scholarly journals Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

2021 ◽  
Vol 11 (18) ◽  
pp. 8314
Author(s):  
Jozafina Milicaj ◽  
Colleen D. Castro ◽  
Nadiya Jaunbocus ◽  
Erika A. Taylor
Keyword(s):  
Bacterial Infections ◽  
Visual Cues ◽  
Large Scale ◽  
Lipid A ◽  
Biosynthetic Enzymes ◽  
Bacterial Cells ◽  
Site Specific ◽  
E Coli ◽  
Key Steps

The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

Integrated mass spectrometry-based multi-omics for elucidating mechanisms of bacterial virulence

2021 ◽  
Author(s):  
Lok Man ◽  
William P. Klare ◽  
Ashleigh L. Dale ◽  
Joel A. Cain ◽  
Stuart J. Cordwell
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Lipid A ◽  
Phenotypic Characterization ◽  
Post Translational Modification ◽  
Form Of Life ◽  
Antimicrobial Interventions

Despite being considered the simplest form of life, bacteria remain enigmatic, particularly in light of pathogenesis and evolving antimicrobial resistance. After three decades of genomics, we remain some way from understanding these organisms, and a substantial proportion of genes remain functionally unknown. Methodological advances, principally mass spectrometry (MS), are paving the way for parallel analysis of the proteome, metabolome and lipidome. Each provides a global, complementary assay, in addition to genomics, and the ability to better comprehend how pathogens respond to changes in their internal (e.g. mutation) and external environments consistent with infection-like conditions. Such responses include accessing necessary nutrients for survival in a hostile environment where co-colonizing bacteria and normal flora are acclimated to the prevailing conditions. Multi-omics can be harnessed across temporal and spatial (sub-cellular) dimensions to understand adaptation at the molecular level. Gene deletion libraries, in conjunction with large-scale approaches and evolving bioinformatics integration, will greatly facilitate next-generation vaccines and antimicrobial interventions by highlighting novel targets and pathogen-specific pathways. MS is also central in phenotypic characterization of surface biomolecules such as lipid A, as well as aiding in the determination of protein interactions and complexes. There is increasing evidence that bacteria are capable of widespread post-translational modification, including phosphorylation, glycosylation and acetylation; with each contributing to virulence. This review focuses on the bacterial genotype to phenotype transition and surveys the recent literature showing how the genome can be validated at the proteome, metabolome and lipidome levels to provide an integrated view of organism response to host conditions.

scholarly journals Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jolanta Krucinska ◽  
Michael N. Lombardo ◽  
Heidi Erlandsen ◽  
Akram Hazeen ◽  
Searle S. Duay ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Antibacterial Properties ◽  
High Energy ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Promising Target ◽  
Antibiotic Discovery ◽  
Growth And Reproduction

AbstractMany years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

scholarly journals Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 1002 ◽  
Author(s):  
Chunmeng Xu ◽  
Lingjun Tang ◽  
Youxiang Liang ◽  
Song Jiao ◽  
Huimin Yu ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Large Scale ◽  
Nitrile Hydratase ◽  
Fusion Expression ◽  
Solvent Tolerance ◽  
Rhodococcus Ruber ◽  
Bacterial Cells ◽  
E Coli ◽  
Nitrilase Activity ◽  
Stability Enhancement

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32–47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

scholarly journals Shedding new light on opsin evolution

2011 ◽  
Vol 279 (1726) ◽  
pp. 3-14 ◽  
Author(s):  
Megan L. Porter ◽  
Joseph R. Blasic ◽  
Michael J. Bok ◽  
Evan G. Cameron ◽  
Thomas Pringle ◽  
...  
Keyword(s):  
Evolutionary History ◽  
Large Scale ◽  
Current Model ◽  
Expression Patterns ◽  
Model Organisms ◽  
Biological Functions ◽  
Site Specific ◽  
History Of ◽  
Functional Features

