for four successive periods of 3 sec/ml each. After each period of cavitation, a 1-ml aliquot was withdrawn and transferred into a 4-ml centrifuge tube at 0 C. The volume of suspension was then reduced to 12 ml, and cavitation was continued for a total of 1 min/ml. All centrifugations were carried out at 1 to 5 C.Heterocyst Preparations. Twenty-two-milliliter batches of algal suspension containing about 250 ,ug Chl/ml were cavitated for 11 sec/ml, left 10 min at 24 C, diluted with 60 ml of distilled water (2-4 C), and centrifuged for 2 min at 1500g.The pellets were combined and washed three times with 20 ml of growth medium (2-4 C). The final pellet was resuspended in phosphate buffer with supplements (Table IA). In another set of experiments (Table IB)

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“…The activity of isocitrate dehydrogenase was several fold greater after growth of Escherichia coli and Pseudomonas aeruginosa on acetate rather than glucose (Pearce & Carr, unpublished). In other aspects of intermediary metabolism Anabaetta variabilis has been shown not to exercise control over enzyme biosynthesis but only on existing enzyme molecules by end-product interaction (Carr, 1967;Pearce & Carr, 1967a;Pearce & Carr, 1968;Hood & Carr, 1968). The failure of A. variabilis and other bluegreen algae to increase growth rate in the presence of substrates known to be assimilated and metabolized could be due, at least in part, to lack of control at the transcription level.…”

Section: Discussionmentioning

confidence: 99%

The Incomplete Tricarboxylic Acid Cycle in the Blue-green Alga Anabaena Variabilis

Pearce

Leach

Carr

1969

Journal of General Microbiology

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153

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SUMMARYThe presence of an incomplete tricarboxylic acid cycle in Anabaenu vuriabilis and Anacystis nidulans is described. These blue-green algae lack both a-oxoglutarate dehydrogenase and succinyl-CoA synthetase. Succinyl-CoA was formed in extracts of A. variabilis by 3-ketoacyl CoA-transferase using acetoacetyl-CoA as CoA donor. The activities of the other tricarboxylic cycle enzymes were measured in extracts prepared from autotrophic organisms and from those grown in the presence of acetate. No alterations in activity indicative of enzyme repression or de-repression were observed. These results are discussed in relation to possible reasons for autotrophic behaviour.

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The Incorporation and Metabolism of Glucose by Anabaena variabilis

Cited by 101 publications

References 28 publications

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

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

Activities of Enzymes of the Oxidative and the Reductive Pentose Phosphate Pathways in Heterocysts of a Blue-Green Alga

The Incomplete Tricarboxylic Acid Cycle in the Blue-green Alga Anabaena Variabilis

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