scholarly journals The A component (SmhA) of a tripartite pore-forming toxin from Serratia marcescens: expression, purification and crystallographic analysis

2020 ◽  
Vol 76 (12) ◽  
pp. 577-582
Author(s):  
Alicia M. Churchill-Angus ◽  
Svetlana E. Sedelnikova ◽  
Thomas H. B. Schofield ◽  
Patrick J. Baker
Keyword(s):  
Serratia Marcescens ◽  
Electron Density ◽  
Crystallographic Analysis ◽  
Gram Negative Bacteria ◽  
Human Pathogen ◽  
Gram Negative ◽  
Anomalous Data ◽  
Density Map ◽  
Opportunistic Human Pathogen ◽  
Electron Density Map

Tripartite α-pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α-pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced.

scholarly journals TonB dependent uptake of β-lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia

2019 ◽  
Author(s):  
Karina Calvopiña ◽  
Punyawee Dulyayangkul ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Cross Resistance ◽  
Gram Negative Bacteria ◽  
Potential Utility ◽  
Human Pathogen ◽  
Gram Negative ◽  
Outer Membrane Porins ◽  
Lactam Antibiotic ◽  
Energy Transducer ◽  
Opportunistic Human Pathogen

AbstractThe β-lactam antibiotic ceftazidime is one of only a handful of drugs with proven clinical efficacy against the opportunistic human pathogen Stenotrophomonas maltophilia, Here, we show that mutations in the energy transducer TonB, encoded by smlt0009 in S. maltophilia, confer ceftazidime resistance. This breaks the dogma that β-lactams enter Gram-negative bacteria only by passive diffusion through outer membrane porins. By confirming cross-resistance of Smlt0009 mutants with a siderophore-conjugated lactivicin antibiotic, we reveal that attempts to improve penetration of antimicrobials into Gram negative bacteria by conjugating them with TonB substrates is likely to select β-lactam resistance in S. maltophilia, increasing its clinical threat. Furthermore, we show that S. maltophilia clinical isolates that have an Smlt0009 mutation already exist. Remarkably, therefore, β-lactam use is already eroding the potential utility of currently experimental siderophore-conjugated antimicrobials against this species.

scholarly journals Characterisation of a tripartite α-pore forming toxin from Serratia marcescens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alicia M. Churchill-Angus ◽  
Thomas H. B. Schofield ◽  
Thomas R. Marlow ◽  
Svetlana E. Sedelnikova ◽  
Jason S. Wilson ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Serratia Marcescens ◽  
Conformational Change ◽  
Pore Formation ◽  
Gram Negative Bacteria ◽  
Human Pathogen ◽  
Gram Negative ◽  
The Family

AbstractTripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S. marcescens and propose a mechanism of pore assembly. We present the structure of soluble SmhA, as well as the soluble and pore forms of SmhB. We show that the β-tongue soluble structure is well conserved in the family and propose two conserved latches between the head and tail domains that are broken on the soluble to pore conformational change. Using the structures of individual components, sequence analysis and docking predictions we illustrate how the A, B and C protomers would assemble on the membrane to produce a complete tripartite α-PFT pore.

scholarly journals Packaging Covered with Antiviral and Antibacterial Coatings Based on ZnO Nanoparticles Supplemented with Geraniol and Carvacrol

2021 ◽  
Vol 22 (4) ◽  
pp. 1717
Author(s):  
Małgorzata Mizielińska ◽  
Paweł Nawrotek ◽  
Xymena Stachurska ◽  
Magdalena Ordon ◽  
Artur Bartkowiak
Keyword(s):  
Synergistic Effect ◽  
Moderate Activity ◽  
Gram Negative Bacteria ◽  
Human Pathogen ◽  
Safety Measures ◽  
Gram Negative ◽  
Airborne Viruses ◽  
Respiratory Droplets ◽  
Active Substances ◽  
External Coating

The purpose of the study was to obtain an external coating based on nanoparticles of ZnO, carvacrol, and geraniol that could be active against viruses such as SARS-Co-V2. Additionally, the synergistic effect of the chosen substances in coatings was analyzed. The goal of the study was to measure the possible antibacterial activity of the coatings obtained. Testing antiviral activity with human pathogen viruses, such as SARS-Co-V2, requires immense safety measures. Bacteriophages such as phi 6 phage represent good surrogates for the study of airborne viruses. The results of the study indicated that the ZC1 and ZG1 coatings containing an increased amount of geraniol or carvacrol and a very small amount of nanoZnO were found to be active against Gram-positive and Gram-negative bacteria. It is also important that a synergistic effect between these active substances was noted. This explains why polyethylene (PE) films covered with the ZC1 or ZG1 coatings (as internal coatings) were found to be the best packaging materials to extend the quality and freshness of food products. The same coatings may be used as the external coatings with antiviral properties. The ZC1 and ZG1 coatings showed moderate activity against the phi 6 phage that has been selected as a surrogate for viruses such as coronaviruses. It can be assumed that coatings ZG1 and ZC1 will also be active against SARS-CoV-2 that is transmitted via respiratory droplets.

