scholarly journals Simulated Diffusion of Phosphorylated CheY through the Cytoplasm of Escherichia coli

2005 ◽  
Vol 187 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Karen Lipkow ◽  
Steven S. Andrews ◽  
Dennis Bray
Keyword(s):  
Escherichia Coli ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Three Dimensional Model ◽  
Large Protein ◽  
E Coli ◽  
Flagellar Motors ◽  
Apparent Diffusion ◽  
A Cell ◽  
Mutant Cells

ABSTRACT We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis. A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With Smoldyn, we constructed a three-dimensional model of an E. coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.

scholarly journals Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

2008 ◽  
Vol 52 (8) ◽  
pp. 2909-2914 ◽  
Author(s):  
Stéphanie Matrat ◽  
Alexandra Aubry ◽  
Claudine Mayer ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Primary Structure ◽  
Three Dimensional ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Intrinsic Resistance ◽  
E Coli

ABSTRACT The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

scholarly journals Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

2020 ◽  
Vol 295 (46) ◽  
pp. 15454-15463 ◽  
Author(s):  
Chelsey R. Fontenot ◽  
Homyra Tasnim ◽  
Kathryn A. Valdes ◽  
Codrina V. Popescu ◽  
Huangen Ding
Keyword(s):  
Escherichia Coli ◽  
Iron Content ◽  
Iron Homeostasis ◽  
Ferric Uptake Regulator ◽  
Intracellular Iron ◽  
Free Iron ◽  
E Coli ◽  
Genes Encoding ◽  
Fur Protein ◽  
Mutant Cells

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron–sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

scholarly journals Escherichia coli Harboring a Natural IncF Conjugative F Plasmid Develops Complex Mature Biofilms by Stimulating Synthesis of Colanic Acid and Curli

2008 ◽  
Vol 190 (22) ◽  
pp. 7479-7490 ◽  
Author(s):  
Thithiwat May ◽  
Satoshi Okabe
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Three Dimensional ◽  
F Plasmid ◽  
Form Complex ◽  
Global Regulators ◽  
E Coli ◽  
Colanic Acid ◽  
Regulatory Systems ◽  
Initial Deposition

ABSTRACT It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F+ cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.

scholarly journals Efficient Escherichia coli (E. coli) Trapping and Retrieval from Bodily Fluids via a Three-Dimensional (3D) Microbeads Stacked Nano-Device

2019 ◽  
Author(s):  
Xinye Chen ◽  
Abbi miller ◽  
Shengting Cao ◽  
Yu Gan ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Flow Rate ◽  
Magnetic Beads ◽  
Three Dimensional ◽  
E Coli ◽  
Bodily Fluids ◽  
3D Space ◽  
Fluidic Device ◽  
Computational Fluid Dynamics Cfd ◽  
On Chip

<div>A micro- and nano-fluidic device stacked with magnetic beads is developed to efficiently trap, concentrate, and retrieve Escherichia coli (E. coli) from bacteria suspension</div><div>and pig plasma. The small voids between the magnetic beads are used to physically isolate the bacteria in the device. We use computational fluid dynamics (CFD), 3D</div><div>tomography technology, and machine learning to probe and explain the bead stacking in a small 3D space with various flow rates. A combination of beads with different sizes is utilized to achieve a high capture efficiency of ~86% with a flow rate of 50 μL/min. Leveraging the high deformability of this device, the E. coli sample is retrieved from the designated bacteria suspension by applying a higher flow rate, followed by rapid magnetic separation. This unique function is also utilized to concentrate E. coli from the original bacteria suspension. An on-chip concentration</div><div>factor of ~11× is achieved by inputting 1,300 μL of the E. coli sample and then concentrating it in 100 μL buffer.</div><div>Importantly, this multiplexed, miniaturized, inexpensive, and transparent device is easy to fabricate and operate, making it ideal for pathogen separation in both laboratory and pointof- care (POC) settings.</div>

scholarly journals Efficient Escherichia coli (E. coli) Trapping and Retrieval from Bodily Fluids via a Three-Dimensional (3D) Microbeads Stacked Nano-Device

