scholarly journals Expression of a Gene Encoding the Carrot HSP17.7 in Escherichia coli Enhances Cell Viability and Protein Solubility Under Heat Stress

HortScience ◽  
2009 ◽  
Vol 44 (3) ◽  
pp. 866-869 ◽  
Author(s):  
Hyesoon Kim ◽  
Yeh-Jin Ahn
Keyword(s):  
Escherichia Coli ◽  
Heat Stress ◽  
Protein Solubility ◽  
Small Heat Shock Protein ◽  
Immunoblot Analysis ◽  
Transformed Cells ◽  
Bacterial Cells ◽  
Native Page ◽  
E Coli ◽  
Inducible Protein

DcHSP17.7, a small heat shock protein from carrot (Daucus carota L.), was expressed in Escherichia coli to examine its functional mechanism under heat stress. When transformed cells expressing DcHSP17.7 were exposed to 50 °C for 1 h, the number of viable cells was ≈4-fold higher than that of control. When the amount of soluble proteins was compared, it was more than twofold higher in transformed cells expressing DcHSP17.7 than that in control, suggesting that DcHSP17.7 may function as a molecular chaperone preventing heat-inducible protein degradation. Native-PAGE followed by immunoblot analysis showed that in transformed E. coli, DcHSP17.7 was present in an oligomeric complex, ≈300 kDa in molecular mass, on isopropyl b-D-thiogalactopyranoside treatment. However, the complex rapidly disappeared when bacterial cells were exposed to heat stress. In carrot, DcHSP17.7 was found in the similar-sized complex (≈300 kDa), but only during heat stress (40 °C), suggesting that the functional structure of DcHSP17.7 may be different in transformed E. coli from that in carrot.

scholarly journals Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli

2016 ◽  
Vol 11 (9) ◽  
pp. 1934578X1601100 ◽  
Author(s):  
Takahiro Kato ◽  
Jung-Bum Lee ◽  
Futoshi Taura ◽  
Fumiya Kurosaki
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Immunoblot Analysis ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Transformed Cells ◽  
Bacterial Cells ◽  
E Coli ◽  
Enhanced Production ◽  
Escherichia Coli Cells

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and farnesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 μg/mL culture), however, the concentration of the compound elevated to 0.08 μg/mL culture in the cells transformed by GS plus FPS δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 μg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient 6-guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

scholarly journals DcHsp17.7, a Small Heat Shock Protein from Carrot, Is Upregulated under Cold Stress and Enhances Cold Tolerance by Functioning as a Molecular Chaperone

HortScience ◽  
2010 ◽  
Vol 45 (3) ◽  
pp. 469-474 ◽  
Author(s):  
Na-Hyun Song ◽  
Yeh-Jin Ahn
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Functional Properties ◽  
Molecular Chaperone ◽  
Small Heat Shock Protein ◽  
Immunoblot Analysis ◽  
Transformed Cells ◽  
E Coli

The expression profile and functional properties of DcHsp17.7, a small heat shock protein from carrot (Daucus carota L.), were examined under cold stress. Immunoblot analysis showed that low temperature (2 °C) induced DcHsp17.7 in vegetative tissues. Differential accumulation of the transcript and protein under the cold suggests that expression of DcHsp17.7 might be controlled at the transcriptional and/or translational levels. To examine the functional properties of DcHsp17.7, the gene was expressed in Escherichia coli. When exposed to 2 °C for 10 days, transformed cells expressing DcHsp17.7 showed 115% cell viability, whereas control cells recorded 24%, suggesting that DcHsp17.7 can confer cold tolerance. The amount of soluble protein under the cold was 83% in transformed cells expressing DcHsp17.7, whereas the control cells showed only 52%, suggesting that DcHsp17.7 functions as a molecular chaperone preventing cold-induced protein degradation. Native-polyacrylamide analysis revealed that DcHsp17.7 was found in two oligomeric complexes (≈160 and 240 kDa) and possibly multiple complexes (from 300 to 450 kDa) in cold-stressed carrot and transformed E. coli, respectively. During prolonged cold stress, these complexes disappeared and then reappeared, suggesting that the dissociation and reassociation of DcHsp17.7 complexes might be important for the function of the protein.

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

Thermal and Starvation Stress Response of Escherichia coli O157:H7 Isolates Selected from Agricultural Environments

2016 ◽  
Vol 79 (10) ◽  
pp. 1673-1679 ◽  
Author(s):  
ACHYUT ADHIKARI ◽  
ANDY BARY ◽  
CRAIG COGGER ◽  
CALEB JAMES ◽  
GÜLHAN ÜNLÜ ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Stress ◽  
Stress Response ◽  
Weibull Model ◽  
Stress Conditions ◽  
Inactivation Kinetics ◽  
Escherichia Coli O157 ◽  
Starvation Stress ◽  
E Coli ◽  
Response To Stress

