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Sample records for coli cell surface

  1. Cell surface growth in Escherichia coli: distribution of matrix protein.

    PubMed Central

    Begg, K J

    1978-01-01

    Autoradiography of cell envelope "ghosts" from Escherichia coli was used to demonstrate that newly synthesized molecules of "matrix" protein are inserted at random locations over the entire surface of the outer membrane and that, once inserted, these molecules are not thereafter conserved in any fixed spatial location. Images PMID:355219

  2. DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES BY IMMOBILIZED ESCHERICHIA COLI EXPRESSING ORGANOPHOSPHORUS HYDROLASE ON CELL SURFACE. (R823663)

    EPA Science Inventory

    An improved whole-cell technology for detoxifying organophosphate nerve agents was recently developed based on genetically engineered Escherichia coli with organophosphorus hydrolase anchored on the surface. This article reports the immobilization of these novel biocatalys...

  3. Surface cell density effects on Escherichia coli gene expression during cell attachment.

    PubMed

    Mauter, Meagan S; Mauter, Meagan; Fait, Aaron; Elimelech, Menachem; Herzberg, Moshe

    2013-06-18

    Escherichia coli attachment to a surface initiates a complex series of interconnected signaling and regulation pathways that promote biofilm formation and maturation. The present work investigates the effect of deposited cell density on E. coli cell physiology, metabolic activity, and gene expression in the initial stages of biofilm development. Deposited cell density is controlled by exploiting the relationship between ionic strength and bacterial attachment efficiency in a packed bed column. Distinct differences in cell transcriptome are analyzed by comparing sessile cultures at two different cell surface densities and differentiating ionic strength effects by analyzing planktonic cultures in parallel. Our results indicate that operons regulating trypotophan production and the galactitol phosphotransferase system (including dihydroxyacetone phosphate synthesis) are strongly affected by cell density on the surface. Additional transcriptome and metabolomic impacts of cell density on succinate, proline, and pyroglutamic acid systems are also reported. These results are consistent with the hypothesis that surface cell density plays a major role in sessile cell physiology, commencing with the first stage of biofilm formation. These findings improve our understanding of biofilm formation in natural and engineered environmental systems and will contribute to future work ranging from pathogen migration in the environment to control of biofouling on engineered surfaces.

  4. Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins

    PubMed Central

    2014-01-01

    In this work we report synthetic adhesins (SAs) enabling the rational design of the adhesion properties of E. coli. SAs have a modular structure comprising a stable β-domain for outer membrane anchoring and surface-exposed immunoglobulin domains with high affinity and specificity that can be selected from large repertoires. SAs are constitutively and stably expressed in an E. coli strain lacking a conserved set of natural adhesins, directing a robust, fast, and specific adhesion of bacteria to target antigenic surfaces and cells. We demonstrate the functionality of SAs in vivo, showing that, compared to wild type E. coli, lower doses of engineered E. coli are sufficient to colonize solid tumors expressing an antigen recognized by the SA. In addition, lower levels of engineered bacteria were found in non-target tissues. Therefore, SAs provide stable and specific adhesion capabilities to E. coli against target surfaces of interest for diverse applications using live bacteria. PMID:25045780

  5. Cell preparation methods influence Escherichia coli D21g surface chemistry and transport in saturated sand

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of cell preparation methods on the surface chemistry and deposition of Escherichia coli D21g was investigated over a range of ionic strength conditions. The cell preparation methods, which were considered, included filtration and centrifugation (at various speeds and for different duratio...

  6. Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

    PubMed Central

    Hiemstra, H; de Hoop, M J; Inouye, M; Witholt, B

    1986-01-01

    The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface. Images PMID:3531164

  7. Optimization of the whole-cell catalytic activity of recombinant Escherichia coli cells with surface-immobilized organophosphorus hydrolase.

    PubMed

    Zhang, Hongxing; Li, Qianqian; Ye, Ting; Zhang, Zhen; Li, Lin

    2013-04-01

    Previous studies have verified the feasibility of using Escherichia coli systems that display organophosphorous hydrolase (OPH) on the cell surface as whole-cell catalysts. However, the inefficient display of the enzyme on cell surfaces remains unaddressed. In the present study, multiple optimization experiments on full-length and truncated ice nucleation protein anchors, E. coli host cells, culture media, and culture conditions were performed to optimize whole-cell OPH enzymatic activity. The results show that apart from the dramatic effect of isopropyl-beta-D-thiogalactoside concentration and culture temperature, the coordination between the anchor protein, culture media, and host cells is essential for highly efficient OPH display. Under optimal conditions, namely, culturing in M9 medium, 20 degrees C induction temperature, 0.1 mmol l(-1) IPTG, and 100 micromol I(-1) Co2+, the engineered E. coli strain MB109-406 that expresses the fusion enzyme lnaK-N-OPH exhibited a whole-cell OPH activity of 0.62 U mg(-1) x cell d.wt. This result is much higher than that of several currently available OPH-displaying systems, which shows the potential of the current system for further large-scale industrial or environmental applications. PMID:24620599

  8. Separation of Escherichia coli Bacteria from Peripheral Blood Mononuclear Cells Using Standing Surface Acoustic Waves

    PubMed Central

    2013-01-01

    A microfluidic device was developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. In this device, two identical surface acoustic waves (SAWs) generated by interdigital transducers (IDTs) propagated toward a microchannel, which accordingly built up a standing surface acoustic wave (SSAW) field across the channel. A numerical model, coupling a piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SSAW-based particle manipulation. It was found that the pressure nodes across the channel were individual planes perpendicular to the solid substrate. In the separation experiments, two side sheath flows hydrodynamically focused the injected particle or cell mixtures into a very narrow stream along the centerline. Particles flowing through the SSAW field experienced an acoustic radiation force that highly depends on the particle properties. As a result, dissimilar particles or cells were laterally attracted toward the pressure nodes at different magnitudes, and were eventually switched to different outlets. Two types of fluorescent microspheres with different sizes were successfully separated using the developed device. In addition, Escherichia coli bacteria premixed in peripheral blood mononuclear cells (PBMCs) were also efficiently isolated using the SSAW-base separation technique. Flow cytometric analysis on the collected samples found that the purity of separated E. coli bacteria was 95.65%. PMID:23968497

  9. Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus.

    PubMed

    Pyo, Hyun-Mi; Kim, In-Joong; Kim, Seong-Hee; Kim, Hyun-Soo; Cho, Soo-Dong; Cho, In-Soo; Hyun, Bang-Hun

    2009-03-23

    Porcine epidemic diarrhea virus (PEDV) is a causative agent of severe diarrhea which leads to death in piglets. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease in Korea. In this study, we developed a prophylactic candidate using single-chain Fvs to prevent the PEDV infection. ScFvs of mouse monoclonal antibody which was verified to neutralize PEDV was expressed in Escherichia coli expression system. After the confirmation of PEDV neutralizing activity of purified recombinant scFvs by VN test, scFvs were expressed on the surface of E. coli cells. The signal sequence and autotransporter beta domain of protease IgA (IgAP) of Neisseria gonorrhoeae were introduced to endow scFvs with the direction to the cell surface and the support as a transmembrane domain. 5x10(6)CFU of E. coli expressing scFvs against PEDV showed promising result of 94% foci reduction compared to wild type E. coli. This result demonstrated that E. coli expressing scFvs on the cell surface retained functional potency of parent antibody and therefore blocked PEDV infection into target cells in vitro. This in vitro assay result proposes the perspective of recombinant E. coli cells expressing scFvs as a novel prophylactic against PEDV infection. PMID:19428826

  10. Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior

    PubMed Central

    Gustavsson, Martin; Hörnström, David; Lundh, Susanna; Belotserkovsky, Jaroslav; Larsson, Gen

    2016-01-01

    Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants. PMID:27782179

  11. Interaction force measurement between E. coli cells and nanoparticles immobilized surfaces by using AFM

    SciTech Connect

    Zhang, Wen; Chen, Yongsheng

    2011-01-01

    To better understand environmental behaviors of nanoparticles (NPs), we used the atomic force microscopy (AFM) to measure interaction forces between E. coli cells and NPs immobilized on surfaces in an aqueous environment. The results showed that adhesion force strength was significantly influenced by particle size for both hematite ( -Fe2 O3 ) and corundum ( -Al2 O3 ) NPs whereas the effect on the repulsive force was not observed. The adhesion force decreased from 6.3 0.7 nN to 0.8 0.4 nN as hematite NPs increased from 26 nm to 98 nm in diameter. Corundum NPs exhibited a similar dependence of adhesion force on particle size. The Johnson Kendall Roberts (JKR) model was employed to estimate the contact area between E. coli cells and NPs, and based on the JKR model a new model that considers local effective contact area was developed. The prediction of the new model matched the size dependence of adhesion force in experimental results. Size effects on adhesion forces may originate from the difference in local effective contact areas as supported by our model. These findings provide fundamental information for interpreting the environmental behaviors and biological interactions of NPs, which barely have been addressed.

  12. Isolation of a potential anchoring motif based on proteome analysis of Escherichia coli and its use for cell surface display.

    PubMed

    Yim, Sung Sun; An, Seul Ji; Han, Mee-Jung; Choi, Jae Woong; Jeong, Ki Jun

    2013-06-01

    For bacterial cell surface display, the target protein needs to be linked to an anchoring motif, and it is essential to choose an appropriate anchoring motif for efficient and stable display of the protein on the cell surface. To isolate a potential anchoring motif that would allow a stable and enhanced display of target proteins on the surface of an Escherichia coli host, we analyzed the outer membrane proteome of E. coli. On the basis of this proteomic analysis, the outer membrane protein X (OmpX), which has a small, monomeric β-barrel structure and is highly expressed, was selected as a potential anchoring motif. The role of OmpX as an anchoring motif for cell surface display was demonstrated using three important industrial enzymes: endoxylanase, lipase, and alkaline phosphatase. Two different positions (Lys(122), Val(160)) in the extracellular loops of OmpX were examined for C-terminal fusion, and the biological activities and localization of the displayed enzymes were analyzed. All three enzymes examined were efficiently displayed on the E. coli cell surface with high activity. These results reveal that the use of OmpX as an anchoring motif is an efficient method to display functional enzymes on the surface of an E. coli host.

  13. How Escherichia coli lands and forms cell clusters on a surface: a new role of surface topography

    PubMed Central

    Gu, Huan; Chen, Aaron; Song, Xinran; Brasch, Megan E.; Henderson, James H.; Ren, Dacheng

    2016-01-01

    Bacterial response to surface topography during biofilm formation was studied using 5 μm tall line patterns of poly (dimethylsiloxane) (PDMS). Escherichia coli cells attached on top of protruding line patterns were found to align more perpendicularly to the orientation of line patterns when the pattern narrowed. Consistently, cell cluster formation per unit area on 5 μm wide line patterns was reduced by 14-fold compared to flat PDMS. Contrasting the reduced colony formation, cells attached on narrow patterns were longer and had higher transcriptional activities, suggesting that such unfavorable topography may present a stress to attached cells. Results of mutant studies indicate that flagellar motility is involved in the observed preference in cell orientation on narrow patterns, which was corroborated by the changes in cell rotation pattern before settling on different surface topographies. These findings led to a set of new design principles for creating antifouling topographies, which was validated using 10 μm tall hexagonal patterns. PMID:27412365

  14. How Escherichia coli lands and forms cell clusters on a surface: a new role of surface topography.

    PubMed

    Gu, Huan; Chen, Aaron; Song, Xinran; Brasch, Megan E; Henderson, James H; Ren, Dacheng

    2016-01-01

    Bacterial response to surface topography during biofilm formation was studied using 5 μm tall line patterns of poly (dimethylsiloxane) (PDMS). Escherichia coli cells attached on top of protruding line patterns were found to align more perpendicularly to the orientation of line patterns when the pattern narrowed. Consistently, cell cluster formation per unit area on 5 μm wide line patterns was reduced by 14-fold compared to flat PDMS. Contrasting the reduced colony formation, cells attached on narrow patterns were longer and had higher transcriptional activities, suggesting that such unfavorable topography may present a stress to attached cells. Results of mutant studies indicate that flagellar motility is involved in the observed preference in cell orientation on narrow patterns, which was corroborated by the changes in cell rotation pattern before settling on different surface topographies. These findings led to a set of new design principles for creating antifouling topographies, which was validated using 10 μm tall hexagonal patterns. PMID:27412365

  15. Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface.

    PubMed Central

    Gómez-Miguel, M J; Moriyón, I; López, J

    1987-01-01

    In an enzyme-linked immunosorbent assay (ELISA), purified Brucella abortus and Escherichia coli peptidoglycan-linked lipoproteins gave a strong cross-reaction with sera from rabbits hyperimmunized with the heterologous lipoprotein. When smooth E. coli cells were used as ELISA antigens, the immunological cross-reaction was not observed unless the cells were treated to remove lipopolysaccharide and other outer membrane components. In contrast, intact cells from smooth strains of B. abortus and Brucella melitensis bound anti-lipoprotein immunoglobulin G, and the controls performed by ELISA showed that this reaction was not due to antibodies to the lipopolysaccharide, group 3 outer membrane proteins, or porins. Electron microscopy of cells labeled with antilipoprotein serum and protein A-colloidal gold showed specific labeling of smooth cells from both B. abortus and B. melitensis, even though unspecific labeling by nonimmune serum was observed with rough B. abortus. These results confirm the close similarity between E. coli and Brucella peptidoglycan-linked lipoproteins and show that, in contrast to E. coli, the lipoprotein of B. abortus and B. melitensis is partially exposed on the surface of smooth cells. Images PMID:2432014

  16. Cell surface display of poliovirus receptor on Escherichia coli, a novel method for concentrating viral particles in water.

    PubMed

    Abbaszadegan, Morteza; Alum, Absar; Abbaszadegan, Hamed; Stout, Valerie

    2011-08-01

    The lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface of Escherichia coli cells by using the ice nucleation protein (INP) gene. The hPVR gene was ligated to the 3' end of the INP gene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane of E. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineered E. coli cells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.

  17. Enhanced colonization of rpoS-deficient Escherichia coli cells on solid surfaces by reinforced csgA gene expression.

    PubMed

    Nguyen, Minh Hong; Ojima, Yoshihiro; Taya, Masahito

    2014-01-01

    Scanning electron microscopy revealed that the rpoS-deficient cells of E. coli K-12 BW25113 (ΔrpoS) increased the number of flagella on the cell surfaces. However, the quantitative analysis of cell colonization showed that the increased number of flagella on ΔrpoS cell surfaces did not cause the enhancement of cell colonization on the surfaces of polyvinyl chloride (PVC), polypropylene (PP) and polystyrene (PS) after 24 h of incubation at 37℃. To facilitate the enhanced expression of curli, the csgA gene was introduced into the ΔrpoS cells. The transformed cells rich in flagella and curli on the cell surfaces were found to make colonies 2-3 times larger than both the wild type and ΔrpoS cells on the PVC, PP and PS surfaces at 37℃. It was thus verified that the reinforcement of csgA gene in the ΔrpoS cells induced the enhanced colonization on the solid surfaces with the increased flagellum and curli expressions. PMID:25252647

  18. The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

    PubMed Central

    Lai, C F; Gong, S C; Esteban, M

    1991-01-01

    The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. Images PMID:1985213

  19. Creation of a Cellooligosaccharide-Assimilating Escherichia coli Strain by Displaying Active Beta-Glucosidase on the Cell Surface via a Novel Anchor Protein ▿

    PubMed Central

    Tanaka, Tsutomu; Kawabata, Hitomi; Ogino, Chiaki; Kondo, Akihiko

    2011-01-01

    We demonstrated direct assimilation of cellooligosaccharide using Escherichia coli displaying beta-glucosidase (BGL). BGL from Thermobifida fusca YX (Tfu0937) was displayed on the E. coli cell surface using a novel anchor protein named Blc. This strain was grown successfully on 0.2% cellobiose, and the optical density at 600 nm (OD600) was 1.05 after 20 h. PMID:21742905

  20. A comparison of the surface nanostructure from two different types of gram-negative cells: Escherichia coli and Rhodobacter sphaeroides.

    PubMed

    Oestreicher, Zachery; Taoka, Azuma; Fukumori, Yoshihiro

    2015-05-01

    Bacteria have been studied using different microscopy methods for many years. Recently, the developments of high-speed atomic force microscopy have opened the doors to study bacteria in new ways due to the fact that it uses much less force on the sample while imaging. This makes the high-speed atomic force microscope an indispensable technique for imaging the surface of living bacterial cells because it allows for the high-resolution visualization of surface proteins in their natural condition without disrupting the cell or the activity of the proteins. Previous work examining living cells of Magnetospirillum magneticum AMB-1 demonstrated that the surface of these bacteria was covered with a net-like structure that is mainly composed of porin molecules. However, it was unclear whether or not this feature was unique to other living bacteria. In this study we used the high-speed atomic force microscope to examine the surface of living cells of Escherichia coli and Rhodobacter sphaeroides to compare their structure with that of M. magneticum. Our research clearly demonstrated that both of these types of cells have an outer surface that is covered in a network of nanometer-sized holes similar to M. magneticum. The diameter of the holes was 8.0±1.5 nm for E. coli and 6.6±1.1 nm for R. sphaeroides. The results in this paper confirm that this type of outer surface structure exists in other types of bacteria and it is not unique to Magnetospirillum.

  1. Development of an autofluorescent whole-cell biocatalyst by displaying dual functional moieties on Escherichia coli cell surfaces and construction of a coculture with organophosphate-mineralizing activity .

    PubMed

    Yang, Chao; Zhu, Yaran; Yang, Jijian; Liu, Zheng; Qiao, Chuanling; Mulchandani, Ashok; Chen, Wilfred

    2008-12-01

    Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites. PMID:18952884

  2. Effects of Lipopolysaccharide Biosynthesis Mutations on K1 Polysaccharide Association with the Escherichia coli Cell Surface

    PubMed Central

    Jiménez, Natalia; Senchenkova, Sofya N.; Knirel, Yuriy A.; Pieretti, Giuseppina; Corsaro, Maria M.; Aquilini, Eleonora; Regué, Miguel; Merino, Susana

    2012-01-01

    The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determined in a series of in-frame nonpolar core biosynthetic mutants from Escherichia coli KT1094 (K1, R1 core lipopolysaccharide [LPS] type) for which the major core oligosaccharide structures were determined. Cell-bound K1 capsule was absent from mutants devoid of phosphoryl modifications on l-glycero-d-manno-heptose residues (HepI and HepII) of the inner-core LPS and reduced in mutants devoid of phosphoryl modification on HepII or devoid of HepIII. In contrast, in all of the mutants, K1 polysaccharide was found in culture supernatants. These results were confirmed by using a mutant with a deletion spanning from the hldD to waaQ genes of the waa gene cluster to which individual genes were reintroduced. A nuclear magnetic resonance (NMR) analysis of core LPS from HepIII-deficient mutants showed an alteration in the pattern of phosphoryl modifications. A cell extract containing both K1 capsule polysaccharide and LPS obtained from an O-antigen-deficient mutant could be resolved into K1 polysaccharide and core LPS by column chromatography only when EDTA and deoxycholate (DOC) buffer were used. These results suggest that the K1 polysaccharide remains cell associated by ionically interacting with the phosphate-negative charges of the core LPS. PMID:22522903

  3. Inactivation of stressed Escherichia coli O157:H7 cells on the surfaces of rocket salad leaves by chlorine and peroxyacetic acid.

    PubMed

    Al-Nabulsi, Anas A; Osaili, Tareq M; Obaidat, Heba M; Shaker, Reyad R; Awaisheh, Saddam S; Holley, Richard A

    2014-01-01

    Because Escherichia coli O157:H7 has been frequently associated with many foodborne outbreaks caused by consumption of leafy greens (lettuce, spinach, and celery), this study investigated the ability of deionized water, chlorine, and peroxyacetic acid to detach or inactivate stressed and unstressed cells of E. coli O157:H7 contaminating the surfaces of rocket salad leaves. E. coli O157:H7 cells stressed by acid, cold, starvation, or NaCl exposure, as well as unstressed cells, were inoculated on the surfaces of rocket salad leaves at 4°C. The effectiveness of two sanitizers (200 ppm of chlorine and 80 ppm of peroxyacetic acid) and deionized water for decontaminating the leaves treated with stressed and unstressed E. coli O157:H7 were evaluated during storage at 10 or 25°C for 0.5, 1, 3, and 7 days. It was found that washing with 80 ppm of peroxyacetic acid was more effective and reduced unstressed and stressed cells of E. coli O157:H7 by about 1 log CFU per leaf on the leaves. There was no apparent difference in the ability of stressed and unstressed cells to survive surface disinfection with the tested agents. Treatments to reduce viable E. coli O157:H7 cells on rocket leaves stored at 25°C were more effective than when used on those stored at 10°C. Washing with peroxyacetic acid or chlorine solution did not ensure the safety of rocket leaves, but such treatments could reduce the likelihood of water-mediated transfer of E. coli O157:H7 during washing and subsequent processing.

  4. Inactivation of stressed Escherichia coli O157:H7 cells on the surfaces of rocket salad leaves by chlorine and peroxyacetic acid.

    PubMed

    Al-Nabulsi, Anas A; Osaili, Tareq M; Obaidat, Heba M; Shaker, Reyad R; Awaisheh, Saddam S; Holley, Richard A

    2014-01-01

    Because Escherichia coli O157:H7 has been frequently associated with many foodborne outbreaks caused by consumption of leafy greens (lettuce, spinach, and celery), this study investigated the ability of deionized water, chlorine, and peroxyacetic acid to detach or inactivate stressed and unstressed cells of E. coli O157:H7 contaminating the surfaces of rocket salad leaves. E. coli O157:H7 cells stressed by acid, cold, starvation, or NaCl exposure, as well as unstressed cells, were inoculated on the surfaces of rocket salad leaves at 4°C. The effectiveness of two sanitizers (200 ppm of chlorine and 80 ppm of peroxyacetic acid) and deionized water for decontaminating the leaves treated with stressed and unstressed E. coli O157:H7 were evaluated during storage at 10 or 25°C for 0.5, 1, 3, and 7 days. It was found that washing with 80 ppm of peroxyacetic acid was more effective and reduced unstressed and stressed cells of E. coli O157:H7 by about 1 log CFU per leaf on the leaves. There was no apparent difference in the ability of stressed and unstressed cells to survive surface disinfection with the tested agents. Treatments to reduce viable E. coli O157:H7 cells on rocket leaves stored at 25°C were more effective than when used on those stored at 10°C. Washing with peroxyacetic acid or chlorine solution did not ensure the safety of rocket leaves, but such treatments could reduce the likelihood of water-mediated transfer of E. coli O157:H7 during washing and subsequent processing. PMID:24405996

  5. Development of a whole-cell biocatalyst/biosensor by display of multiple heterologous proteins on the Escherichia coli cell surface for the detoxification and detection of organophosphates.

    PubMed

    Liu, Ruihua; Yang, Chao; Xu, Yingming; Xu, Ping; Jiang, Hong; Qiao, Chuanling

    2013-08-14

    This paper reports the codisplay of organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH)-green fluorescent protein (GFP) fusion on the cell surface of Escherichia coli using the truncated ice nucleation protein (INPNC) and Lpp-OmpA as the anchoring motifs. The surface localization of both OPH and MPH-GFP was demonstrated by cell fractionation, Western blot analysis, protease accessibility experiment, and immunofluorescence microscopy. Anchorage of the foreign proteins on the outer membrane neither inhibits cell growth nor affects cell viability. The recombinant strain can be used as a whole-cell biocatalyst and showed a broader substrate range than strains expressing either OPH or MPH. A mixture of six organophosphorus pesticides (OPs) (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes the recombinant strain a promising candidate for detoxification of OPs. The fluorescence of surface-displayed GFP is very sensitive to environmental pH change. Because hydrolysis of OPs by OPH or MPH generates protons, the recombinant E. coli could be used as a whole-cell biosensor for the rapid detection of OPs by evaluating fluorescence changes as a function of OP concentrations. PMID:23875606

  6. Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion.