2019 ◽  
Author(s):  
Xinye Chen ◽  
Abbi miller ◽  
Shengting Cao ◽  
Yu Gan ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Flow Rate ◽  
Magnetic Beads ◽  
Three Dimensional ◽  
E Coli ◽  
Bodily Fluids ◽  
3D Space ◽  
Fluidic Device ◽  
Computational Fluid Dynamics Cfd ◽  
On Chip

<div>A micro- and nano-fluidic device stacked with magnetic beads is developed to efficiently trap, concentrate, and retrieve Escherichia coli (E. coli) from bacteria suspension</div><div>and pig plasma. The small voids between the magnetic beads are used to physically isolate the bacteria in the device. We use computational fluid dynamics (CFD), 3D</div><div>tomography technology, and machine learning to probe and explain the bead stacking in a small 3D space with various flow rates. A combination of beads with different sizes is utilized to achieve a high capture efficiency of ~86% with a flow rate of 50 μL/min. Leveraging the high deformability of this device, the E. coli sample is retrieved from the designated bacteria suspension by applying a higher flow rate, followed by rapid magnetic separation. This unique function is also utilized to concentrate E. coli from the original bacteria suspension. An on-chip concentration</div><div>factor of ~11× is achieved by inputting 1,300 μL of the E. coli sample and then concentrating it in 100 μL buffer.</div><div>Importantly, this multiplexed, miniaturized, inexpensive, and transparent device is easy to fabricate and operate, making it ideal for pathogen separation in both laboratory and pointof- care (POC) settings.</div>

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP

1988 ◽  
Vol 8 (3) ◽  
pp. 1206-1215
Author(s):  
C F Clarke ◽  
K Cheng ◽  
A B Frey ◽  
R Stein ◽  
P W Hinds ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Protein Complexes ◽  
P53 Protein ◽  
Immunoaffinity Column ◽  
E Coli ◽  
Shock Proteins

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.

scholarly journals Microtubular apparatus of melanophores: three dimensional organization

1978 ◽  
Vol 76 (3) ◽  
pp. 605-614 ◽  
Author(s):  
M Schliwa
Keyword(s):  
Three Dimensional ◽  
Cell Process ◽  
Serial Sections ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Cell Region ◽  
Thin Sectioning ◽  
A Cell ◽  
Central Apparatus ◽  
Pterophyllum Scalare

Microtubular organization in the melanophores of the angelfish, Pterophyllum scalare, has been studied by serial thin sectioning. The course of microtubules has been followed in sets of transverse serial sections taken from the centrosphere and a segment of a cell process, respectively. Microtubules arise from a prominent zone in the cell center, the central apparatus, which is composed of numerous, small, electron-dense aggregates. the number of these loosely distributed densities is highest in the center of the centrosphere, but they may also be found at its periphery. Microtubules insert into, or becomes part of, the dense material, or at least start in its vicinity. Dense aggregates may be separated from centrioles by several micrometers rather than only being closely associated with these organelles. At some distance from the organizing zone, most of the microtubules gradually assume a cortical arrangement, i.e., take a course within about 100 nm of the limiting membrane. Serial sections were used to trace all microtubules within a 6μm-long segment of a cell process. 94 percent of the microtubules observed in this segment run its entire length; it is conceivable, therefore that a considerable number of microtubules extend between the initiating site in the centrosphere and the outermost cell region. A three-dimensional model of the 6μm-long segment reveals that, despite changes in the cell process outline, microtubules maintain a strictly cortical arrangement which gives the impression of a microtubule "palisade" lining the cortex of the cell process. The features of the microtubular apparatus of angelfish melanophores are discussed in relation to factors controlling microtubule initiation and distribution.