Opsin proteins are essential molecules in mediating the ability of animals to detect and use light for diverse biological functions. Therefore, understanding the evolutionary history of opsins is key to understanding the evolution of light detection and photoreception in animals. As genomic data have appeared and rapidly expanded in quantity, it has become possible to analyse opsins that functionally and histologically are less well characterized, and thus to examine opsin evolution strictly from a genetic perspective. We have incorporated these new data into a large-scale, genome-based analysis of opsin evolution. We use an extensive phylogeny of currently known opsin sequence diversity as a foundation for examining the evolutionary distributions of key functional features within the opsin clade. This new analysis illustrates the lability of opsin protein-expression patterns, site-specific functionality (i.e. counterion position) and G-protein binding interactions. Further, it demonstrates the limitations of current model organisms, and highlights the need for further characterization of many of the opsin sequence groups with unknown function.

scholarly journals Optimal Dissociation Methods Differ for N- and O-glycopeptides

2020 ◽  
Author(s):  
Nicholas Riley ◽  
Stacy A. Malaker ◽  
Marc D. Driessen ◽  
Carolyn Bertozzi
Keyword(s):  
Large Scale ◽  
Collision Energy ◽  
Electron Transfer Dissociation ◽  
Method Performance ◽  
Site Specific ◽  
Advantages And Disadvantages ◽  
Beam Type ◽  
Supplemental Activation ◽  
Glycopeptide Analysis

<p><a>Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.</a></p>

scholarly journals Phage–Bacteria Interactions in Potential Applications of Bacteriophage vB_EfaS-271 against Enterococcus faecalis

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 318
Author(s):  
Gracja Topka-Bielecka ◽  
Bożena Nejman-Faleńczyk ◽  
Sylwia Bloch ◽  
Aleksandra Dydecka ◽  
Agnieszka Necel ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Mammalian Cells ◽  
Bacterial Infections ◽  
Phage Therapy ◽  
Bacterial Species ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Potential Applications ◽  
High Level

Phage therapy is one of main alternative option for antibiotic treatment of bacterial infections, particularly in the era of appearance of pathogenic strains revealing resistance to most or even all known antibiotics. Enterococcus faecalis is one of such pathogens causing serious human infections. In the light of high level of biodiversity of bacteriophages and specificity of phages to bacterial species or even strains, development of effective phage therapy depend, between others, on identification and characterization of a large collection of these viruses, including understanding of their interactions with host bacterial cells. Recently, isolation of molecular characterization of bacteriophage vB_EfaS-271, infecting E. faecalis strains have been reported. In this report, phage–host interactions are reported, including ability of vB_EfaS-271 to infect bacteria forming biofilms, efficiency of eliminating bacterial cells from cultures depending on multiplicity of infection (m.o.i.), toxicity of purified phage particles to mammalian cells, and efficiency of appearance of phage-resistant bacteria. The presented results indicate that vB_EfaS-271 can significantly decrease number of viable E. faecalis cells in biofilms and in liquid cultures and reveals no considerable toxicity to mammalian cells. Efficiency of formation of phage-resistant bacteria was dependent on m.o.i. and was higher when the virion-cell ratio was as high as 10 than at low (between 0.01 and 0.0001) m.o.i. values. We conclude that vB_EfaS-271 may be considered as a candidate for its further use in phage therapy.

scholarly journals Antibacterial Activities and Characterization of Novel Inhibitors of LpxC

2002 ◽  
Vol 46 (6) ◽  
pp. 1793-1799 ◽  
Author(s):  
John M. Clements ◽  
Fanny Coignard ◽  
Ian Johnson ◽  
Stephen Chandler ◽  
Shilpa Palan ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Lipid A ◽  
Antibacterial Activities ◽  
Vitro Assay ◽  
Gram Negative ◽  
E Coli ◽  
Antibacterial Target ◽  
Derivatives Of

ABSTRACT Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria. Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A. Inhibitors of LpxC have previously been shown to have antibiotic activities. We have screened a metalloenzyme inhibitor library for antibacterial activities against an Escherichia coli strain with reduced LpxC activity. From this screen, a series of sulfonamide derivatives of the α-(R)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay. Leads from this series showed gram-negative selective activities against members of the Enterobacteriaceae, Serratia marcescens, Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia cepacia. BB-78484 was bactericidal against E. coli, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis. E. coli mutants with decreased susceptibility to BB-78484 were selected. Analysis of these mutants revealed that resistance arose as a consequence of mutations in the fabZ or lpxC genes. These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials.

