EFFECTS OF HOMOLOGOUS AND HETEROLOGOUS ANTISERA ON THE GLYCEROL DEHYDROGENASES OF HALOPHILIC AND NON-HALOPHILIC BACTERIA

1955 ◽  
Vol 1 (4) ◽  
pp. 262-265 ◽  
Author(s):  
R. M. Baxter ◽  
N. E. Gibbons
Keyword(s):  
Escherichia Coli ◽  
Halophilic Bacteria ◽  
E Coli ◽  
Crude Extracts ◽  
Minimal Quantity ◽  
Original Preparation

Antisera against the glycerol dehydrogenases of Escherichia coli and Vibrio costicolus, prepared by injecting the enzymes into rabbits, precipitated the homologous but not the heterologous enzymes. When the enzyme from E. coli was completely precipitated from the supernatant by a minimal quantity of antiserum, about half the activity could be demonstrated in the precipitate. The activity of the precipitate was progressively reduced by increasing amounts of antiserum. Crude extracts of V. costicolus oxidized butanediol as well as glycerol. The antiserum reduced the oxidation of glycerol to a greater extent than the oxidation of butanediol indicating the presence of two enzymes in the original preparation. Antisera against the E. coli and V. costicolus enzymes were inactive against the P. salinaria enzyme. An attempt to prepare antibodies against the enzyme from Pseudomonas salinaria was not successful.

scholarly journals Conversion of Methylglyoxal to Acetol by Escherichia coli Aldo-Keto Reductases

2005 ◽  
Vol 187 (16) ◽  
pp. 5782-5789 ◽  
Author(s):  
Junsang Ko ◽  
Insook Kim ◽  
Seokho Yoo ◽  
Bumchan Min ◽  
Kyungmin Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biochemical Study ◽  
Cell Types ◽  
Open Reading Frames ◽  
Toxic Metabolite ◽  
E Coli ◽  
H Nmr ◽  
Crude Extracts

ABSTRACT Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of 1H nuclear magnetic resonance (1H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with 1H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of these genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.

THE GLYCEROL DEHYDROGENASES OF PSEUDOMONAS SALINARIA, VIBRIO COSTICOLUS, AND ESCHERICHIA COLI IN RELATION TO BACTERIAL HALOPHILISM

1954 ◽  
Vol 32 (1) ◽  
pp. 206-217 ◽  
Author(s):  
R. M. Baxter ◽  
N. E. Gibbons
Keyword(s):  
Escherichia Coli ◽  
Sodium Chloride ◽  
Potassium Chloride ◽  
Salt Concentration ◽  
Halophilic Bacteria ◽  
Molar Concentration ◽  
Moderately Halophilic ◽  
E Coli ◽  
High Concentrations ◽  
Chloride Concentrations

Glycerol dehydrogenases from the extremely halophilic Pseudomonas salinaria and the moderately halophilic Vibrio costicolus are described and compared with the corresponding enzyme from the nonhalophilic Escherichia coli. The properties of all three enzymes are similar except their responses to salt concentration. The enzymes from E. coli and V. costicolus are most active at sodium chloride concentrations of about 0.25 M and 0.5 M respectively; that from P. salinaria is not only most active in the presence of 1.5 M NaCl but is irreversibly inactivated in the absence of salt. All three enzymes are more active in the presence of potassium chloride than of sodium chloride at any given molar concentration. These results suggest that the extremely halophilic bacteria contain high concentrations of salt and that their enzymes function maximally at these high concentrations. In contrast the moderately halophilic organisms contain relatively little salt and their enzymes are more comparable with those of nonhalophiles.

scholarly journals Metabolic Engineering of Escherichia Coli for Ectoine Production with the Fermentation Strategy of Supplementing Amino Donor

2020 ◽  
Author(s):  
Hao Zhang ◽  
Ming Zhao ◽  
Yingsheng Dong ◽  
Yingying Cao ◽  
Yanqin Ma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ammonium Sulfate ◽  
Metabolic Flux ◽  
Halophilic Bacteria ◽  
Amino Acid Derivative ◽  
E Coli ◽  
Amino Donor ◽  
Ectoine Biosynthesis ◽  
Ectoine Synthesis ◽  
Engineered Strain