    PubMed

    Narita, Junya; Okano, Kenji; Tateno, Toshihiro; Tanino, Takanori; Sewaki, Tomomitsu; Sung, Moon-Hee; Fukuda, Hideki; Kondo, Akihiko

    2006-05-01

    We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.

  7. Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis.

    PubMed

    van Bloois, Edwin; Winter, Remko T; Janssen, Dick B; Fraaije, Marco W

    2009-06-01

    Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes. PMID:19224207

  8. Going for baroque at the Escherichia coli K1 cell surface.

    PubMed

    King, Michael R; Steenbergen, Susan M; Vimr, Eric R

    2007-05-01

    Phase variation is usually thought of as the stochastic switching between alternatively expressed ('on') and unexpressed ('off') phenotypic states. However, coupling synthesis of a monotonous homopolysaccharide to a mechanism of random but incomplete chemical modification produces almost infinite structural variation. Potentially limitless variability implies that evolution can produce highly ornate or extravagant flourishes reminiscent of the baroque style. Here, we describe an analysis of capsular polysialic acid form variation in Escherichia coli K1, demonstrating that the large number of variant structures is controlled by a single contingency locus. The mechanism for generating maximum structural diversity from maximal genetic parsimony is conferred by a simple translational switch carried on a K1-specific prophage. PMID:17418577

  9. Single-cell Characterization of Autotransporter-mediated Escherichia coli Surface Display of Disulfide Bond-containing Proteins*

    PubMed Central

    Ramesh, Balakrishnan; Sendra, Victor G; Cirino, Patrick C; Varadarajan, Navin

    2012-01-01

    Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal β-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal β-barrel and the threaded α-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering. PMID:23019324

  10. Identification of Cell Surface-Exposed Proteins Involved in the Fimbria-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

    PubMed Central

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P.; Ruiz-Perez, Fernando

    2014-01-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC. PMID:24516112

  11. Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-04-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

  12. Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Navarro-Garcia, Fernando; Nava-Acosta, Raul; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-04-01

    Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC. PMID:24516112

  13. Effects of cell surface characteristics and manure-application practices on Escherichia coli populations in the subsurface: A three-farm study

    NASA Astrophysics Data System (ADS)

    Salvucci, A. E.; Elton, M.; Siler, J. D.; Zhang, W.; Richards, B. K.; Geohring, L. D.; Warnick, L. D.; Hay, A. G.; Steenhuis, T.

    2010-12-01

    The introduction of microbial pathogens into the environment from untreated manure represents a threat to water quality and human health. Thus, understanding the effect of manure management strategies is imperative to effectively mitigate the inadvertent release of pathogens, particularly in subsurface environments where they can be transported through macropores to the groundwater or through agricultural tile line to open water bodies. The production of cell-surface biomolecules is also suspected to play an important role in the environmental survival and transport of enterobacterial pathogens. This study collected Escherichia coli samples from three dairy farms with artificial tile drainage systems and active manure spreading in the Central New York region over a three-month period. Sampling targeted four potential source locations on each farm: (i) cow housing, (ii) manure storage facilities, (iii) field soil, and (iv) subsurface drainage effluent. Over 2800 E. coli isolates were recovered and consequently analyzed for the cell surface components, cellulose and curli, traits associated with increased environmental survival, altered transport and pathogenicity. The E. coli isolates from locations i-iii displayed highly variable curli and cellulose-producing communities, while isolates collected from subsurface runoff on each farm had stable curli and cellulose production communities over all sampling dates. Furthermore, the method of manure application to the fields influenced the population characteristics found in drainage effluent isolates. Incorporation of manure into the soil was correlated to isolate populations largely deficient of curli and cellulose; whereas farms that only surface-applied manure were correlated to isolate populations of high curli and cellulose production. The production of curli and cellulose has previously been shown to be a response to environmental stress on the cell. Therefore, incorporation of manure directly into the soil appears

  14. Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride.

    PubMed

    Nakayama, Motokazu; Shigemune, Naofumi; Tsugukuni, Takashi; Tokuda, Hajime; Miyamoto, Takahisa

    2011-07-01

    We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy.

  15. Surface Characteristics and Adhesion Behavior of Escherichia coli O157:H7: Role of Extracellular Macromolecules

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surface macromolecule cleavage experiments were conducted on enterohaemorrhagic Escherichia coli O157:H7 cells to investigate the influence of these macromolecules on cell surface properties. Electrophoretic mobility, hydrophobicity, and titration experiments were carried out on proteinase K treate...

  16. The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling.

    PubMed

    Ray, Rupa; de Ridder, Gustaaf G; Eu, Jerry P; Paton, Adrienne W; Paton, James C; Pizzo, Salvatore V

    2012-09-21

    GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.

  17. A bioluminescence ATP assay for estimating surface hydrophobicity and membrane damage of Escherichia coli cells treated with pulsed electric fields

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pulse Electric Field (PEF) treatments, a non-thermal process have been reported to injure and inactivate bacteria in liquid foods. However, the effect of this treatment on bacterial cell surface charge and hydrophobicity has not been investigated. Apple juice (AJ, pH 3.8) purchased from a wholesale ...

  18. Inactivation of Escherichia coli planktonic cells by multi-walled carbon nanotubes in suspensions: Effect of surface functionalization coupled with medium nutrition level.

    PubMed

    Chi, Mu-Fan; Wu, Wei-Ling; Du, Yuchin; Chin, Ching-Ju M; Lin, Chu-Ching

    2016-11-15

    While earlier studies have identified the antibacterial activity of carbon nanotubes (CNTs) and proposed that cell membrane damage by direct contact with CNTs is likely the main toxicity mechanism, the relative importance of chemical versus physical properties of CNTs in controlling their bacterial cytotoxicity is understudied. Given that CNT is commonly modified via acid treatment to enhance its dispersivity and surface chemistry, in this study commercially available multi-walled carbon nanotubes (MWCNTs) with high purity were processed carefully by acid reflux, resulting in differences in surface charge of MWCNTs without altering their physical properties. The surface condition of MWCNTs was also modified by adsorption of organic matter to compare bacterial toxicity of functionalized and non-functionalized MWCNTs in suspensions. Results show that although overall electrostatic repulsion and steric obstruction resulted from surface modifications led to elevated dispersivity of MWCNTs and mitigated toxicity on planktonic Escherichia coli cultures, no correlation between the dispersivity and bacterial toxicity of MWCNTs was observed, suggesting that dispersity alone may not be a proper index to estimate the CNT antibacterial effect on planktonic cells in the aqueous phase. In addition, viability recovery of MWCNT-treated cells was observed to be nutrition level-dependent, implying that availability of proper nutrients may be another important factor to be considered when assessing the ecotoxicity of CNTs in the aquatic system. PMID:27450343

  19. Changes in the biocide susceptibility of Staphylococcus epidermidis and Escherichia coli cells associated with rapid attachment to plastic surfaces.

    PubMed

    Das, J R; Bhakoo, M; Jones, M V; Gilbert, P

    1998-05-01

    Differences in opacity between wells of a microtitre plate containing different volumes of inoculated growth medium reflected planktonic growth without any contribution from cells attached at the well surface. Simple algebra and a knowledge of the dependence of optical density upon sample path length (volume) for suspensions of differing cell density enables the generation of growth curves for attached populations (biofilms). In this manner, minimum inhibitory concentrations (MICs) were determined at various stages of growth (0-20 h), both for cells growing attached to the bases of the plate wells and, simultaneously, for cells growing in suspension above them. Biocides included cetrimide, polyhexamethylene biguanide, peracetic acid, phenoxyethanol and chloroxylenol. Results, expressed as planktonic:biofilm MIC ratios, showed susceptibility to change, not only as a function of attachment and biofilm formation, but also with respect to the nature of the chemical agent. In some instances, changes in susceptibility greater than twofold occurred immediately on attachment and could occur in the presence of biocide concentrations which exceeded the MIC.

  20. Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response.

    PubMed

    Lacey, Melissa M; Partridge, Jonathan D; Green, Jeffrey

    2010-09-01

    The Escherichia coli K-12 yfgF gene encodes a protein with domains associated with cyclic di-GMP signalling: GGDEF (associated with diguanylate cyclase activity) and EAL (associated with cyclic di-GMP phosphodiesterase activity). Here, it is shown that yfgF is expressed under anaerobic conditions from a class II FNR (regulator of fumarate and nitrate reduction)-dependent promoter. Anaerobic expression of yfgF is greatest in stationary phase, and in cultures grown at 28 degrees C, suggesting that low growth rates promote yfgF expression. Mutation of yfgF resulted in altered cell surface properties and enhanced sensitivity when anaerobic cultures were exposed to peroxides. The purified YfgF GGDEF-EAL (YfgF(GE)) and EAL (YfgF(E)) domains possessed cyclic di-GMP-specific phosphodiesterase activity, but lacked diguanylate cyclase activity. However, the catalytically inactive GGDEF domain was required for YfgF(GE) dimerization and enhanced cyclic di-GMP phosphodiesterase activity in the presence of physiological concentrations of Mg(2+). The cyclic di-GMP phosphodiesterase activity of YfgF(GE) and YfgF(E) was inhibited by the product of the reaction, 5'-phosphoguanylyl-(3'-5')-guanosine (pGpG). Thus, it is shown that the yfgF gene encodes an anaerobic cyclic di-GMP phosphodiesterase that is involved in remodelling the cell surface of E. coli K-12 and in the response to peroxide shock, with implications for integrating three global regulatory networks, i.e. oxygen regulation, cyclic di-GMP signalling and the oxidative stress response.

  1. Interactions of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) recovered from bovine hide and carcass with human cells and abiotic surfaces.

    PubMed

    Matheus-Guimarães, Cecilia; Gonçalves, Evanilde Maria; Cabilio Guth, Beatriz E

    2014-03-01

    Different structures related to biofilm formation by Shiga toxin-producing Escherichia coli (STEC), particularly O157 strains, have been described, but there are few data regarding their involvement in non-O157 strains. The aim of this study was to determine the ability of 14 O157 and 8 non-O157 strains isolated from bovine hide and carcass to interact with biotic and abiotic surfaces and also to evaluate the role of different adhesins. Biofilm formation assays showed that four O157 and two non-O157 strains were able to adhere to glass, and that only one O157 strain adhered to polystyrene. Reverse transcriptase-polymerase chain reaction was carried out using biofilm-forming strains to determine the expression of antigen 43 (Ag43), curli, type 1 fimbriae, STEC autotransporter contributing to biofilm formation (Sab), calcium-binding antigen 43 homologue (Cah), and autotransporter protein of enterohemorrhagic E. coli (EhaA). Most of these structures were expressed under biofilm conditions. However, the lack of Ag43 in one non-O157 strain, as well as Cah and EhaA in two O157 strains, suggests that other adhesins are involved in biofilm formation in these strains. Despite the fact that adherence to HeLa cells was detected in 20 strains (91%), it was not possible to correlate biofilm formation with adherence patterns. Invasiveness in T84 and Caco-2 cells was observed in four and three O157 strains, respectively. Altogether, we showed that there are different sets of genes involved in the interactions of STEC with biotic and abiotic surfaces. Interestingly, one O157 strain that was able to form biofilm on both glass and polystyrene also adhered to and invaded human cells, indicating an important route for its persistence in the environment and interaction with the host. Additionally, the ability of non-O157 strains not carrying the LEE pathogenicity island to form biofilm highlights an industrial and health problem that cannot be neglected.

  2. Impact of cranberry on Escherichia coli cellular surface characteristics

    SciTech Connect

    Johnson, Brandy J.; Malanoski, Anthony P.; Ligler, Frances S.

    2008-12-19

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  3. Impact of cranberry on Escherichia coli cellular surface characteristics.

    PubMed

    Johnson, Brandy J; Lin, Baochuan; Dinderman, Michael A; Rubin, Robert A; Malanoski, Anthony P; Ligler, Frances S

    2008-12-19

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  4. Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine Moiety

    PubMed Central

    Park, Joseph P.; Choi, Min-Jung; Kim, Se Hun

    2014-01-01

    Mussels attach to virtually all types of inorganic and organic surfaces in aqueous environments, and catecholamines composed of 3,4-dihydroxy-l-phenylalanine (DOPA), lysine, and histidine in mussel adhesive proteins play a key role in the robust adhesion. DOPA is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we displayed the adhesive moiety of DOPA-histidine on Escherichia coli surfaces using outer membrane protein W as an anchoring motif for the first time. Localization of catecholamines on the cell surface was confirmed by Western blot and immunofluorescence microscopy. Furthermore, cell-to-cell cohesion (i.e., cellular aggregation) induced by the displayed catecholamine and synthesis of gold nanoparticles on the cell surface support functional display of adhesive catecholamines. The engineered E. coli exhibited significant adhesion onto various material surfaces, including silica and glass microparticles, gold, titanium, silicon, poly(ethylene terephthalate), poly(urethane), and poly(dimethylsiloxane). The uniqueness of this approach utilizing the engineered sticky E. coli is that no chemistry for cell attachment are necessary, and the ability of spontaneous E. coli attachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine yielded sticky E. coli that can be used as a new type of engineered microbe for various emerging fields, such as whole living cell attachment on versatile material surfaces, cell-to-cell communication systems, and many others. PMID:24123747

  5. Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

    PubMed Central

    Ko, Hyeok-Jin; Park, Eunhye; Song, Joseph; Yang, Taek Ho; Lee, Hee Jong; Kim, Kyoung Heon

    2012-01-01

    Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104 molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli. PMID:22344647

  6. Bacterial cell surface hydrophobicity properties in the mediation of in vitro adhesion by the rabbit enteric pathogen Escherichia coli strain RDEC-1.

    PubMed Central

    Drumm, B; Neumann, A W; Policova, Z; Sherman, P M

    1989-01-01

    The role of hydrophobicity in the attachment of enteropathogens to gastrointestinal mucosa is controversial. In vitro binding of Escherichia coli RDEC-1 to rabbit intestine is dependent on the expression of pili. We examined in vitro adherence of piliated RDEC-1 after altering either the hydrophobicity of the organisms, the hydrophobicity of the substrate for attachment, or the surface tension of the suspending liquid. Hydrophobicity of RDEC-1 was determined using four complementary methods. In each assay piliated RDEC-1 demonstrated relatively more hydrophobic properties compared with both organisms grown to suppress pilus expression and a mutant that cannot express mannose-resistant pili. When piliated RDEC-1 were pretreated with tetramethyl urea to disrupt hydrophobic bonds surface hydrophobicity decreased. Concurrently, bacterial adherence to rabbit ileal microvillus membranes, mucus and mucin was reduced. Binding of piliated organisms to hydrophobic surfaces was significantly higher compared to both nonpiliated bacteria and the adherence of piliated RDEC-1 to relatively hydrophilic surfaces. Addition of propanol reduced the surface tension of the suspending liquid, and decreased adhesion of piliated RDEC-1 to polystyrene by 80%. Conversely, adherence of piliated organisms to a hydrophilic surface increased 12-fold after lowering the surface tension of the suspending liquid. We conclude that hydrophobic properties have a role in mediating in vitro adherence of this E. coli enteric pathogen. Images PMID:2572606

  7. The surface protease ompT serves as Escherichia coli K1 adhesin in binding to human brain micro vascular endothelial cells.

    PubMed

    Wan, Lei; Guo, Yan; Hui, Chang-Ye; Liu, Xiao-Lu; Zhang, Wen-Bing; Cao, Hong; Cao, Hong

    2014-05-01

    Escherichia coli (E. coli) K1 is the most common bacteria that cause meningitis in the neonatal period. But it's not entirely clear about how E. coli crosses the blood-brain barrier. The features of the ompT deletion in meningitic E. coli infection were texted in vitro. In comparison with the parent strain, the isogenic ompT deletion mutant was significantly less adhesive to human brain microvascular endothelial cells (HBMEC). The adhesion-deficient phenotype of the mutant was restored to the level of the wild-type by complementing with low-level OmpT expression plasmid. Interestingly, the adhesion was enhanced by point mutation at the OmpT proposed catalytic residue D85. Compared with the poor adhesive activity of bovine serum albumin-coated fluorescent beads, recombinant OmpT or catalytically inactive variant of OmpT-coated beads bound to HBMEC monolayer effectively. Our study suggests that OmpT is important for bacterial adhesion while entering into central nervous system, and the adhesion does not involve in the proteolytic activity of OmpT.

  8. Identification of Coli Surface Antigen 23, a Novel Adhesin of Enterotoxigenic Escherichia coli

    PubMed Central

    Del Canto, Felipe; Botkin, Douglas J.; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P.; Levine, Myron M.; Stine, O. Colin; Pop, Mihai

    2012-01-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains. PMID:22645287

  9. Identification of Coli Surface Antigen 23, a novel adhesin of enterotoxigenic Escherichia coli.

    PubMed

    Del Canto, Felipe; Botkin, Douglas J; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P; Levine, Myron M; Stine, O Colin; Pop, Mihai; Torres, Alfredo G; Vidal, Roberto

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains. PMID:22645287

  10. Identification of Coli Surface Antigen 23, a novel adhesin of enterotoxigenic Escherichia coli.

    PubMed

    Del Canto, Felipe; Botkin, Douglas J; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P; Levine, Myron M; Stine, O Colin; Pop, Mihai; Torres, Alfredo G; Vidal, Roberto

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.

  11. A Whole-Cell Surface Plasmon Resonance Sensor Based on a Leucine Auxotroph of Escherichia coli Displaying a Gold-Binding Protein: Usefulness for Diagnosis of Maple Syrup Urine Disease.

    PubMed

    Woo, Min-Ah; Park, Jung Hun; Cho, Daeyeon; Sim, Sang Jun; Kim, Moon Il; Park, Hyun Gyu

    2016-03-01

    We developed a whole-cell surface plasmon resonance (SPR) sensor based on a leucine auxotroph of Escherichia coli displaying a gold-binding protein (GBP) in response to cell growth and applied this sensor to the diagnosis of maple syrup urine disease, which is represented by the elevated leucine level in blood. The leucine auxotroph was genetically engineered to grow displaying GBP in a proportion to the concentration of target amino acid leucine. The GBP expressed on the surface of the auxotrophs directly bound to the golden surface of an SPR chip without the need for any additional treatment or reagents, which consequently produced SPR signals used to determine leucine levels in a test sample. Gold nanoparticles (GNPs) were further applied to the SPR system, which significantly enhanced the signal intensity up to 10-fold by specifically binding to GBP expressed on the cell surface. Finally, the diagnostic utility of our system was demonstrated by its employment in reliably determining different statuses of maple syrup urine disease based on a known cutoff level of leucine. This new approach based on an amino acid-auxotrophic E. coli strain expressing a GBP that binds to an SPR sensor holds great promise for detection of other metabolic diseases of newborn babies including homocystinuria and phenylketonuria, which are also associated with abnormal levels of amino acids. PMID:26828904

  12. Surface properties of the Vero cytotoxin-producing Escherichia coli O157:H7.

    PubMed Central

    Sherman, P; Soni, R; Petric, M; Karmali, M

    1987-01-01

    Strains of Escherichia coli serotype O157:H7 are Vero cytotoxin-producing enteric pathogens which have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. In addition to toxin production, adherence of many pathogenic bacteria to intestinal mucosal surfaces is a critical primary step in the pathogenesis of diarrheal diseases. Although E. coli serotype O157:H7 organisms adhere to intestinal epithelia of orally infected animals in a pattern morphologically identical to that previously described in adherent, effacing E. coli infections, the mechanisms of bacterial adherence are not known. To determine the cell surface adhesins which mediate attachment of E. coli O157:H7 to epithelial surfaces, we evaluated the surface properties of these organisms. Five strains isolated from children with the hemolytic uremic syndrome were grown both in broth cultures and on agar media. Adherence and invasion of E. coli O157:H7 in Intestine 407 and HEp-2 epithelial cell lines was quantitated using an enteroinvasive E. coli strain (serotype O164:NM) as a control. Cell surface properties of E. coli O157:H7 were evaluated by agglutination of a series of erythrocytes, transmission electron microscopy, DEAE-ion-exchange chromatography, and hydrophobic interaction chromatography. E. coli O157:H7 strains adhered to but did not invade either Intestine 407 or HEp-2 cells. Homologous O157:H7 rabbit antiserum blocked attachment of bacteria to tissue culture cells, in contrast to heterologous antiserum and preimmune rabbit serum, which did not inhibit attachment of E. coli O157:H7. None of the five O15:H7 isolates mediated mannose-resistant hemagglutination under any of the in vitro culture conditions. One isolate mediated mannose-sensitive hemagglutination after serial passage in broth cultures. Pili and fibrillae were not visualized by electron microscopy on nonhemagglutinating organisms, but pili were demonstrated on the one

  13. Characterization of adhesion associated surface properties of uropathogenic Escherichia coli.

    PubMed

    Bartková, G; Ciznár, I; Lehotská, V; Kernová, T

    1994-01-01

    Escherichia coli was isolated from the urine of patients with pyelonephritis, with urinary tract infections other than pyelonephritis and with asymptomatic bacteriuria. Surface properties of the strains were analyzed by the salting-out aggregation test (SAT), hydrophobic interaction chromatography (HIC), Congo red binding (Crb), agglutination of erythrocytes (MRHA) and latex particles covered by digalactoside (PF) and by adherence to tissue culture cells. In addition, a DNA probe for the pap gene was used. The DNA probe detected the highest proportion of strains with pap gene in the group of patients with pyelonephritis, lower in the urinary tract infections other than pyelonephritis and the lowest in the group with asymptomatic bacteriuria. Tests for P-fimbriae (PF, MRHA) showed a similar distribution. Hydrophobicity measured by SAT and by HIC did not show differences among the tested groups of strains. The results suggest that factors other than the P-fimbriae and hydrophobicity may contribute to the persistence of E. coli in the urinary tract.

  14. Biofouling on polymeric heat exchanger surfaces with E. coli and native biofilms.

    PubMed

    Pohl, S; Madzgalla, M; Manz, W; Bart, H J

    2015-01-01

    The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials. PMID:26479780

  15. Biofouling on polymeric heat exchanger surfaces with E. coli and native biofilms.

    PubMed

    Pohl, S; Madzgalla, M; Manz, W; Bart, H J

    2015-01-01

    The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials.

  16. Flexible polymers suppress wobbling and tumbling of E. coli cells

    NASA Astrophysics Data System (ADS)

    Koser, Alison; Arratia, Paulo

    2014-11-01

    The run-and-tumble dynamics of swimming E. coli has been extensively studied. In this talk, we experimentally investigate the role of polymer concentration on the swimming dynamics of E. coli using tracking methods. We find that the addition of small amount of polymer to water drastically changes the run-and-tumble behavior of E. coli cells, enhancing translation while hindering rotational diffusion. Here, the cells are suspended in dilute solutions of carboxy-methyl cellulose (CMC) and imaged in a liquid film away from surfaces. The addition of polymer molecules to the fluid (water) leads to cell trajectories that are highly correlated in time; cells move in nearly straight lines and rotational diffusion is greatly reduced. By varying the polymer molecular weight, we show that trajectories are a result of two mechanisms: (1) suppression of cell wobbling due to elasticity and (2) enhancement of run times due to viscosity. Our experiments show that this combination of increased speed and suppressed reorientation dramatically changes overall cell dynamics in the presence of polymers.

