scholarly journals Control of n-Butanol Induced Lipidome Adaptations in E. coli

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 286
Author(s):  
Aike Jeucken ◽  
Miaomiao Zhou ◽  
Marc M. S. M. Wösten ◽  
Jos F. Brouwers
Keyword(s):  
Lipid Membranes ◽  
Internal Combustion Engines ◽  
Large Scale ◽  
Clean Energy ◽  
Sublethal Concentration ◽  
Smooth Transition ◽  
Butanol Tolerance ◽  
E Coli ◽  
Lipid Species ◽  
Bacterial Lipid

The versatile compound n-butanol is one of the most promising biofuels for use in existing internal combustion engines, contributing to a smooth transition towards a clean energy society. Furthermore, n-butanol is a valuable resource to produce more complex molecules such as bioplastics. Microbial production of n-butanol from waste materials is hampered by the biotoxicity of n-butanol as it interferes with the proper functioning of lipid membranes. In this study we perform a large-scale investigation of the complete lipid-related enzyme machinery and its response to exposure to a sublethal concentration of n-butanol. We profiled, in triplicate, the growth characteristics and phospholipidomes of 116 different genetic constructs of E. coli, both in the presence and absence of 0.5% n-butanol (v/v). This led to the identification of 230 lipid species and subsequently to the reconstruction of the network of metabolites, enzymes and lipid properties driving the homeostasis of the E. coli lipidome. We were able to identify key lipids and biochemical pathways leading to altered n-butanol tolerance. The data led to new conceptual insights into the bacterial lipid metabolism which are discussed.

The Impact of Antibacterial Peptides on Bacterial Lipid Membranes Depends on Stage of Growth

2020 ◽  
Author(s):  
Tzong-Hsien Lee ◽  
Vinzenz Hofferek ◽  
Marc-antoine Sani ◽  
Frances Separovic ◽  
Gavin Reid ◽  
...  
Keyword(s):  
Growth Phase ◽  
Lipid Membranes ◽  
Stationary Growth Phase ◽  
Exponential Growth Phase ◽  
Antibacterial Peptides ◽  
Stationary Growth ◽  
E Coli ◽  
Bacterial Lipid ◽  
Supported Lipid Membranes ◽  
The Impact

The impact of maculatin 1.1 (Mac1) on the mechanical properties of supported lipid membranes derived from exponential growth phase (EGP) and stationary growth phase (SGP) E. coli lipid extracts was...

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

Large-scale comparison of E. coli levels determined by culture and a qPCR method (EPA Draft Method C) in Michigan towards the implementation of rapid, multi-site beach testing

2021 ◽  
Vol 184 ◽  
pp. 106186
Author(s):  
Richard Haugland ◽  
Kevin Oshima ◽  
Mano Sivaganesan ◽  
Alfred Dufour ◽  
Manju Varma ◽  
...  
Keyword(s):  
Large Scale ◽  
E Coli ◽  
Qpcr Method

scholarly journals Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

2021 ◽  
Vol 9 (2) ◽  
pp. 310
Author(s):  
Masayuki Hashimoto ◽  
Yi-Fen Ma ◽  
Sin-Tian Wang ◽  
Chang-Shi Chen ◽  
Ching-Hao Teng
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Caenorhabditis Elegans ◽  
Large Scale ◽  
Iron Acquisition ◽  
Infection Model ◽  
High Throughput Analysis ◽  
E Coli ◽  
C Elegans ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

scholarly journals Enzyme-catalyzed biodegradation of penicillin fermentation residues by β-lactamase OtLac from Ochrobactrum tritici

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Peng Wang ◽  
Chen Shen ◽  
Qinqin Cong ◽  
Kaili Xu ◽  
Jialin Lu
Keyword(s):  
Large Scale ◽  
In Vitro Study ◽  
Scale Removal ◽  
E Coli ◽  
Penicillin V ◽  
Highly Active ◽  
Steady State Kinetics ◽  
Biodegradation Process ◽  
Km Value