Abstract Background: Ectoine, a compatible solute, has broad application prospects in food biotechnology, agriculture, medicine, and cosmetics because of its protective action on biological compounds. Industrially, ectoine is produced by halophilic bacteria in a complex process. Recently, various works focus on improving ectoine production by using engineered strains, but there are still problems of low yield and low ectoine production efficiency.Results: To overcome the drawback, a final metabolic engineered strain E. coli ET08 was constructed by eliminating lysine synthesis branch and by-product metabolic pathways, and ectoine production reached 10.2 g/L through culture medium optimization. Compared with nitrate, addition of ammonium salt contributed more to the ectoine synthesis. Furthermore, the ammonium sulphate boosted more ectoine titers than ammonium chloride and sodium glutamate. The analysis of transcriptional levels revealed that the ammonium sulfate enhanced ectoine biosynthesis by enhancing metabolic flux toward ectoine biosynthesis and providing affluent synthetic precursors. Ultimately, the ectoine production and yield of the E. coli ET08 reached 36.5 g/L and 0.3 g/g glucose with supplementing amino donor in a 7.5 L bioreactor.Conclusions: a novel potential metabolic engineered Escherichia coli for ectoine production was constructed. optimizing amino donor and analyzing the transcription levels conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis. It is the first report about the effect of exogenous amino donor on ectoine fermentation by metabolic engineered strain. The maximum ectoine production and yield from glucose synthesized by E. coli were obtained by two-stage feeding fermentation with supplementing amino donor. It provides a novel strategy for the synthesis of ectoine by engineered strain in industry. This research provides the basis for an effective process for ectoine production, together with the further applications of ectoine in food and cosmetics, and could also be used to produce other high value amino acid derivative.

THE GLYCEROL DEHYDROGENASES OF PSEUDOMONAS SALINARIA, VIBRIO COSTICOLUS, AND ESCHERICHIA COLI IN RELATION TO BACTERIAL HALOPHILISM

1954 ◽  
Vol 32 (3) ◽  
pp. 206-217 ◽  
Author(s):  
R. M. Baxter ◽  
N. E. Gibbons
Keyword(s):  
Escherichia Coli ◽  
Sodium Chloride ◽  
Potassium Chloride ◽  
Salt Concentration ◽  
Halophilic Bacteria ◽  
Molar Concentration ◽  
Moderately Halophilic ◽  
E Coli ◽  
High Concentrations ◽  
Chloride Concentrations

Glycerol dehydrogenases from the extremely halophilic Pseudomonas salinaria and the moderately halophilic Vibrio costicolus are described and compared with the corresponding enzyme from the nonhalophilic Escherichia coli. The properties of all three enzymes are similar except their responses to salt concentration. The enzymes from E. coli and V. costicolus are most active at sodium chloride concentrations of about 0.25 M and 0.5 M respectively; that from P. salinaria is not only most active in the presence of 1.5 M NaCl but is irreversibly inactivated in the absence of salt. All three enzymes are more active in the presence of potassium chloride than of sodium chloride at any given molar concentration. These results suggest that the extremely halophilic bacteria contain high concentrations of salt and that their enzymes function maximally at these high concentrations. In contrast the moderately halophilic organisms contain relatively little salt and their enzymes are more comparable with those of nonhalophiles.

scholarly journals N-acetyl-d-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-d-mannosamine and pyruvate

1995 ◽  
Vol 308 (2) ◽  
pp. 501-505 ◽  
Author(s):  
L B Rodríguez-Aparicio ◽  
M A Ferrero ◽  
A Reglero
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Decarboxylase ◽  
Acid Synthesis ◽  
Neuraminic Acid ◽  
Chromatographic Peak ◽  
E Coli ◽  
Crude Extracts ◽  
Lyase Activity ◽  
Escherichia Coli K1

