scholarly journals Characterization of an Escherichia coli O157:H7 O-Antigen Deletion Mutant and Effect of the Deletion on Bacterial Persistence in the Mouse Intestine and Colonization at the Bovine Terminal Rectal Mucosa

2008 ◽  
Vol 74 (16) ◽  
pp. 5015-5022 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Maryann K. Watkins ◽  
Scott A. Minnich ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Cell Membrane ◽  
Membrane Permeability ◽  
Sodium Dodecyl ◽  
Rectal Mucosa ◽  
Cell Membrane Permeability ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Δper mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Δper mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Δper mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Δper mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.

scholarly journals Role of Escherichia coli O157:H7 Virulence Factors in Colonization at the Bovine Terminal Rectal Mucosa

2006 ◽  
Vol 74 (8) ◽  
pp. 4685-4693 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Hannah J. Knecht ◽  
Jie Li ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Clinical Manifestations ◽  
Rectal Mucosa ◽  
Wild Type ◽  
E Coli ◽  
Healthy Cattle ◽  
Life Threatening ◽  
K 12 ◽  
The Impact

ABSTRACT The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ∼92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, Δtir, and Δeae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.

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 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.

Chemical Composition, Antibacterial Activity, and Mechanism of Action of the Essential Oil from Amomum kravanh

2014 ◽  
Vol 77 (10) ◽  
pp. 1740-1746 ◽  
Author(s):  
WEN-RUI DIAO ◽  
LIANG-LIANG ZHANG ◽  
SAI-SAI FENG ◽  
JIAN-GUO XU
Keyword(s):  
Antibacterial Activity ◽  
Chemical Composition ◽  
Essential Oil ◽  
Cell Membrane ◽  
Membrane Permeability ◽  
Foodborne Pathogens ◽  
Cell Membrane Permeability ◽  
Gas Chromatography Mass Spectrometry ◽  
E Coli ◽  
Transmission Electron

Amomum kravanh is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of A. kravanh fruits was analyzed by gas chromatography–mass spectrometry, and 34 components were identified. 1,8-Cineole (68.42%) was found to be the major component, followed by α-pinene (5.71%), α-terpinene (2.63%), and β-pinene (2.41%). The results of antibacterial tests showed that the sensitivities to the essential oil of different foodborne pathogens tested were different based on the Oxford cup method, MIC, and MBC assays, and the essential oil exhibited the best antibacterial activity against Bacillus subtilis, a gram-positive bacterium, and Escherichia coli, a gram-negative bacterium. Growth in the presence of Amomum kravanh at the MIC, as measured by monitoring optical density over time, demonstrated that the essential oil was bacteriostatic after 12 h to both B. subtilis and E. coli. Observations of cell membrane permeability, cell constituent release assay, and transmission electron microscopy indicated that this essential oil may disrupt the cell wall and cell membrane permeability, leading to leakage of intracellular constituents in both B. subtilis and E. coli.

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 Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

2006 ◽  
Vol 75 (4) ◽  
pp. 1661-1666 ◽  
Author(s):  
Theresa Deland Ho ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Genetic Manipulation ◽  
Enterohemorrhagic Escherichia Coli ◽  
Deletion Strain ◽  
Growth Defect ◽  
Intestinal Colonization ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

scholarly journals The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

2006 ◽  
Vol 281 (43) ◽  
pp. 32303-32309 ◽  
Author(s):  
Taeko Komoda ◽  
Neuza S. Sato ◽  
Steven S. Phelps ◽  
Naoki Namba ◽  
Simpson Joseph ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Functional Role ◽  
Elongation Factor ◽  
Normal Growth ◽  
Wild Type ◽  
Reading Frame ◽  
E Coli ◽  
Translational Activity ◽  
A Site

Helix 38 (H38) in 23 S rRNA, which is known as the “A-site finger (ASF),” is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

scholarly journals Escherichia coli robustly expresses ATP synthase at growth rate maximizing concentrations

2021 ◽  
Author(s):  
Iraes Rabbers ◽  
Frank J Bruggeman
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Protein Expression ◽  
Genetic Variants ◽  
Evolutionary Adaptation ◽  
Wild Type ◽  
Short Term ◽  
E Coli ◽  
Fitness Maximization

AbstractImproved protein expression is an important evolutionary adaptation of bacteria. A key question is whether evolution has led to optimal protein expression that maximizes immediate growth rate (short-term fitness) across conditions. Alternatively, fitter genetic variants could display suboptimal short-term fitness, because they cannot do better or because they strive for long-term fitness maximization by, for instance, anticipating future conditions. To answer this question, we focus on the ATP-producing enzyme F1F0 H+-ATPase, which is an abundant enzyme and ubiquitously expressed across conditions. We tested the optimality of H+-ATPase expression in Escherichia coli across 27 different nutrient conditions. In all tested conditions, wild-type E. coli expresses its H+- ATPase remarkably close to optimal concentrations that maximize immediate growth rate. This work indicates that bacteria can achieve robust optimal protein expression for immediate growth- rate.

scholarly journals Determining the Optimal Thymidine Concentration for Growing Thy−Escherichia coli Strains

1998 ◽  
Vol 180 (11) ◽  
pp. 2992-2994 ◽  
Author(s):  
Felipe Molina ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Growth Medium ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Mg1655 ◽  
E Coli ◽  
Replication Time ◽  
Detectable Difference

ABSTRACT Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy− strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy− Escherichia coli strain. Using this method, we found that the E. coli “wild-type” strain MG1655 has a partial Thy− phenotype.

scholarly journals ANTIBACTERIAL AND ANTIBIOFILM ACTIVITIES OF TRANS-CINNAMALDEHYDE NANOEMULSION AGAINST ESCHERICHIA COLI

2019 ◽  
pp. 301-304
Author(s):  
UTKRISHTA L RAJ ◽  
MEGHA GAUTAM ◽  
SHWETA DANG ◽  
REEMA GABRANI
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Membrane Permeability ◽  
Mode Of Action ◽  
Skin Irritation ◽  
Ruthenium Red ◽  
Cell Membrane Permeability ◽  
Antibiofilm Activity ◽  
E Coli ◽  
Bacterial Cell Membrane

Objective: Trans-cinnamaldehyde (TC) has shown antimicrobial activity against various microorganisms, but its direct use has some disadvantages such as skin irritation, low bioavailability, and low solubility. The objective of the present work was to develop the oil-in-water nanoemulsions (NEs) of TC to enhance its antimicrobial activity against Escherichia coli. Methods: The NEs of TC were prepared using triton x-100 and isopropyl alcohol as surfactant and cosurfactant. The developed NE was studied for size, zeta potential, and stability. NEs were evaluated for antimicrobial and antibiofilm activity against E. coli as indicator organism. NEs possible mode of action on E. coli was assessed by scanning electron microscopy (SEM). Results: Stable NEs of TC exhibited a particle size of 210 nm and were able to inhibit the growth of planktonic as well as biofilm cultures of E. coli at 67 μg/ml. The ruthenium red staining indicated the inhibition of glycoprotein layer formation in extracellular matrix after treating with NE. TC-NE exhibited substantial decrease in E. coli growth as well as its viability at its minimum inhibitory concentration as determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of action of cinnamaldehyde through β-galactosidase assay on E. coli ML-35p strain indicated that it altered the bacterial cell membrane permeability. SEM results showed the presence of holes on the cell wall of the E. coli in the presence of TC-NE. Conclusions: TC-NEs exhibited enhanced antimicrobial activity and were effective against E. coli biofilm. They also exhibited microbicidal activity and altered E. coli membrane permeability.

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