scholarly journals The mechanoelectrical response of the cytoplasmic membrane of Vibrio cholerae

2013 ◽  
Vol 142 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Ian Rowe ◽  
Merina Elahi ◽  
Anwar Huq ◽  
Sergei Sukharev
Keyword(s):  
Vibrio Cholerae ◽  
Cytoplasmic Membrane ◽  
Mechanosensitive Channel ◽  
Inward Rectification ◽  
Electrogenic Transport ◽  
E Coli ◽  
Esherichia Coli ◽  
Membrane Components

Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p0.5MscS/p0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.

scholarly journals Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2405-2420 ◽  
Author(s):  
Anu Daniel ◽  
Aparna Singh ◽  
Lynette J. Crowther ◽  
Paula J. Fernandes ◽  
Wiebke Schreiber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Complete Characterization ◽  
Type Iv Pili ◽  
Amino Terminus ◽  
Type Iv ◽  
Membrane Components ◽  
Nucleotide Binding Proteins

Typical enteropathogenic Escherichia coli strains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by the bfp operon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly.

scholarly journals Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP, LacY, ProW, AqpZ, MscS, and MscL

2009 ◽  
Vol 192 (4) ◽  
pp. 912-924 ◽  
Author(s):  
Tatyana Romantsov ◽  
Andrew R. Battle ◽  
Jenifer L. Hendel ◽  
Boris Martinac ◽  
Janet M. Wood
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Cytoplasmic Membrane ◽  
Protein Localization ◽  
General Characteristic ◽  
Mechanosensitive Channel ◽  
E Coli ◽  
Polar Localization ◽  
Cytoplasmic Membrane Proteins

ABSTRACT Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).

scholarly journals In situ genotyping of a pooled strain library after characterizing complex phenotypes

2017 ◽  
Author(s):  
Michael J. Lawson ◽  
Daniel Camsund ◽  
Jimmy Larsson ◽  
Özden Baltekin ◽  
David Fange ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Detailed Characterization ◽  
E Coli ◽  
Complex Phenotypes

So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strain’s genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

scholarly journals Preparation and Characterization of Chitosan Nanocomposite Based on Nanoscale Silver and Nanomontmorillonite

2019 ◽  
Vol 2 (2) ◽  
pp. 5-12 ◽  
Author(s):  
Fatemeh- Sadat Ebnerasool ◽  
Negar Motakef Kazemi
Keyword(s):  
Food Packaging ◽  
Solution Method ◽  
In Situ Synthesis ◽  
Diffusion Method ◽  
X Ray ◽  
E Coli ◽  
Packaging Industry ◽  
Scanning Electron

The chitosan nanocomposites were rapidly prepared by simple solution method. This biopolymer matrix was modified by prepared nanoscale silver (Ag) using in situ synthesis from precursor and nanomontmorillonite (NMMT). The samples were characterized by Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), and energy dispersive x-ray spectroscopy (EDX). The water vapor properties (WVP) of nanocomposites were investigated using gravimetric standard. The antibacterial activity of nanocomposite was measured by the well diffusion method on Muller–Hinton Agar against Escherichia coli (E. coli) by zone inhibition. Based on the obtained results, the nanocomposite can have a good candidate for different applications and food packaging industry.

scholarly journals Infrared Characterization of the Bidirectional Oxygen-Sensitive [Nife]-Hydrogenase from E. Coli

2018 ◽  
Author(s):  
Moritz Senger ◽  
Konstanin Laun ◽  
Basem Soboh ◽  
Sven Timo Stripp
Keyword(s):  
Escherichia Coli ◽  
Green Chemistry ◽  
Ftir Spectroscopy ◽  
Biological Macromolecules ◽  
E Coli ◽  
Gas Processing ◽  
H2 Generation ◽  
Oxygen Sensitive

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional “O2-sensitive” [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ ATR FTIR spectroscopy under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., “O2-tolerant” [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the “unready” Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis.

scholarly journals In Situ Characterization of Mycobacterial Growth Inhibition by Lytic Enzymes Expressed in Vectorized E. coli

