scholarly journals THE EFFECTS OF MAMMALIAN AND OTHER CATIONIC POLYPEPTIDES ON THE CYTOCHEMICAL CHARACTER OF BACTERIAL CELLS

1961 ◽  
Vol 114 (6) ◽  
pp. 1063-1078 ◽  
Author(s):  
John K. Spitznagel
Keyword(s):  
Differential Staining ◽  
Bacterial Cells ◽  
Surface Layers ◽  
High Ph ◽  
E Coli ◽  
Detergent Action ◽  
Cell Components

Cationic polypeptides interact with bacterial cells of E. coli and B. anthracis. They confer upon the cells some of the characteristics of cationic particles. Since bacterial cells usually behave as anions, acidic dyes at high pH levels differentiate between cells which have and those which have not interacted with cationic polypeptides. Under the conditions of these experiments it appeared that cationic polypeptides tend to be sorbed in highest concentration in the surface layers of the cells. Electrovalent binding to anionic cell components and detergent action are probably among the mechanisms involved in the interaction between the polypeptides such as histones and bacterial cells. The differential staining of bacterial cells which have interacted with cationic polypeptides is feasible and reasonably selective. It should be useful in determining whether bacterial cells interact with host cationic polypeptides in vitro.

scholarly journals SELECTIVE "STAINING" FOR ELECTRON MICROGRAPHY

1942 ◽  
Vol 76 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Stuart Mudd ◽  
Thomas F. Anderson
Keyword(s):  
Heavy Metals ◽  
Cell Walls ◽  
Differential Staining ◽  
Bacterial Cells ◽  
Selective Staining ◽  
Electron Micrography ◽  
Chemical Effects ◽  
Microchemical Analysis ◽  
Cell Components ◽  
Selective Chemical

The physical basis of contrast and image formation in electron micrography is considered in relation to the possibility of recording selective chemical effects on cell components. A technology of selective microchemical analysis, equivalent to differential staining, is suggested as practicable in electron micrography. Electron pictures of bacteria after exposure to salts of heavy metals have shown the bacterial inner protoplasm, but not the cell walls, to be selectively darkened; shrinkage, coagulation, or escape of protoplasm from the injured cells may result and be recorded in the electron micrographs. Recording of the action of germicidal agents on individual bacterial cells is indicated as one promising field of application of microchemical analysis with the aid of the electron microscope.

scholarly journals Microencapsulation of Enteric Bacteriophages in a pH-Responsive Solid Oral Dosage Formulation Using a Scalable Membrane Emulsification Process

Pharmaceutics ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 475 ◽  
Author(s):  
Vinner ◽  
Richards ◽  
Leppanen ◽  
Sagona ◽  
Malik
Keyword(s):  
Epithelial Cells ◽  
Prolonged Exposure ◽  
Oral Dosage ◽  
Bacterial Cells ◽  
Ph Responsive ◽  
Ion Milling ◽  
Membrane Emulsification ◽  
E Coli ◽  
Emulsification Process

A scalable low-shear membrane emulsification process was used to produce microencapsulated Escherichia coli-phages in a solid oral dosage form. Uniform pH-responsive composite microparticles (mean size ~100 µm) composed of Eudragit® S100 and alginate were produced. The internal microstructure of the gelled microcapsules was studied using ion-milling and imaging, which showed that the microparticles had a solid internal core. The microencapsulation process significantly protected phages upon prolonged exposure to a simulated gastric acidic environment. Encapsulated phages that had been pre-exposed to simulated gastric acid were added to actively growing bacterial cells using in vitro cell cultures and were found to be effective in killing E. coli. Encapsulated phages were also shown to be effective in killing actively growing E. coli in the presence of human epithelial cells. Confocal microscopy images showed that the morphology of encapsulated phage-treated epithelial cells was considerably better than controls without phage treatment. The encapsulated phages were stable during refrigerated storage over a four-week period. The process of membrane emulsification is highly scalable and is a promising route to produce industrial quantities of pH-responsive oral solid dosage forms suitable for delivering high titres of viable phages to the gastrointestinal tract.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

scholarly journals Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

2002 ◽  
Vol 68 (10) ◽  
pp. 4894-4899 ◽  
Author(s):  
Jörg Schirmer ◽  
Hans-Joachim Wieden ◽  
Marina V. Rodnina ◽  
Klaus Aktories
Keyword(s):  
Peptide Mapping ◽  
Elongation Factor ◽  
Bacillus Sphaericus ◽  
Elongation Factor Tu ◽  
Bacterial Cells ◽  
Single Chain ◽  
E Coli ◽  
Adp Ribosylation ◽  
Flight Mass Spectrometry

ABSTRACT The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ∼97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ∼45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.

scholarly journals Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator inPseudomonas aeruginosa

2018 ◽  
Vol 115 (12) ◽  
pp. 3150-3155 ◽  
Author(s):  
Neil G. Greene ◽  
Coralie Fumeaux ◽  
Thomas G. Bernhardt
Keyword(s):  
Cell Wall ◽  
Drug Targets ◽  
Bacterial Cells ◽  
Acinetobacter Baylyi ◽  
Gram Negative ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Synthase Regulation

