scholarly journals The Escherichia coli Efflux Pump TolC Promotes Aggregation of Enteroaggregative E. coli 042

2007 ◽  
Vol 76 (3) ◽  
pp. 1247-1256 ◽  
Author(s):  
Naoko Imuta ◽  
Junichiro Nishi ◽  
Koichi Tokuda ◽  
Rika Fujiyama ◽  
Kunihiro Manago ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Microtiter Plate ◽  
Virulence Plasmid ◽  
Wild Type ◽  
Planktonic Cells ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Aggregative Adherence

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

BaeR participates in cephalosporins susceptibility by regulating the expression level of outer membrane proteins in Escherichia coli

2020 ◽  
Author(s):  
Shuaiyang Wang ◽  
Chunbo You ◽  
Fareed Qumar Memon ◽  
Geyin Zhang ◽  
Yawei Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Expression Level ◽  
Antibiotics Resistance ◽  
Dilution Method ◽  
Expression Levels ◽  
E Coli

Abstract The two-component system BaeSR participates in antibiotics resistance of Escherichia coli. To know whether the outer membrane proteins involve in the antibiotics resistance mediated by BaeSR, deletion of acrB was constructed and the recombined plasmid p-baeR was introduced into E. coli K12 and K12△acrB. Minimum inhibitory concentrations (MICs) of antibacterial agents were determined by 2-fold broth micro-dilution method. Gene expressions related with major outer membrane proteins and multidrug efflux pump-related genes were determined by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the MICs of K12ΔacrB to the tested drugs except for gentamycin and amikacin decreased 2- to 16.75-folds compared with those of K12. When BaeR was overexpressed, the MICs of K12ΔacrB/p-baeR to ceftiofur and cefotaxime increased 2.5- and 2-fold, respectively, compared with their corresponding that of K12△acrB. At the same time, the expression levels of ompC, ompF, ompW, ompA and ompX showed significant reduction in K12ΔacrB/p-baeR as compared with K12△acrB. Moreover, the expression levels of ompR, marA, rob and tolC also significantly ‘decreased’ in K12ΔacrB/p-baeR. These findings indicated that BaeR overproduction can decrease cephalosporins susceptibility in acrB-free E. coli by decreasing the expression level of outer membrane proteins.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

2001 ◽  
Vol 183 (7) ◽  
pp. 2259-2264 ◽  
Author(s):  
Yan Wei ◽  
Amy C. Vollmer ◽  
Robert A. LaRossa
Keyword(s):  
Escherichia Coli ◽  
Mitomycin C ◽  
Transcriptional Activation ◽  
Efflux Pump ◽  
Wild Type ◽  
Potent Inducer ◽  
E Coli ◽  
Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

scholarly journals Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

2020 ◽  
Author(s):  
Sara Abdollahi ◽  
Mohammad Hossein Morowvat ◽  
Amir Savardashtaki ◽  
Cambyz Irajie ◽  
Sohrab Najafipour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Microtiter Plate ◽  
Arginine Deiminase ◽  
Expression Vectors ◽  
Efficacy Evaluation ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Host Strains

Abstract Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. This study attempted to evaluate the five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

scholarly journals Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

2010 ◽  
Vol 78 (6) ◽  
pp. 2377-2384 ◽  
Author(s):  
Supraja Puttamreddy ◽  
Nancy A. Cornick ◽  
F. Chris Minion
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Microtiter Plate ◽  
Virulence Plasmid ◽  
Microtiter Plate Assay ◽  
Severe Diarrhea ◽  
Wide Range ◽  
Intergenic Regions ◽  
Biofilm Development

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

scholarly journals Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli.

1981 ◽  
Vol 77 (2) ◽  
pp. 121-135 ◽  
Author(s):  
H Nikaido ◽  
E Y Rosenberg
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Specific Class ◽  
Wild Type ◽  
Intact Cells ◽  
E Coli ◽  
Low Concentrations ◽  
Transmembrane Pores ◽  
Diffusion Rates ◽  
Type Strains

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.

scholarly journals Chemical Composition and Antibacterial Activity of Angelica archangelica Root Essential Oil

2017 ◽  
Vol 12 (2) ◽  
pp. 1934578X1701200 ◽  
Author(s):  
Milica G. Aćimović ◽  
Snežana Đ. Pavlović ◽  
Ana O. Varga ◽  
Vladimir M. Filipović ◽  
Mirjana T. Cvetković ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Essential Oil ◽  
Inhibitory Concentration ◽  
Microtiter Plate ◽  
Microtiter Plate Assay ◽  
Natural Preservative ◽  
E Coli ◽  
Angelica Archangelica ◽  
Root Essential Oil

Roots of wild growing Angelica archangelica L. from Mt. Ozren (Serbia) were subjected to hydrodistillation and GC-MS analysis. The roots contained 0.10% of essential oil with α-pinene (29.7%), δ-3-carene (14.2%), and a mixture of β-phellandrene and limonene (13.2%) as main compounds. The modified resazurin microtiter-plate assay was used to evaluate the antibacterial activity of the essential oil against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration (MIC) values were 14.2 μL/mL for S. aureus and 28.4 μL/mL for E. coli, while the minimum bactericidal concentrations (MBC) were 56.8 μL/mL and 113.6 μL/mL, respectively. According to the obtained results, the angelica root essential oil can be applied as a natural preservative in food and as a natural antibiotic for the treatment of several infectious diseases caused by these two bacteria.

scholarly journals The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1639-1647 ◽  
Author(s):  
Georg Polleichtner ◽  
Christian Andersen
Keyword(s):  
Escherichia Coli ◽  
Single Channel ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Tunnel Entrance

Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure–function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.

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