Identification of the glpT -Encoded sn -Glycerol-3-Phosphate Permease of Escherichia coli , an Oligomeric Integral Membrane Protein

1982 ◽  
Vol 152 (3) ◽  
pp. 1008-1021
Author(s):  
Timothy J. Larson ◽  
Günter Schumacher ◽  
Winfried Boos
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gene Product ◽  
Active Transport ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wild Type ◽  
Dodecyl Sulfate ◽  
Active Transport System

A collection of hybrid plasmids carrying either the wild-type or mutated glpT gene was generated in vitro and used to characterize the glpT -dependent active transport system for sn -glycerol-3-phosphate in Escherichia coli K-12. Restriction endonuclease analysis and recloning of DNA fragments localized glpT to a 3-kilobase pair Pst I- Hpa I segment of DNA. Comparison of DNA carrying glpT-lacZ fusions with DNA carrying intact glpT allowed determination of the direction of transcription. Through characterization of the proteins synthesized by strains harboring hybrid plasmids carrying amber, missense, or deletion mutations in glpT , it was shown that glpT is a promoter-proximal gene in an operon consisting of at least two genes. The gene product of glpT , the sn -glycerol-3-phosphate permease, was found associated with the inner membrane. It could be solubilized by treatment with sodium dodecyl sulfate at 50°C. Its molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was dependent upon sample treatment before electrophoresis. The apparent molecular weight was 44,000 when membrane fractions were heated to 50°C; subsequent treatment at 95°C modified the protein such that it migrated faster (apparent molecular weight = 33,000). Several missense mutations in glpT were negatively dominant over wild-type glpT , indicating that the active form of the permease is multimeric. A gene (named glpQ ) promoter distal to glpT codes for a periplasmic protein. This protein had previously been named GLPT protein to indicate its relationship to the glpT gene. The present report demonstrates that it is not the gene product of glpT and is not required for active transport of sn -glycerol-3-phosphate.

scholarly journals Sodium Dodecyl Sulfate Hypersensitivity of clpP and clpB Mutants of Escherichia coli

2002 ◽  
Vol 68 (8) ◽  
pp. 4117-4121 ◽  
Author(s):  
Soumitra Rajagopal ◽  
Narasimhan Sudarsan ◽  
Kenneth W. Nickerson
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Uronic Acid ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Wild Type ◽  
E Coli ◽  
Dodecyl Sulfate ◽  
Shock Proteins ◽  
Western Immunoblot Analysis

ABSTRACT We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.

On the Evolution of an Oligocephalic Enzyme. Glutamine- Chorismate-Amidotransferase-Free Anthranilate Phosphoribosyltransferases from Mutant Strains of Salmonella typhimurium

1978 ◽  
Vol 33 (3-4) ◽  
pp. 235-244 ◽  
Author(s):  
Manfred Grieshaber
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Salmonella Typhimurium ◽  
Sodium Dodecyl ◽  
Single Component ◽  
Wild Type ◽  
Molecular Weights ◽  
Mutant Strains ◽  
Dodecyl Sulfate

(1) A procedure has been described for the purification of two glutamine-chorismate-amido- transferase-free anthranilate phosphoribosyltransferases from mutant strains TAX6trpR782 and trpAB1653trpR782 of Salmonella typhimurium.(2) The native enzymes tend to aggregate forming polymers of molecular weights 333,000 in the case of TAXtrpR782 and 220,000 and larger than 1X106 in the case of trpABI653trpR782. In the presence of sodium dodecyl sulfate the polymer of trpAB1653trpR782 dissociates into a single component with molecular weight of 72,000.(3) In contrast to anthranilate phosphoribosyltransferase of the wild type component II, the glutamine-chorismate-amidotransferase-free proteins do not complex with component I. They do however show catalytical similarities with the wild type with respect to anthranilate phosphoribosyltransferase activity.

scholarly journals Plasmid Curing of Escherichia coli Cells with Ethidium Bromide, Sodium Dodecyl Sulfate and Acridine Orange

1970 ◽  
Vol 27 (1) ◽  
pp. 28-31 ◽  
Author(s):  
MA Zaman ◽  
MH Pasha ◽  
MZ Akhter
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Acridine Orange ◽  
Ethidium Bromide ◽  
Sodium Dodecyl ◽  
Drug Resistant ◽  
Plasmid Curing ◽  
Wild Type ◽  
Dodecyl Sulfate ◽  
I Cells

The plasmid eliminating abilities of acridine orange, ethidium bromide and sodium dodecyl sulfate were investigated on multi drug resistant Escherichia coli from urinary tract infection specimens. Three different concentrations of each curing agent (Et-Br, SDS and AO) were used. The frequencies of cured cells were 5.55 % (with 50 μg/ml) and 11.76 % (with 75 μg/ml) for acridine orange, 14.29 % (with 100 μg/ml), 21.05 % (with 100 μg/ ml), 17.65 % (with 125 μg/ml) for ethidium bromide and 7.4 % (with 10 % w/v) & 6.67 % (with 10 % w/v) for sodium dodecyl sulfate. However, no cured cells were obtained from 100 μg/ml acridine orange, 75 μg/ml ethidium bromide and 8 and 12 % SDS. Analysis of profiles of wild type and plasmid cured strains by electrophoresis yielded bands of varying sizes for wild type cells, but none were obtained for Et-Br cured cells. Acridine orange treated cells could eliminate only plasmids of 2.7 MDa and another smaller than 2 MDa. Key Words: Plasmid curing; Escherichia coli; Ethidium Bromide; Sodium Dodecyl Sulfate; Acridine Orange. DOI: http://dx.doi.org/10.3329/bjm.v27i1.9165 BJM 2010; 27(1): 28-31

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis of Proteins

2021 ◽  
Vol 2021 (12) ◽  
pp. pdb.prot102228
Author(s):  
Clara L. Kielkopf ◽  
William Bauer ◽  
Ina L. Urbatsch
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Size ◽  
Apparent Molecular Weight ◽  
Protein Markers ◽  
Sds Page ◽  
Dodecyl Sulfate

Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (β-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or ∼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS–polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.

Discrimination of live and dead cells of Escherichia coli using propidium monoazide after sodium dodecyl sulfate treatment

Food Control ◽  
2017 ◽  
Vol 71 ◽  
pp. 79-82 ◽  
Author(s):  
Hajime Takahashi ◽  
Yun Gao ◽  
Satoko Miya ◽  
Takashi Kuda ◽  
Bon Kimura
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Propidium Monoazide ◽  
Dodecyl Sulfate

scholarly journals Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

1982 ◽  
Vol 152 (1) ◽  
pp. 166-174
Author(s):  
J A Mulder ◽  
G Venema
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Magnesium Ions ◽  
Polyacrylamide Gels ◽  
Wild Type ◽  
Molecular Weights ◽  
Defective Mutant ◽  
Mutant Strains

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

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