scholarly journals Identification of a Phosphotransferase System of Escherichia coli Required for Growth on N-Acetylmuramic Acid

2004 ◽  
Vol 186 (8) ◽  
pp. 2385-2392 ◽  
Author(s):  
Ulrike Dahl ◽  
Tina Jaeger ◽  
Bao Trâm Nguyen ◽  
Julia M. Sattler ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Energy Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Glucose Transport System ◽  
Single Polypeptide

ABSTRACT We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIAGlc), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His6 fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

scholarly journals Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

2006 ◽  
Vol 74 (4) ◽  
pp. 2233-2244 ◽  
Author(s):  
Alison S. Low ◽  
Francis Dziva ◽  
Alfredo G. Torres ◽  
Jessenya L. Martinez ◽  
Tracy Rosser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
Induced Expression ◽  
E Coli ◽  
Extracellular Matrix Components ◽  
K 12

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

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.

scholarly journals Alterations in Protein Expression Caused by thehha Mutation in Escherichia coli: Influence of Growth Medium Osmolarity

1999 ◽  
Vol 181 (10) ◽  
pp. 3018-3024 ◽  
Author(s):  
Carlos Balsalobre ◽  
Jörgen Johansson ◽  
Bernt Eric Uhlin ◽  
Antonio Juárez ◽  
Francisco J. Muñoa
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Gel Electrophoresis ◽  
Catabolite Repression ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Wild Type ◽  
Phosphotransferase System ◽  
New Family ◽  
Medium Osmolarity

ABSTRACT The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the hha mutation on the overall protein pattern of Escherichia coli cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the hhamutation. The number of proteins whose expression was altered inhha cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the hha allele, both OmpA and protein IIAGlc of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of thelacZ gene was also modified in hha mutants. This was the case, suggesting that at least some of the pleiotropic effects of the hha mutation could be caused by its effect on the catabolite repression system.

scholarly journals Media Optimization for Production of Recombinant Carrier Protein (CRM197) in Escherichia coli

2021 ◽  
Vol 13 (1) ◽  
pp. 283-297
Author(s):  
S. Shukla ◽  
D. Mishra
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Luria Bertani ◽  
Lag Phase ◽  
Carrier Protein ◽  
Scale Production ◽  
E Coli ◽  
Defined Media

Since the advent of vaccines, the mankind has benefited from the same and has been able to curb the mortality rate around the globe. Amongst different types of available vaccines, polysaccharide based vaccines are very widely used against various infectious diseases. The polysaccharide vaccines need to be conjugated with a carrier protein to make the vaccine more immunogenic. Recombinant Escherichia coli cells are the organism of choice for large scale production of a carrier protein because of its widely studied scientific aspects. In the present study, for proof of concept, the recombinant E. coli cells were cultured in Luria-Bertani media to check the expression of rCRM197. At 80L scale, it was observed that when recombinant E. coli cells were grown in a chemically defined media, it resulted in inconsistent growth and a long lag phase. When the defined media was supplemented with yeast extract, the lag phase of the culture was substantially reduced and the maximum growth of the culture was achieved. Protein expression was checked using SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) and Western blot technique. The optimized media resulted in a robust fermentation process to achieve high cell density and maximum biomass for the production of recombinant protein.

Isolation and Purification of Colicin ECL 12, a Bacteriocin That Inhibits Strains of Escherichia coli O157:H7†

1997 ◽  
Vol 60 (12) ◽  
pp. 1520-1528 ◽  
Author(s):  
WANDA J. LYON ◽  
DENNIS G. OLSON
Keyword(s):  
Escherichia Coli ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Viable Cell ◽  
Exchange Chromatography ◽  
Isolation And Purification ◽  
E Coli ◽  
Log Reduction ◽  
Cell Counts

A swine fecal isolate, identified as Escherichia coli ECL12, was found to produce an antimicrobial substance designated as colicin ECL12. Colicin ECL12 was inhibitory against 20 strains of E. coli O157:H7 previously isolated from both human and bovine feces. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Colicin ECL12 was sensitive to several proteolytic enzymes. Adsorption of colicin ECL12 to sensitive cells of E. coli O157:H7 was bactericidal, resulting in a 2 log reduction in viable cell counts. Colicin ECL12 was purified from strain ECL12 by cell extraction and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of colicin ECL12 resolved a single protein with a molecular weight of approximately 65,000.

scholarly journals COMPARISON OF EXTRACELLULAR SECRETION OF RECOMBINANT HUMAN EPIDERMAL GROWTH FACTOR USING TORA AND PELB SIGNAL PEPTIDES IN ESCHERICHIA COLI BL21 (DE3)

2019 ◽  
pp. 81-84 ◽  
Author(s):  
RIMA MELATI ◽  
ANNISA INDRIYANI ◽  
SHABARNI GAFFAR ◽  
SRIWIDODO ◽  
IMAN PERMANA MAKSUM
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Signal Peptides ◽  
Extracellular Expression ◽  
E Coli ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.

Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

1980 ◽  
Vol 29 (2) ◽  
pp. 704-713 ◽  
Author(s):  
Henk Gankema ◽  
Jan Wensink ◽  
Pieter A. M. Guinée ◽  
Wim H. Jansen ◽  
Bernard Witholt
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Sucrose Density Gradient ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Freeze Fracture Electron Microscopy ◽  
K 12

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.

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