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoproteinEcLpoB was identified as a critical activator ofEscherichia coliPBP1b (EcPBP1b), one of the major PG synthases of this organism. Activation ofEcPBP1b is mediated through the association ofEcLpoB with a regulatory domain onEcPBP1b called UB2H. Notably,Pseudomonas aeruginosaalso encodes PBP1b (PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potentialPaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPaPBP1b. We named this protein LpoP and found that it interacts directly withPaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatPaLpoP-PaPBP1b as well as an equivalent protein pair fromAcinetobacter baylyican fully substitute forEcLpoB-EcPBP1b inE. colifor PG synthesis. Furthermore, we show that amino acid changes inPaPBP1b that bypass thePaLpoP requirement map to similar locations in the protein as changes promotingEcLpoB bypass inEcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

scholarly journals Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court
Keyword(s):  
Dna Assembly ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Engineering Technology ◽  
E Coli ◽  
Linear Dna ◽  
Recombination System ◽  
Genetic Engineering Technology

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

scholarly journals 60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5620 ◽  
Author(s):  
Sergey Levitskii ◽  
Ksenia Derbikova ◽  
Maria V. Baleva ◽  
Anton Kuzmenko ◽  
Andrey V. Golovin ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Sedimentation Coefficient ◽  
Initiation Factor ◽  
Dynamic State ◽  
Bacterial Cells ◽  
E Coli ◽  
Bacterial Ribosome

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of the ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for “60S state” formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.

scholarly journals Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Marcin Krupka ◽  
Marta Sobrinos-Sanguino ◽  
Mercedes Jiménez ◽  
Germán Rivas ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
High Surface ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Attachment ◽  
Lipid Surface ◽  
Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

Preparation of Silver Sulfadiazine Loaded Pectin-Zinc Nanocomposites for Improvement of Antioxidant, Antibacterial and Care Cutaneous Wound Care During the Perioperative Period

2020 ◽  
Vol 10 (1) ◽  
pp. 1-7
Author(s):  
Linjing Chen ◽  
Fangfang Wang ◽  
Tao Luan ◽  
Shuhong Jiang ◽  
Feifei Yu ◽  
...  
Keyword(s):  
Wound Infection ◽  
Silver Sulfadiazine ◽  
Resistant Bacteria ◽  
Antioxidant Enzyme Activities ◽  
Bacterial Cells ◽  
Tem Analysis ◽  
Cutaneous Wound ◽  
E Coli ◽  
Antioxidant Agents

Medicinal care for cutaneous wound becomes difficult due to the emergence of antibiotic resistant bacteria. The development of a new cost-effective drug based on silver sulfadiazine loaded with pectin-zinc nanocomposites offers better therapeutics in wound healing besides its role in enhancing antioxidant and antibacterial activities. In this study, silver sulfadiazine loaded with pectin-zinc nanocomposites (AgSD-PC-Zn) was produced and its antioxidant and antimicrobial properties were tested against wound infection causing bacterial pathogens (S. aureus and E. coli). The synthesized AgSD-PC-Zn composite was biophysically characterized. TEM analysis confirmed the spherical shape of NPs. AgSD-PC-Zn composite exhibited a greater antibacterial effect against S. aureus and E. coli. The number of dead bacterial cells was higher after exposure to AgSD-PC-Zn composite. Furthermore, in vitro antioxidant enzyme activities (DPPH and H2O2) were increased with AgSDPC-Zn composite. A rapid healing of the skin wounds in mouse was noticed with AgSD-PC-Zn composite treatment. This study concludes that the newly formed AgSD-PC-Zn composite will act as a strong antimicrobial and antioxidant agents for treating skin wound infection.

scholarly journals In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Vuong Van Hung Le ◽  
Catrina Olivera ◽  
Julian Spagnuolo ◽  
Ieuan G. Davies ◽  
Jasna Rakonjac
Keyword(s):  
Nitric Oxide ◽  
Growth Inhibition ◽  
Fold Increase ◽  
Sodium Deoxycholate ◽  
Efflux Pumps ◽  
Bacterial Cells ◽  
Bacterial Killing ◽  
E Coli

Abstract Background Antimicrobial combinations have been proven as a promising approach in the confrontation with multi-drug resistant bacterial pathogens. In the present study, we identify and characterize a synergistic interaction of broad-spectrum nitroreductase-activated prodrugs 5-nitrofurans, with a secondary bile salt, sodium deoxycholate (DOC) in growth inhibition and killing of enterobacteria. Results Using checkerboard assay, we show that combination of nitrofuran furazolidone (FZ) and DOC generates a profound synergistic effect on growth inhibition in several enterobacterial species including Escherichia coli, Salmonella enterica, Citrobacter gillenii and Klebsiella pneumoniae. The Fractional Inhibitory Concentration Index (FICI) for DOC-FZ synergy ranges from 0.125 to 0.35 that remains unchanged in an ampicillin-resistant E. coli strain containing a β-lactamase-producing plasmid. Findings from the time-kill assay further highlight the synergy with respect to bacterial killing in E. coli and Salmonella. We further characterize the mechanism of synergy in E. coli K12, showing that disruption of the tolC or acrA genes that encode components of multidrug efflux pumps causes, respectively, a complete or partial loss, of the DOC-FZ synergy. This finding indicates the key role of TolC-associated efflux pumps in the DOC-FZ synergy. Overexpression of nitric oxide-detoxifying enzyme Hmp results in a three-fold increase in FICI for DOC-FZ interaction, suggesting a role of nitric oxide in the synergy. We further demonstrate that DOC-FZ synergy is largely independent of NfsA and NfsB, the two major activation enzymes of the nitrofuran prodrugs. Conclusions This study is to our knowledge the first report of nitrofuran-deoxycholate synergy against Gram-negative bacteria, offering potential applications in antimicrobial therapeutics. The mechanism of DOC-FZ synergy involves FZ-mediated inhibition of TolC-associated efflux pumps that normally remove DOC from bacterial cells. One possible route contributing to that effect is via FZ-mediated nitric oxide production.

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