scholarly journals Metabolomics of Escherichia coli Treated with the Antimicrobial Carbon Monoxide-Releasing Molecule CORM-3 Reveals Tricarboxylic Acid Cycle as Major Target

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Sandra M. Carvalho ◽  
Joana Marques ◽  
Carlos C. Romão ◽  
Lígia M. Saraiva
Keyword(s):  
Escherichia Coli ◽  
Carbon Monoxide ◽  
Tricarboxylic Acid Cycle ◽  
Redox Homeostasis ◽  
Glutamate Synthase ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Bacterial Cells ◽  
Content Type ◽  
Iron Sulfur

ABSTRACT In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli. By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.

scholarly journals Blocks in Tricarboxylic Acid Cycle of Salmonella enterica Cause Global Perturbation of Carbon Storage, Motility, and Host-Pathogen Interaction

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Janina Noster ◽  
Nicole Hansmeier ◽  
Marcus Persicke ◽  
Tzu-Chiao Chao ◽  
Rainer Kurre ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Tricarboxylic Acid Cycle ◽  
Glycogen Synthesis ◽  
Tca Cycle ◽  
Carbon Fluxes ◽  
Tricarboxylic Acid ◽  
Cellular Functions ◽  
Content Type ◽  
Acid Cycle ◽  
Serovar Typhimurium

ABSTRACT The tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S. Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (ΔfumABC) elicited a unique metabolic profile. Alongside fumarate, S. Typhimurium ΔfumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S. Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S. Typhimurium and cellular functions and virulence. IMPORTANCE We performed perturbation analyses of the tricarboxylic acid cycle of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium. The defect of fumarase activity led to accumulation of fumarate but also resulted in a global alteration of carbon fluxes, leading to increased storage of glycogen. Gross alterations were observed in proteome and metabolome compositions of fumarase-deficient Salmonella. In turn, these changes were linked to aberrant motility patterns of the mutant strain and resulted in highly increased phagocytic uptake by macrophages. Our findings indicate that basic cellular functions and specific virulence functions in Salmonella critically depend on the proper function of the primary metabolism.

scholarly journals A Dysfunctional Tricarboxylic Acid Cycle Enhances Fitness of Staphylococcus epidermidis During β-Lactam Stress

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Vinai Chittezham Thomas ◽  
Lauren C. Kinkead ◽  
Ashley Janssen ◽  
Carolyn R. Schaeffer ◽  
Keith M. Woods ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Citric Acid ◽  
Cell Surface ◽  
Staphylococcus Epidermidis ◽  
Citric Acid Cycle ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Content Type ◽  
Acid Cycle

ABSTRACT A recent controversial hypothesis suggested that the bactericidal action of antibiotics is due to the generation of endogenous reactive oxygen species (ROS), a process requiring the citric acid cycle (tricarboxylic acid [TCA] cycle). To test this hypothesis, we assessed the ability of oxacillin to induce ROS production and cell death in Staphylococcus epidermidis strain 1457 and an isogenic citric acid cycle mutant. Our results confirm a contributory role for TCA-dependent ROS in enhancing susceptibility of S. epidermidis toward β-lactam antibiotics and also revealed a propensity for clinical isolates to accumulate TCA cycle dysfunctions presumably as a way to tolerate these antibiotics. The increased protection from β-lactam antibiotics could result from pleiotropic effects of a dysfunctional TCA cycle, including increased resistance to oxidative stress, reduced susceptibility to autolysis, and a more positively charged cell surface. IMPORTANCE Staphylococcus epidermidis, a normal inhabitant of the human skin microflora, is the most common cause of indwelling medical device infections. In the present study, we analyzed 126 clinical S. epidermidis isolates and discovered that tricarboxylic acid (TCA) cycle dysfunctions are relatively common in the clinical environment. We determined that a dysfunctional TCA cycle enables S. epidermidis to resist oxidative stress and alter its cell surface properties, making it less susceptible to β-lactam antibiotics.

scholarly journals Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

2016 ◽  
Vol 82 (19) ◽  
pp. 5960-5968 ◽  
Author(s):  
Tomohiro Shimada ◽  
Kan Tanaka
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Monitoring System ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Bacterial Cells ◽  
Bacterial Luciferase ◽  
Content Type ◽  
Time Dynamics ◽  
The Tca Cycle

ABSTRACTRegulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employingVibrio fischeriluciferase to probe the intracellular metabolic condition inEscherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used aVibrio fischeriluciferase monitoring system to probe the intracellular metabolic condition inEscherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.

scholarly journals Inactivation of the Tricarboxylic Acid Cycle Aconitase Gene from Streptomyces viridochromogenesTü494 Impairs Morphological and Physiological Differentiation

1999 ◽  
Vol 181 (22) ◽  
pp. 7131-7135 ◽  
Author(s):  
D. Schwartz ◽  
S. Kaspar ◽  
G. Kienzlen ◽  
K. Muschko ◽  
W. Wohlleben
Keyword(s):  
Escherichia Coli ◽  
Tricarboxylic Acid Cycle ◽  
Aerial Mycelium ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
The Tca Cycle

ABSTRACT The tricarboxylic acid (TCA) cycle aconitase gene acnAfrom Streptomyces viridochromogenes Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of acnA to complement the aconitase-deficient Escherichia coli mutant JRG3259. An acnA mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals HISTOCHEMICAL INVESTIGATIONS ON THE IN VIVO EFFECTS OF FLUORIDE ON TRICARBOXYLIC ACID CYCLE DEHYDROGENASES FROM PELARGONIUM ZONALE: PART II

1967 ◽  
Vol 15 (4) ◽  
pp. 202-206
Author(s):  
C. JAMES LOVELACE ◽  
GENE W. MILLER
Keyword(s):  
Sodium Fluoride ◽  
Reductase Activity ◽  
Tricarboxylic Acid Cycle ◽  
Leaf Tissue ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Pelargonium Zonale ◽  
Acid Cycle ◽  
In Vivo Effects

In vivo effects of fluoride on tricarboxylic acid (TCA) cycle dehydrogenase enzymes of Pelargonium zonale were studied using p-nitro blue tetrazoleum chloride. Plants were exposed to 17 ppb HF, and enzyme activities in treated plants were compared to those in controls. Leaves of control plants were incubated in 5 x 10–3 M sodium fluoride. Injuries observed in fumigation and solution experiments were similar. Leaf tissue subjected to HF or sodium fluoride evidenced less succinic p-nitro blue tetrazoleum reductase activity than did control tissue. Other TCA cycle dehydrogenase enzymes were not observably affected by the fluoride concentrations used in these experiments. Excised leaves cultured in 5 x 10–3 M sodium fluoride exhibited less succinic p-nitro blue tetrazoleum reductase activity after 24 hr than did leaves cultured in 5 x 10–3 M sodium chloride.

scholarly journals Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amin Zargar ◽  
David N. Quan ◽  
Karen K. Carter ◽  
Min Guo ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Cell Contact ◽  
Bacterial Cells ◽  
Content Type ◽  
Phenotypic Differences ◽  
E Coli ◽  
Autoinducer 2 ◽  
Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

scholarly journals Current Advances in the Biodegradation and Bioconversion of Polyethylene Terephthalate

2021 ◽  
Vol 10 (1) ◽  
pp. 39
Author(s):  
Xinhua Qi ◽  
Wenlong Yan ◽  
Zhibei Cao ◽  
Mingzhu Ding ◽  
Yingjin Yuan
Keyword(s):  
Polyethylene Terephthalate ◽  
Microbial Consortium ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Microbial Consortia ◽  
Plastic Pollution ◽  
One Step ◽  
Complex Polymers ◽  
The One

Polyethylene terephthalate (PET) is a widely used plastic that is polymerized by terephthalic acid (TPA) and ethylene glycol (EG). In recent years, PET biodegradation and bioconversion have become important in solving environmental plastic pollution. More and more PET hydrolases have been discovered and modified, which mainly act on and degrade the ester bond of PET. The monomers, TPA and EG, can be further utilized by microorganisms, entering the tricarboxylic acid cycle (TCA cycle) or being converted into high value chemicals, and finally realizing the biodegradation and bioconversion of PET. Based on synthetic biology and metabolic engineering strategies, this review summarizes the current advances in the modified PET hydrolases, engineered microbial chassis in degrading PET, bioconversion pathways of PET monomers, and artificial microbial consortia in PET biodegradation and bioconversion. Artificial microbial consortium provides novel ideas for the biodegradation and bioconversion of PET or other complex polymers. It is helpful to realize the one-step bioconversion of PET into high value chemicals.

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

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