  17. Development of functionalised polyelectrolyte capsules using filamentous Escherichia coli cells

    PubMed Central

    2012-01-01

    Background Escherichia coli is one of the best studied microorganisms and finds multiple applications especially as tool in the heterologous production of interesting proteins of other organisms. The heterologous expression of special surface (S-) layer proteins caused the formation of extremely long E. coli cells which leave transparent tubes when they divide into single E. coli cells. Such natural structures are of high value as bio-templates for the development of bio-inorganic composites for many applications. In this study we used genetically modified filamentous Escherichia coli cells as template for the design of polyelectrolyte tubes that can be used as carrier for functional molecules or particles. Diversity of structures of biogenic materials has the potential to be used to construct inorganic or polymeric superior hybrid materials that reflect the form of the bio-template. Such bio-inspired materials are of great interest in diverse scientific fields like Biology, Chemistry and Material Science and can find application for the construction of functional materials or the bio-inspired synthesis of inorganic nanoparticles. Results Genetically modified filamentous E. coli cells were fixed in 2% glutaraldehyde and coated with alternating six layers of the polyanion polyelectrolyte poly(sodium-4styrenesulfonate) (PSS) and polycation polyelectrolyte poly(allylamine-hydrochloride) (PAH). Afterwards we dissolved the E. coli cells with 1.2% sodium hypochlorite, thus obtaining hollow polyelectrolyte tubes of 0.7 μm in diameter and 5–50 μm in length. For functionalisation the polyelectrolyte tubes were coated with S-layer protein polymers followed by metallisation with Pd(0) particles. These assemblies were analysed with light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. Conclusion The thus constructed new material offers possibilities for diverse applications like novel catalysts or metal

  18. Subnanometric Roughness Affects the Deposition and Mobile Adhesion of Escherichia coli on Silanized Glass Surfaces.

    PubMed

    Sharma, Sumedha; Jaimes-Lizcano, Yuly Andrea; McLay, Ryan B; Cirino, Patrick C; Conrad, Jacinta C

    2016-05-31

    We investigate the deposition and transient adhesion of Escherichia coli on alkyl and fluoroalkyl silanized glass surfaces of different carbon chain lengths. The rate at which bacteria deposit onto these surfaces decreases as the shear stress is increased from 3 to 67 mPa, but trends in the deposition rate across all surfaces cannot be predicted from extended DLVO calculations of the interaction potential. As the surface root-mean-square (rms) roughness increases, the deposition rate increases and the percentage of motile tethered cells decreases. Furthermore, on surfaces of root-mean-square roughness of less than 0.2 nm, bacteria exhibit mobile adhesion, for which surface-associated cells linearly translate distances greater than approximately 1.5 times their average body length along the flow direction. E. coli bacteria with and without flagella exhibit mobile adhesion, indicating that this behavior is not driven by these appendages. Cells that express fimbriae do not exhibit mobile adhesion. These results suggest that even subnanoscale roughness can influence the deposition and transient adhesion of bacteria and imply that strategies to reduce frictional interactions by making cells or surfaces smoother may help to control the initial fouling of surfaces by E. coli bacteria. PMID:27158837

  19. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli

    PubMed Central

    Njoroge, Samuel M.; Boinett, Christine J.; Madé, Laure F.; Ouko, Tom T.; Fèvre, Eric M.; Thomson, Nicholas R.; Kariuki, Samuel

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described. PMID:26187892

  20. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    PubMed

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described. PMID:26187892

  1. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    PubMed

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described.

  2. Toward Network Biology in E. coli Cell.

    PubMed

    Mori, Hirotada; Takeuchi, Rikiya; Otsuka, Yuta; Bowden, Steven; Yokoyama, Katsushi; Muto, Ai; Libourel, Igor; Wanner, Barry L

    2015-01-01

    E. coli has been a critically important model research organism for more than 50 years, particularly in molecular biology. In 1997, the E. coli draft genome sequence was published. Post-genomic techniques and resources were then developed that allowed E. coli to become a model organism for systems biology. Progress made since publication of the E. coli genome sequence will be summarized.

  3. The relation between Escherichia coli surface functional groups’ composition and their physicochemical properties

    PubMed Central

    Hamadi, Fatima; Latrache, Hassan; Zahir, Hafida; Elghmari, Abderrahmene; Timinouni, Mohamed; Ellouali, Mostapha

    2008-01-01

    Escherichia coli surface characteristics including hydrophobicity, electrophoretic mobility and surface functional groups’ composition were investigated. These characteristics were determined respectively by water contact angle measurements, microelectrophoresis and x-ray photoelectron spectroscopy (XPS). The relation between the physicochemical properties and functional groups’ composition was also examined. The electrophoretic mobility at pH 7 appeared to be governed on the cell surface by the (O=C) functional groups. The cell surface’s hydrophilicity was associated with high levels of (C-(O.N)) and (OH- (C-O-C)) functional groups, whereas the cell surface’s hydrophobicity was associated with (C-(C,H)) functional groups. PMID:24031168

  4. Nanomechanical motion of Escherichia coli adhered to a surface.

    PubMed

    Lissandrello, C; Inci, F; Francom, M; Paul, M R; Demirci, U; Ekinci, K L

    2014-09-15

    Nanomechanical motion of bacteria adhered to a chemically functionalized silicon surface is studied by means of a microcantilever. A non-specific binding agent is used to attach Escherichia coli (E. coli) to the surface of a silicon microcantilever. The microcantilever is kept in a liquid medium, and its nanomechanical fluctuations are monitored using an optical displacement transducer. The motion of the bacteria couples efficiently to the microcantilever well below its resonance frequency, causing a measurable increase in the microcantilever fluctuations. In the time domain, the fluctuations exhibit large-amplitude low-frequency oscillations. In corresponding frequency-domain measurements, it is observed that the mechanical energy is focused at low frequencies with a 1/f(α) -type power law. A basic physical model is used for explaining the observed spectral distribution of the mechanical energy. These results lay the groundwork for understanding the motion of microorganisms adhered to surfaces and for developing micromechanical sensors for bacteria.

  5. Nanomechanical motion of Escherichia coli adhered to a surface

    NASA Astrophysics Data System (ADS)

    Lissandrello, C.; Inci, F.; Francom, M.; Paul, M. R.; Demirci, U.; Ekinci, K. L.

    2014-09-01

    Nanomechanical motion of bacteria adhered to a chemically functionalized silicon surface is studied by means of a microcantilever. A non-specific binding agent is used to attach Escherichia coli (E. coli) to the surface of a silicon microcantilever. The microcantilever is kept in a liquid medium, and its nanomechanical fluctuations are monitored using an optical displacement transducer. The motion of the bacteria couples efficiently to the microcantilever well below its resonance frequency, causing a measurable increase in the microcantilever fluctuations. In the time domain, the fluctuations exhibit large-amplitude low-frequency oscillations. In corresponding frequency-domain measurements, it is observed that the mechanical energy is focused at low frequencies with a 1/fα-type power law. A basic physical model is used for explaining the observed spectral distribution of the mechanical energy. These results lay the groundwork for understanding the motion of microorganisms adhered to surfaces and for developing micromechanical sensors for bacteria.

  6. Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae.

    PubMed

    Kuehni-Boghenbor, Kathrin; Jordi, Helene A; Frey, Joachim; Vilei, Edy M; Favre, Didier; Stoffel, Michael H

    2012-06-01

    Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae. PMID:22607531

  7. Programming Surface Chemistry with Engineered Cells.

    PubMed

    Zhang, Ruihua; Heyde, Keith C; Scott, Felicia Y; Paek, Sung-Ho; Ruder, Warren C

    2016-09-16

    We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery. PMID:27203116

  8. Programming Surface Chemistry with Engineered Cells.

    PubMed

    Zhang, Ruihua; Heyde, Keith C; Scott, Felicia Y; Paek, Sung-Ho; Ruder, Warren C

    2016-09-16

    We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery.

  9. Failed Escape: Solid Surfaces Prevent Tumbling of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Molaei, Mehdi; Barry, Michael; Stocker, Roman; Sheng, Jian

    2014-08-01

    Understanding how bacteria move close to surfaces is crucial for a broad range of microbial processes including biofilm formation, bacterial dispersion, and pathogenic infections. We used digital holographic microscopy to capture a large number (>103) of three-dimensional Escherichia coli trajectories near and far from a surface. We found that within 20 μm from a surface tumbles are suppressed by 50% and reorientations are largely confined to surface-parallel directions, preventing escape of bacteria from the near-surface region. A hydrodynamic model indicates that the tumble suppression is likely due to a surface-induced reduction in the hydrodynamic force responsible for the flagellar unbundling that causes tumbling. These findings imply that tumbling does not provide an effective means to escape trapping near surfaces.

  10. Bioremediation of organophosphorus pesticides by surface-expressed carboxylesterase from mosquito on Escherichia coli.

    PubMed

    Zhang, Jianliang; Lan, Wensheng; Qiao, Chuanling; Jiang, Hong; Mulchandani, Ashok; Chen, Wilfred

    2004-01-01

    The insecticide resistance-associated esterase, carboxylesterase B1 (CaE B1), from mosquito was used to degrade the organophosphorus compounds. To eradicate the need for enzyme purification and minimize the resistance to mass transport of the substrate and product across the cell membranes, the CaE B1 was displayed on the cell surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP). The presence of CaE B1 on the bacterial cell surface was verified by SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy. More than 50% of active CaE B1 is exported across the membrane and anchored onto the cell surface as determined by proteinase accessibility and cell fractionation experiments. In contrast, only a 6% drop in activity for proteinase K-treated cells was detected from E.coli cells containing pET-B1. From the degradation experiment, more than 80% of the malathion was degraded by whole cells containing plasmid pUC-NC-B1. Constitutive expression of CaE B1 on the surface using INPNC resulted in no cell lysis, and the suspended cultures also exhibited good stability. Because of their high biodegradation activity and superior stability, these "live biocatalysts" are promising for detoxification of organophosphorus pesticides. PMID:15458345

  11. An Escherichia coli Mutant That Makes Exceptionally Long Cells

    PubMed Central

    Newman, Elaine B.

    2015-01-01

    ABSTRACT Although Escherichia coli is a very small (1- to 2-μm) rod-shaped cell, here we describe an E. coli mutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. These extremely elongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division. IMPORTANCE Escherichia coli is usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usual E. coli cell. E. coli filaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects of E. coli physiology that are difficult to investigate with small cells. PMID:25691528

  12. Inactivation of Escherichia coli on PTFE surfaces by diffuse coplanar surface barrier discharge

    NASA Astrophysics Data System (ADS)

    Tučeková, Zlata; Koval'ová, Zuzana; Zahoranová, Anna; Machala, Zdenko; Černák, Mirko

    2016-08-01

    The non-equilibrium plasma of diffuse coplanar surface barrier discharge (DCSBD) was tested for decontamination of bacteria Escherichia coli on polymer surfaces. We investigated the optical parameters of DCSBD plasma generated in synthetic air with different relative humidity. Our study was provided to estimate the main plasma components active during the DCSBD plasma degradation of E. coli contamination prepared on polytetrafluoroethylene (PTFE, Teflon) surface, in ambient air at atmospheric pressure. The DCSBD plasma was characterized by means of electrical measurements and optical emission spectroscopy. The inactivation of E. coli bacteria was evaluated by standard microbiological cultivation (CFU plate counting). The experimental results of the germicidal efficiency obtained for short plasma exposure times proved the effectiveness of DCSBD plasma for the polymer surface decontamination. Contribution to the topical issue "6th Central European Symposium on Plasma Chemistry (CESPC-6)", edited by Nicolas Gherardi, Ester Marotta and Cristina Paradisi

  13. Potential of Escherichia coli O157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment.

    PubMed

    Marouani-Gadri, Nesrine; Firmesse, Olivier; Chassaing, Danielle; Sandris-Nielsen, Dennis; Arneborg, Nils; Carpentier, Brigitte

    2010-11-15

    Our aim was to assess the potential of Escherichia coli O157:H7 to persist in a processing environment. We studied E. coli behaviour under conditions modelling those of meat plants to establish one initial bacterial load that allows persistence and another that does not. Polyurethane coupons (3.5 cm²) were contaminated once with E. coli in meat exudate before being subjected daily to a cleaning product and a disinfectant, both at half the recommended in-use concentrations, and a further soiling with the exudate. This procedure aimed to model what occurs in harbourage sites. Because previous experiments showed that persistence could not be achieved at 15°C (temperature of slaughter halls), we incubated the coupons at 20°C. Viable cells were determined by ethidium monoazide-qPCR (EMA-qPCR). When the first chemical treatment (CT) was applied to 24-hour biofilms with 5.4 log CFU/cm² cells were no longer detectable after the first week. However, on 66-hour biofilms with 6.7 log CFU/cm², after initially decreasing, E. coli numbers reached 6.6 log CFU/cm² and 8.3 log viable cells/cm² on the 11th day. When E. coli was cultured with a Comamonas testosteroni previously shown to increase E. coli biofilm formation, and subjected to CT on alternate days, E. coli stabilized at 4.6 log CFU/cm² before the CT, from the 5th day of the experiment. The killing and detachment effects of the CT decreased over time and PCR quantification detected a resumption of growth after 2 days (CT on alternate days) or 3 days (daily CT). Intracellular pH (pHi) of individual cells was determined during an experiment in which the CT was applied on alternate days. The proportion of cells with no proton gradient towards the environment (pHi ≤ 5.4) increased after the CT as expected. But during the first week of the experiment only, a further increase in this proportion occurred 24 h after the CT, suggesting that some of the surviving viable but non-culturable cells finally died. This study

  14. Effects of ELF magnetic field in combination with Iron(III) chloride (FeCl3) on cellular growth and surface morphology of Escherichia coli (E. coli).

    PubMed

    Esmekaya, Meric A; Acar, S Ipek; Kıran, Fadime; Canseven, Ayşe G; Osmanagaoglu, Ozlem; Seyhan, Nesrin

    2013-04-01

    This study investigated the effects of extremely low frequency (ELF) magnetic field with/without iron(III) chloride (FeCl3) on bacterial growth and morphology. The ELF exposures were carried out using a pair of Helmholtz coil-based ELF exposure system which was designed to generate 50 Hz sinusoidal magnetic field. The field was approximately uniform throughout the axis of the coil pair. The samples which were treated or non-treated with different concentrations FeCl3 were exposed to 50 Hz, 2 millitesla (mT) magnetic field for 24 h. ELF effect on viability was assessed in terms of viable colony counts (in colony-forming unit per milliliter) with the standard plate count technique. Scanning electron microscopy was used to investigate the magnetic field effect on surface morphology of Escherichia coli. No significant results were seen in terms of cell viability between ELF and sham-exposed bacterial strains. Similarly, FeCl3 treatment did not change cell viability of E. coli samples. However, we observed some morphological changes on E. coli cell surfaces. Pore formations and membrane destruction were seen on the surface of 24 h ELF field-exposed cells. We concluded that ELF magnetic field exposure at 2 mT does not affect cell viability; however, it may affect bacterial surface morphology.

  15. Tsr Chemoreceptor Interacts With IL-8 Provoking E. coli Transmigration Across Human Lung Epithelial Cells.

    PubMed

    Han, Bing; Li, Manshu; Xu, Yonghao; Islam, Diana; Khang, Julie; Del Sorbo, Lorenzo; Lee, Warren; Szaszi, Katalin; Zhong, Nanshan; Slutsky, Arthur S; Li, Yimin; Zhang, Haibo

    2016-01-01

    Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections. PMID:27506372

  16. Tsr Chemoreceptor Interacts With IL-8 Provoking E. coli Transmigration Across Human Lung Epithelial Cells

    PubMed Central

    Han, Bing; Li, Manshu; Xu, Yonghao; Islam, Diana; Khang, Julie; Del Sorbo, Lorenzo; Lee, Warren; Szaszi, Katalin; Zhong, Nanshan; Slutsky, Arthur S.; Li, Yimin; Zhang, Haibo

    2016-01-01

    Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections. PMID:27506372

  17. Tsr Chemoreceptor Interacts With IL-8 Provoking E. coli Transmigration Across Human Lung Epithelial Cells.

    PubMed

    Han, Bing; Li, Manshu; Xu, Yonghao; Islam, Diana; Khang, Julie; Del Sorbo, Lorenzo; Lee, Warren; Szaszi, Katalin; Zhong, Nanshan; Slutsky, Arthur S; Li, Yimin; Zhang, Haibo

    2016-01-01

    Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections.

  18. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  19. Quantum dot enabled detection of Escherichia coli using a cell-phone.

    PubMed

    Zhu, Hongying; Sikora, Uzair; Ozcan, Aydogan

    2012-06-01

    We report a cell-phone based Escherichia coli (E. coli) detection platform for screening of liquid samples. In this compact and cost-effective design attached to a cell-phone, we utilize anti-E. coli O157:H7 antibody functionalized glass capillaries as solid substrates to perform a quantum dot based sandwich assay for specific detection of E. coli O157:H7 in liquid samples. Using battery-powered inexpensive light-emitting-diodes (LEDs) we excite/pump these labelled E. coli particles captured on the capillary surface, where the emission from the quantum dots is then imaged using the cell-phone camera unit through an additional lens that is inserted between the capillary and the cell-phone. By quantifying the fluorescent light emission from each capillary tube, the concentration of E. coli in the sample is determined. We experimentally confirmed the detection limit of this cell-phone based fluorescent imaging and sensing platform as ∼5 to 10 cfu mL(-1) in buffer solution. We also tested the specificity of this E. coli detection platform by spiking samples with different species (e.g., Salmonella) to confirm that non-specific binding/detection is negligible. We further demonstrated the proof-of-concept of our approach in a complex food matrix, e.g., fat-free milk, where a similar detection limit of ∼5 to 10 cfu mL(-1) was achieved despite challenges associated with the density of proteins that exist in milk. Our results reveal the promising potential of this cell-phone enabled field-portable and cost-effective E. coli detection platform for e.g., screening of water and food samples even in resource limited environments. The presented platform can also be applicable to other pathogens of interest through the use of different antibodies.

  20. Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains.

    PubMed

    Sommer, Ulf; Petersen, Jørgen; Pfeiffer, Michael; Schrotz-King, Petra; Morsczeck, Christian

    2010-10-01

    Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.

  1. Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells.

    PubMed

    Hossain, Sk Tofajjen; Mukherjee, Samir Kumar

    2013-09-15

    The present study endeavours to assess the toxic effect of synthesized CdS nanoparticles (NPs) on Escherichia coli and HeLa cells. The CdS NPs were characterized by DLS, XRD, TEM and AFM studies and the average size of NPs was revealed as ∼3 nm. On CdS NPs exposure bacterial cells changed morphological features to filamentous form and damage of the cell surface was found by AFM study. The expression of two conserved cell division components namely ftsZ and ftsQ in E. coli was decreased both at transcriptional and translational levels upon CdS NPs exposure. CdS NPs inhibited proper cell septum formation without affecting the nucleoid segregation. Viability of HeLa cells declined with increasing concentration of CdS NPs and the IC₅₀ value was found to be 4 μg/mL. NPs treated HeLa cells showed changed morphology with condensed and fragmented nuclei. Increased level of reactive oxygen species (ROS) was found both in E. coli and HeLa cells on CdS NPs exposure. The inverse correlation between declined cell viabilities and elevated ROS level suggested that oxidative stress seems to be the key event by which NPs induce toxicity both in E. coli and HeLa cells.

  2. Chlorine-induced damage to surface adhesions during sublethal injury of enterotoxigenic Escherichia coli.

    PubMed Central

    Walsh, S M; Bissonnette, G K

    1983-01-01

    A comparison of the adhesive ability of noninjured and chlorine-injured enterotoxigenic Escherichia coli was made by in vitro attachment to human peripheral leukocytes. Chlorination selected for noninjured cells with greater capabilities for colonizing the small intestine. Injured populations exhibited reduced association with leukocytes. Maximum reduction was seen in populations with greater than 80% injury. These cells demonstrated less adhesive ability than nonpiliated populations. Electron micrographs suggested that reduced adhesive ability was due to the loss of surface structures as a consequence of sublethal chlorination. The data imply a reduced ability among chlorine-injured pathogens to colonize the small intestine and initiate disease. Images PMID:6133503

  3. Enhanced surface colonization by Escherichia coli O157:H7 in biofilms formed by an Acinetobacter calcoaceticus isolate from meat-processing environments.

    PubMed

    Habimana, Olivier; Heir, Even; Langsrud, Solveig; Asli, Anette Wold; Møretrø, Trond

    2010-07-01

    A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 x 10(6) microm(3)) in a dynamic cocultured biofilm than in a monoculture (3.0 x 10(3) microm(3)), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.

  4. Binding studies of antimicrobial peptides to Escherichia coli cells.

    PubMed

    Avitabile, Concetta; D'Andrea, Luca D; Saviano, Michele; Olivieri, Michele; Cimmino, Amelia; Romanelli, Alessandra

    2016-09-01

    Understanding the mechanism of action of antimicrobial peptides is pivotal to the design of new and more active peptides. In the last few years it has become clear that the behavior of antimicrobial peptides on membrane model systems does not always translate to cells; therefore the need to develop methods aimed at capturing details of the interactions of peptides with bacterial cells is compelling. In this work we analyzed binding of two peptides, namely temporin B and TB_KKG6A, to Escherichia coli cells and to Escherichia coli LPS. Temporin B is a natural peptide active against Gram positive bacteria but inactive against Gram negative bacteria, TB_KKG6A is an analogue of temporin B showing activity against both Gram positive and Gram negative bacteria. We found that binding to cells occurs only for the active peptide TB_KKG6A; stoichiometry and affinity constant of this peptide toward Escherichia coli cells were determined.

  5. Binding studies of antimicrobial peptides to Escherichia coli cells.

    PubMed

    Avitabile, Concetta; D'Andrea, Luca D; Saviano, Michele; Olivieri, Michele; Cimmino, Amelia; Romanelli, Alessandra

    2016-09-01

    Understanding the mechanism of action of antimicrobial peptides is pivotal to the design of new and more active peptides. In the last few years it has become clear that the behavior of antimicrobial peptides on membrane model systems does not always translate to cells; therefore the need to develop methods aimed at capturing details of the interactions of peptides with bacterial cells is compelling. In this work we analyzed binding of two peptides, namely temporin B and TB_KKG6A, to Escherichia coli cells and to Escherichia coli LPS. Temporin B is a natural peptide active against Gram positive bacteria but inactive against Gram negative bacteria, TB_KKG6A is an analogue of temporin B showing activity against both Gram positive and Gram negative bacteria. We found that binding to cells occurs only for the active peptide TB_KKG6A; stoichiometry and affinity constant of this peptide toward Escherichia coli cells were determined. PMID:27450805

  6. Reactive oxygen species in plasma against E. coli cells survival rate

    NASA Astrophysics Data System (ADS)

    Zhou, Ren-Wu; Zhang, Xian-Hui; Zong, Zi-Chao; Li, Jun-Xiong; Yang, Zhou-Bin; Liu, Dong-Ping; Yang, Si-Ze

    2015-08-01

    In this paper, we report on the contrastive analysis of inactivation efficiency of E. coli cells in solution with different disinfection methods. Compared with the hydrogen peroxide solution and the ozone gas, the atmospheric-pressure He plasma can completely kill the E. coli cells in the shortest time. The inactivation efficiency of E. coli cells in solution can be well described by using the chemical reaction rate model. X-ray photoelectron spectroscopy (XPS) analysis shows that the C-O or C=O content of the inactivated E. coli cell surface by plasma is predominantly increased, indicating the quantity of oxygen-containing species in plasma is more than those of two other methods, and then the C-C or C-H bonds can be broken, leading to the etching of organic compounds. Analysis also indicates that plasma-generated species can play a crucial role in the inactivation process by their direct reactions or the decompositions of reactive species, such as ozone into OH radicals in water, then reacting with E. coli cells. Project supported by the Natural Science Foundation of Fujian Province, China (Grant No. 2014J01025), the National Natural Science Foundation of China (Grant No. 11275261), and the Funds from the Fujian Provincial Key Laboratory for Plasma and Magnetic Resonance, China.

  7. Effect of curli expression and hydrophobicity of E. coli O157:H7 on attachment to fresh produce surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation, and attachment to cut and intact fresh produce surfaces. Methods and Results: Five E. coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation, and attachment of E. co...

