RegX3 Controls Glyoxylate Shunt and Mycobacteria Survival by Directly Regulating the Transcription of Isocitrate Lyase Gene in Mycobacterium smegmatis

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl-CoA was shown to increase the binding affinity of RamB to its binding sites and enable RamB to bind to RamBS2 that is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1: MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis . Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis , this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis - and trans -acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that the similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis .

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

Microbial Cell Factories ◽

10.1186/s12934-021-01529-y ◽

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.

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Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis

Chinese Medical Journal ◽

10.1097/00029330-200806020-00015 ◽

2008 ◽

Vol 121 (12) ◽

pp. 1114-1119 ◽

Cited By ~ 11

Author(s):

Jun-ming LI ◽

Na LI ◽

Dao-yin ZHU ◽

La-gen WAN ◽

Yong-lin HE ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cell Apoptosis ◽

Mycobacterium Smegmatis ◽

Isocitrate Lyase

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Increase in glyoxylate shunt enzymes during cellular morphogenesis in Myxococcus xanthus

Canadian Journal of Microbiology ◽

10.1139/m71-036 ◽

1971 ◽

Vol 17 (2) ◽

pp. 209-211 ◽

Cited By ~ 5

Author(s):

J. Bland ◽

Wu-Kuang Yeh ◽

D. White ◽

A. Hendricks

Keyword(s):

Actinomycin D ◽

Isocitrate Lyase ◽

Myxococcus Xanthus ◽

Malate Synthase ◽

Glyoxylate Shunt ◽

Cellular Morphogenesis

Isocitrate lyase (EC. 4.1.3.1) and malate synthase (EC. 4.1.3.2) increase during microcyst formation in Myxococcus xanthus. The increase in activity is inhibited by chloramphenicol and actinomycin-D.

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Pseudomonas aeruginosa Twitching Motility-Mediated Chemotaxis towards Phospholipids and Fatty Acids: Specificity and Metabolic Requirements

Journal of Bacteriology ◽

10.1128/jb.00129-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4038-4049 ◽

Cited By ~ 44

Author(s):

Rhea M. Miller ◽

Andrew P. Tomaras ◽

Adam P. Barker ◽

Dennis R. Voelker ◽

Edward D. Chan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Alternative Energy ◽

Isocitrate Lyase ◽

Malate Synthase ◽

Glyoxylate Shunt ◽

Twitching Motility ◽

Long Chain Fatty Acid ◽

Type Iv ◽

Energy Taxis ◽

Lipid Metabolism Genes

ABSTRACT Pseudomonas aeruginosa demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of P. aeruginosa (i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via β-oxidation and the glyoxylate shunt. It was discovered that P. aeruginosa exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase (ΔaceA) and malate synthase (ΔaceB), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of P. aeruginosa up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the ΔaceA and ΔaceB mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.

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Comparison of glycolytic, pentose phosphate pathway, glyoxylate shunt, Krebs' cycle enzymes in Ganeo tigrinum parasitizing hibernating and non-hibernating Rana cyanophlyctis and R. tigrina

Journal of Helminthology ◽

10.1017/s0022149x00007896 ◽

1983 ◽

Vol 57 (1) ◽

pp. 59-68 ◽

Cited By ~ 1

Author(s):

P. N. Sharma ◽

Sushila Mandawat

Keyword(s):