Two enzymes have been found to be involved in bacterial N-acetyl-D-neuraminic acid (NeuAc) synthesis: NeuAc synthase, which condenses N-acetyl-L,D-mannosamine and phosphoenolpyruvate, and NeuAc lyase or NeuAc aldolase, which condenses N-acetyl-D-mannosamine and pyruvate. When we used Escherichia coli K1 crude extracts, we observed the generation of NeuAc in the presence of N-acetylmannosamine and both phosphoenolpyruvate (NeuAc synthase activity) or pyruvate (NeuAc lyase activity). However, when crude extracts were fractionated by Sephacryl S-200 chromatography, NeuAc synthase activity disappeared. A chromatographic peak of NeuAc synthase activity was detected when column fractions were re-tested in the presence of the active NeuAc lyase peak. Furthermore, crude extracts converted phosphoenolpyruvate into pyruvate. Pyruvate depletion, due to the addition of pyruvate decarboxylase to the NeuAc synthase reaction mixture, blocked NeuAc formation. Moreover, after NeuAc lyase immunoprecipitation no NeuAc synthase was detected. These findings suggest that NeuAc synthase is not present in E. coli K1 and therefore that NeuAc lyase is the only enzyme responsible for NeuAc synthesis in this bacterium.

Cloning and Sequencing of the Histidine Decarboxylase Gene from Photobacterium phosphoreum and its Functional Expression in Escherichia coli

2006 ◽  
Vol 69 (8) ◽  
pp. 1768-1776 ◽  
Author(s):  
HIDEAKI MORII ◽  
KENTARO KASAMA ◽  
RAUL HERRERA-ESPINOZA
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Histidine Decarboxylase ◽  
Halophilic Bacteria ◽  
Trna Synthetase ◽  
Amino Acid Sequences ◽  
Photobacterium Phosphoreum ◽  
Bootstrap Support ◽  
Amino Acid Permease ◽  
E Coli

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10°C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5′-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.

scholarly journals UJI ANTIBAKTERI EKSTRAK DAN FRAKSI SPONS TERHADAP Escherichia coli DAN Staphylococcus aureus SERTA POTENSINYA TERHADAP AKTIVITAS ANTI-UV

2019 ◽  
Vol 7 (3) ◽  
pp. 196
Author(s):  
Monika Gabrielle ◽  
Deiske A Sumilat ◽  
Veibe Warouw ◽  
Remy EP Mangindaan ◽  
Chatrien AC Sinjal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Crude Extract ◽  
Diffusion Method ◽  
Water Fraction ◽  
E Coli ◽  
Self Defense ◽  
Crude Extracts ◽  
Water Fractions

Sponge organisms produce bioactive compounds that are toxic as a means of self-defense. The compound is known to have the potential as an antibacterial and anti-UV which can absorb sunlight with the potential to be used as a material for making sunscreen. The purpose of this study was to obtain antibacterial activity against Escherichia coli and Staphylococcus aureus bacteria and anti-UV activity from crude extracts and sponges fractions. The antibacterial test done by agar diffusion method (Kirby and Bauer diffusion disc) and the crude extract and the active fraction of antibacterial compounds were tested in a UV spectrophotometer to see its anti-UV activity. As a result, 4 species of sponges were extracted and partitioned into water fractions, methanol fractions, and n-hexane fractions. All samples were tested for antibacterial activity and the results showed antibacterial activity against S. aureus by Plakortis sp. in crude extract (9 mm) and water fraction (8.6 mm), Agelas sp. in crude extract (7 mm) and in E. coli bacteria shown by Plakortis sp. in crude extract (12.6 mm) and water fraction (9 mm), Liosina sp. in the water fraction (7.6 mm), Haliclona sp. in the water fraction (8 mm) and Agelassp. in crude extract (10.3 mm). Crude extracts and water fractions were tested using a UV spectrophotometer for anti-UV testing, the results showed that crude extract and all the water fractions of four species sponge could absorb UV-B (λ 290-320 nm) and UV-A (λ 320-400 nm).Keywords: sponges, antibacterial, partition, anti-UV

scholarly journals Metabolic Engineering of Escherichia Coli for Ectoine Production With the Fermentation Strategy of Supplementing Amino Donor

