Metabolic control in Acinetobacter sp. II. Effect of C4 versus C2 substrates on alpha-ketoglutarate dehydrogenase synthesis

1970 ◽  
Vol 16 (9) ◽  
pp. 817-820 ◽  
Author(s):  
Robert N. Sturm ◽  
Norma J. Herman ◽  
Emily J. Bell
Keyword(s):  
Control Mechanism ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Substrate Oxidation ◽  
Enzyme Synthesis ◽  
Lag Period ◽  
Alpha Ketoglutarate Dehydrogenase ◽  
Acetate Medium ◽  
Ketoglutarate Dehydrogenase

The synthesis of alpha-ketoglutarate dehydrogenase by a species of Acinetobacter growing in the presence of C4 compounds (succinate or malate) and in the presence of a C2 compound (acetate), as sole carbon sources, has been investigated. The rate of synthesis of this enzyme is increased rapidly when cells are inoculated into a succinate medium, and growth is initiated essentially without a lag period. The enzyme is synthesized after some lag period in the presence of malate and growth begins as the rate of enzyme synthesis begins to increase. On the contrary, growth begins immediately upon inoculation of the cells into an acetate medium. After a few hours of growth the level of alpha-ketoglutarate dehydrogenase begins to fall and apparent repression of synthesis occurs. These results are discussed in the light of isocitrate lyase levels in the cells at the same time periods and the evidence indicates that when levels of alpha-ketoglutarate dehydrogenase are high, those of isocitrate lyase are low. This suggests a control mechanism regulating the concurrent operation of the tricarboxylic acid cycle and the glyoxylate by-pass. Data are presented also which correlate substrate oxidation by succinate-grown cells and the cellular levels of alpha-ketoglutarate dehydrogenase.

Influence of RpoN on isocitrate lyase activity in Pseudomonas aeruginosa

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1201-1210 ◽  
Author(s):  
Jessica M. Hagins ◽  
Jessica A. Scoffield ◽  
Sang-Jin Suh ◽  
Laura Silo-Suh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metabolic Pathways ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Acute Infection ◽  
Carbon Sources ◽  
Pulmonary Infections ◽  
Lyase Activity ◽  
Glyoxylate Pathway ◽  
Knockout Mutation

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in patients with cystic fibrosis (CF). The metabolic pathways utilized by P. aeruginosa during these infections, which can persist for decades, are poorly understood. Several lines of evidence suggest that the glyoxylate pathway, which utilizes acetate or fatty acids to replenish intermediates of the tricarboxylic acid cycle, is an important metabolic pathway for P. aeruginosa adapted to the CF lung. Isocitrate lyase (ICL) is one of two major enzymes of the glyoxylate pathway. In a previous study, we determined that P. aeruginosa is dependent upon aceA, which encodes ICL, to cause disease on alfalfa seedlings and in rat lungs. Expression of aceA in PAO1, a P. aeruginosa isolate associated with acute infection, is regulated by carbon sources that utilize the glyoxyate pathway. In contrast, expression of aceA in FRD1, a CF isolate, is constitutively upregulated. Moreover, this deregulation of aceA occurs in other P. aeruginosa isolates associated with chronic infection, suggesting that high ICL activity facilitates adaptation of P. aeruginosa to the CF lung. Complementation of FRD1 with a PAO1 clone bank identified that rpoN negatively regulates aceA. However, the deregulation of aceA in FRD1 was not due to a knockout mutation of rpoN. Regulation of the glyoxylate pathway by RpoN is likely to be indirect, and represents a unique regulatory role for this sigma factor in bacterial metabolism.

Metabolic control in Acinetobacter sp. I. Effect of C4 versus C2 and C3 substrates on isocitrate lyase synthesis

1970 ◽  
Vol 16 (8) ◽  
pp. 769-774 ◽  
Author(s):  
Norma J. Herman ◽  
Emily J. Bell
Keyword(s):  
Carbon Source ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Enzyme Synthesis ◽  
Synthetase Activity ◽  
The Glyoxylate Cycle