2014 ◽  
Vol 3 (12) ◽  
pp. 932-934 ◽  
Author(s):  
Iva Atanaskovic ◽  
Amel Camélia Bencherif ◽  
Matthew Deyell ◽  
Sebastián Jaramillo-Riveri ◽  
Marguerite Benony ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Lytic Enzymes ◽  
In Situ Characterization ◽  
E Coli ◽  
Mycobacterial Growth

scholarly journals Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

2021 ◽  
Author(s):  
Zachary Morrison ◽  
Alexander Eddenden ◽  
Adithya S Subramanian ◽  
P. Lynne Howell ◽  
mark nitz
Keyword(s):  
Biofilm Formation ◽  
Chain Termination ◽  
Salvage Pathway ◽  
Biofilm Inhibition ◽  
E Coli ◽  
Substrate Analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

scholarly journals A Multifunctional ATP-Binding Cassette Transporter System from Vibrio cholerae Transports Vibriobactin and Enterobactin

1999 ◽  
Vol 181 (24) ◽  
pp. 7588-7596 ◽  
Author(s):  
Elizabeth E. Wyckoff ◽  
Ana-Maria Valle ◽  
Stacey L. Smith ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Iron Transport ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Biosynthetic Gene Cluster ◽  
Transport Systems ◽  
E Coli ◽  
Amino Terminal ◽  
Atp Binding Cassette Transporter ◽  
Membrane Lipoprotein

ABSTRACT Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD,viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [3H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations infepB, fepD, fepG, orfepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or inviuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.

Synthesis and characterization of silver cluster particles in hydrogels

2015 ◽  
Vol 35 (4) ◽  
pp. 303-309
Author(s):  
Aman Deo ◽  
Syed Sauban Ghani
Keyword(s):  
Silver Cluster ◽  
Maximum Size ◽  
Spectrophotometric Analysis ◽  
E Coli ◽  
Hydrogel Network ◽  
Microscopy Analysis ◽  
Antibacterial Material ◽  
Spectroscopy Studies

Abstract A chitosan-based hydrogel network was crosslinked with genipin; the crosslinked and uncrosslinked hydrogels were prepared and studied. Uniformly distributed silver cluster particles (AgCPs) were prepared using these hydrogel networks as a carrier via in situ reduction of silver nitrate (AgNO3) in the presence of sodium borohydride (NaBH4) as a reducing agent; UV irradiation reduction was also done. Fourier transform infrared (FTIR) spectroscopy studies of the hydrogels gave results on the decree of crosslinking and some indication on the presence of the AgCPs. The spectrophotometric analysis indicated the presence of AgCPs, as a peak appeared around 400 nm. The microscopy analysis gave images of the presence of AgCPs and their size; it showed that the crosslinked hydrogels have uniformly distributed AgCPs and as the crosslinking increased, the AgCPs size decreased. The studies on the increasing concentration of AgNO3 solution gave an increase AgCPs size; the maximum size of AgCPs clusters was ∼0.2 micron in uncrosslinked chitosan. It was demonstrated, by using Escherichia coli (E. coli) bacterium, that the AgCPs hydrogel can be effectively employed as antibacterial material.

scholarly journals In Situ Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic Escherichia coli and Vibrio cholerae

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Yasmin Ara Begum ◽  
Hanna A. Rydberg ◽  
Kaisa Thorell ◽  
Young-Keun Kwak ◽  
Lei Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Bacterial Load ◽  
Enterotoxigenic Escherichia Coli ◽  
Dose Dependency ◽  
E Coli ◽  
Three Samples ◽  
Globally Distributed

The cause of diarrheal disease is usually determined by screening for several microorganisms by various methods, and sole detection is used to assign the agent as the cause of disease. However, it has become increasingly clear that many infections are caused by coinfections with several pathogens and that the dose of the infecting pathogen is important. We quantified the absolute numbers of enterotoxigenicE. coli(ETEC) andVibrio choleraedirectly in diarrheal fluid. We noted several events where both pathogens were found but also a large dose dependency. In three samples, we found ETEC as the only pathogen sought for. These isolates belonged to globally distributed ETEC clones and were the dominating species in stool with active toxin expression. This suggests that certain superior virulent ETEC lineages are able to outcompete the gut microbiota and be the sole cause of disease and hence need to be specifically monitored.

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