  8. Attachment of Escherichia coli O157:H7 to abiotic surfaces of cooking utensils.

    PubMed

    Tsuji, Makiko; Yokoigawa, Kumio

    2012-04-01

    We examined the attachment of enterohemorrhagic Escherichia coli O157:H7 to abiotic surfaces of cooking utensils. When the cell suspension in 0.85% NaCl (about 100 cells/mL, 10 mL) was contacted with various abiotic surfaces (square pieces, 25 cm²) at 25 °C for 20 min, the number of attached cells varied depending on the types of abiotic materials. The pathogen well attached to stainless steel (about 50 cells/25 cm²), pure titanium (35 to 45 cells/25 cm²), and glass (about 20 cells/25 cm²), but little attached to aluminum foil and plastics, irrespective of strains used. Fewer cells (below 10 cells/25 cm²) attached to stainless steel, pure titanium, and glass surfaces conditioned with aseptically sliced beef (sirloin) and autoclaved beef tallow at 25 °C for 20 min, but bovine serum albumin did not reduce the number of attached cells. The cells grown at 15 °C to the stationary phase (OD660 = about 2.8) less attached to the abiotic surfaces than those grown at 25 °C and 37 °C. When we pretreated the cells at 37 °C for 2 h with 50 μM N-hexanoyl-L-homoserine lactone (HHL), the number of cells attached to stainless steel was reduced by 70%. The number of cells attached to cooking utensils seemed to change depending on types of abiotic materials, adhesion of beef tallow to abiotic surfaces, growth temperature of the pathogen, and HHL-producing bacteria. PMID:22515247

  9. A study of the surface charging properties of a standard strain of Escherichia coli (ATCC 11775) in aqueous solutions.

    PubMed

    Li, Xinpeng; Xue, Xinkai; Pashley, Richard M

    2015-11-01

    In this study, the Escherichia coli (E. coli) strain ATCC 11775 was studied to determine its surface charging properties in a range of different aqueous salt solutions, with the aim of evaluating its potential as a monitor organism for water treatment. Zeta potential measurements were carried out in various solutions containing: NaCl, CaCl2, MgSO4, ZnSO4 and C14TAB, at different pH values and concentrations. Interestingly, it was found that the zeta potential of this strain of E. coli remained fairly constant at pH values over about 6, in 1mM NaCl solutions. In order to explain the cell surface charging properties, a simple, mass action surface ionization model was developed. This model indicates that the surface charging of these E. coli cells can be modeled simply using the ionization behavior of the acid groups in the common anionic membrane lipid phosphatidylserine (PS). There appeared to be no specific, strong adsorption of either divalent anions or cations, until high salt concentrations, above about 0.1M. The results suggest that at high concentrations both Ca(2+) and SO4(2-) ions are strongly adsorbed at the cell surface. However reduction of the magnitude of the surface electrostatic potential, due to Ca(2+) ion adsorption, did not appear to cause any cellular binding. In comparison, cationic surfactant was strongly adsorbed by the cell membrane surface, even at concentrations of 0.1mM, and light scattering studies indicated that the adsorption of the surfactant appeared to lyse the cell membrane and release internal cellular materials leading to a significant reduction in cell size.

  10. Enterohaemorrhagic Escherichia coli inhibits recycling endosome function and trafficking of surface receptors

    PubMed Central

    Clements, Abigail; Stoneham, Charlotte A; Furniss, R Christopher D; Frankel, Gad

    2014-01-01

    Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate many cell processes by injecting effector proteins from the bacteria into the host cell via a Type III secretion system. In this paper we report that the effector protein EspG disrupts recycling endosome function. In particular, we found that following transferrin binding and endocytosis EspG reduces recycling of the transferrin receptor (TfR), the prototypical recycling protein, from an intracellular location to the cell surface, resulting in an accumulation of TfR within the cell. The surface levels of three receptors [TfR, epidermal growth factor receptor (EGFR) and β1 integrin] were tested and found to be reduced dependent on EspG translocation. Furthermore, disruption of recycling endosome function and the reduced surface presentation of receptors was dependent on the previously reported RabGAP activity and ARF binding ability of EspG. This paper therefore supports the previous hypothesis that EspG acts as an enzyme scaffold perturbing cell signalling events, in this case altering recycling endosome function and cell surface receptor levels during infection. PMID:24898821

  11. How much territory can a single E. coli cell control?

    PubMed Central

    El-Hajj, Ziad W.; Newman, Elaine B.

    2015-01-01

    Bacteria have been traditionally classified in terms of size and shape and are best known for their very small size. Escherichia coli cells in particular are small rods, each 1–2 μ. However, the size varies with the medium, and faster growing cells are larger because they must have more ribosomes to make more protoplasm per unit time, and ribosomes take up space. Indeed, Maaløe’s experiments on how E. coli establishes its size began with shifts between rich and poor media. Recently much larger bacteria have been described, including Epulopiscium fishelsoni at 700 μm and Thiomargarita namibiensis at 750 μm. These are not only much longer than E. coli cells but also much wider, necessitating considerable intracellular organization. Epulopiscium cells for instance, at 80 μm wide, enclose a large enough volume of cytoplasm to present it with major transport problems. This review surveys E. coli cells much longer than those which grow in nature and in usual lab cultures. These include cells mutated in a single gene (metK) which are 2–4 × longer than their non-mutated parent. This metK mutant stops dividing when slowly starved of S-adenosylmethionine but continues to elongate to 50 μm and more. FtsZ mutants have been routinely isolated as long cells which form during growth at 42°C. The SOS response is a well-characterized regulatory network that is activated in response to DNA damage and also results in cell elongation. Our champion elongated E. coli is a metK strain with a further, as yet unidentified mutation, which reaches 750 μm with no internal divisions and no increase in width. PMID:25954251

  12. Surface pasteurization of whole fresh cantaloupes inoculated with Salmonella poona or Escherichia coli.

    PubMed

    Annous, Bassam A; Burke, Angela; Sites, Joseph E

    2004-09-01

    Numerous outbreaks of salmonellosis by Salmonella Poona have been associated with the consumption of cantaloupe. Commercial washing processes for cantaloupe are limited in their ability to inactivate or remove this human pathogen. Our objective was to develop a commercial-scale surface pasteurization process to enhance the microbiological safety of cantaloupe. Populations of indigenous bacteria recovered from cantaloupes that were surface pasteurized at 96, 86, or 76 degrees C for 2 to 3 min were significantly (P < 0.05) lower than those of the controls. Whole cantaloupes, surface inoculated with Salmonella Poona RM 2350 or Escherichia coli ATCC 25922 to a final cell concentration of ca. 5 log CFU/cm2 were stored at 4 degrees C or room temperature (RT = 19+/-1 degrees C) for up to 72 h before processing. Treatments at 76 degrees C for 2 to 3 min at 24 h postinoculation resulted in a reduction in excess of 5 log CFU/cm2 of Salmonella Poona and E. coli populations. Cantaloupes that were surface pasteurized and stored at 4 degrees C for 21 days retained their firmness qualities and had no visible mold growth compared with the controls, which became soft and moldy. These results indicate that surface pasteurization will enhance the microbiological safety of cantaloupes and will extend the shelf life of this commodity as well. Storage of untreated inoculated cantaloupes at RT for 24 to 72 h postinoculation caused a significant (P < 0.05) increase in Salmonella Poona and E. coli populations compared with storage at 4 degrees C. This indicates that cantaloupes should be refrigerated as soon as possible following harvest to suppress the growth of any possible contaminant on the rind.

  13. Roles of ionic strength and biofilm roughness on adhesion kinetics of Escherichia coli onto groundwater biofilm grown on PVC surfaces.

    PubMed

    Janjaroen, Dao; Ling, Fangqiong Q; Ling, Fangqiong; Monroy, Guillermo; Derlon, Nicolas; Morgenroth, Eberhard; Mogenroth, Eberhard; Boppart, Stephen A; Liu, Wen-Tso; Nguyen, Thanh H

    2013-05-01

    Mechanisms of Escherichia coli attachment on biofilms grown on PVC coupons were investigated. Biofilms were grown in CDC reactors using groundwater as feed solution over a period up to 27 weeks. Biofilm physical structure was characterized at the micro- and meso-scales using Scanning Electron Microscopy (SEM) and Optical Coherence Tomography (OCT), respectively. Microbial community diversity was analyzed with Terminal Restricted Fragment Length Polymorphism (T-RFLP). Both physical structure and microbial community diversity of the biofilms were shown to be changing from 2 weeks to 14 weeks, and became relatively stable after 16 weeks. A parallel plate flow chamber coupled with an inverted fluorescent microscope was also used to monitor the attachment of fluorescent microspheres and E. coli on clean PVC surfaces and biofilms grown on PVC surfaces for different ages. Two mechanisms of E. coli attachment were identified. The adhesion rate coefficients (kd) of E. coli on nascent PVC surfaces and 2-week biofilms increased with ionic strength. However, after biofilms grew for 8 weeks, the adhesion was found to be independent of solution chemistry. Instead, a positive correlation between kd and biofilm roughness as determined by OCT was obtained, indicating that the physical structure of biofilms could play an important role in facilitating the adhesion of E. coli cells.

  14. Roles of ionic strength and biofilm roughness on adhesion kinetics of Escherichia coli onto groundwater biofilm grown on PVC surfaces

    PubMed Central

    Janjaroen, Dao; Ling, Fangqiong; Monroy, Guillermo; Derlon, Nicolas; Mogenroth, Eberhard; Boppart, Stephen A.; Liu, Wen-Tso; Nguyen, Thanh H.

    2013-01-01

    Mechanisms of Escherichia coli attachment on biofilms grown on PVC coupons were investigated. Biofilms were grown in CDC reactors using groundwater as feed solution over a period up to 27 weeks. Biofilm physical structure was characterized at the micro- and meso-scales using Scanning Electron Microscopy (SEM) and Optical Coherence Tomography (OCT), respectively. Microbial community diversity was analyzed with Terminal Restricted Fragment Length Polymorphism (T-RFLP). Both physical structure and microbial community diversity of the biofilms were shown to be changing from 2 weeks to 14 weeks, and became relatively stable after 16 weeks. A parallel plate flow chamber coupled with an inverted fluorescent microscope was also used to monitor the attachment of fluorescent microspheres and E. coli on clean PVC surfaces and biofilms grown on PVC surfaces for different ages. Two mechanisms of E. coli attachment were identified. The adhesion rate coefficients (kd) of E. coli on nascent PVC surfaces and 2-week biofilms increased with ionic strength. However, after biofilms grew for 8 weeks, the adhesion was found to be independent of solution chemistry. Instead, a positive correlation between kd and biofilm roughness as determined by OCT was obtained, indicating that the physical structure of biofilms could play an important role in facilitating the adhesion of E. coli cells. PMID:23497979

  15. Accelerated Escherichia coli inactivation in the dark on uniform copper flexible surfaces.

    PubMed

    Rtimi, Sami; Sanjines, Rosendo; Bensimon, Michël; Pulgarin, César; Kiwi, John

    2014-06-01

    The bacterial inactivation of Escherichia coli on Cu/CuO-polyester surfaces prepared by direct current magnetron sputtering was investigated in the dark and under actinic light (360 nm≤ λ ≤ 720 nm; 4.1 mW/cm(2)) as used commonly in hospital facilities. In the dark, complete bacterial inactivation (6log10 reduction) was observed within 150 min and under actinic light within 45 min. Sputtered samples led to nanoparticulate uniform Cu/CuO films ~70 nm thick. The deposition rate used was 2.2×10(15) atoms/cm(2) s as determined by profilometry. X-ray fluorescence was used to determine the sample Cu-content and transmission electron microscopy determined Cu-particles ~20 ± 5 nm in size. The film optical absorption was observed to increase with Cu-content of the sample by diffuse reflection spectroscopy. The bacterial inactivation involved redox processes between Cu/CuO-polyester and the bacteria as observed by x-ray photoelectron spectroscopy. During sample recycling, the amount of Cu-release was determined by inductively coupled plasma-mass spectroscopy. The values required for E. coli inactivation were below the cytotoxicity level threshold allowed for mammalian cells. The E. coli inactivation by Cu/CuO-polyester seems to involve an oligodynamic effect since bacterial inactivation was achieved at very low Cu-concentrations. PMID:24985216

  16. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  17. Comparison of temperature effects on E. coli, Salmonella, and Enterococcus survival in surface waters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the dependencies of survival rates on temperature for indicator organisms E. coli and Enterococcus and the pathogen Salmonella in surface waters. A database consisting of 86 survival datasets from peer-reviewed papers on inactivation of E. coli, Salmonella...

  18. Comparing Temperature Effects on E. Coli, Salmonella, and Enterococcus Survival in Surface Waters

    EPA Science Inventory

    The objective of this study was to compare dependency of survival rates on temperature for indicator organisms E. coli and Enterococcus and the pathogen Salmonella in surface waters. A database of 86 survival datasets from peer-reviewed papers on inactivation of E. coli, Salmonel...

  19. Immobilization of bioluminescent Escherichia coli cells using natural and artificial fibers treated with polyethyleneimine.

    PubMed

    Chu, Yi-Fang; Hsu, Chia-Hua; Soma, Pavan K; Lo, Y Martin

    2009-07-01

    Biosensors based on whole-cell bioluminescence have the potential to become a cost-effective alternative to conventional detection methods upon validation of target selectivity and sensitivity. However, quantitative analysis of bioluminescence is greatly hindered due to lack of control over the total number of cells in a suspending culture. In this study, the effect of surface properties of genetically engineered luminous E. coli cells and fibrous matrices on the immobilization capacity and effectiveness under various environmental conditions were characterized. Four different fibers, including cotton, polyester, viscose rayon, and silk, were investigated. Although cell adhesion was observed on untreated viscose and cotton fibers, viscose fiber pretreated with 0.667% polyethyleneimine (PEI) was found capable of immobilizing the most viable E. coli DPD2234 cells, followed by viscose treated with 0.33% and 1% PEI. The cells immobilized on PEI-treated viscose remained viable and yielded 20% or more bioluminescence signals immediately upon contact with the inducer up to 72 h without feeding nutrients to the cells, suggesting that viscose treated with 0.667% PEI could provide a stable immobilization mechanism for bioluminescent E. coli cells with long sensing period, quick response time, and good signal reproducibility.

  20. Exchange of E. coli from the foreshore reservoir to surface waters during intensified wave conditions

    NASA Astrophysics Data System (ADS)

    Malott, S. S.; Vogel, L. J.; Edge, T.; O'Carroll, D. M.; Robinson, C. E.

    2014-12-01

    In recent years a number of studies have suggested that foreshore sand and porewater can act as a non-point source of microbial contamination to adjacent surface waters. Fecal indicator bacteria (FIB) can be released from the sand into the surface water through sand erosion or wave-induced porewater flows leading to FIB detachment. Although regression models often show that there is a strong correlation between wave events and high E. coli in surface waters, there is limited understanding of the mechanisms by which E. coli is transported from the subsurface foreshore reservoir (sand and porewater) to surface waters during wave events. An improved understanding of the transport mechanisms will facilitate the development of better water quality exceedences predictions. Detailed groundwater flow, sand level and E. coli measurements were conducted at Ipperwash Beach, Lake Huron (Ontario) for three wave events during the 2014 bathing season to evaluate the relative contribution of sand erosion and wave-induced pore water flow in transporting E. coli from the subsurface reservoir to the shallow waters. As expected, results indicate increased E. coli concentrations in ankle and waist deep surface water during periods of increased wave activity (wave height > 0.5m). Considerable sand erosion from the foreshore may have contributed to these increased surface water concentrations. The E. coli concentrations in the foreshore reservoir generally decreased as the wave height intensified, while E. coli concentrations in upshore sand and porewater locations increased.

  1. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    SciTech Connect

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    2015-01-15

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.

  2. Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

    PubMed Central

    MacKay, P; Pasek, M; Magazin, M; Kovacic, R T; Allet, B; Stahl, S; Gilbert, W; Schaller, H; Bruce, S A; Murray, K

    1981-01-01

    Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals. PMID:6170067

  3. Contribution of the highly conserved EaeH surface protein to enterotoxigenic Escherichia coli pathogenesis.

    PubMed

    Sheikh, Alaullah; Luo, Qingwei; Roy, Koushik; Shabaan, Salwa; Kumar, Pardeep; Qadri, Firdausi; Fleckenstein, James M

    2014-09-01

    Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of diarrheal illness worldwide. These pathogens disproportionately afflict children in developing countries, where they cause substantial morbidity and are responsible for hundreds of thousands of deaths each year. Although these organisms are important targets for enteric vaccines, most development efforts to date have centered on a subset of plasmid-encoded fimbrial adhesins known as colonization factors and heat-labile toxin (LT). Emerging data suggest that ETEC undergoes considerable changes in its surface architecture, sequentially deploying a number of putative adhesins during its interactions with the host. We demonstrate here that one putative highly conserved, chromosomally encoded adhesin, EaeH, engages the surfaces of intestinal epithelial cells and contributes to bacterial adhesion, LT delivery, and colonization of the small intestine. PMID:24935979

  4. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4.

    PubMed

    Nagy, Attila; Xu, Yunfeng; Bauchan, Gary R; Shelton, Daniel R; Nou, Xiangwu

    2016-07-16

    The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce.

  5. Prevalence of Escherichia coli in surface waters of Southeast Asian cities.

    PubMed

    Widmer, Kenneth; Van Ha, Nguyen Thi; Vinitnantharat, Soydoa; Sthiannopkao, Suthipong; Wangsaatmaja, Setiawan; Prasetiati, Maria Angela Novi; Thanh, Nguyen Cong; Thepnoo, Kasame; Sutadian, Arief Dhany; Thao, Huynh Thi Thanh; Fapyane, Deby; San, Vibol; Vital, Pierangeli; Hur, Hor-Gil

    2013-11-01

    Surface water samples were collected from rivers which fed into large urban areas within Vietnam, Indonesia, Cambodia, and Thailand and were processed to enumerate Escherichia coli. Selected isolates were further characterized using PCR to detect the presence of specific virulence genes. Analyzing the four countries together, the approximate mean cfu/100 ml for E. coli counts in the dry season were log 4.3, while counts in the wet season were log 2.8. Of the 564 E. coli isolates screened for the presence of pathogenic genes, 3.9 % possessed at least one virulence gene. The most common pathogenic types found were Shiga toxin-producing E. coli isolates. These results reinforce the importance of monitoring urban surface waters for fecal contamination, that E. coli in these water environments may serve as opportunistic pathogens, and may help in determining the impact water usage from these rivers have on the public health of urban populations in Southeast Asia.

  6. Type II secretory pathway for surface secretion of DraD invasin from the uropathogenic Escherichia coli Dr+ strain.

    PubMed

    Zalewska-Piatek, Beata; Bury, Katarzyna; Piatek, Rafal; Bruzdziak, Piotr; Kur, Józef

    2008-07-01

    The virulence of the uropathogenic Escherichia coli Dr(+) IH11128 strain is associated with the presence of Dr fimbrial structures and a DraD invasin which can act as a fimbrial capping domain at the bacterial cell surface. However, a recent study suggests that the DraD protein is surface exposed in two forms: fimbria associated and fimbria nonassociated (prone to interaction with the N-terminal extension of the DraE protein located on the fimbrial tip). The actual mechanism of DraD surface secretion is presently unknown. We identified a previously unrecognized type II secretory pathway (secreton) in the uropathogenic E. coli Dr(+) strain which is well conserved among gram-negative bacteria and used mainly for secretion of virulence determinants. An active secreton is composed of 12 to 15 different proteins, among which GspD functions as an outer-membrane channel to permit extrusion of proteins in a folded state. Therefore, we inactivated the pathway by inserting the group II intron into a gspD gene of the type II secretion machinery by site-specific recombination. DraD secretion by the E. coli Dr(+) and gspD mutant strains was determined by immunofluorescence microscopy (with antibodies raised against DraD) and an assay of cell binding between bacteria and HeLa cells. The specificity of DraD-mediated bacterial binding for the integrin receptor was confirmed by examination of the adhesion of DraD-coated beads to HeLa cells in the presence and absence of alpha(5)beta(1) monoclonal antibodies. The investigations that we performed showed that type II secretion in E. coli Dr(+) strains leads to DraD translocation at the bacterial cell surfaces.

  7. Adherence of streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa to fibronectin-coated and uncoated epithelial cells.

    PubMed Central

    Abraham, S N; Beachey, E H; Simpson, W A

    1983-01-01

    The relationship between the variability in the fibronectin (Fn) content on human buccal epithelial cells and the capacity of the cells to bind gram-positive (Streptococcus pyogenes) or gram-negative (Escherichia coli or Pseudomonas aeruginosa) bacteria was investigated. Adhesion experiments performed with mixtures of epithelial cells and mixed suspensions of either S. pyogenes and E. coli or S. pyogenes and P. aeruginosa exhibited three major populations of buccal cells: one of these was able to bind S. pyogenes (gram positive) but neither of the gram-negative bacteria; a second population was able to bind the gram-negative but not the gram-positive bacteria; and a third was able to bind various numbers of both types of organisms. Further adhesion experiments performed with a mixture of epithelial cells and a mixed suspension of S. pyrogens, E. coli, and fluoresceinconjugated methacrylate beads coated with immune immunoglobulin G directed against Fn revealed that the epithelial cells recognizing the gram-positive bacteria were rich in Fn, whereas those recognizing the gram-negative organisms were poor in Fn. Immunoelectron microscopy confirmed that cells of S. pyogenes bound to epithelial cells coated with Fn, whereas cells of E. coli bound to epithelial cells lacking Fn. These results suggest that Fn on the surfaces of epithelial cells may modulate the ecology of the human oropharyngeal cavity, especially with respect to the colonization of these surfaces by pathogenic gram-negative or gram-positive bacteria. Images PMID:6411621

  8. pH changes during in vitro adherence of Escherichia coli to HeLa cells.

    PubMed Central

    McCabe, K; Mann, M D; Bowie, M D

    1994-01-01

    Escherichia coli-induced acidic pH conditions were observed during the in vitro adherence of E. coli to HeLa cells. No pH changes occurred in the absence of adherence. This suggests that adherence affects the function or interaction of HeLa cells and E. coli. PMID:7927801

  9. Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

    PubMed Central

    Ahmad, Zulfiqar; Laughlin, Thomas F.; Kady, Ismail O.

    2015-01-01

    We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase. PMID:25996607

  10. RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants

    PubMed Central

    Ju, Wenting; Moyne, Anne-Laure; Marco, Maria L.

    2016-01-01

    The capacity to distinguish between living and dead cells is an important, but often unrealized, attribute of rapid detection methods for foodborne pathogens. In this study, the numbers of enterohemorrhagic Escherichia coli O157:H7 after inoculation onto Romaine lettuce plants and on plastic (abiotic) surfaces were measured over time by culturing, and quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, and reverse transcriptase (RT)-qPCR targeting E. coli O157:H7 gapA, rfbE, eae, and lpfA genes and gene transcripts. On Romaine lettuce plants incubated at low relative humidity, E. coli O157:H7 cell numbers declined 107-fold within 96 h according to culture-based assessments. In contrast, there were no reductions in E. coli levels according to qPCR and only 100- and 1000-fold lower numbers per leaf by RT-qPCR and PMA-qPCR, respectively. Similar results were obtained upon exposure of E. coli O157:H7 to desiccation conditions on a sterile plastic surface. Subsequent investigation of mixtures of living and dead E. coli O157:H7 cells strongly indicated that PMA-qPCR detection was subject to false-positive enumerations of viable targets when in the presence of 100-fold higher numbers of dead cells. RT-qPCR measurements of killed E. coli O157:H7 as well as for RNaseA-treated E. coli RNA confirmed that transcripts from dead cells and highly degraded RNA were also amplified by RT-qPCR. These findings show that neither PMA-qPCR nor RT-qPCR provide accurate estimates of bacterial viability in environments where growth and survival is limited. PMID:26955370

  11. RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants.

    PubMed

    Ju, Wenting; Moyne, Anne-Laure; Marco, Maria L

    2016-01-01

    The capacity to distinguish between living and dead cells is an important, but often unrealized, attribute of rapid detection methods for foodborne pathogens. In this study, the numbers of enterohemorrhagic Escherichia coli O157:H7 after inoculation onto Romaine lettuce plants and on plastic (abiotic) surfaces were measured over time by culturing, and quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, and reverse transcriptase (RT)-qPCR targeting E. coli O157:H7 gapA, rfbE, eae, and lpfA genes and gene transcripts. On Romaine lettuce plants incubated at low relative humidity, E. coli O157:H7 cell numbers declined 10(7)-fold within 96 h according to culture-based assessments. In contrast, there were no reductions in E. coli levels according to qPCR and only 100- and 1000-fold lower numbers per leaf by RT-qPCR and PMA-qPCR, respectively. Similar results were obtained upon exposure of E. coli O157:H7 to desiccation conditions on a sterile plastic surface. Subsequent investigation of mixtures of living and dead E. coli O157:H7 cells strongly indicated that PMA-qPCR detection was subject to false-positive enumerations of viable targets when in the presence of 100-fold higher numbers of dead cells. RT-qPCR measurements of killed E. coli O157:H7 as well as for RNaseA-treated E. coli RNA confirmed that transcripts from dead cells and highly degraded RNA were also amplified by RT-qPCR. These findings show that neither PMA-qPCR nor RT-qPCR provide accurate estimates of bacterial viability in environments where growth and survival is limited. PMID:26955370

  12. Low intensity infrared laser induces filamentation in Escherichia coli cells

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Presta, G. A.; Geller, M.; Paoli, F.