ABSTRACT Pathogens exposed to agricultural production environments are subject to multiple stresses that may alter their survival under subsequent stress conditions. The objective of this study was to examine heat and starvation stress response of Escherichia coli O157:H7 strains isolated from agricultural matrices. Seven E. coli O157:H7 isolates from different agricultural matrices—soil, compost, irrigation water, and sheep manure—were selected, and two ATCC strains were used as controls. The E. coli O157:H7 isolates were exposed to heat stress (56°C in 0.1% peptone water for up to 1 h) and starvation (in phosphate-buffered saline at 37°C for 15 days), and their survival was examined. GInaFiT freeware tool was used to perform regression analyses of the surviving populations. The Weibull model was identified as the most appropriate model for response of the isolates to heat stress, whereas the biphasic survival curves during starvation were fitted using the double Weibull model, indicating the adaptation to starvation or a resistant subpopulation. The inactivation time during heating to achieve the first decimal reduction time (δ) calculated with the Weibull parameters was the highest (45 min) for a compost isolate (Comp60A) and the lowest (28 min) for ATCC strain 43895. Two of the nine isolates (ATCC 43895 and a manure isolate) had β < 1, indicating that surviving populations adapted to heat stress, and six strains demonstrated downward concavity (β > 1), indicating decreasing heat resistance over time. The ATCC strains displayed the longest δ2 (>1,250 h) in response to starvation stress, compared with from 328 to 812 h for the environmental strains. The considerable variation in inactivation kinetics of E. coli O157:H7 highlights the importance of evaluating response to stress conditions among individual strains of a specific pathogen. Environmental isolates did not exhibit more robust response to stress conditions in this study compared with ATCC strains.

scholarly journals Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amin Zargar ◽  
David N. Quan ◽  
Karen K. Carter ◽  
Min Guo ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Cell Contact ◽  
Bacterial Cells ◽  
Content Type ◽  
Phenotypic Differences ◽  
E Coli ◽  
Autoinducer 2 ◽  
Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

scholarly journals HypVW is an HOCl-Sensing Two Component System in Escherichia coli

2021 ◽  
Author(s):  
Sara El Hajj ◽  
Camille Henry ◽  
Alexandra Vergnes ◽  
Laurent Loiseau ◽  
Brasseur Gael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hypochlorous Acid ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Bacterial Cells ◽  
E Coli ◽  
Two Component ◽  
Redox Switch ◽  
Two Component Systems ◽  
Methionine Residues

Two component systems (TCS) are signalling pathways that allow bacterial cells to sense, respond and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, YedVW has been recently showed to be involved in the regulation of msrPQ, encoding for the periplasmic methionine sulfoxide reductase system. In this study, we demonstrate that hypochlorous acid (HOCl) induces the expression of msrPQ in a YedVW dependant manner, whereas H2O2, NO and paraquat (a superoxide generator) do not. Therefore, YedV appears to be an HOCl-sensing histidine kinase. Based on this finding, we proposed to rename this system HypVW.  Moreover, using a directed mutagenesis approach, we show that Met residues located in the periplasmic loop of HypV (formerly YedV) are important for its activity. Given that HOCl oxidizes preferentially Met residues, we bring evidences that HypV could be activated via the reversible oxidation of its methionine residues, thus conferring to MsrPQ a role in switching HypVW off. Based on these results, we propose that the activation of HypV by HOCl could occur through a Met redox switch. HypVW appears to be the first characterized TCS able to detect HOCl in E. coli. This study represents an important step in understanding the mechanisms of reactive chlorine species resistance in prokaryotes.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Changes in Cell Surface Properties of Shiga Toxigenic Escherichia coli by Quercus infectoria G. Olivier

2009 ◽  
Vol 72 (8) ◽  
pp. 1699-1704 ◽  
Author(s):  
SUPAYANG PIYAWAN VORAVUTHIKUNCHAI ◽  
SAKOL SUWALAK
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Surface Properties ◽  
Cell Surface Hydrophobicity ◽  
Ethanolic Extract ◽  
Bacterial Cells ◽  
Electron Microscopic ◽  
Cell Surface Properties ◽  
E Coli ◽  
Quercus Infectoria

The effects of Quercus infectoria (family Fagaceae) nutgalls on cell surface properties of Shiga toxigenic Escherichia coli (STEC) were investigated with an assay of microbial adhesion to hydrocarbon. The surface of bacterial cells treated with Q. infectoria exhibited a higher level of cell surface hydrophobicity (CSH) toward toluene than did the surface of untreated cells. With 50% ethanolic extract, the CSH of the three strains of STEC O157:H7 treated with 4× MIC of the extract resulted in moderate or strong hydrophobicity, whereas at 2× MIC and MIC, the CSH of only one strain of E. coli O157:H7 was significantly affected. The 95% ethanolic extract had a significant effect on CSH of all three strains at both 4× MIC and 2× MIC but not at the MIC. The effect on bacterial CSH was less pronounced with the other STEC strains. At 4× MIC, the 50% ethanolic extract increased the CSH of all non-O157 STEC strains significantly. At 2× MIC and 4× MIC, the 95% ethanolic extract affected the CSH of E. coli O26:H11 significantly but did not affect E. coli O111:NM or E. coli O22. Electron microscopic examination revealed the loss of pili in the treated cells. The ability of Q. infectoria extract to modify hydrophobic domains enables this extract to partition the lipids of the bacterial cell membrane, rendering the membrane more permeable and allowing leakage of ions and other cell contents, which leads to cell death. Further studies are required to evaluate the effects of Q. infectoria extract in food systems or in vivo and provide support for the use of this extract as a food additive for control of these STEC pathogens.

scholarly journals Utilization of Mn-Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Thi-Diem Bui ◽  
Quang-Liem Nguyen ◽  
Thi-Bich Luong ◽  
Van Thuan Le ◽  
Van-Dat Doan
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Quantum Dots ◽  
Fluorescent Labeling ◽  
Core Shell ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Methicillin Resistant ◽  
E Coli ◽  
Mn Doped

In this study, Mn-doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti-E. coli antibody and anti-MRSA antibody via protein A supported by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria-Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

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