scholarly journals An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance

2007 ◽  
Vol 4 (1) ◽  
pp. 53-56 ◽  
Author(s):  
Brittany A Morrison ◽  
Daniel H Shain
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Atp Hydrolysis ◽  
Gene Knockout ◽  
Compensatory Mechanism ◽  
Wild Type ◽  
Intracellular Atp ◽  
Atp Levels ◽  
Glacier Ice ◽  
Mesophilic Bacterium

Disparate psychrophiles (e.g. glacier ice worms, bacteria, algae and fungi) elevate steady-state intracellular ATP levels as temperatures decline, which has been interpreted as a compensatory mechanism to offset reductions in molecular motion and Gibb's free energy of ATP hydrolysis. In this study, we sought to manipulate steady-state ATP levels in the mesophilic bacterium, Escherichia coli , to investigate the relationship between cold temperature survivability and elevated intracellular ATP. Based on known energetic pathways and feedback loops, we targeted the AMP nucleotidase ( amn ) gene, which is thought to encode the primary AMP degradative enzyme in prokaryotes. By knocking out amn in wild-type E. coli DY330 cells using recombineering methodology, we generated a mutant (AMNk) that elevated intracellular ATP levels by more than 30% across its viable temperature range. As temperature was lowered, the relative ATP disparity between AMNk and DY330 cells increased to approximately 66% at 10°C, and was approximately 100% after storage at 0°C for 5–7 days. AMNk cells stored at 0°C for 7 days displayed approximately fivefold higher cell viability than wild-type DY330 cells treated in the same manner.

scholarly journals Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

2006 ◽  
Vol 188 (13) ◽  
pp. 4759-4768 ◽  
Author(s):  
Bonnie B. Stephens ◽  
Star N. Loar ◽  
Gladys Alexandre
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Azospirillum Brasilense ◽  
Model Organism ◽  
Spatial Gradient ◽  
Wild Type ◽  
Temporal Gradient ◽  
E Coli ◽  
Significant Difference

ABSTRACT It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

scholarly journals Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia

2020 ◽  
Vol 318 (2) ◽  
pp. L356-L365 ◽  
Author(s):  
Catharina van Heusden ◽  
Brian Button ◽  
Wayne H. Anderson ◽  
Agathe Ceppe ◽  
Lisa C. Morton ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Steady State ◽  
Atp Hydrolysis ◽  
Fluid Secretion ◽  
Pathological Feature ◽  
Airway Surface Liquid ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Atp Levels ◽  
Surface Liquid

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl− channel controlled in part by the adenosine A2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y2 receptor (P2Y2R) that activates Ca2+-regulated Cl− channels (CaCC/TMEM16) and inhibits Na+ absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10− (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.

scholarly journals Quinolone Resistance Due to Reduced Target Enzyme Expression

2003 ◽  
Vol 185 (23) ◽  
pp. 6883-6892 ◽  
Author(s):  
Dilek Ince ◽  
David C. Hooper
Keyword(s):  
Steady State ◽  
Quinolone Resistance ◽  
Start Codon ◽  
Cell Multiplication ◽  
Compensatory Mechanism ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Resistance Phenotype ◽  
Allelic Exchange ◽  
Steady State Levels

ABSTRACT We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus. A single-step mutant of wild-type S. aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four- and four- to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type. Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G→A) 13 bp upstream of the start codon of parE. Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance. Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed. The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18. The steady-state levels of topA transcripts were similar in the various strains. Growth competition experiments performed at 30, 37, and 41°C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41°C. However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts. Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype.

scholarly journals α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites

1999 ◽  
Vol 343 (1) ◽  
pp. 135-138 ◽  
Author(s):  
Toyoki AMANO ◽  
Tadashi MATSUI ◽  
Eiro MUNEYUKI ◽  
Hiroyuki NOJI ◽  
Kiyotaka HARA ◽  
...  
Keyword(s):  
Steady State ◽  
Atp Hydrolysis ◽  
Catalytic Site ◽  
Wild Type ◽  
Thermophilic Bacillus ◽  
F1 Atpase ◽  
Type Complex ◽  
Catalytic Sites ◽  
Phase Activity ◽  
Hydrolysis Activity