    2011-10-01

    Low intensity continuous wave and pulsed emission modes laser is used in treating many diseases and the resulting biostimulative effect on tissues has been described, yet the photobiological basis is not well understood. The aim of this wok was to evaluate, using bacterial filamentation assay, effects of laser on Escherichia coli cultures in exponential and stationary growth phase. E. coli cultures, proficient and deficient on DNA repair, in exponential and stationary growth phase, were exposed to low intensity infrared laser, aliquots were spread onto microscopic slides, stained by Gram method, visualized by optical microscopy, photographed and percentage of bacterial filamentation were determined. Low intensity infrared laser with therapeutic fluencies and different emission modes can induce bacterial filamentation in cultures of E. coli wild type, fpg/ mutM, endonuclease III and exonuclease III mutants in exponential and stationary growth phase. This study showed induction of bacterial, filamentation in E. coli cultures expose to low intensity infrared laser and attention to laser therapy protocols, which should take into account fluencies, wavelengths, tissue conditions, and genetic characteristics of cells before beginning treatment.

  13. Escherichia coli K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities.

    PubMed

    Korea, Charalampia-Georgia; Badouraly, Réana; Prevost, Marie-Christine; Ghigo, Jean-Marc; Beloin, Christophe

    2010-07-01

    Commensal and pathogenic Escherichia coli adherence to host and environmental surfaces is mediated by a variety of adhesins. Although extensively studied as a model bacterium, 34% of the genes in the E. coli K-12 genome have no known function. We hypothesized that some of them may correspond to functional adhesins. We characterized E. coli K-12 ycb, ybg, yfc, yad, yra, sfm and yeh operons, which display sequence and organizational homologies to type 1 fimbriae exported by the chaperone/usher pathway. We showed that, although these operons are poorly expressed under laboratory conditions, six of them are nevertheless functional when expressed, and promote adhesion to abiotic and/or epithelial cell surfaces. While the studied fimbriae display different binding specificities, we obtained evidence of synergy/interference with other adhesins such as Ag43 or type 1 fimbriae. We showed that their expression is under the negative control of H-NS and, except for yad, subjected to cAMP receptor protein-mediated activation and carbon catabolite repression. These results therefore demonstrate that ycb, yfc, yad, yra, sfm and yeh operons encode cryptic but functional fimbriae adhesins whose expression following environmental modifications could contribute to E. coli's ability to adhere to and colonize a wide diversity of surfaces in its various ecological niches.

  14. Optimizing Escherichia coli's metabolism for fuel cell applications

    NASA Astrophysics Data System (ADS)

    Nieves, Ismael U.

    In the last few years there have been many publications about applications that center on the generation of electrons from bacterial cells. These applications take advantage of the catabolic diversity of microbes to generate electrical power. The practicality of these applications depends on the microorganism's ability to effectively donate electrons, either directly to the electrode or indirectly through the use of a mediator. After establishing the limitations of electrical output in microbial fuel cells (MFCs) imposed by the bacterial cells, a spectrophotometric assay measuring the indirect reduction of the electronophore neutral red via iron reduction was used to measure electron production from Escherichia coli resting cells. Using this assay I identified NADH dehydrogenase I as a likely site of neutral red reduction. The only previously reported site of interaction between E. coli cells and NR is at the hydrogenases. Although we cannot rule out the possibility that NR is reduced by soluble hydrogenases in the cytoplasm, this previous report indicated that hydrogenase activity does not account for all of the NR reduction activity. Supporting this, data in this thesis suggest that the hydrogenases play a small role in NR reduction. It seems that NR reduction is largely taking place within the cytoplasmic membrane of the bacterial cells, serving as a substrate of enzymes that typically reduce quinones. Furthermore, it seems that under the experimental conditions used here, E. coli's catabolism of glucose is rather inefficient. Instead of using the complete TCA cycle, the bacterial cells are carrying out fermentation, leading to incomplete oxidation of the fuel and low yields of electrons. The results obtained from the TC31 strain suggest that eliminating fermentation pathways to improve NR reduction was the correct approach. Following up on this a new strain was created, KN02, which, in addition to the mutations on strain TC31, lacks acetate kinase activity.

  15. Surface functionalisation of TiO2 evanescent waveguide sensor for E.coli monitoring

    NASA Astrophysics Data System (ADS)

    Purniawan, A.; Pandraud, G.; Vakalopoulos, K. A.; French, P. J.; Sarro, P. M.

    2012-04-01

    This work reports the surface functionalisation of evanescent waveguide sensors to immobilise E. coli. In biosensors, the surface functionalisation is an important treatment to ensure that the sensor properly detects the cells of interest. In this paper, we study the thin film surface functionalisation of a TiO2 evanescent waveguide sensor and their effect on light transmission for the early detection of E. coli in post colon surgery. TiO2 deposited using atomic layer deposition (ALD) is used as waveguide material. Four layers are used in the functionalisation : the self-assembled monolayer (SAM), the protein, 1-ethyl-3-(3-dimethylaminopropyl) (EDC) and the antibodies. Aminopropyltriethoxysilane (APTES) is used as SAM and reacts with -OH group (hydroxyl). The -OH group must be provided on substrate. In order to have the proper -OH group we deposited 10 nm SiO2 on the waveguides using PECVD and then treated the samples in oxygen plasma chamber for 2 minutes to create the groups. Afterward APTES is immediately applied on the surface after every layers of the functionalisation process. The second layer (Protein A) of the functionalisation is then put on APTES as interlayer. EDC is used as crosslink agent between APTES and antibodies. The light of Superluminescent light emitting diodes (SLEDs) (λ = 1.3 μm, 400 mA) is channelled using an optical fibre into the functionalised waveguides. The transmitted light is measured with a photodiode. The sensitivity of the sensor was evaluated using several different drain fluid concentrations in medium.

  16. Amber mutation affecting the length of Escherichia coli cells.

    PubMed Central

    Martínez-Salas, E; Vicente, M

    1980-01-01

    An amber mutation in a newly found gene (wee) of Escherichia coli has been isolated from strain OV-2, which harbors a temperature-sensitive suppressor. At 42 degrees C cells of the mutant, OV-25, increased in mass and deoxyribonucleic acid content and divided at normal rates, compared with the wild type under the same growth conditions. Total cell length increased under the restrictive conditions, although at a slightly lower rate. Values of mean cell length and cell volume, contrary to what would be expected from the increment in the rate of increase in particles, mass, and deoxyribonucleic acid, became at 42 degrees C smaller than those found in the wild type. A parallel increase in protein content per length and cell density and a loss of viability were found to occur after four generations at the restrictive temperature. The behavior of strain OV-25 in the absence of the wee gene product could be interpreted in terms of either a faulty regulation of the elongation processes or their abnormal coordination with the cell cycle. The genetic location of the wee gene has been found to be at 83.5 min on the E. coli genetic map. PMID:7000749

  17. Dielectrophoretic Capture of E. coli Cells at Nanoelectrode Arrays

    PubMed Central

    Syed, Lateef U; Liu, Jianwei; Price, Alex; Li, Yi-fen; Culbertson, Christopher; Li, Jun

    2012-01-01

    This paper reports capture and detection of pathogenic bacteria based on AC dielectrophoresis (DEP) and electrochemical impedance spectroscopy (EIS) employing an embedded vertically aligned carbon nanofiber (VACNF) nanoelectrode array (NEA) vs. a macroscopic indium tin oxide (ITO) transparent electrode in “points-and-lid” configuration. The nano-DEP device was fabricated using photolithography processes to define an exposed active region on a randomly distributed NEA and a microfluidic channel on ITO to guide the flow of labeled E. coli cells, respectively, and then bond them into a fluidic chip. A high frequency (100 kHz) AC field was applied to generate positive DEP at the tips of exposed CNFs. Enhanced electric field gradient was achieved due to reduction in electrode size down to nanometer scale which helped to overcome the large hydrodynamic drag force experienced by E. coli cells at high flow velocities (up to 1.6 mm/sec). This DEP device was able to effectively capture a significant number of E. coli. Significant decrease in the absolute impedance (|Z|) at the NEA was observed by EIS experiments. The results obtained in this study suggest the possibility of integration of a fully functional electronic device for rapid, reversible and label-free capture and detection of pathogenic bacteria. PMID:21823128

  18. Antibiotic-resistant E. coli in surface water and groundwater in dairy operations in Northern California.

    PubMed

    Li, Xunde; Watanabe, Naoko; Xiao, Chengling; Harter, Thomas; McCowan, Brenda; Liu, Yingjia; Atwill, Edward R

    2014-02-01

    Generic Escherichia coli was isolated from surface water and groundwater samples from two dairies in Northern California and tested for susceptibility to antibiotics. Surface samples were collected from flush water, lagoon water, and manure solids, and groundwater samples were collected from monitoring wells. Although E. coli was ubiquitous in surface samples with concentrations ranging from several hundred thousand to over a million colony-forming units per 100 mL of surface water or per gram of surface solids, groundwater under the influence of these high surface microbial loadings had substantially fewer bacteria (3- to 7-log10 reduction). Among 80 isolates of E. coli tested, 34 (42.5%) were resistant to one or more antibiotics and 22 (27.5%) were multi-antibiotic resistant (resistant to ≥3 antibiotics), with resistance to tetracycline, cefoxitin, amoxicillin/clavulanic acid, and ampicillin being the most common. E. coli isolates from the calf hutch area exhibited the highest levels of multi-antibiotic resistance, much higher than isolates in surface soil solids from heifer and cow pens, flush alleys, manure storage lagoons, and irrigated fields. Among E. coli isolates from four groundwater samples, only one sample exhibited resistance to ceftriaxone, chloramphenicol, and tetracycline, indicating the potential of groundwater contamination with antibiotic-resistant bacteria from dairy operations.

  19. [Immobilization of E. coli cells in polyacrylamide-based microporous cryogels].

    PubMed

    Lusta, K A; Starostina, N G; Gorkina, N B; Fikhte, B A; Lozinskiĭ, V I

    1988-01-01

    E. coli cells were immobilized in polyacrylamide cryogel by three ways: (1) introduction of cells in the reaction mixture followed by cryopolymerization; (2) the filling of the cryogel pores followed by cell fixation with diluted glutaric dialdehyde (GDA), and (3) the filling of the macropores of the polymeric matrix with modified surface. The ultrastructure of the gels and immobilized cells as well as distribution of attachment of the cells immobilized by different techniques were studied. The first type of immobilization was characterized by the highest quantity of the biomass in the gel (by protein) and by a sharp decrease of the cell viability. The second failed to retain the cells in the pores, and the GDA treatment significantly decreased the viability index. The latter technique was the mildest and completely maintained the viability of the population. However, the biomass content was lower as compared to the first type of immobilization, but could be considerably increased by the GDA treatment.

  20. The effect of solvent polarity on the molecular surface properties and adhesion of Escherichia coli.

    PubMed

    Abu-Lail, Nehal I; Camesano, Terri A

    2006-08-01

    The elasticity and molecular surface characteristics of Escherichia coli JM109 were investigated via atomic force microscopy (AFM) in solvents expressing different polarities. The nature of bacterial adhesion and surface characteristics was probed in formamide, water, and methanol, with dielectric constants of 111, 80, and 33, respectively. Solvent polarity affected the elasticity of the bacterium, the conformation of the cell surface biopolymers, the height of the surface biopolymers, and measured adhesion forces between the bacterium and silicon nitride. By applying the Hertz model to force-indentation data, we determined that the Young's modulus was greatest in the least polar solvent, with values of 182 +/- 34.6, 12.8 +/- 0.1, and 0.8 +/- 0.3 MPa in methanol, water, and formamide, respectively. The thickness of the biopolymer brush layer on the bacterial surface was quantified using a steric model, and these values increased as polarity increased, with values of 27, 93, and 257 nm in methanol, water, and formamide, respectively. The latter results suggest that highly polar conditions favor extension of the biopolymer brush layer. Cross-sectional analysis performed on tapping mode images of the bacterial cells in methanol, water, and formamide further supported this hypothesis. The image height values are larger, since the image analysis measures the height of the bacterium and the polymer layer, but the trend with respect to solvent polarity was the same as was obtained from the steric model of the brush length. Measured adhesion forces scaled inversely with solvent polarity, with greatest adhesion observed in the least polar solvent, methanol. The combined conformational changes to the bacterial surface and biopolymer layer result in different presentations of macromolecules to a substrate surface, and therefore affect the adhesion forces between the bacterial molecules and the substrate. These results suggest that polarity of the solvent environment can be

  1. Comparison of Helicobacter pylori and attaching-effacing Escherichia coli adhesion to eukaryotic cells.

    PubMed Central

    Dytoc, M; Gold, B; Louie, M; Huesca, M; Fedorko, L; Crowe, S; Lingwood, C; Brunton, J; Sherman, P

    1993-01-01

    Adhesion of Helicobacter pylori was reported previously to be morphologically identical to "attaching and effacing" Escherichia coli. Therefore, the aim of the present study was to define the adhesion phenotype of H. pylori LC-11 to HEp-2, KATO-III, HEL, and CHO tissue culture cells. By using both staining of F-actin with fluorescein-labeled phalloidin and ultrastructural analysis, diffuse bacterial adhesion to discrete microvillus-denuded regions of the plasma membrane was observed in each of the infected cell lines. However, strain LC-11 did not induce formation of F-actin adhesion pedestals on the eukaryotic cells. H. pylori was negative by colony blot hybridization with an E. coli attaching and effacing gene probe. Elevations in inositol triphosphates followed infection of HEp-2 cells with H. pylori (405% of control values +/- 147%; P < 0.05). To correlate the observed histopathology with expression of the H. pylori phosphatidylethanolamine receptor, a thin-layer chromatography overlay-binding assay was used to identify receptors in each of the cell lines. H. pylori adhered to eukaryotic cells regardless of the presence (HEp-2, KATO-III, and CHO cells) or absence (HEL cells) of the lipid receptor as detected under the assay conditions. However, in comparison to cell lines that possess the phosphatidylethanolamine receptor, HEL cells demonstrated less quantitative H. pylori binding. These findings suggest that mechanisms distinct from E. coli enteropathogens underlie the adhesion of H. pylori to mucosal surfaces. In addition to the phosphatidylethanolamine H. pylori receptor, another host factor(s) likely mediates the attachment of H. pylori to human eukaryotic cells. Images PMID:8380793

  2. The Effect of Enterohemorrhagic E. coli Infection on the Cell Mechanics of Host Cells

    PubMed Central

    Chen, Yin-Quan; Su, Pin-Tzu; Chen, Yu-Hsuan; Wei, Ming-Tzo; Huang, Chien-Hsiu; Osterday, Kathryn; del Álamo, Juan C.; Syu, Wan-Jr; Chiou, Arthur

    2014-01-01

    Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity. PMID:25369259

  3. Surface cell immobilization within perfluoroalkoxy microchannels

    NASA Astrophysics Data System (ADS)

    Stojkovič, Gorazd; Krivec, Matic; Vesel, Alenka; Marinšek, Marjan; Žnidaršič-Plazl, Polona

    2014-11-01

    Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

  4. Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli.

    PubMed Central

    Blasco, B; Pisabarro, A G; de Pedro, M A

    1988-01-01

    The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process. PMID:3141382

  5. Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface.

    PubMed

    Yoshihara, Akinori; Nobuhira, Noritaka; Narahara, Hisaya; Toyoda, Syunsuke; Tokumoto, Hayato; Konishi, Yasuhiro; Nomura, Toshiyuki

    2015-07-01

    The effects of the presence or absence of microbial flagella and the microbial motility on the colloidal behaviors of microbial cells were quantitatively evaluated. The microbial cell attachment and detachment processes on a glass surface were observed directly using a parallel-plate flow chamber. Wild-type, flagellar paralyzed, and nonflagellated Escherichia coli strains were used as model microbial cells. In the cell attachment tests, the microbial adhesion rate in a 160mM NaCl solution was approximately 10 times higher than that in a 10mM solution, for all E. coli strains. The colloidal behavior of the microbial cells agreed well with the predictions of the DLVO theory. In addition, the microbial flagella and motility did not significantly affect the cell attachment, regardless of the existence of a potential barrier between the cell and the glass substratum. In the cell detachment tests, the cumulative number of microbial cells detached from the glass substratum with increasing flow rate was fit well with the Weibull distribution function. The list of strains arranged in order of increasing median drag force required to remove them was nonflagellated strain, flagellar paralyzed strain, and wild-type strain. These results indicated that the flagella and the flagellar motility inhibited the cell detachment from the glass substratum. Furthermore, a large external force would likely be required to inhibit the microbial adhesion in the early stage of the biofilm formation.

  6. Pathogenic Escherichia coli producing Extended-Spectrum β-Lactamases isolated from surface water and wastewater.

    PubMed

    Franz, Eelco; Veenman, Christiaan; van Hoek, Angela H A M; de Roda Husman, Ana; Blaak, Hetty

    2015-01-01

    To assess public health risks from environmental exposure to Extended-Spectrum β-Lactamases (ESBL)-producing bacteria, it is necessary to have insight in the proportion of relative harmless commensal variants and potentially pathogenic ones (which may directly cause disease). In the current study, 170 ESBL-producing E. coli from Dutch wastewater (n = 82) and surface water (n = 88) were characterized with respect to ESBL-genotype, phylogenetic group, resistance phenotype and virulence markers associated with enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), extraintesinal E. coli (ExPEC), and Shiga toxin-producing E. coli (STEC). Overall, 17.1% of all ESBL-producing E. coli were suspected pathogenic variants. Suspected ExPECs constituted 8.8% of all ESBL-producing variants and 8.3% were potential gastrointestinal pathogens (4.1% EAEC, 1.8% EPEC, 1.2% EIEC, 1.2% ETEC, no STEC). Suspected pathogens were significantly associated with ESBL-genotype CTX-M-15 (X(2) = 14.7, P < 0.001) and phylogenetic group B2 (X(2) = 23.5, P < 0.001). Finally, 84% of the pathogenic ESBL-producing E. coli isolates were resistant to three or more different classes of antibiotics. In conclusion, this study demonstrates that the aquatic environment is a potential reservoir of E. coli variants that combine ESBL-genes, a high level of multi-drug resistance and virulence factors, and therewith pose a health risk to humans upon exposure.

  7. Pathogenic Escherichia coli producing Extended-Spectrum β-Lactamases isolated from surface water and wastewater

    PubMed Central

    Franz, Eelco; Veenman, Christiaan; van Hoek, Angela H. A. M.; Husman, Ana de Roda; Blaak, Hetty

    2015-01-01

    To assess public health risks from environmental exposure to Extended-Spectrum β-Lactamases (ESBL)-producing bacteria, it is necessary to have insight in the proportion of relative harmless commensal variants and potentially pathogenic ones (which may directly cause disease). In the current study, 170 ESBL-producing E. coli from Dutch wastewater (n = 82) and surface water (n = 88) were characterized with respect to ESBL-genotype, phylogenetic group, resistance phenotype and virulence markers associated with enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), extraintesinal E. coli (ExPEC), and Shiga toxin-producing E. coli (STEC). Overall, 17.1% of all ESBL-producing E. coli were suspected pathogenic variants. Suspected ExPECs constituted 8.8% of all ESBL-producing variants and 8.3% were potential gastrointestinal pathogens (4.1% EAEC, 1.8% EPEC, 1.2% EIEC, 1.2% ETEC, no STEC). Suspected pathogens were significantly associated with ESBL-genotype CTX-M-15 (X2 = 14.7, P < 0.001) and phylogenetic group B2 (X2 = 23.5, P < 0.001). Finally, 84% of the pathogenic ESBL-producing E. coli isolates were resistant to three or more different classes of antibiotics. In conclusion, this study demonstrates that the aquatic environment is a potential reservoir of E. coli variants that combine ESBL-genes, a high level of multi-drug resistance and virulence factors, and therewith pose a health risk to humans upon exposure. PMID:26399418

  8. Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species.

    PubMed Central

    Dean, C R; Ward, O P

    1991-01-01

    Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C. Different E. coli strains manifested different lysis sensitivities when treated at 45 degrees C. When the lysis rates of E. coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis. The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E. coli cells. A close relation was observed between levels of lysis activity and proteolytic activity. A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E. coli cells. It was concluded that Bacillus proteases played a major role in the observed E. coli lysis. PMID:1892379

  9. Tumbling and quasi-tumbling motions of E.coli over a solid surface under shear flows

    NASA Astrophysics Data System (ADS)

    Molaei, Mehdi; Sheng, Jian

    2015-11-01

    Flow shear is known to alter bacterial motility by inducing Jeffery Orbit, rheotaxis, and trapping cells in the high shear region. Over a solid surface flow shear Interferes with hydrodynamic interaction of cells with solid surface. Our previous study shows that in the quiescent condition the tumbles of wild E.coli are suppressed and tumbling reorientation of cells is restricted to the surface parallel direction. In the current study, we exposed bacteria to the well controlled shear flows inside a microchannel and applying Digital Holography Microscopy to track them over time. The results show that flow shear promotes tumbling of E.coli and preserve reorientation of the cells during tumbles. Our hydrodynamic model indicates that in the low shear levels the tumble enhancement is due to shear induced flagella unbundling, while in the high shear flow regime, Jeffery Orbit causes rapid cell re-orientation which causes quasi-tumbles with similar angular displacement one would expect during a tumbling. NIH, GoMRI.

  10. Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

    PubMed Central

    2014-01-01

    Background Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli. Results The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents. Conclusions Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic

  11. Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying.

    PubMed

    Bang, Jihyun; Hong, Ayoung; Kim, Hoikyung; Beuchat, Larry R; Rhee, Min Suk; Kim, Younghoon; Ryu, Jee-Hoon

    2014-11-17

    We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P ≤ 0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO₂) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood>plastic>glass>stainless steel. The synergistic lethal effects of sequential ClO₂ and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO₂ (200 μg/ml, 10 min), rinsed with water, and subsequently dried at 43% relative humidity and 22 °C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4 CFU/coupon within 6 h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0 log CFU/coupon, respectively, after 12 h of drying. This indicates that ClO₂ treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on food-contact surfaces in food processing, distribution, and preparation environments.

  12. Production L-tryptophan by Escherichia coli cells.

    PubMed

    Bang, W G; Lang, S; Sahm, H; Wagner, F

    1983-04-01

    Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.