The comparative effects of various substrates serving as sole carbon and energy source or as a supplemental nutrient on the synthesis of isocitrate lyase by a species of Acinetobacter have been investigated. Previous work has shown that succinate, as carbon source, allows some late, limited induction of enzyme synthesis. No increase in synthesis is seen above the basal level, however, in cultures growing in a medium containing L-malate as a sole carbon source. The addition of acetate to cultures growing in media containing either of the C4 intermediates results in rapid enzyme induction. Further, Acinetobacter grows very well in pyruvate medium and isocitrate lyase is synthesized to a significant extent, indicating that the glyoxylate cycle is acting anaplerotically under these conditions. Phosphoenolpyruvate synthetase activity has been demonstrated in this organism; levels comparable to those observed in Escherichia coli have been detected; the levels of NAD- and NADP-linked "malic enzyme" and phosphoenolpyruvate carboxykinase, enzymes functioning in C4 to C3 conversion, do not fluctuate with the various carbon sources tested; i.e. no correlation between the in vitro specific activity of these enzymes and the levels of isocitrate lyase activity may be made. All of the data are consistent with the hypothesis that, in this aerobic organism, as opposed to the facultative E. coli, the C4 intermediates of the tricarboxylic acid cycle may be more direct "coarse" control metabolites regulating the rate of the glyoxylate cycle.

scholarly journals A 27 kDa protein binds to a positive and a negative regulatory sequence in the promoter of the ICL1 gene from Saccharomyces cerevisiae

1998 ◽  
Vol 329 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Isabel ORDIZ ◽  
Pilar HERRERO ◽  
Rosaura RODICIO ◽  
M. Juana GANCEDO ◽  
Fernando MORENO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Repression ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Regulatory Protein ◽  
Carbon Sources ◽  
Promoter Sequence ◽  
Regulatory Sequence ◽  
Glucose Binding ◽  
Upstream Activating Sequence

Isocitrate lyase, encoded by ICL1, is one of the key enzymes of the glyoxylate pathway, which operates as an anaplerotic route for replenishing the tricarboxylic acid cycle; it is required for growth of Saccharomyces cerevisiae on carbon sources such as ethanol, but is dispensable when fermentable carbon sources are available. The positive regulation of the ICL1 gene by an upstream activating sequence (UAS) element located between -397 and -388 has been previously reported. In this paper we show that the ICL1 promoter sequence 5ʹ-AGTCCGGACTAGCATCCCAG-3ʹ located between -261 and -242 contains an upstream repressing sequence (URS) element. We have identified and partially purified a 27 kDa protein that binds specifically to both the UAS and URS sequences of the ICL1 promoter. For both UAS and URS, binding requires the protein Snf1 (Cat1), a protein kinase essential for the derepression of genes repressed by glucose. Binding does not take place with extracts from glucose-grown strains, unless they lack Mig1, a negative regulatory protein involved in glucose repression.

scholarly journals Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claudia Durall ◽  
Kateryna Kukil ◽  
Jeffrey A. Hawkes ◽  
Alessia Albergati ◽  
Peter Lindblad ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Tca Cycle ◽  
Malate Synthase ◽  
Glyoxylate Shunt ◽  
Control Strain ◽  
Genes Encoding ◽  
Pcc 6803

Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and .alpha.-ketoglutarate dehydrogenase complexes of Escherichia coli

Biochemistry ◽  
1981 ◽  
Vol 20 (16) ◽  
pp. 4555-4560 ◽  
Author(s):  
Larry R. Stepp ◽  
Dennis M. Bleile ◽  
Donald K. McRorie ◽  
Flora H. Pettit ◽  
Lester J. Reed
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Alpha Ketoglutarate Dehydrogenase ◽  
Ketoglutarate Dehydrogenase

Acetate metabolism in Escherichia coli

1978 ◽  
Vol 24 (2) ◽  
pp. 149-153 ◽  
Author(s):  
T. M. Lakshmi ◽  
Robert B. Helling
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Citrate Synthase ◽  
Isocitrate Lyase ◽  
Glyoxylate Cycle ◽  
Acetate Metabolism ◽  
Wild Type ◽  
Intermediary Metabolites ◽  
Lyase Activity ◽  
Acetate Medium ◽  
The Glyoxylate Cycle

Levels of several intermediary metabolites were measured in cells grown in acetate medium in order to test the hypothesis that the glyoxylate cycle is repressed by phosphoenolpyruvate (PEP). Wild-type cells had less PEP than either isocitrate dehydrogenase – deficient cells (which had greater isocitrate lyase activity than the wild type) or isocitrate dehydrogenase – deficient, citrate synthase – deficient cells (which are poorly inducible). Thus induction of the glyoxylate cycle is more complicated than a simple function of PEP concentration. No correlation between enzyme activity and the level of oxaloacetate, pyruvate, or citrate was found either. Citrate was synthesized in citrate synthase – deficient mutants, possibly via citrate lyase.

Sign in / Sign up

Export Citation Format

Share Document