Abstract Background Biodegradation of antibiotics is a promising method for the large-scale removal of antibiotic residues in the environment. However, the enzyme that is involved in the biodegradation process is the key information to be revealed. Results In this study, the beta-lactamase from Ochrobactrumtritici that mediates the biodegradation of penicillin V was identified and characterized. When searching the proteins of Ochrobactrumtritici, the β-lactamase (OtLac) was identified. OtLac consists of 347 amino acids, and predicted isoelectric point is 7.0. It is a class C β-lactamase according to BLAST analysis. The coding gene of OtLac was amplified from the genomic DNA of Ochrobactrumtritici. The OtLac was overexpressed in E. coli BL21 (DE3) and purified with Ni2+ column affinity chromatography. The biodegradation ability of penicillin V by OtLac was identified in an in vitro study and analyzed by HPLC. The optimal temperature for OtLac is 32 ℃ and the optimal pH is 7.0. Steady-state kinetics showed that OtLac was highly active against penicillin V with a Km value of 17.86 μM and a kcat value of 25.28 s−1 respectively. Conclusions OtLac demonstrated biodegradation activity towards penicillin V potassium, indicating that OtLac is expected to degrade penicillin V in the future.

scholarly journals Cloning, expression, and analysis of the group 2 allergen from Dermatophagoides farinae from China

2010 ◽  
Vol 82 (4) ◽  
pp. 941-951 ◽  
Author(s):  
Cui Yu-bao ◽  
Ying Zhou ◽  
Shi Weihong ◽  
Ma Guifang ◽  
Li Yang ◽  
...  
Keyword(s):  
Large Scale ◽  
Alpha Helix ◽  
Random Coil ◽  
Reference Sequence ◽  
Scale Production ◽  
Dermatophagoides Farinae ◽  
E Coli ◽  
Large Scale Production ◽  
Solid Foundation ◽  
Group 2

To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3% identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the hole cell lysate. s estiated by chroatography, about 3.86 g of the recobinant product as obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86%), an extended strand (30.82%), and a random coil (49.32%). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatent of allergic disorders.

The Research of Wasting Wind Power and Methods of its Consumption Based on that of Fuxin

2014 ◽  
Vol 986-987 ◽  
pp. 622-629
Author(s):  
Tian Long Shao ◽  
Jian Zhang ◽  
Xu Nan Zhao
Keyword(s):  
Power System ◽  
Wind Power ◽  
Large Scale ◽  
Clean Energy ◽  
Power Grids ◽  
Grid Structure ◽  
Wind Power Integration

As a kind of renewable clean energy, the constant access of wind power to power grids is bound to have a great impact on the power system. Based on the grid structure in Fuxin, this paper will state the difficulty of peak regulation and the matter of wasting wind power caused by the large-scale wind power integration and put forward some reasonable methods for using the wasting wind power in the heating in winter. The relevant results indicate that capacity of local consumption of wasting wind power can be improved. Under the circumstances, it can be conductive to solve the problem of wasting wind power results from the difficulty of peak regulation as well as inspire the power system planners.

Territorial Governance of EU Cross-Border Renewable Energy Cooperation: A Soluble or Turbulent Model in the Current Framework?

2021 ◽  
Vol 2 (1) ◽  
pp. 79-97
Author(s):  
Melis Aras
Keyword(s):  
Renewable Energy ◽  
Large Scale ◽  
Planning Process ◽  
Renewable Energy Sources ◽  
Local Level ◽  
Energy Transition ◽  
Clean Energy ◽  
Legal Framework ◽  
Development Planning ◽  
Cross Border

The energy transition in Europe requires not only the implementation of technological innovations to reduce carbon emissions but also the decentralised extension of these innovations throughout the continent, as demonstrated by the ‘Clean Energy for All Europeans’ package. However, decentralised energy generation, and specifically electricity generation, as it gives rise to new players and interactions, also requires a review of the energy planning process. In this sense, governance becomes the key concept for understanding the implementation of the energy transition in a territory. This is particularly visible in a cross-border setting, especially considering cross-border cooperation in the development of renewable energy sources (RES) provides the necessary elements to determine the criteria of local regulation between the different levels of governance. In light of the current legal framework in France, this paper presents the institutional framework of the multi-level governance of the RES development planning process. It concludes that it is quite conceivable for the rationales of governance at the local level (decentralisation) and the large-scale operation of a large interconnected network (Europeanisation) to coexist.

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