A Comparison of Two Algorithms for Electron-Density Map Improvement by Introduction of Atomicity: Skeletonization, and Map Sorting Followed by Refinement

1998 ◽  
Vol 54 (1) ◽  
pp. 81-85 ◽  
Author(s):  
F. M. D. Vellieux
Keyword(s):  
Electron Density ◽  
Initial Density ◽  
Acta Cryst ◽  
Density Maps ◽  
Resolution Electron ◽  
Crystallographic Symmetry ◽  
Density Map ◽  
Ornithine Transcarbamoylase ◽  
Electron Density Map ◽  
Pseudo Atom

A comparison has been made of two methods for electron-density map improvement by the introduction of atomicity, namely the iterative skeletonization procedure of the CCP4 program DM [Cowtan & Main (1993). Acta Cryst. D49, 148–157] and the pseudo-atom introduction followed by the refinement protocol in the program suite DEMON/ANGEL [Vellieux, Hunt, Roy & Read (1995). J. Appl. Cryst. 28, 347–351]. Tests carried out using the 3.0 Å resolution electron density resulting from iterative 12-fold non-crystallographic symmetry averaging and solvent flattening for the Pseudomonas aeruginosa ornithine transcarbamoylase [Villeret, Tricot, Stalon & Dideberg (1995). Proc. Natl Acad. Sci. USA, 92, 10762–10766] indicate that pseudo-atom introduction followed by refinement performs much better than iterative skeletonization: with the former method, a phase improvement of 15.3° is obtained with respect to the initial density modification phases. With iterative skeletonization a phase degradation of 0.4° is obtained. Consequently, the electron-density maps obtained using pseudo-atom phases or pseudo-atom phases combined with density-modification phases are much easier to interpret. These tests also show that for ornithine transcarbamoylase, where 12-fold non-crystallographic symmetry is present in the P1 crystals, G-function coupling leads to the simultaneous decrease of the conventional R factor and of the free R factor, a phenomenon which is not observed when non-crystallographic symmetry is absent from the crystal. The method is far less effective in such a case, and the results obtained suggest that the map sorting followed by refinement stage should be by-passed to obtain interpretable electron-density distributions.

Electron-density maps

2002 ◽  
Author(s):  
David Blow
Keyword(s):  
Fourier Transform ◽  
Electron Density ◽  
Three Dimensional ◽  
Two Dimensions ◽  
Unit Cell ◽  
Density Maps ◽  
Wave Cycle ◽  
Density Map ◽  
The Fourier Transform ◽  
Electron Density Map

When everything has been done to make the phases as good as possible, the time has come to examine the image of the structure in the form of an electron-density map. The electron-density map is the Fourier transform of the structure factors (with their phases). If the resolution and phases are good enough, the electron-density map may be interpreted in terms of atomic positions. In practice, it may be necessary to alternate between study of the electron-density map and the procedures mentioned in Chapter 10, which may allow improvements to be made to it. Electron-density maps contain a great deal of information, which is not easy to grasp. Considerable technical effort has gone into methods of presenting the electron density to the observer in the clearest possible way. The Fourier transform is calculated as a set of electron-density values at every point of a three-dimensional grid labelled with fractional coordinates x, y, z. These coordinates each go from 0 to 1 in order to cover the whole unit cell. To present the electron density as a smoothly varying function, values have to be calculated at intervals that are much smaller than the nominal resolution of the map. Say, for example, there is a protein unit cell 50 Å on a side, at a routine resolution of 2Å. This means that some of the waves included in the calculation of the electron density go through a complete wave cycle in 2 Å. As a rule of thumb, to represent this properly, the spacing of the points on the grid for calculation must be less than one-third of the resolution. In our example, this spacing might be 0.6 Å. To cover the whole of the 50 Å unit cell, about 80 values of x are needed; and the same number of values of y and z. The electron density therefore needs to be calculated on an array of 80×80×80 points, which is over half a million values. Although our world is three-dimensional, our retinas are two-dimensional, and we are good at looking at pictures and diagrams in two dimensions.

scholarly journals Optimization of Agitation Rate in Bioreactor Increases Chitinase Activity of Serratia marcescens PT6

2020 ◽  
Vol 147 ◽  
pp. 03019
Author(s):  
Amara Faiz Wriahusna ◽  
Niswah Umhudloh Dzakiyya ◽  
Indun Dewi Puspita ◽  
Sri Pudjiraharti
Keyword(s):  
Serratia Marcescens ◽  
Bacterial Growth ◽  
Agar Medium ◽  
Chitinase Activity ◽  
Colloidal Chitin ◽  
Gram Negative Bacteria ◽  
Agitation Rate ◽  
Anova Analysis ◽  
Gram Negative ◽  
Shrimp Pond

Serratia marcescens PT6 is a Gram-negative bacteria isolated from shrimp pond sediment that capable of producing chitinase. This study aimed to observe the effect of agitation rate on growth and chitinase activity of S. marcescens PT-6 in a bioreactor. The production of chitinase was done in 1.5 l bioreactor using colloidal chitin broth at the condition of pH 7, the temperature of 30°C, aeration of 0.04 vvm, and variation of agitation rate (200, 350, 500 rpm). Bacterial growth was measured by colonies counting in agar medium, while chitinase activity was measured by means of colorimetric every day for four days incubation. The results of ANOVA analysis show that the agitation rate had no effect on bacterial growth, but a significant effect (P<0.05) was observed on chitinase activity. The highest growth and chitinase activity were obtained at 200 rpm, with the highest chitinase activity of 0.006 ± 0.001 U/ml was at day-2. This study implies that the optimized agitation rate in the bioreactor increased the chitinase activity produced by S. marcescens PT-6.

scholarly journals Electron density map as an aid in the sequencing of peptides.

1973 ◽  
Vol 70 (6) ◽  
pp. 1793-1794 ◽  
Author(s):  
M. J. Adams ◽  
G. C. Ford ◽  
P. J. Lentz ◽  
A. Liljas ◽  
M. G. Rossmann
Keyword(s):  
Electron Density ◽  
Density Map ◽  
Electron Density Map
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