Homogeneous preparations of α3β3γ complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP-. Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover.

scholarly journals mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

2013 ◽  
Vol 79 (20) ◽  
pp. 6362-6368 ◽  
Author(s):  
Ying Xu ◽  
Bing Chen ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Transport Activity ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent ◽  
K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

scholarly journals Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Chunhua Zhao ◽  
Zhao Lin ◽  
Hongjun Dong ◽  
Yanping Zhang ◽  
Yin Li
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Physiological Role ◽  
Glucose Consumption ◽  
General Strategy ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Intracellular Atp ◽  
Atp Levels

ABSTRACT Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli. PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA::Tn5 mutant in a screen for increased yield of an end product derived from pyruvate (n-butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n-butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products.

scholarly journals In Vivo 31P Nuclear Magnetic Resonance Investigation of Tellurite Toxicity in Escherichia coli

2004 ◽  
Vol 70 (12) ◽  
pp. 7342-7347 ◽  
Author(s):  
Elke M. Lohmeier-Vogel ◽  
Shiela Ung ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Ph Gradient ◽  
Resistance Determinants ◽  
Intracellular Atp ◽  
Atp Levels ◽  
Glycolytic Intermediates ◽  
Nuclear Magnetic ◽  
Resistant Cells

ABSTRACT Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by 31P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by 31P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.

scholarly journals A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for profiling bacterial membrane transporters

Microbiology ◽  
2021 ◽  
Author(s):  
Jesus Enrique Salcedo-Sora ◽  
Srijan Jindal ◽  
Steve O'Hagan ◽  
Douglas B. Kell
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Dyes ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Single Gene ◽  
Wild Type ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Effects Of Ph ◽  
Small Collection

Our previous work demonstrated that two commonly used fluorescent dyes that were accumulated by wild-type Escherichia coli MG1655 were differentially transported in single-gene knockout strains, and also that they might be used as surrogates in flow cytometric transporter assays. We summarize the desirable properties of such stains, and here survey 143 candidate dyes. We eventually triage them (on the basis of signal, accumulation levels and cost) to a palette of 39 commercially available and affordable fluorophores that are accumulated significantly by wild-type cells of the ‘Keio’ strain BW25113, as measured flow cytometrically. Cheminformatic analyses indicate both their similarities and their (much more considerable) structural differences. We describe the effects of pH and of the efflux pump inhibitor chlorpromazine on the accumulation of the dyes. Even the ‘wild-type’ MG1655 and BW25113 strains can differ significantly in their ability to take up such dyes. We illustrate the highly differential uptake of our dyes into strains with particular lesions in, or overexpressed levels of, three particular transporters or transporter components (yhjV, yihN and tolC). The relatively small collection of dyes described offers a rapid, inexpensive, convenient and informative approach to the assessment of microbial physiology and phenotyping of membrane transporter function.

Escherichia coli polypeptide controlled by the lon (capR) ATP hydrolysis-dependent protease and possibly involved in cell division

1982 ◽  
Vol 152 (2) ◽  
pp. 919-923
Author(s):  
J M Schoemaker ◽  
G W Henderson ◽  
A Markovitz
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Wild Type Strain ◽  
Atp Hydrolysis ◽  
Sos Response ◽  
Wild Type ◽  
Two Dimensional Gel Electrophoresis ◽  
In The Wild ◽  
Conditional Inhibition ◽  
K 12

Mutation in the gene lon (capR) of Escherichia coli K-12 causes conditional inhibition of cell division. Two-dimensional gel electrophoresis was used to compare polypeptides from isogenic capR+ and capR strains. One polypeptide was present in the capR strain but absent in the wild-type strain, and it was proteolyzed when the pure capR+ protease was added to the capR extract. This polypeptide could only be detected in the capR strain when cell division was inhibited, and its synthesis was independent of the SOS response.

Sign in / Sign up

Export Citation Format

Share Document