  13. Ferritinophagy drives uropathogenic Escherichia coli persistence in bladder epithelial cells.

    PubMed

    Bauckman, Kyle A; Mysorekar, Indira U

    2016-05-01

    Autophagy is a cellular recycling pathway, which in many cases, protects host cells from infections by degrading pathogens. However, uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections (UTIs), persist within the urinary tract epithelium (urothelium) by forming reservoirs within autophagosomes. Iron is a critical nutrient for both host and pathogen, and regulation of iron availability is a key host defense against pathogens. Iron homeostasis depends on the shuttling of iron-bound ferritin to the lysosome for recycling, a process termed ferritinophagy (a form of selective autophagy). Here, we demonstrate for the first time that UPEC shuttles with ferritin-bound iron into the autophagosomal and lysosomal compartments within the urothelium. Iron overload in urothelial cells induces ferritinophagy in an NCOA4-dependent manner causing increased iron availability for UPEC, triggering bacterial overproliferation and host cell death. Addition of even moderate levels of iron is sufficient to increase and prolong bacterial burden. Furthermore, we show that lysosomal damage due to iron overload is the specific mechanism causing host cell death. Significantly, we demonstrate that host cell death and bacterial burden can be reversed by inhibition of autophagy or inhibition of iron-regulatory proteins, or chelation of iron. Together, our findings suggest that UPEC persist in host cells by taking advantage of ferritinophagy. Thus, modulation of iron levels in the bladder may provide a therapeutic avenue to controlling UPEC persistence, epithelial cell death, and recurrent UTIs.

  14. Green biosynthesis of biocompatible CdSe quantum dots in living Escherichia coli cells

    NASA Astrophysics Data System (ADS)

    Yan, Zhengyu; Qian, Jing; Gu, Yueqing; Su, Yilong; Ai, Xiaoxia; Wu, Shengmei

    2014-03-01

    A green and efficient biosynthesis method to prepare fluorescence-tunable biocompatible cadmium selenide quantum dots using Escherichia coli cells as biological matrix was proposed. Decisive factors in biosynthesis of cadmium selenide quantum dots in a designed route in Escherichia coli cells were elaborately investigated, including the influence of the biological matrix growth stage, the working concentration of inorganic reactants, and the co-incubation duration of inorganic metals to biomatrix. Ultraviolet-visible, photoluminescence, and inverted fluorescence microscope analysis confirmed the unique optical properties of the biosynthesized cadmium selenide quantum dots. The size distribution of the nanocrystals extracted from cells and the location of nanocrystals foci in vivo were also detected seriously by transmission electron microscopy. A surface protein capping layer outside the nanocrystals was confirmed by Fourier transform infrared spectroscopy measurements, which were supposed to contribute to reducing cytotoxicity and maintain a high viability of cells when incubating with quantum dots at concentrations as high as 2 μM. Cell morphology observation indicated an effective labeling of living cells by the biosynthesized quantum dots after a 48 h co-incubation. The present work demonstrated an economical and environmentally friendly approach to fabricating highly fluorescent quantum dots which were expected to be an excellent fluorescent dye for broad bio-imaging and labeling.

  15. [Evaluation of Staphylococcus aureus and Escherichia coli biofilm formation on the surface of polypropylene mesh].

    PubMed

    Reśliński, Adrian; Mikucka, Agnieszka; Kwiecińska-Piróg, Joanna; Głowacka, Katarzyna; Gospodarek, Eugenia; Dabrowiecki, Stanisław

    2011-01-01

    A serious complication of hernioplasty with the use of a biomaterial implant is deep surgical site infection (SSI) encompassing the implant. Among the most common etiological factors of deep SSI in patients after hernioplasty are Staphylococcus aureus and Escherichia coli strains, which may create a biofilm on the surface of synthetic implants. The aim of this study was assessment of biofilm formation by S. aureus and E. coli on the surface ofpolypropylene mesh. The study included 108 strains (62 S. aureus and 46 E. coli) from the collection of Department of Microbiology Collegium Medicum im. L. Rydygier in Bydgoszcz, Nicolaus Copernicus University in Torun (CM UMK). Evaluation of biofilm formation was performed using the method of reduction of 2,3,5-triphenyltetrazolium chloride (TTC) and a scanning electron microscope. In the group of S. aureus strains, 88.7% isolates formed biofilm very strongly, 1.6% strongly, and 9.7% poor. Among E. coli strains, 54.3% isolates were characterized by very strong biofilm formation, while 45.7% strong biofilm formation. Strains ofS. aureus strongly than E. coli form a biofilm on the surface of monofilament polypropylene mesh.

  16. Uropathogenic Escherichia coli Epigenetically Manipulate Host Cell Death Pathways.

    PubMed

    Zhang, Zhengguo; Wang, Ming; Eisel, Florian; Tchatalbachev, Svetlin; Chakraborty, Trinad; Meinhardt, Andreas; Bhushan, Sudhanshu

    2016-04-01

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in human. It is well established that UPEC can subvert innate immune responses, but the role of UPEC in interfering with host cell death pathways is not known. Here, we show that UPEC abrogates activation of the host cell prosurvival protein kinase B signaling pathway, which results in the activation of mammalian forkhead box O (FOXO) transcription factors. Although FOXOs were localized in the nucleus and showed increased DNA-binding activity, no change in the expression levels of FOXO target genes were observed. UPEC can suppress BIM expression induced by LY249002, which results in attenuation of caspase 3 activation and blockage of apoptosis. Mechanistically, BIM expression appears to be epigenetically silenced by a decrease in histone 4 acetylation at the BIM promoter site. Taken together, these results suggest that UPEC can epigenetically silence BIM expression, a molecular switch that prevents apoptosis.

  17. Measuring cell wall elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

    SciTech Connect

    Beckmann, Melissa; Venkataraman, Sankar; Doktycz, Mitchel John; Nataro, James P; Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P

    2006-07-01

    Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

  18. Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane.

    PubMed Central

    Fehniger, T E; Radolf, J D; Walfield, A M; Cunningham, T M; Miller, J N; Lovett, M A

    1986-01-01

    A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy. Images PMID:3516880

  19. Estimation of the biofilm formation of Escherichia coli K-12 by the cell number.

    PubMed

    Narisawa, Naoki; Furukawa, Soichi; Ogihara, Hirokazu; Yamasaki, Makari

    2005-01-01

    We developed a method of estimating the biofilm formation of Escherichia coli K-12 strains in microtiter-plate wells by the cell number. Regression lines between the cell number and absorbance of crystal violet that stained the E. coli biofilm consisted of high and low slope lines, respectively.

  20. Role of Extracellular Structures of Escherichia coli O157:H7 in Initial Attachment to Biotic and Abiotic Surfaces

    PubMed Central

    Nagy, Attila; Mowery, Joseph; Bauchan, Gary R.; Wang, Lili; Nichols-Russell, Lydia

    2015-01-01

    Infection by human pathogens through the consumption of fresh, minimally processed produce and solid plant-derived foods is a major concern of the U.S. and global food industries and of public health services. Enterohemorrhagic Escherichia coli O157:H7 is a frequent and potent foodborne pathogen that causes severe disease in humans. Biofilms formed by E. coli O157:H7 facilitate cross-contamination by sheltering pathogens and protecting them from cleaning and sanitation operations. The objective of this research was to determine the role that several surface structures of E. coli O157:H7 play in adherence to biotic and abiotic surfaces. A set of isogenic deletion mutants lacking major surface structures was generated. The mutant strains were inoculated onto fresh spinach and glass surfaces, and their capability to adhere was assessed by adherence assays and fluorescence microscopy methods. Our results showed that filament-deficient mutants bound to the spinach leaves and glass surfaces less strongly than the wild-type strain did. We mimicked the switch to the external environment—during which bacteria leave the host organism and adapt to lower ambient temperatures of cultivation or food processing—by decreasing the temperature from 37°C to 25°C and 4°C. We concluded that flagella and some other cell surface proteins are important factors in the process of initial attachment and in the establishment of biofilms. A better understanding of the specific roles of these structures in early stages of biofilm formation can help to prevent cross-contaminations and foodborne disease outbreaks. PMID:25956766

  1. Effect of serogroup, surface material and disinfectant on biofilm formation by avian pathogenic Escherichia coli.

    PubMed

    Oosterik, Leon H; Tuntufye, Huruma N; Butaye, Patrick; Goddeeris, Bruno M

    2014-12-01

    Avian pathogenic Escherichia coli (APEC) are responsible for significant economic losses in the poultry industry and are difficult to eradicate. Biofilm formation by APEC has the potential to reduce the efficacy of cleaning and disinfection. In this study, biofilm formation on materials used in poultry facilities by APEC strains from laying hens was determined. APEC strains were analysed for an association between biofilm forming capacity and O serogroup. The abilities of two routinely used disinfectants, hydrogen peroxide (H2O2) and a quaternary ammonium compound (QAC), to kill adherent cells of two strong APEC biofilm producers (05/503 and 04/40) and a non-biofilm producer (05/293) on polystyrene (PS) and polyvinylchloride (PVC) surfaces were tested. Most APEC strains were moderate (PS) or strong biofilm producers (polypropylene, PP, and PVC). Strains in serogroup O2 more often belonged to the moderate (PS) or strong (PP and PVC) biofilm producers than to other groups, while most O78 strains were weak biofilm producers. O78 strains were stronger biofilm producers on stainless steel than on PP and PVC, while O2 strains were stronger biofilm producers on PP and PVC. A concentration of 1% H2O2 killed all adherent bacteria of strains 05/503 and 04/40 on PP and PVC, while 0.5% H2O2 killed all adherent bacteria of strain 05/293. QAC at a concentration of 0.01% killed all adherent cells of strains 05/503, 04/40 and 05/293 under equal conditions. In conclusion, biofilm formation by APEC was affected by serogroup and surface material, and inactivation of APEC was dependent on the disinfectant and surface material.

  2. Cell-surface carbohydrates of Entamoeba invadens.

    PubMed

    Ribeiro, S; Soares, R M; Alviano, C S; Da Silva, E F; De Souza, W; Angluster, J

    1997-01-01

    Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc. PMID:9342747

  3. Protein folding in the cell envelope of Escherichia coli.

    PubMed

    De Geyter, Jozefien; Tsirigotaki, Alexandra; Orfanoudaki, Georgia; Zorzini, Valentina; Economou, Anastassios; Karamanou, Spyridoula

    2016-01-01

    While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines. PMID:27573113

  4. Rapid detection of Escherichia coli and Salmonella typhimurium by surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Su, Lan; Zhang, Ping; Zheng, Da-wei; Wang, Yang-jun-qi; Zhong, Ru-gang

    2015-03-01

    In this paper, the surface-enhanced Raman scattering (SERS) is used as an analytical tool for the detection and identification of pathogenic bacteria of Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium). Compared with normal Raman signal, the intensity of SERS signal is greatly enhanced. After processing all SERS data, the obvious differences between the SERS spectra of two species are determined. And applying the chemometric tools of principal component analysis and hierarchical cluster analysis (PCA-HCA), the SERS spectra of two species are distinguished more accurately. The results indicate that SERS analysis can provide a rapid and sensitive method for the detection of pathogenic bacteria.

  5. Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages.

    PubMed

    Tawil, Nancy; Sacher, Edward; Mandeville, Rosemonde; Meunier, Michel

    2012-01-01

    Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections.

  6. SOS response dynamics in Escherichia coli bacterial cells upon ultraviolet irradiation

    NASA Astrophysics Data System (ADS)

    Belov, O. V.; Krasavin, E. A.; Parkhomenko, A. Yu.

    2009-05-01

    A mathematical model of induced mutations in Escherichia coli bacterial cells upon ultraviolet irradiation is developed. The concentration dynamics of inducible protein complexes synthesized in the course of the SOS response of E. coli is described. The mutation induction at translesion synthesis is studied. The solutions to the model are based on experimental data concerning the expression of the main genes of the SOS system of E. coli bacteria.

  7. Overexpression of the waaZ Gene Leads to Modification of the Structure of the Inner Core Region of Escherichia coli Lipopolysaccharide, Truncation of the Outer Core, and Reduction of the Amount of O Polysaccharide on the Cell Surface

    PubMed Central

    Frirdich, Emilisa; Lindner, Buko; Holst, Otto; Whitfield, Chris

    2003-01-01

    The waa gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in Escherichia coli and Salmonella. Homologs of the waaZ gene product are encoded by the waa gene clusters of Salmonella enterica and E. coli strains with the K-12 and R2 core types. Overexpression of WaaZ in E. coli and S. enterica led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an E. coli isolate with an R1 core (waaZ is lacking from the type R1 waa gene cluster) with a copy of the waaZ gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core. PMID:12591884

  8. Binding characteristics of S fimbriated Escherichia coli to isolated brain microvascular endothelial cells.

    PubMed Central

    Stins, M. F.; Prasadarao, N. V.; Ibric, L.; Wass, C. A.; Luckett, P.; Kim, K. S.

    1994-01-01

    To assess the role of S fimbriae in the pathogenesis of Escherichia coli meningitis, transformants of E. coli strains with or without S fimbriae plasmid were compared for their binding to microvessel endothelial cells isolated from bovine brain cortices (BMEC). The BMEC's displayed a cobblestone appearance, were positive for factor VIII, carbonic anhydrase IV, took up fluorescent-labeled acetylated low density lipoprotein, and exhibited gamma glutamyl transpeptidase activity. Binding of S fimbriated E. coli to BMEC was approximately threefold greater than nonfimbriated E. coli Similarly S fimbriated E. coli bound to human brain endothelial cells approximately threefold greater than nonfimbriated E. coli. Binding was reduced approximately 60% by isolated S fimbriae and about 80% by anti-S adhesin antibody. Mutating the S adhesin gene resulted in a complete loss of the binding, whereas mutagenesis of the major S fimbriae subunit gene sfaA did not significantly affect binding. Pretreatment of BMEC with neuraminidase or prior incubation of S fimbriated E. coli with NeuAc alpha 2,3-sialyl lactose completely abolished binding. These findings indicate that S fimbriated E. coli bind to NeuAc alpha 2,3-galactose containing glycoproteins on brain endothelial cells via a lectin-like activity of SfaS adhesin. This might be an important early step in the penetration of bacteria across the blood-brain barrier in the development of E. coli meningitis. Images Figure 6 PMID:7977653

  9. Reduction of Adherence of E. coli O157:H7 to HEp-2 Cells and to Bovine Large Intestinal Mucosal Explants by Colicinogenic E. coli

    PubMed Central

    Etcheverría, A. I.; Arroyo, G. H.; Alzola, R.; Parma, A. E.

    2011-01-01

    Enterohemorrhagic E. coli strains (EHEC) had emerged as foodborne pathogens and cause in human diarrhea and hemolytic-uremic syndrome. Because of the widespread distribution of EHEC serotypes and O157 and non-O157 in cattle population, its control will require interventions at the farm level such as the administration of probiotics that produce inhibitory metabolites. E. coli O157:H7 shows tissue tropisms for the gastrointestinal tract (GIT) of cattle. The aim of this study was to test the ability of a colicinogenic E. coli (isolated from bovine) to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to GIT of cattle. We inoculated HEp-2 cells and bovine colon explants with both kinds of strains. Colicinogenic E. coli was able to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to bovine tissues. PMID:23724308

  10. Corona discharges with water electrospray for Escherichia coli biofilm eradication on a surface.

    PubMed

    Kovalova, Zuzana; Leroy, Magali; Kirkpatrick, Michael J; Odic, Emmanuel; Machala, Zdenko

    2016-12-01

    Low-temperature plasma (cold), a new method for the decontamination of surfaces, can be an advantageous alternative to the traditional chemical methods, autoclave or dry heat. Positive and negative corona discharges in air were tested for the eradication of 48-h Escherichia coli biofilms grown on glass slides. The biofilms were treated by cold corona discharge plasma for various exposure times. Water electrospray from the high voltage electrode was applied in some experiments. Thermostatic cultivation of the biofilm, and confocal laser scanning microscopy (CLSM) of the biofilm stained with fluorescent dyes were used for biocidal efficiency quantification. Up to 5 log10 reduction of bacterial concentration in the biofilm was measured by thermostatic cultivation after exposure to both corona discharges for 15min. This decontamination efficiency was significantly enhanced by simultaneous water electrospray through the plasma. CLSM showed that the live/dead ratio after treatment remained almost constant inside the biofilm; only cells on the top layers of the biofilm were affected. DAPI fluorescence showed that biofilm thickness was reduced by about 1/3 upon exposure to the corona discharges with electrospray for 15min. The biofilm biomass loss by about 2/3 was confirmed by crystal violet assay. PMID:27261097

  11. Corona discharges with water electrospray for Escherichia coli biofilm eradication on a surface.

    PubMed

    Kovalova, Zuzana; Leroy, Magali; Kirkpatrick, Michael J; Odic, Emmanuel; Machala, Zdenko

    2016-12-01

    Low-temperature plasma (cold), a new method for the decontamination of surfaces, can be an advantageous alternative to the traditional chemical methods, autoclave or dry heat. Positive and negative corona discharges in air were tested for the eradication of 48-h Escherichia coli biofilms grown on glass slides. The biofilms were treated by cold corona discharge plasma for various exposure times. Water electrospray from the high voltage electrode was applied in some experiments. Thermostatic cultivation of the biofilm, and confocal laser scanning microscopy (CLSM) of the biofilm stained with fluorescent dyes were used for biocidal efficiency quantification. Up to 5 log10 reduction of bacterial concentration in the biofilm was measured by thermostatic cultivation after exposure to both corona discharges for 15min. This decontamination efficiency was significantly enhanced by simultaneous water electrospray through the plasma. CLSM showed that the live/dead ratio after treatment remained almost constant inside the biofilm; only cells on the top layers of the biofilm were affected. DAPI fluorescence showed that biofilm thickness was reduced by about 1/3 upon exposure to the corona discharges with electrospray for 15min. The biofilm biomass loss by about 2/3 was confirmed by crystal violet assay.

  12. Prevalence of Escherichia coli O157:H7 in surface water near cattle feedlots.

    PubMed

    Tanaro, José D; Piaggio, Mercedes C; Galli, Lucía; Gasparovic, Alejandra M C; Procura, Francisco; Molina, Demián A; Vitón, Mauro; Zolezzi, Gisela; Rivas, Marta

    2014-12-01

    Between April 2009 and July 2011, 311 surface water samples in 48 cattle feedlots distributed in an area of about 67,000 km(2) were analyzed to examine the environmental dissemination of Escherichia coli O157:H7. Samples were taken inside and outside the pens, exposed and not exposed to runoff from corrals, near the feedlots. Two types of samples were defined: (1) exposed surface waters (ESW; n=251), downstream from cattle pens; and (2) nonexposed surface waters (NESW; n=60), upstream from cattle pens. By multiplex PCR, 177 (70.5%) ESW samples were rfb(O157)-positive, and 62 (24.7%) E. coli O157, and 32 (12.7%) Shiga toxin-producing E. coli (STEC) O157:H7 strains were isolated. In the NESW samples, 36 (60.0%) were rfb(O157)- positive, and 9 (15.0%) E. coli O157, and 6 (10.0%) STEC O157:H7 strains were isolated. These results showed that the environmental surface waters exposed to liquid discharges from intensive livestock operations tended to be contaminated with more STEC O157:H7 than NESW. However, no significant difference was found. This fact emphasizes the relevance of other horizontal routes of transmission, as the persistence of E. coli in the environment resulting from extensive livestock farming. By XbaI-PFGE, some patterns identified are included in the Argentine Database of E. coli O157, corresponding to strains isolated from hemolytic uremic syndrome and diarrhea cases, food, and animals, such as AREXHX01.0022, second prevalent pattern in Argentina, representing 5.5% of the total database. In the study area, characterized by the abundance of waterways, pathogens contained in feedlot runoff could reach recreational waters and also contaminate produce through irrigation, increasing the potential dissemination of STEC O157:H7 and the risk of human infections. The control of runoff systems from intensive livestock is necessary, but other alternatives should be explored to solve the problem of the presence of E. coli O157 in the aquatic rural environment

  13. Prevalence of Escherichia coli O157:H7 in surface water near cattle feedlots.

    PubMed

    Tanaro, José D; Piaggio, Mercedes C; Galli, Lucía; Gasparovic, Alejandra M C; Procura, Francisco; Molina, Demián A; Vitón, Mauro; Zolezzi, Gisela; Rivas, Marta

    2014-12-01

    Between April 2009 and July 2011, 311 surface water samples in 48 cattle feedlots distributed in an area of about 67,000 km(2) were analyzed to examine the environmental dissemination of Escherichia coli O157:H7. Samples were taken inside and outside the pens, exposed and not exposed to runoff from corrals, near the feedlots. Two types of samples were defined: (1) exposed surface waters (ESW; n=251), downstream from cattle pens; and (2) nonexposed surface waters (NESW; n=60), upstream from cattle pens. By multiplex PCR, 177 (70.5%) ESW samples were rfb(O157)-positive, and 62 (24.7%) E. coli O157, and 32 (12.7%) Shiga toxin-producing E. coli (STEC) O157:H7 strains were isolated. In the NESW samples, 36 (60.0%) were rfb(O157)- positive, and 9 (15.0%) E. coli O157, and 6 (10.0%) STEC O157:H7 strains were isolated. These results showed that the environmental surface waters exposed to liquid discharges from intensive livestock operations tended to be contaminated with more STEC O157:H7 than NESW. However, no significant difference was found. This fact emphasizes the relevance of other horizontal routes of transmission, as the persistence of E. coli in the environment resulting from extensive livestock farming. By XbaI-PFGE, some patterns identified are included in the Argentine Database of E. coli O157, corresponding to strains isolated from hemolytic uremic syndrome and diarrhea cases, food, and animals, such as AREXHX01.0022, second prevalent pattern in Argentina, representing 5.5% of the total database. In the study area, characterized by the abundance of waterways, pathogens contained in feedlot runoff could reach recreational waters and also contaminate produce through irrigation, increasing the potential dissemination of STEC O157:H7 and the risk of human infections. The control of runoff systems from intensive livestock is necessary, but other alternatives should be explored to solve the problem of the presence of E. coli O157 in the aquatic rural environment.

  14. Surfacing of domestic wastewater applied to soil through drip tubing and reduction in numbers of Escherichia coli.

    PubMed

    Franti, J M; Weaver, R W; McInnes, K J

    2002-09-01

    Drip tubing is a technology that is increasing in use. The effectiveness of such systems in distributing the wastewater uniformly through the soil matrix, providing adequate removal of bacteria from wastewater, and keeping wastewater from reaching the soil surface has not been adequately evaluated. Experiments were conducted at two sites that had used drip tubing for approximately 3 years. This 3-year-old drip tubing and newly installed tubing were used in this investigation. A solution containing Brilliant Blue FCF dye and Escherichia coli, at an approximate concentration of 1 x 10(6) cells ml(-1), was applied to the sites through drip emitters. Reduction i n Escherichia coli populations reaching the soil surface was generally less than 10%. The route of travel for the solution reaching the soil surface was consistently along preferential flow paths and not uniformly through the soil matrix. Instances of water reaching the soil surface for drip tubing installed at 15 cm was nearly 50%. Increasing burial of the drip tubing from 15 to 30 cm nearly eliminated water surfacing. A 31 per emitter dose of water, immediately following drip line installation later increased instances of water reaching the soil surface for drip tubing buried at 30 cm. The volume of water applied per dose had little effect on the number of times water reached the soil surface. Inherent soil structural characteristics limited the drip tubing's ability to uniformly distribute water and adsorb bacteria. Drip tubing installation to 30 cm may be an important practice to reduce public health hazards from the likelihood of wastewater surfacing.

  15. Surfacing of domestic wastewater applied to soil through drip tubing and reduction in numbers of Escherichia coli.

    PubMed

    Franti, J M; Weaver, R W; McInnes, K J

    2002-09-01

    Drip tubing is a technology that is increasing in use. The effectiveness of such systems in distributing the wastewater uniformly through the soil matrix, providing adequate removal of bacteria from wastewater, and keeping wastewater from reaching the soil surface has not been adequately evaluated. Experiments were conducted at two sites that had used drip tubing for approximately 3 years. This 3-year-old drip tubing and newly installed tubing were used in this investigation. A solution containing Brilliant Blue FCF dye and Escherichia coli, at an approximate concentration of 1 x 10(6) cells ml(-1), was applied to the sites through drip emitters. Reduction i n Escherichia coli populations reaching the soil surface was generally less than 10%. The route of travel for the solution reaching the soil surface was consistently along preferential flow paths and not uniformly through the soil matrix. Instances of water reaching the soil surface for drip tubing installed at 15 cm was nearly 50%. Increasing burial of the drip tubing from 15 to 30 cm nearly eliminated water surfacing. A 31 per emitter dose of water, immediately following drip line installation later increased instances of water reaching the soil surface for drip tubing buried at 30 cm. The volume of water applied per dose had little effect on the number of times water reached the soil surface. Inherent soil structural characteristics limited the drip tubing's ability to uniformly distribute water and adsorb bacteria. Drip tubing installation to 30 cm may be an important practice to reduce public health hazards from the likelihood of wastewater surfacing. PMID:12361375

  16. Morphological and nanostructural surface changes in Escherichia coli over time, monitored by atomic force microscopy.