scholarly journals Green Synthesis and Characterization of Silver Nanoparticles using Crude Extract of Crotalaria burhia

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Khalil Ahmad ◽  
Raeesa Noor ◽  
Muhammad Younus ◽  
Akram Chohan ◽  
Ume Habiba ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Antibacterial Activity ◽  
Zeta Potential ◽  
Green Synthesis ◽  
Crude Extract ◽  
Bacterial Infections ◽  
Synthesis And Characterization ◽  
Phytochemical Analysis ◽  
E Coli

Background: Appearance of antibiotic resistance has raised the demand to find alternative therapies and modified drug delivery system of medicinal plants to treat bacterial infections. Objective: The aim of this study is the green synthesis and characterization of silver nanoparticles by using crude extract of Crotalaria burhia and to evaluate their antibacterial potential. Methods: The roots and stems of plant were used to prepare the crude extract. The phytochemical analysis of different compounds in extract was performed. 1mM AgNO3 and different concentrations of plant extract were used for the green synthesis of silver nanoparticles. The particles size and zeta potential were measured by zeta sizer while surface morphology of silver nanoparticles was observed with Scanning Electron Microscope (SEM). The antibacterial activity of silver nanoparticles was performed by 96 well microdilution plate method. Results: The particle size and zeta potential of optimized formulation was 92 nm and -24.8 mV. The SEM analysis showed that silver nanoparticles are irregular and spherical shape. The antibacterial activity showed that MIC value of silver nanoparticles was lower for E. coli than S. aureus. Conclusion: Silver nanoparticles possess potent bactericidal activity against E. coli and moderate activity against S. aureus. It had been concluded that these nanoparticles can be used against multi-drug resistant bacterial infections.

scholarly journals PRODUCTION AND CHARACTERIZATION OF RECOMBINANT PROTEINS CONTAINING BET V 2 ANTIGENIC EPITOPES OF BETULA PENDULA

2012 ◽  
Vol 9 (6) ◽  
pp. 28-31
Author(s):  
A V Chernysheva ◽  
V V Tyutyaeva ◽  
A V Pivovarova ◽  
I V Andreev ◽  
M N Sankov ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Inclusion Bodies ◽  
Birch Pollen ◽  
Elisa Test ◽  
Recombinant Allergen ◽  
Bacterial Cells ◽  
E Coli ◽  
Immunological Tests ◽  
Linear Epitopes

Aim of investigation. Production of immunologically active recombinant protein of Bet v 2 allergen ofbirch pollen. Materials and methods. mRNA was isolated from a sample ofbirch pollen. cDNA library was derived using SMART technology. Gene Bet v 2 was amplified by means of PCR with primers from the cDNA. The resulting PCR fragment of the gene was cloned into the vector pET29b(+). The recombinant protein Bet v 2 was expressed in cells E. coli, transformed with a plasmid. The recombinant protein was purified using NiNTA agarose. Immunological activity of the recombinant protein Bet v 2 was measured by ELISA and immunoblot methods. Results. The production system of the recombinant allergen Bet v 2 preparation suitable for immunological tests was developed during the research project. In the first phase allergen Bet v 2 gene was cloned from birch pollen collected in Russia. The gene was inserted into the vector pET29(+) for expression in bacterial cells. The expression cell strain E. coli was obtained with this plasmid. The synthesis of the recombinant protein that accumulates in inclusion bodies was activated in bacterial cells. The procedure of recombinant protein Bet v 2 isolation from inclusion bodies was developed by one round of chromatography purification. The recombinant protein isolation was carried out by chromatography on Niagarose. The highly purified preparation of the recombinant allergen was obtained as a result. The recognition of the recombinant protein Bet v 2 by sera varied in ELISA, indicating a different degree of patients sensitization to this allergen. In the immunoblot test the preparation was active only in 15% of cases. Apparently, reactive epitopes of the allergen are mainly conformational ones and are active in ELISA test, whereas linear epitopes, that are active in immunoblot, are in the minority. Conclusion.The system for production of recombinant allergen Bet v 2 preparation suitable for immunological tests has been developed.

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