2020 ◽  
Author(s):  
Hao Zhang ◽  
Yingying Cao ◽  
Yingsheng Dong ◽  
Ming Zhao ◽  
Yanqin Ma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ammonium Sulfate ◽  
Metabolic Flux ◽  
Halophilic Bacteria ◽  
Amino Acid Derivative ◽  
E Coli ◽  
Amino Donor ◽  
Ectoine Biosynthesis ◽  
Ectoine Synthesis ◽  
Engineered Strain

Abstract Background: Ectoine, a compatible solute, has broad application prospects in food biotechnology, agriculture, medicine, and cosmetics because of its protective action on biological compounds. Industrially, ectoine is produced by halophilic bacteria in a complex process. Recently, various works focus on improving ectoine production by using engineered strains, but there are still problems of low yield and low ectoine production efficiency.Results: To overcome the drawback, a final metabolic engineered strain E. coli ET08 was constructed by eliminating lysine synthesis branch and by-product metabolic pathways, and ectoine production reached 10.2 g/L through culture medium optimization. Compared with nitrate, addition of ammonium salt contributed more to the ectoine synthesis. Furthermore, the ammonium sulphate boosted more ectoine titers than ammonium chloride and sodium glutamate. The analysis of transcriptional levels revealed that the ammonium sulfate enhanced ectoine biosynthesis by enhancing metabolic flux toward ectoine biosynthesis and providing affluent synthetic precursors. Ultimately, the ectoine production and yield of the E. coli ET08 reached 36.5 g/L and 0.3 g/g glucose with supplementing amino donor in a 7.5 L bioreactor.Conclusions: a novel potential metabolic engineered Escherichia coli for ectoine production was constructed. optimizing amino donor and analyzing the transcription levels conclude that ammonium sulfate, as the optimal amino donor, has a positive effect on ectoine synthesis. It is the first report about the effect of exogenous amino donor on ectoine fermentation by metabolic engineered strain. The maximum ectoine production and yield from glucose synthesized by E. coli were obtained by two-stage feeding fermentation with supplementing amino donor. It provides a novel strategy for the synthesis of ectoine by engineered strain in industry. This research provides the basis for an effective process for ectoine production, together with the further applications of ectoine in food and cosmetics, and could also be used to produce other high value amino acid derivative.

Determination of the levels of HPr and enzyme I of the phosphoenolpyruvate–sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium

1983 ◽  
Vol 61 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Roshan L. Mattoo ◽  
E. Bruce Waygood
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Specific Activity ◽  
Phosphotransferase System ◽  
E Coli ◽  
Crude Extracts ◽  
Activity Measurements ◽  
A Cell ◽  
Enzyme I

The levels of histidine-containing protein HPr and enzyme I of the phosphoenolpyruvate–sugar phosphotransferase system of Escherichia coli strains 1100, NC3, W3110, and P650 and Salmonella typhimurium strains SB3507 and LJ144 have been determined by quantitative sugar phosphorylation assay and immunochemically. The levels have been determined for cells grown on minimal salts with glucose, fructose, mannitol, glycerol, and lactate and on nutrient broth. All determinations indicate a two- to three-fold change in the levels of enzyme I and HPr between growth on hexoses, which gave the higher levels, and the other growth substrates. The highest levels were not always found in glucose-grown cells. Antibodies were produced in rabbits using purified proteins from E. coli P650. The activity measurements and immunochemically determined enzyme I protein gave specific activities in the crude extracts of E. coli strains which were similar to that of the pure enzyme. The wild-type S. typhimurium enzyme I in crude extracts did not have the same immunochemical reactivity, although there was a considerable cross-reaction and the specific activity appeared to be half that of pure enzyme I. The HPr from both E. coli and S. typhimurium behaved identically and, although the immunoprecipitation was weak, it did indicate that HPr assays may not be as reliable as the enzyme I assays. The relative amounts of enzyme I and HPr found indicate that there are between 10- and 20-fold more HPr molecules in a cell than enzyme I subunits which form active dimers.

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

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