    PubMed

    Gammoudi, Ibtissem; Mathelie-Guinlet, Marion; Morote, Fabien; Beven, Laure; Moynet, Daniel; Grauby-Heywang, Christine; Cohen-Bouhacina, Touria

    2016-05-01

    The present study aims at evaluating intrinsic changes in Escherichia coli (E. coli) surface over time, by Atomic Force Microscopy (AFM). For that purpose, bacteria were immobilized on mica or on mica previously functionalized by the deposition of a polyelectrolyte multilayer cushion. AFM images reveal that E. coli population goes through different stages. Firstly, after a week, the number of healthy bacteria decreases resulting in a release of cellular components which likely become, in turn, a nutrition source for increasing the healthy population after around two weeks. Finally, after one month, most of the bacteria is dead. Our study shows a transition of a healthy rod-shaped bacterium to a dead collapsed one. Most importantly, along with the morphological evolution of bacteria, are the structure changes and the mechanical properties of their outer membrane, emphasized by AFM phase images with very high resolution. Indeed, the surface of healthy bacteria is characterized by a phase separation pattern, thereafter mentioned as "ripples". Bacterial ageing goes along with the loss of this organized structure, turning into circular areas with irregular boundaries. These changes are likely caused by a re-organization, due to external stress, of mainly lipopolysaccharides (LPS) present in the outer membrane of E. coli. PMID:26878286

  17. Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource.

    PubMed

    Ko, Kyong-Cheol; Lee, Binna; Cheong, Dae-Eun; Han, Yunjon; Choi, Jong Hyun; Song, Jae Jun

    2015-11-01

    A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

  18. Effects of saponins against clinical E. coli strains and eukaryotic cell line.

    PubMed

    Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

    2012-01-01

    Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12-50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. PMID:22500084

  19. Modeling ultraviolet-induced SOS response in translesion synthesis-deficient cells of Escherichia Coli bacteria

    NASA Astrophysics Data System (ADS)

    Belov, O. V.

    2011-01-01

    A quantitative analysis is performed of SOS response induced by ultraviolet radiation in Escherichia coli bacterial cells with the disordered function of translesion synthesis. The dynamics of the concentration of the basic SOS proteins is estimated for the recA, umuD, and umuC mutants of E. coli. The estimation is based on the model approaches developed earlier.

  20. Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology

    PubMed Central

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Background Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes. Objectives This investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene in Escherichia coli BL21 (DE3). Materials and Methods The hES gene was amplified, cloned, and expressed in the E. coli expression system. Three factors, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, post-induction time, and cell density before induction, were selected as important factors. The mRNA expression level was determined using real-time PCR. The expression levels of hES mRNA under the different growth conditions were analyzed. SDS-PAGE and western blot analyses were carried out for further confirmation of interest-gene expression. Results A maximum rhES mRNA level of 376.16% was obtained under the following conditions: 0.6 mM IPTG, 7 hours post-induction time, and 0.9 cell density before induction. The level of rhES mRNA was significantly correlated with post-induction time, IPTG concentration, and cell density before induction (P < 0.05). The expression of the hES gene was confirmed by western blot. Conclusions The obtained results indicate that RSM is an effective method for the optimization of culture conditions for hES gene expression in E. coli. PMID:27800134

  1. Inhibition of Escherichia coli and Proteus mirabilis adhesion and biofilm formation on medical grade silicone surface.

    PubMed

    Wang, Rong; Neoh, Koon Gee; Shi, Zhilong; Kang, En-Tang; Tambyah, Paul Anantharajah; Chiong, Edmund

    2012-02-01

    Silicone has been utilized extensively for biomedical devices due to its excellent biocompatibility and biodurability properties. However, its surface is easily colonized by bacteria which will increase the probability of nosocomial infection. In the present work, a hydrophilic antimicrobial carboxymethyl chitosan (CMCS) layer has been grafted on medical grade silicone surface pre-treated with polydopamine (PDA). The increase in hydrophilicity was confirmed from contact angle measurement. Bacterial adhesion tests showed that the PDA-CMCS coating reduced the adhesion of Escherichia coli and Proteus mirabilis by ≥ 90%. The anti-adhesion property was preserved even after the aging of the functionalized surfaces for 21 days in phosphate-buffered saline (PBS), and also after autoclaving at 121°C for 20 min. Both E. coli and P. mirabilis readily form biofilms on the pristine surface under static and flow conditions but with the PDA-CMCS layer, biofilm formation is inhibited. The flow experiments indicated that it is more difficult to inhibit biofilm formation by the highly motile P. mirabilis as compared to E. coli. No significant cytotoxicity of the modified substrates was observed with 3T3 fibroblasts. PMID:21956834

  2. Inhibition of Escherichia coli and Proteus mirabilis adhesion and biofilm formation on medical grade silicone surface.

    PubMed

    Wang, Rong; Neoh, Koon Gee; Shi, Zhilong; Kang, En-Tang; Tambyah, Paul Anantharajah; Chiong, Edmund

    2012-02-01

    Silicone has been utilized extensively for biomedical devices due to its excellent biocompatibility and biodurability properties. However, its surface is easily colonized by bacteria which will increase the probability of nosocomial infection. In the present work, a hydrophilic antimicrobial carboxymethyl chitosan (CMCS) layer has been grafted on medical grade silicone surface pre-treated with polydopamine (PDA). The increase in hydrophilicity was confirmed from contact angle measurement. Bacterial adhesion tests showed that the PDA-CMCS coating reduced the adhesion of Escherichia coli and Proteus mirabilis by ≥ 90%. The anti-adhesion property was preserved even after the aging of the functionalized surfaces for 21 days in phosphate-buffered saline (PBS), and also after autoclaving at 121°C for 20 min. Both E. coli and P. mirabilis readily form biofilms on the pristine surface under static and flow conditions but with the PDA-CMCS layer, biofilm formation is inhibited. The flow experiments indicated that it is more difficult to inhibit biofilm formation by the highly motile P. mirabilis as compared to E. coli. No significant cytotoxicity of the modified substrates was observed with 3T3 fibroblasts.

  3. Cell surface engineering of industrial microorganisms for biorefining applications.

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed.

  4. Cell surface engineering of industrial microorganisms for biorefining applications.

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed. PMID:26070720

  5. Atomic force microscopy study on the attachment of E. coli and S. aureus to a patterned surface of different materials

    NASA Astrophysics Data System (ADS)

    Zhang, Hailong; Komaromy, Andras; Boysen, Reinhard I.; Rius, Gemma; Borrise, Xavier; Perez-Murano, Francesc; Hearn, Milton T. W.; Nicolau, Dan V.

    2007-12-01

    Many bacterial species are able to colonize the surfaces of biomedical tools or devices and form biofilms creating a source of infection and other deleterious effects. Biofilms constitute environments in which bacteria grow and are protected from the host's immune system and antimicrobial medications. The bacterial adhesion, which is an important and first step in biofilm formation, is influenced by several physico-chemical and topographical factors at the interfaces between the bacterial cell and the surface. Therefore, the mechanism of initial adhesion needs to be investigated to better understand the events of anchorage and film formation as bacteria colonise surfaces. In this work, atomic force microscopy (AFM) in the tapping mode of imaging has been employed to investigate the attachment of bacteria onto a structured surface patterned with different hydrophilic and hydrophobic areas. The interactions of Escherichia coli and Staphylococcus aureus with these structures were also monitored by fluorescence microscopy. AFM was successfully employed for the study of the cell responses to both nanotopography and the surface chemistry via observation of various cell functions; including extracellular polymeric substance (EPS) mediated cellular adhesion.

  6. Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells.

    PubMed

    Kawasaki, Y; Tazume, S; Shimizu, K; Matsuzawa, H; Dosako, S; Isoda, H; Tsukiji, M; Fujimura, R; Muranaka, Y; Isihida, H

    2000-02-01

    Adherence is an essential and prerequisite step for the colonization of mucosal surfaces by enterotoxigenic Escherichia coli (ETEC). We studied the effect of bovine lactoferrin (BLF) on the adherence of ETEC to human epithelial cells in vitro, and to intestinal mucosa of ICR germfree mice in vivo. In the in vitro study, BLF was found to inhibit the adherence of ETEC. This adhesion-inhibiting activity of BLF was found to lessen with decreasing BLF concentration, but the data obtained suggest a positive inhibitory effect of BLF against the adhesion of ETEC cells. In the in vivo study, the counts of adherent bacteria in various sections of the intestinal tract (duodenum, jejunoileum, and large intestine) were lower in the BLF group than in the control group, suggesting the possible action of BLF as an intestinal tract adherence-blocking agent with regards to ETEC.

  7. Structure of the cell surface.

    PubMed

    Singer, S J

    1982-01-01

    The cell surface is the locus for many important biochemical functions of cells and for the interactions of cells with one another and with their environment. The structure of the cell surface may be thought of as three-layered, with a central plasma membrane to which certain macromolecular components are attached on the outer face (the exoskeleton) and other components on the inner face (the membrane cytoskeleton). In the last decade, the basic molecular structure of the plasma membrane has been elucidated and can be represented by the fluid mosaic model as a first approximation. The binding of specific integral proteins of the membrane to individual peripheral proteins outside or inside the cell is most likely the basis for the three-layered structure of the cell surface. Studies of the last several years on the molecular structures of these three-layered cell surfaces of cultured normal fibroblasts and of fibroblasts transformed by oncogenic viruses are beginning to shed light on the molecular mechanisms responsible for changes in cell shape, adhesiveness, and in contact inhibition of motility associated with neoplastic transformation.

  8. Survival of artificially inoculated Escherichia coli and Salmonella Typhimurium on the surface of raw poultry products subjected to crust freezing.

    PubMed

    Chaves, B D; Han, I Y; Dawson, P L; Northcutt, J K

    2011-12-01

    Escherichia coli and Salmonella spp. are ubiquitous in the poultry production environment, and hence, their transmission to poultry products is of concern. Industry has widely used freezing as a strategy to halt pathogen growth, and more recently, crust freezing has been suggested as a means to improve mechanical operations, quality, and safety of poultry products. The purpose of this study was to evaluate the effect of crust freezing on the survival of Escherichia coli and Salmonella Typhimurium that were artificially inoculated on the surface of raw poultry products with or without adhering skin. Ampicillin-resistant (AR) E. coli JM 109 and nalidixic acid-resistant (NAR) Salmonella Typhimurium were used in the experiments. A set of cultures was subjected to cold-shock stress by storage at 4°C for 10 d. After being either cold-shocked or non-cold-shocked, commercial chicken breasts without skin and chicken thighs with skin were inoculated in separate experiments with each bacterium. Samples were crust frozen at -85°C for 20 min or completely frozen at -85°C for 60 min. The E. coli and Salmonella Typhimurium were recovered on appropriate selective and nonselective media containing the corresponding antibiotic. Log reductions and extent of injury were calculated and treatments were compared using ANOVA. No significant differences were observed in the reduction of cold-shocked or non-cold-shocked bacteria on products with or without skin that were crust or completely frozen. The average reduction for E. coli was 0.15 log(10) cfu/mL of rinse, and for Salmonella Typhimurium 0.10 log(10) cfu/mL of rinse; therefore, none of the final reductions were greater than the desired target (1 log). Bacterial cell injury was not significantly different (P > 0.05) among any of the treatments. Data showed no practical significance for initial reduction of these pathogens from crust freezing and thus, this technology should not be considered as a strategy for the reduction of E

  9. Penicillin-binding site on the Escherichia coli cell envelope

    SciTech Connect

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-08-01

    The binding of /sup 35/S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin.

  10. Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli.

    PubMed

    Gujral, Tarunmeet; Kumar, Anoop; Priyamvada, Shubha; Saksena, Seema; Gill, Ravinder K; Hodges, Kim; Alrefai, Waddah A; Hecht, Gail A; Dudeja, Pradeep K

    2015-12-15

    Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl(-)/HCO3(-) exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl(-)/HCO3(-) exchange activity was measured as DIDS-sensitive (125)I(-) uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl(-)/HCO3(-) exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.

  11. Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

    PubMed Central

    Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

    2002-01-01

    We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

  12. Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.

    PubMed

    Alonso, Jose L; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A; Hernández, Javier

    2002-10-01

    We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

  13. Phenotypic and Genotypic Characterization of Escherichia coli Isolated from Untreated Surface Waters

    PubMed Central

    Janezic, Kristopher J; Ferry, Blake; Hendricks, Eric W; Janiga, Brian A; Johnson, Tiffany; Murphy, Samantha; Roberts, Morgan E; Scott, Sarah M; Theisen, Alexandra N; Hung, Kai F; Daniel, Steven L

    2013-01-01

    A common member of the intestinal microbiota in humans and animals is Escherichia coli. Based on the presence of virulence factors, E. coli can be potentially pathogenic. The focus of this study was to isolate E. coli from untreated surface waters (37 sites) in Illinois and Missouri and determine phenotypic and genotypic diversity among isolates. Water samples positive for fecal coliforms based on the Colisure® test were streaked directly onto Eosin Methylene Blue (EMB) agar (37°C) or transferred to EC broth (44.5°C). EC broth cultures producing gas were then streaked onto EMB agar. Forty-five isolates were identified as E. coli using API 20E and Enterotube II identification systems, and some phenotypic variation was observed in metabolism and fermentation. Antibiotic susceptibility of each isolate was also determined using the Kirby-Bauer Method. Differential responses to 10 antimicrobial agents were seen with 7, 16, 2, and 9 of the isolates resistant to ampicillin, cephalothin, tetracycline, and triple sulfonamide, respectively. All of the isolates were susceptible or intermediate to amoxicillin, ciprofloxacin, polymyxin B, gentamicin, imipenem, and nalidixic acid. Genotypic variation was assessed through multiplex Polymerase Chain Reaction for four virulence genes (stx1 and stx2 [shiga toxin], eaeA [intimin]; and hlyA [enterohemolysin]) and one housekeeping gene (uidA [β-D-glucuronidase]). Genotypic variation was observed with two of the isolates possessing the virulence gene (eaeA) for intimin. These findings increase our understanding of the diversity of E. coli in the environment which will ultimately help in the assessment of this organism and its role in public health. PMID:23539437

  14. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

    PubMed

    Frankel, G; Lider, O; Hershkoviz, R; Mould, A P; Kachalsky, S G; Candy, D C; Cahalon, L; Humphries, M J; Dougan, G

    1996-08-23

    Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin. PMID:8702771

  15. Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

    PubMed

    Zhang, Zhen; Tang, Rongxin; Bian, Lu; Mei, Meng; Li, Chunhua; Ma, Xiangdong; Yi, Li; Ma, Lixin

    2016-01-01

    Ice nucleation protein (INP) is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N) to improve its surface display efficiency for human Arginase1 (ARG1). Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1) by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg) to L-Ornithine (L-Orn) in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells. PMID:27479442

  16. Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli

    PubMed Central

    Zhang, Zhen; Tang, Rongxin; Bian, Lu; Mei, Meng; Li, Chunhua; Ma, Xiangdong; Yi, Li; Ma, Lixin

    2016-01-01

    Ice nucleation protein (INP) is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N) to improve its surface display efficiency for human Arginase1 (ARG1). Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1) by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg) to L-Ornithine (L-Orn) in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells. PMID:27479442

  17. Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions.

    PubMed

    Kansal, Rita; Rasko, David A; Sahl, Jason W; Munson, George P; Roy, Koushik; Luo, Qingwei; Sheikh, Alaullah; Kuhne, Kurt J; Fleckenstein, James M

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens. PMID:23115039

  18. Adhesion of E. coli to silver- or copper-coated porous clay ceramic surfaces

    NASA Astrophysics Data System (ADS)

    Yakub, I.; Soboyejo, W. O.

    2012-06-01

    Porous ceramic water filters (CWFs), produced by sintering a mixture of clay and a combustible material (such as woodchips), are often used in point-of-use water filtration systems that occlude microbes by size exclusion. They are also coated with colloidal silver, which serves as a microbial disinfectant. However, the adhesion of microbes to porous clay surfaces and colloidal silver coated clay surfaces has not been studied. This paper presents the results of atomic force microscopy (AFM) measurements of the adhesion force between Escherichia coli bacteria, colloidal silver, and porous clay-based ceramic surfaces. The adhesion of silver and copper nanoparticles is also studied in control experiments on these alternative disinfectant materials. The adhesive force between the wide range of possible bi-materials was measured using pull-off measurements during force microscopy. These were combined with measurements of AFM tip radii/substrate roughness that were incorporated into adhesion models to obtain the adhesion energies for the pair wise interaction. Of the three antimicrobial metals studied, the colloidal silver had the highest affinity for porous ceramic surface (125 ± 32 nN and ˜0.29 J/m2) while the silver nanoparticles had the highest affinity for E. coli bacteria (133 ± 21 nN and ˜0.39 J/m2). The implications of the results are then discussed for the design of ceramic water filter that can purify water by adsorption and size exclusion.

  19. Probing roles of lipopolysaccharide, type 1 fimbria, and colanic acid in the attachment of Escherichia coli strains on inert surfaces.

    PubMed

    Chao, Yuanqing; Zhang, Tong

    2011-09-20

    The roles of bacterial surface polymers in reversible (phase I) and irreversible (phase II) attachment (i.e., lipopolysaccharides (LPS), type 1 fimbria, and capsular colanic acid (CA)) were investigated in situ by combining fluorescence microscopy and atomic force microscopy. Fluorescence microscopy was used to evaluate the phase I attachment by counting the total number of cells on the substrata, and AFM was applied to image the phase II cells and measure the lateral detachment force to characterize phase II attachment. Also, by comparing the number of cells in phases I and II, the transformation ratio was calculated and used as an index to evaluate the roles of different polymers in the attachment process. Escherichia coli K-12 and its six mutants, which had different surface polymers in terms of LPS structures, CA contents, and type 1 fimbriae, were used as the test strains. Six different materials were applied as substrata, including glass, two metals (aluminum and stainless steel), and three plastics (polyvinyl chloride, polycarbonate, and polyethylene). The results indicated that LPS significantly enhanced phases I and II attachment as well as the transformation ratio from phase I to II. Like LPS, type 1 fimbriae largely increased the phase I attachment and the transformation ratio; however, they did not significantly influence the adhesion strength in phase II. CA had a negative effect on attachment in phases I and II by decreasing the adhered number of cells and the lateral detachment force, respectively, but had no influence on the transformation ratio. PMID:21842859

  20. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

    PubMed

    Ojima, Yoshihiro; Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-09-01

    Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.

  1. Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

    PubMed

    Ojima, Yoshihiro; Nguyen, Minh Hong; Yajima, Reiki; Taya, Masahito

    2015-09-01

    Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells. PMID:26092467

  2. Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

    NASA Technical Reports Server (NTRS)

    Kacena, M. A.; Smith, E. E.; Todd, P.

    1999-01-01

    The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

  3. Comparative Study of Various E. coli Strains for Biohydrogen Production Applying Response Surface Methodology

    PubMed Central

    Bakonyi, Péter; Nemestóthy, Nándor; Bélafi-Bakó, Katalin

    2012-01-01

    The proper strategy to establish efficient hydrogen-producing biosystems is the biochemical, physiological characterization of hydrogen-producing microbes followed by metabolic engineering in order to give extraordinary properties to the strains and, finally, bioprocess optimization to realize enhanced hydrogen fermentation capability. In present paper, it was aimed to show the utility both of strain engineering and process optimization through a comparative study of wild-type and genetically modified E. coli strains, where the effect of two major operational factors (substrate concentration and pH) on bioH2 production was investigated by experimental design and response surface methodology (RSM) was used to determine the suitable conditions in order to obtain maximum yields. The results revealed that by employing the genetically engineered E. coli (DJT 135) strain under optimized conditions (pH: 6.5; Formate conc.: 1.25 g/L), 0.63 mol H2/mol formate could be attained, which was 1.5 times higher compared to the wild-type E. coli (XL1-BLUE) that produced 0.42 mol H2/mol formate (pH: 6.4; Formate conc.: 1.3 g/L). PMID:22666156

  4. A rapid detection method of Escherichia coli by surface enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Tao, Feifei; Peng, Yankun; Xu, Tianfeng

    2015-05-01

    Conventional microbiological detection and enumeration methods are time-consuming, labor-intensive, and giving retrospective information. The objectives of the present work are to study the capability of surface enhanced Raman scattering (SERS) to detect Escherichia coli (E. coli) using the presented silver colloidal substrate. The obtained results showed that the adaptive iteratively reweighed Penalized Least Squares (airPLS) algorithm could effectively remove the fluorescent background from original Raman spectra, and Raman characteristic peaks of 558, 682, 726, 1128, 1210 and 1328 cm-1 could be observed stably in the baseline corrected SERS spectra of all studied bacterial concentrations. The detection limit of SERS could be determined to be as low as 0.73 log CFU/ml for E. coli with the prepared silver colloidal substrate. The quantitative prediction results using the intensity values of characteristic peaks were not good, with the correlation coefficients of calibration set and cross validation set of 0.99 and 0.64, respectively.

  5. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  6. Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods

    PubMed Central

    Ramasamy, Mohankandhasamy; Yi, Dong Kee; An, Seong Soo A

    2015-01-01

    Escherichia coli O157:H7 (O157) is a Gram negative and highly virulent bacteria found in food and water sources, and is a leading cause of chronic diseases worldwide. Diagnosis and prevention from the infection require simple and rapid analysis methods for the detection of pathogens, including O157. Endogenous membrane peroxidase, an enzyme present on the surface of O157, was used for the colorimetric detection of bacteria by catalytic oxidation of the peroxidase substrate. In this study, we have analyzed the impact of the synthesized bare gold nanorods (AuNRs) and silica-coated AuNRs on the growth of E. coli O157. Along with the membrane peroxidase activity of O157, other bacteria strains were analyzed. Different concentrations of nanorods were used to analyze the growth responses, enzymatic changes, and morphological alterations of bacteria by measuring optical density, 3,3′,5,5′-tetramethylbenzidine assay, flow cytometry analysis, and microscopy studies. The results revealed that O157 showed higher and continuous membrane peroxidase activity than other bacteria. Furthermore, O157 treated with bare AuNRs showed a decreased growth rate in comparison with the bacteria with surface modified AuNRs. Interestingly, silica-coated AuNRs favored the growth of bacteria and also increased membrane peroxidase activity. This result can be particularly important for the enzymatic analysis of surface treated AuNRs in various microbiological applicants. PMID:26347081

  7. Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods.

    PubMed

    Ramasamy, Mohankandhasamy; Yi, Dong Kee; An, Seong Soo A

    2015-01-01

    Escherichia coli O157:H7 (O157) is a Gram negative and highly virulent bacteria found in food and water sources, and is a leading cause of chronic diseases worldwide. Diagnosis and prevention from the infection require simple and rapid analysis methods for the detection of pathogens, including O157. Endogenous membrane peroxidase, an enzyme present on the surface of O157, was used for the colorimetric detection of bacteria by catalytic oxidation of the peroxidase substrate. In this study, we have analyzed the impact of the synthesized bare gold nanorods (AuNRs) and silica-coated AuNRs on the growth of E. coli O157. Along with the membrane peroxidase activity of O157, other bacteria strains were analyzed. Different concentrations of nanorods were used to analyze the growth responses, enzymatic changes, and morphological alterations of bacteria by measuring optical density, 3,3',5,5'-tetramethylbenzidine assay, flow cytometry analysis, and microscopy studies. The results revealed that O157 showed higher and continuous membrane peroxidase activity than other bacteria. Furthermore, O157 treated with bare AuNRs showed a decreased growth rate in comparison with the bacteria with surface modified AuNRs. Interestingly, silica-coated AuNRs favored the growth of bacteria and also increased membrane peroxidase activity. This result can be particularly important for the enzymatic analysis of surface treated AuNRs in various microbiological applicants. PMID:26347081

  8. Cell-density dependent effects of low-dose ionizing radiation on E. coli cells.

    PubMed

    Alipov, E D; Shcheglov, V S; Sarimov, R M; Belyaev, I Ya

    2003-01-01

    The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.