scholarly journals RNA minihelices as model substrates for ATP/CTP:tRNA nucleotidyltransferase

1997 ◽  
Vol 327 (3) ◽  
pp. 847-851 ◽  
Author(s):  
Zengji LI ◽  
Yue SUN ◽  
L. David THURLOW
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Single Base ◽  
Three Dimensional Structure ◽  
E Coli ◽  
Base Identity

Twenty-one RNA minihelices, resembling the coaxially stacked acceptor- /T-stems and T-loop found along the top of a tRNA's three-dimensional structure, were synthesized and used as substrates for ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae. The sequence of nucleotides in the loop varied at positions corresponding to residues 56, 57 and 58 in the T-loop of a tRNA. All minihelices were substrates for both enzymes, and the identity of bases in the loop affected the interaction. In general, RNAs with purines in the loop were better substrates than those with pyrimidines, although no single base identity absolutely determined the effectiveness of the RNA as substrate. RNAs lacking bases near the 5ʹ-end were good substrates for the E. coli enzyme, but were poor substrates for that from yeast. The apparent Km values for selected minihelices were 2-3 times that for natural tRNA, and values for apparent Vmax were lowered 5-10-fold.

Effectiveness and Functional Mechanism of a Multicomponent Sanitizer against Biofilms Formed by Escherichia coli O157:H7 and Five Salmonella Serotypes Prevalent in the Meat Industry

2020 ◽  
Vol 83 (4) ◽  
pp. 568-575
Author(s):  
RONG WANG ◽  
YOU ZHOU ◽  
NORASAK KALCHAYANAND ◽  
DAYNA M. HARHAY ◽  
TOMMY L. WHEELER
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Three Dimensional ◽  
Short Exposure ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
Meat Processing ◽  
E Coli ◽  
Biofilm Removal ◽  
Biofilm Structure

ABSTRACT Biofilm formation by Escherichia coli O157:H7 and Salmonella enterica at meat processing plants poses a potential risk of meat product contamination. Many common sanitizers are unable to completely eradicate biofilms formed by these foodborne pathogens because of the three-dimensional biofilm structure and the presence of bacterial extracellular polymeric substances (EPSs). A novel multifaceted approach combining multiple chemical reagents with various functional mechanisms was used to enhance the effectiveness of biofilm control. We tested a multicomponent sanitizer consisting of a quaternary ammonium compound (QAC), hydrogen peroxide, and the accelerator diacetin for its effectiveness in inactivating and removing Escherichia coli O157:H7 and Salmonella enterica biofilms under meat processing conditions. E. coli O157:H7 and Salmonella biofilms on common contact surfaces were treated with 10, 20, or 100% concentrations of the multicomponent sanitizer solution for 10 min, 1 h, or 6 h, and log reductions in biofilm mass were measured. Scanning electron microscopy (SEM) was used to directly observe the effect of sanitizer treatment on biofilm removal and bacterial morphology. After treatment with the multicomponent sanitizer, viable E. coli O157:H7 and Salmonella biofilm cells were below the limit of detection, and the prevalence of both pathogens was low. After treatment with a QAC-based control sanitizer, surviving bacterial cells were countable, and pathogen prevalence was higher. SEM analysis of water-treated control samples revealed the three-dimensional biofilm structure with a strong EPS matrix connecting bacteria and the contact surface. Treatment with 20% multicomponent sanitizer for 10 min significantly reduced biofilm mass and weakened the EPS connection. The majority of the bacterial cells had altered morphology and compromised membrane integrity. Treatment with 100% multicomponent sanitizer for 10 min dissolved the EPS matrix, and no intact biofilm structure was observed; instead, scattered clusters of bacterial aggregates were detected, indicating the loss of cell viability and biofilm removal. These results indicate that the multicomponent sanitizer is effective, even after short exposure with dilute concentrations, against E. coli O157:H7 and S. enterica biofilms. HIGHLIGHTS

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