1,722,245 research outputs found
AdhE during competence induction.
No full text<p>(<b>A</b>) Expression levels of AdhE and ComGC-FLAG in the RL001 strain following competence induction using an anti-AdhE and anti-FLAG antibodies, respectively. Samples were normalized for total protein content prior to loading on the gel. (<b>B</b>) Transformation efficiency of an <i>adhE</i>-null (strain AD001) mutant relative to the reference strain (strain R1501). The data are representative of two biological replicates, with two repetitions each. An asterisk indicates typical transformation efficiency upon ComGC disruption [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004835#ppat.1004835.ref010" target="_blank">10</a>]. (<b>C-D</b>) T4P-like transformation pili assembled by the <i>ΔadhE</i> strain.</p
Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacteriumsaccharolyticum
Full text linkClostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (?50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level.BionanoscienceApplied Science
Data on Effect of Partial Lockdown amid COVID-19 on Air Quality in Jakarta, Indonesia
No full textThis dataset provides information on the effect of partial lockdown in the capital of Indonesia, Jakarta. The data contains the variation on PM10, SO2, CO, O3, and NO2 concentrations in 5 cities of Jakarta Province. The five cities are Central Jakarta, South Jakarta, East Jakarta, West Jakarta, and North Jakarta. Furthermore, It covers air pollution data from 2018 to 2020 during the lockdown period (January to June). There were nearly 3.7% of data were missing from the original dataset (data.jakarta.go.id) however it was already interpolated in this dataset. The data is presented in Air Pollution Index (API), the only official standard in Indonesia to measure air quality
Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli
No full textThe regulation of the adhE gene, which encodes the trifunctional fermentative acetaldehyde-alcohol dehydrogenase ofEscherichia coli, was investigated by the construction of gene fusions and by two-dimensional protein gel electrophoresis. Both operon and protein fusions of adhE to lacZ were induced 10- to 20-fold by anaerobic conditions, and both fusions were repressed by nitrate, demonstrating that regulation is at the level of transcription. Nitrate repression of 4(adhE-lacZ) expression, as well as of alcohol dehydrogenase enzyme activity, was partly relieved by a mutation in narL. Mutations in rpoN orfirr had no effect on the expression of adhE. Two-dimensional protein gels demonstrated that increases in the amount of adhE protein correlated with increases in enzyme activity, demonstrating that induction was due to synthesis of new protein, not to activation of preexisting protein. When oxidized sugar derivatives such as gluconate or glucuronate were used as carbon sources, the anaerobic expression of4(adhE-lacZ) was greatly reduced, whereas when sugar alcohols such as sorbitol were used, the expression was increased compared with expression when glucose was the carbon source. This observation suggested that induction of0(adhE-lacZ) might depend on the level of reduced NADH, which should be highest with sorbitol-grown cells and lowest with glucuronate-grown cells. When *(adhE-lacZ) was present in a strain deleted for the adhE structural gene, anaerobic expression of *(adhE-lacZ) was approximately 10-fold higher than in an adhE+ strain. Since the presence of alcoho
Enterohaemorrhagic Escherichia coli AdhE spirosome length correlates with enzymatic directionality and is perturbed by salicylidene acylhydrazides
No full textEnterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors
Enterohaemorrhagic Escherichia coli AdhE spirosome length correlates with enzymatic directionality and is perturbed by salicylidene acylhydrazides
Full text linkEnterohaemorrhagic Escherichia coli causes sporadic, and sometimes large-scale, food poisoning outbreaks, for which antibiotic treatment in humans is contraindicated. As an alternative form of therapy, previous studies developed the family of salicylidene acylhydrazide (SA) anti-virulence compounds. One target of the SA compounds is AdhE, an enzyme that converts acetyl-CoA to ethanol and vice versa. AdhE oligomerizes, forming helicoidal filaments, heterogeneous in length, called spirosomes. We show it is possible to only partially fractionate AdhE spirosomes because in vitro they oligomerize in the absence of stimuli, and that spirosome formation is necessary to regulate the direction of AdhE enzymatic reactions. We also show that the SA compound ME0054 binds and perturbs AdhE spirosomes, enhancing the conversion of ethanol to acetyl-CoA. This mechanistic understanding of how ME0054 impacts AdhE function will help in the development of SA compounds as novel anti-virulence inhibitors
Identification of the ‘plaited’ filaments as AdhE spirosomes.
No full text<p><b>(A)</b> Negatively stained purified ‘plaited’ filaments. <b>(B)</b> Left, coomassie stained SDS-PAGE on the purified filament fraction. Center, proteomic analysis of the predominant protein band; Posterior Error Probability (PEP) value for AdhE is 0. Right, Western blot validation using an anti-AdhE antibody (Agrisera). <b>(C)</b> Heterologous expression in <i>E</i>. <i>coli</i> and purification of AdhE<sup>S.pneumoniae</sup>. Left, coomassie stained SDS-PAGE of the affinity purified protein (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004835#ppat.1004835.s005" target="_blank">S2 Table</a>). Right, negatively stained electron microscopy of the purified protein with black arrowheads indicating spontaneously formed spirosomes. <b>(D)</b> Immunopurification of <i>S</i>. <i>pneumoniae</i> spirosomes from a strain carrying an additional competence-inducible FLAG-tagged ectopic copy of the <i>adhE</i> gene (SO007): left, coomassie-stained SDS-PAGE of the eluted fraction in CSP-induced and-uninduced cells (CSP, competence stimulating peptide); right, negative-stain EM on the eluted fraction <b>(E)</b> Top, AdhE domain organization; bottom, high-resolution structural model of a <i>G</i>. <i>thermoglucosidasius</i> spirosome [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004835#ppat.1004835.ref019" target="_blank">19</a>], visualized in PyMOL (Schrödinger). The color of the individual protomers alternate between grayscale and domain-coded color representation; successive AdhE dimers alternate between ribbon and surface representation <b>(F)</b> A representative class average of the ‘plaited’ filaments compared to electron density reprojections of the <i>G</i>. <i>thermoglucosidasius</i> spirosome model [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004835#ppat.1004835.ref019" target="_blank">19</a>]. Scale bars 5nm.</p
Enhanced hydrogen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1
Full text linkEthanol is the main byproduct of anaerobic H-2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD(P)H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3'-adenyltransferase (aadA) gene. As expected, the vector includes the insertion 5'-adhE-aadA-adhE-3'. The amplified DNA fragment 5'-adhE-aadA-adhE-31 from pTA-Str was transformed into K. oxytoca, HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H-2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxytoca HP1 is a potential method for enhancing bacterial H-2-production
High-resolution structure of the alcohol dehydrogenase domain of the bifunctional bacterial enzyme AdhE
Full text linkThe bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Å resolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering
Enhanced hydrogen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1
No full textEthanol is the main byproduct of anaerobic H-2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD(P)H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3'-adenyltransferase (aadA) gene. As expected, the vector includes the insertion 5'-adhE-aadA-adhE-3'. The amplified DNA fragment 5'-adhE-aadA-adhE-31 from pTA-Str was transformed into K. oxytoca, HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H-2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxytoca HP1 is a potential method for enhancing bacterial H-2-production
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