Isocitrate Lyase ◽

Krebs Cycle ◽

Pentose Phosphate ◽

Glyoxylate Shunt ◽

Strong Activity ◽

Key Enzymes ◽

Phosphogluconate Dehydrogenase ◽

Weak Activity ◽

Krebs Cycle Enzymes ◽

Glucose 6 Phosphate Dehydrogenase

AbstractThe histochemical site and distribution of hexokinase, glycogen phosphorylase (GP Rylase), lactate dehydrogenase (LDH) (key enzymes of glycolysis), glucose-6-phosphate dehydrogenase (GPD) and 6-phosphogluconate dehydrogenase (6PGD) (pentose phosphate shunt enzymes), isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and α-ketoglutarate dehydrogenase (α-KDH) (key enzymes of Krebs' cycle), malate synthetase (MS) and isocitrate lyase (IL) (enzymes of glyoxylate shunt) in various tissues of Ganeo tigrinum from hibernating and non-hibernating Rana cyanophlyctis and R. tigrina were studied. Differences in their intensities were revealed. Weak activity of GP Rylase and strong activity of hexokinase in flukes from non-hibernating hosts indicates that they utilize glucose through glycolysis for energy turnover. Intense GP Rylase and weak hexokinase activity in worms from hibernating hosts indicates the utilization of glycogen. Strong activity of IDH, SDH, MDH, α-KGD, MS and IL was demonstrable in the tissues of flukes from non-hibernating hosts, suggesting that Krebs' cycle and glyoxylate shunt, respectively, were operating. Tissues of the fluke from hibernating hosts, on the other hand, displayed positive activity only for SDH and MDH; no activity for MS and IL, the enzymes of glyoxylate shunt, was observed, The activity of the above enzymes was found to be relatively low in worms from hibernating hosts.

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Role of Phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate Lyase Reaction in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01628-06 ◽

2006 ◽

Vol 189 (3) ◽

pp. 1176-1178 ◽

Cited By ~ 14

Author(s):

Tadashi Ogawa ◽

Keiko Murakami ◽

Hirotada Mori ◽

Nobuyoshi Ishii ◽

Masaru Tomita ◽

...

Keyword(s):

Escherichia Coli ◽

Isocitrate Dehydrogenase ◽

Isocitrate Lyase ◽

Tricarboxylic Acid Cycle ◽

Tricarboxylic Acid ◽

Glyoxylate Shunt ◽

Acid Cycle ◽

Uncompetitive Inhibition

ABSTRACT Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki ′ of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

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2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa

International Journal of Molecular Sciences ◽

10.3390/ijms21072490 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2490

Author(s):

Alyssa C. McVey ◽

Sean Bartlett ◽

Mahmud Kajbaf ◽

Annalisa Pellacani ◽

Viviana Gatta ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Isocitrate Lyase ◽

Opportunistic Pathogen ◽

Malate Synthase ◽

Cell Permeability ◽

Titration Calorimetry ◽

Glyoxylate Shunt ◽

Nutrient Sources ◽

Hospital Acquired

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

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The Glyoxylate Cycle Is Involved in White-Opaque Switching in Candida albicans

Journal of Fungi ◽

10.3390/jof7070502 ◽

2021 ◽

Vol 7 (7) ◽

pp. 502

Author(s):

Susana Hidalgo Vico ◽

Daniel Prieto ◽

Rebeca Alonso Monge ◽

Elvira Román ◽

Jesús Pla

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Gastrointestinal Tract ◽

Isocitrate Lyase ◽

Glyoxylate Cycle ◽

Carbon Sources ◽

Glyoxylate Shunt ◽

Phenotypic Analysis ◽

Altered Metabolism ◽

The Glyoxylate Cycle

Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans. The master regulator of the white-opaque transition WOR1 has been implicated in the adaptation to this commensal status. A proteomic analysis of cells overexpressing this transcription factor (WOR1OE) suggested an altered metabolism of carbon sources and a phenotypic analysis confirmed this alteration. The WOR1OE cells are deficient in using trehalose and xylose and are unable to use 2C sources, which is consistent with a reduction in the amount of Icl1, the isocitrate lyase enzyme. The icl1Δ/Δ mutants overexpressing WOR1 are deficient in the production of phloxine B positive cells, a main characteristic of opaque cells, a phenotype also observed in mating type hemizygous mtla1Δ icl1Δ/Δ cells, suggesting the involvement of Icl1 in the adaptation to the commensal state. In fact, icl1Δ/Δ cells have reduced fitness in mouse gastrointestinal tract as compared with essentially isogenic heterozygous ICL1/icl1Δ, but overproduction of WOR1 in an icl1Δ/Δ mutant does not restore fitness. These results implicate the glyoxylate shunt in the adaptation to commensalism of C. albicans by mechanisms that are partially independent of WOR1.

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