  9. Multidrug-Resistant and Extended Spectrum Beta-Lactamase-Producing Escherichia coli in Dutch Surface Water and Wastewater

    PubMed Central

    Blaak, Hetty; Lynch, Gretta; Italiaander, Ronald; Hamidjaja, Raditijo A.; Schets, Franciska M.; de Roda Husman, Ana Maria

    2015-01-01

    Objective The goal of the current study was to gain insight into the prevalence and concentrations of antimicrobial resistant (AMR) Escherichia coli in Dutch surface water, and to explore the role of wastewater as AMR contamination source. Methods The prevalence of AMR E. coli was determined in 113 surface water samples obtained from 30 different water bodies, and in 33 wastewater samples obtained at five health care institutions (HCIs), seven municipal wastewater treatment plants (mWWTPs), and an airport WWTP. Overall, 846 surface water and 313 wastewater E. coli isolates were analysed with respect to susceptibility to eight antimicrobials (representing seven different classes): ampicillin, cefotaxime, tetracycline, ciprofloxacin, streptomycin, sulfamethoxazole, trimethoprim, and chloramphenicol. Results Among surface water isolates, 26% were resistant to at least one class of antimicrobials, and 11% were multidrug-resistant (MDR). In wastewater, the proportions of AMR/MDR E. coli were 76%/62% at HCIs, 69%/19% at the airport WWTP, and 37%/27% and 31%/20% in mWWTP influents and effluents, respectively. Median concentrations of MDR E. coli were 2.2×102, 4.0×104, 1.8×107, and 4.1×107 cfu/l in surface water, WWTP effluents, WWTP influents and HCI wastewater, respectively. The different resistance types occurred with similar frequencies among E. coli from surface water and E. coli from municipal wastewater. By contrast, among E. coli from HCI wastewater, resistance to cefotaxime and resistance to ciprofloxacin were significantly overrepresented compared to E. coli from municipal wastewater and surface water. Most cefotaxime-resistant E. coliisolates produced ESBL. In two of the mWWTP, ESBL-producing variants were detected that were identical with respect to phylogenetic group, sequence type, AMR-profile, and ESBL-genotype to variants from HCI wastewater discharged onto the same sewer and sampled on the same day (A1/ST23/CTX-M-1, B23/ST131/CTX-M-15, D2/ST405/CTX

  10. Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions.

    PubMed

    Costa, Suelen B; Campos, Ana Carolina C; Pereira, Ana Claudia M; de Mattos-Guaraldi, Ana Luiza; Júnior, Raphael Hirata; Rosa, Ana Cláudia P; Asad, Lídia M B O

    2014-09-01

    During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the

  11. Fabrication and characterization of high power dual chamber E. coli microbial fuel cell

    NASA Astrophysics Data System (ADS)

    Lalitha Priya, R.; Ramachandran, T.; Suneesh, P. V.

    2016-09-01

    This work reports the fabrication of a dual chamber microbial fuel cell with E. coli modified graphite as the anode and lead dioxide cathode. At the optimized operating conditions, the cell provided 778 mV open circuit potential, 3.47 mA m-2 of current density and 1660 mW m-2 power density. Morphology of the of E. coli biofilm on the electrode was analysed using AFM and the electrochemical characterization of the fuel cell was carried out using electrochemical impedance spectroscopy (EIS) and polarization curves. The composition of the anode and the time duration for E. coli biofilm formation were varied to obtain maximum power density. The MFC fabricated in this study was found to have improved power density in comparison with other reported fuel cells.

  12. Functional display of triphenylmethane reductase for dye removal on the surface of Escherichia coli using N-terminal domain of ice nucleation protein.

    PubMed

    Gao, Fen; Ding, Haitao; Feng, Zhuo; Liu, Danfeng; Zhao, Yuhua

    2014-10-01

    Traditional biological treatment for triphenylmethane dye effluent is stuck with the inaccessibility of dye molecules to intracellular dye-degrading enzyme, thus a high-efficiency and low-cost method for dye decolorization is highly desirable. Here we established a bioremediation approach to display triphenylmethane reductase (TMR) on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif for triphenylmethane dye decolorization for the first time. Approximately 85% of recombinant protein positioning on the surface of E. coil cells exhibited high activity and stability. The optimal temperature and pH of the surface-displayed TMR are 50 °C and 8.5, respectively. Comparing with other reported microorganisms, the decolorization rate for malachite green of this engineered strain is the highest so far, reaching 640 μmol min(-1) g(-1) dry weight cells. These results indicate that this engineered E. coli strain is a very promising candidate for synthetic dye removal. PMID:25058292

  13. Functional display of triphenylmethane reductase for dye removal on the surface of Escherichia coli using N-terminal domain of ice nucleation protein.

    PubMed

    Gao, Fen; Ding, Haitao; Feng, Zhuo; Liu, Danfeng; Zhao, Yuhua

    2014-10-01

    Traditional biological treatment for triphenylmethane dye effluent is stuck with the inaccessibility of dye molecules to intracellular dye-degrading enzyme, thus a high-efficiency and low-cost method for dye decolorization is highly desirable. Here we established a bioremediation approach to display triphenylmethane reductase (TMR) on the surface of Escherichia coli (E. coli) using N-terminal of ice nucleation protein as anchoring motif for triphenylmethane dye decolorization for the first time. Approximately 85% of recombinant protein positioning on the surface of E. coil cells exhibited high activity and stability. The optimal temperature and pH of the surface-displayed TMR are 50 °C and 8.5, respectively. Comparing with other reported microorganisms, the decolorization rate for malachite green of this engineered strain is the highest so far, reaching 640 μmol min(-1) g(-1) dry weight cells. These results indicate that this engineered E. coli strain is a very promising candidate for synthetic dye removal.

  14. Mechanical damage to Escherichia coli cells in a model of amino-acid crystal fermentation.

    PubMed

    Okutani, Satoshi; Iwai, Takayoshi; Iwatani, Shintaro; Kondo, Kazuya; Osumi, Tsuyoshi; Tsujimoto, Nobuharu; Matsuno, Kiyoshi

    2012-04-01

    We investigated the mechanical damage to the Escherichia coli cell caused by polyvinyl chloride particles as a model of amino-acid crystal fermentation. Our results indicated that the glucose-consumption rate and the intracellular ATP concentration temporarily increased by the mechanical damage, and decreased after considerable damage had occurred on cell membrane. PMID:22153714

  15. E. coli fate and transport in the Happel sphere-in-cell model

    NASA Astrophysics Data System (ADS)

    Nelson, K. E.; Massoudieh, A.; Ginn, T. R.

    2007-06-01

    Rates of mass and gene transfer reactions involving biotic phases are often expressed as proportional to local number densities of bacteria. When the reactions involve attached bacteria, reaction rates depend on local densities of bacteria attached to surfaces. Such may be the case with microbially-facilitated redox reactions involving mineral electron donors and mineral electron receivers (e.g., Sani RK, Peyton BM, Amonette JE, Dohnalkova A. Reoxidation of uranium in the presence of iron(III)-(hydr)oxides under sulfate reducing conditions. Environ Sci Technol 2005;39:2059-66), biofilm formation induced by quorum sensing (Purevdorj B, Costerton JW, Stoodley P. Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa Biofilms. Appl Environ Microbiol 2002;68(9):4457-64) and horizontal gene transfer among attached phase bacteria (Beaudoin DL, Bryers JD, Cunningham AB, Peretti SW. Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. I. Experiments. Biotech Bioeng 1998a;57(3):272-79; Beaudoin DL, Bryers JD, Cunningham AB, Peretti SW. Mobilization of broad host range plasmid from Pseudomonas putida to established biofilm of Bacillus azotoformans. II. Modeling Biotech Bioeng 1998b;57(3):280-86). Here we use the conceptual Happel sphere-in-cell model to determine the microscopic distribution of attached bacteria on idealized spherical grains of porous media, assuming azimuthal symmetry. We extend a Lagrangian model of colloid filtration to investigate the effects of motility of Escherichia coli on attachment rate and on the attachment distribution as a function of location on grain surface. The hydrodynamics of the Happel model is implicitly 3D and represented in 2D polar coordinates under the assumption of axisymmetric flow, while the motility of the E. coli cells is explicitly 3D. The model incorporates the fate and transport processes of colloid filtration theory in

  16. Hydrodynamics of Nutrients and E. coli Deposit and Resuspension in Surface Water

    NASA Astrophysics Data System (ADS)

    Liu, L.; Wang, C.; Wang, J.; Jiang, W.; Xia, Z.

    2015-12-01

    A significant part of nutrients (such as N, P) and Pathogens (E. coli as indicator) attached to sediment or biofilm deposit to bed when they transport in streams to form the secondary contaminant source with much longer lifetime. The deposited contaminants may resuspend and the biofilm where pathogens live may detach from the bed to water body. The deposition and resuspension substantially increase the complexity of the transport process, and make it more difficult and uncertain to accurately simulate and predict the fate and transport of contaminants in surface waters. In this research, a three-dimensional hydrodynamic-water quality model coupled with flume experiments are planned to be utilized to quantify the influence of flow conditions on nitrogen, phosphorous and E. coli deposition and resuspension, and to investigate the impact of shear stress caused by flow on the biostability of biofilm. This research will facilitate to better understand the fate and transport of pathogens in surface waters and provide a theoretical base for pathogen prediction and forecasting.

  17. Attachment of Escherichia coli O157:H7 to the Surfaces and Internal Structures of Apples as Detected by Confocal Scanning Laser Microscopy

    PubMed Central

    Burnett, Scott L.; Chen, Jinru; Beuchat, Larry R.

    2000-01-01

    Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25°C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25°C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 μm below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 μm. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7

  18. Comparison of the organophosphorus hydrolase surface display using InaVN and Lpp-OmpA systems in Escherichia coli.

    PubMed

    Karami, Ali; Latifi, Ali Mohammad; Khodi, Samaneh

    2014-03-28

    The purpose of this study was to compare the ability of an engineered Escherichia coli to degrade chlorpyrifos (Cp) using an organophosphorus hydrolase enzyme by employing the Lpp-OmpA chimera and the N-terminal domain of the ice nucleation protein as anchoring motifs. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by both anchors on the outer membrane. This is the first report on the presentation of OPH on the cell surface by Lpp-OmpA under the control of the T7 promoter. The results showed cell growth in the presence of Cp as the sole source of energy, without growth inhibition, and with higher whole-cell activity for both cells harboring plasmids pENVO and pELMO, at approximately 10,342.85 and 10,857.14 U/mg, respectively. Noticeably, the protein displayed by pELMO was lower than the protein displayed by pENVO. It can be concluded that Lpp-OmpA can display less protein, but more functional OPH protein. These results highlight the high potential, of both engineered bacteria, for use in the bioremediation of pesticide-contaminated sources in the environment. PMID:24150492

  19. Assessment of the effects of holding time and temperature on Escherichia coli densities in surface water samples.

    PubMed

    Pope, Misty L; Bussen, Michelle; Feige, Mary Ann; Shadix, Lois; Gonder, Sharon; Rodgers, Crystal; Chambers, Yildiz; Pulz, Jessica; Miller, Ken; Connell, Kevin; Standridge, Jon

    2003-10-01

    Escherichia coli is a routinely used microbiological indicator of water quality. To determine whether holding time and storage conditions had an effect on E. coli densities in surface water, studies were conducted in three phases, encompassing 24 sites across the United States and four commonly used monitoring methods. During all three phases of the study, E. coli samples were analyzed at time 0 and at 8, 24, 30, and 48 h after sample collection. During phase 1, when 4 degrees C samples were evaluated by Colilert or by placing a membrane onto mFC medium followed by transfer to nutrient agar containing 4-methylumbelliferyl-beta-D-glucuronide (mFC/NA-MUG), three of four sites showed no significant differences throughout the 48-h study. During phase 2, five of seven sites showed no significant difference between time 0 and 24 h by membrane filtration (mFC/NA-MUG). When evaluated by the Colilert method, five of seven sites showed no significant difference in E. coli density between time 0 and 48 h. During phase 3, 8 of 13 sites showed no significant differences in E. coli densities between time 0 and the 48-h holding time, regardless of method. Based on the results of these studies, it appears that if samples are held below 10 degrees C and are not allowed to freeze, most surface water E. coli samples analyzed by commonly used methods beyond 8 h after sample collection can generate E. coli data comparable to those generated within 8 h of sample collection. Notwithstanding this conclusion, E. coli samples collected from surface waters should always be analyzed as soon as possible. PMID:14532081

  20. Lactobacillus acidophilus counteracts enteropathogenic E. coli-induced inhibition of butyrate uptake in intestinal epithelial cells.

    PubMed

    Kumar, Anoop; Alrefai, Waddah A; Borthakur, Alip; Dudeja, Pradeep K

    2015-10-01

    Butyrate, a key short-chain fatty acid metabolite of colonic luminal bacterial action on dietary fiber, serves as a primary fuel for the colonocytes, ameliorates mucosal inflammation, and stimulates NaCl absorption. Absorption of butyrate into the colonocytes is essential for these intracellular effects. Monocarboxylate transporter 1 (MCT1) plays a major role in colonic luminal butyrate absorption. Previous studies (Tan J, McKenzie C, Potamitis M, Thorburn AN, Mackay CR, Macia L. Adv Immunol 121: 91-119, 2014.) showed decreased MCT1 expression and function in intestinal inflammation. We have previously shown (Borthakur A, Gill RK, Hodges K, Ramaswamy K, Hecht G, Dudeja PK. Am J Physiol Gastrointest Liver Physiol 290: G30-G35, 2006.) impaired butyrate absorption in human intestinal epithelial Caco-2 cells due to decreased MCT1 level at the apical cell surface following enteropathogenic E. coli (EPEC) infection. Current studies, therefore, examined the potential role of probiotic Lactobacilli in stimulating MCT1-mediated butyrate uptake and counteracting EPEC inhibition of MCT1 function. Of the five species of Lactobacilli, short-term (3 h) treatment with L. acidophilus (LA) significantly increased MCT1-mediated butyrate uptake in Caco-2 cells. Heat-killed LA was ineffective, whereas the conditioned culture supernatant of LA (LA-CS) was equally effective in stimulating MCT1 function, indicating that the effects are mediated by LA-secreted soluble factor(s). Furthermore, LA-CS increased apical membrane levels of MCT1 protein via decreasing its basal endocytosis, suggesting that LA-CS stimulation of butyrate uptake could be secondary to increased levels of MCT1 on the apical cell surface. LA-CS also attenuated EPEC inhibition of butyrate uptake and EPEC-mediated endocytosis of MCT1. Our studies highlight distinct role of specific LA-secreted molecules in modulating colonic butyrate absorption. PMID:26272259

  1. Integration of AI-2 Based Cell-Cell Signaling with Metabolic Cues in Escherichia coli

    PubMed Central

    Mitra, Arindam; Herren, Christopher D.; Patel, Isha R.; Coleman, Adam; Mukhopadhyay, Suman

    2016-01-01

    The quorum sensing molecule Autoinducer-2 (AI-2) is generated as a byproduct of activated methyl cycle by the action of LuxS in Escherichia coli. AI-2 is synthesized, released and later internalized in a cell-density dependent manner. Here, by mutational analysis of the genes, uvrY and csrA, we describe a regulatory circuit of accumulation and uptake of AI-2. We constructed a single-copy chromosomal luxS-lacZ fusion in a luxS + merodiploid strain and evaluated its relative expression in uvrY and csrA mutants. At the entry of stationary phase, the expression of the fusion and AI-2 accumulation was positively regulated by uvrY and negatively regulated by csrA respectively. A deletion of csrA altered message stability of the luxS transcript and CsrA protein exhibited weak binding to 5’ luxS regulatory region. DNA protein interaction and chromatin immunoprecipitation analysis confirmed direct interaction of UvrY with the luxS promoter. Additionally, reduced expression of the fusion in hfq deletion mutant suggested involvement of small RNA interactions in luxS regulation. In contrast, the expression of lsrA operon involved in AI-2 uptake, is negatively regulated by uvrY and positively by csrA in a cell-density dependent manner. The dual role of csrA in AI-2 synthesis and uptake suggested a regulatory crosstalk of cell signaling with carbon regulation in Escherichia coli. We found that the cAMP-CRP mediated catabolite repression of luxS expression was uvrY dependent. This study suggests that luxS expression is complex and regulated at the level of transcription and translation. The multifactorial regulation supports the notion that cell-cell communication requires interaction and integration of multiple metabolic signals. PMID:27362507

  2. Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli Kills Cultured Human Uroepithelial 5637 Cells by an Apoptotic Mechanism

    PubMed Central

    Mills, Melody; Meysick, Karen C.; O'Brien, Alison D.

    2000-01-01

    Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with

  3. Persistence of Escherichia coli and Salmonella in surface soil following application of liquid hog manure for production of pickling cucumbers.

    PubMed

    Côté, Caroline; Quessy, Sylvain

    2005-05-01

    Liquid hog manure is routinely applied to farm land as a crop fertilizer. However, this practice raises food safety concerns, especially when manure is used on fruit and vegetable crops. The objectives of this project were to evaluate the persistence of Escherichia coli and Salmonella in surface soil after application of liquid hog manure to fields where pickling cucumbers were grown and to verify the microbiological quality of harvested cucumbers. Mineral fertilizers were replaced by liquid hog manure at various ratios in the production of pickling cucumbers in a 3-year field study. The experimental design was a randomized complete block comprising four replicates in sandy loam (years 1, 2, and 3) and loamy sand (year 3). Soil samples were taken at a depth of 20 cm every 2 weeks after June application of organic and inorganic fertilizers. Vegetable samples were also taken at harvest time. Liquid hog manure, soil, and vegetable (washed and unwashed) samples were analyzed for the presence of Salmonella and E. coli. An exponential decrease of E. coli populations was observed in surface soil after the application of manure. The estimated average time required to reach undetectable concentrations of E. coli in sandy loam varied from 56 to 70 days, whereas the absence of E. coli was estimated at 77 days in loamy sand. The maximal Salmonella persistence in soil was 54 days. E. coli and Salmonella were not detected in any vegetable samples.

  4. Human MAIT-cell responses to Escherichia coli: activation, cytokine production, proliferation, and cytotoxicity

    PubMed Central

    Dias, Joana; Sobkowiak, Michał J.; Sandberg, Johan K.; Leeansyah, Edwin

    2016-01-01

    Mucosa-associated invariant T cells are a large and relatively recently described innate-like antimicrobial T-cell subset in humans. These cells recognize riboflavin metabolites from a range of microbes presented by evolutionarily conserved major histocompatibility complex, class I-related molecules. Given the innate-like characteristics of mucosa-associated invariant T cells and the novel type of antigens they recognize, new methodology must be developed and existing methods refined to allow comprehensive studies of their role in human immune defense against microbial infection. In this study, we established protocols to examine a range of mucosa-associated invariant T-cell functions as they respond to antigen produced by Escherichia coli. These improved and dose- and time-optimized experimental protocols allow detailed studies of MR1-dependent mucosa-associated invariant T-cell responses to Escherichia coli pulsed antigen-presenting cells, as assessed by expression of activation markers and cytokines, by proliferation, and by induction of apoptosis and death in major histocompatibility complex, class I-related–expressing target cells. The novel and optimized protocols establish a framework of methods and open new possibilities to study mucosa-associated invariant T-cell immunobiology, using Escherichia coli as a model antigen. Furthermore, we propose that these robust experimental systems can also be adapted to study mucosa-associated invariant T-cell responses to other microbes and types of antigen-presenting cells. PMID:27034405

  5. Identification and functional annotation of mycobacterial septum formation genes using cell division mutants of Escherichia coli.

    PubMed

    Gaiwala Sharma, Sujata S; Kishore, Vimal; Raghunand, Tirumalai R

    2016-01-01

    The major virulence trait of Mycobacterium tuberculosis is its ability to enter a latent state in the face of robust host immunity. Clues to the molecular basis of latency can emerge from understanding the mechanism of cell division, beginning with identification of proteins involved in this process. Using complementation of Escherichia coli mutants, we functionally annotated M. tuberculosis and Mycobacterium smegmatis homologs of divisome proteins FtsW and AmiC. Our results demonstrate that E. coli can be used as a surrogate model to discover mycobacterial cell division genes, and should prove invaluable in delineating the mechanisms of this fundamental process in mycobacteria.

  6. Effect of surface roughness on performance of magnetoelastic biosensors for the detection of Escherichia coli.

    PubMed

    Possan, A L; Menti, C; Beltrami, M; Santos, A D; Roesch-Ely, M; Missell, F P

    2016-01-01

    Escherichia coli are bacteria that must be controlled in the food industry and the hospital sector. Magnetoelastic biosensors offer the promise of rapid identification of these and other harmful antigens. In this work, strips of amorphous Metglas 2826MB3 were cut to size (5 mm × 1 mm) with a microdicing saw and were then coated with thin layers of Cr and Au, as verified by Rutherford backscattering spectroscopy (RBS). Several sensor surfaces were studied: 1) as-cast strip, wheel side; 2) as-cast strip, free surface; and 3) thinned and polished surface. A layer of cystamine was applied to the Au-covered magnetoelastic substrate, forming a self-assembledmonolayer (SAM), followed by antibodies, using a modified Hermanson protocol. The cystamine layer growth was verified by Fourier transform infrared spectroscopy (FTIR) and scanning electronmicroscopy (SEM). The biosensors were exposed to solutions of bacteria and the resonant frequency of the sensors was measured with an impedance analyzer for times up to 100 min. Reductions in the resonant frequency, corresponding to bacteria capture, were measured after optimizing the signal amplitude. For times up to 40 min, high capture rates were observed and thereafter saturation occurred. Saturation values of the frequency shifts were compared with the number of bacteria observed on the sensor using fluorescence microscopy. Parameters associated with capture kinetics were studied for different sensor surfaces. The rough surfaces were found to show a faster response, while the thinned and polished sensors showed the largest frequency shift.

  7. Domain swapping oligomerization of thermostable c-type cytochrome in E. coli cells

    PubMed Central

    Hayashi, Yugo; Yamanaka, Masaru; Nagao, Satoshi; Komori, Hirofumi; Higuchi, Yoshiki; Hirota, Shun

    2016-01-01

    Knowledge on domain swapping in vitro is increasing, but domain swapping may not occur regularly in vivo, and its information in cells is limited. Herein, we show that domain-swapped oligomers of a thermostable c-type cytochrome, Hydrogenobacter thermophilus cyt c552, are formed in E. coli which expresses cyt c552. The region containing the N-terminal α-helix and heme was domain-swapped between protomers in the dimer formed in E. coli. The amount of cyt c552 oligomers increased in E. coli as the cyt c552 concentration was increased, whereas that of high-order oligomers decreased in the order of decrease in protein stability, indicating that domain swapping decreases in cells when the protein stability decreases. Apo cyt c552 was detected in the cyt c552 oligomer formed in E. coli, but not in that of the A5F/M11V/Y32F/Y41E/I76V mutant. The cyt c552 oligomer containing its apo protein may form at the periplasm, since the apo protein detected by mass measurements did not contain the signal peptide. These results show that domain-swapped cyt c552 oligomers were formed in E. coli, owing to the stability of the transient oligomer containing the apo protein before heme attachment. This is an indication that exceedingly stable proteins may have disadvantages forming domain-swapped oligomers in cells. PMID:26838805

  8. Surface water disinfection by chlorination and advanced oxidation processes: Inactivation of an antibiotic resistant E. coli strain and cytotoxicity evaluation.

    PubMed

    Miranda, Andreza Costa; Lepretti, Marilena; Rizzo, Luigi; Caputo, Ivana; Vaiano, Vincenzo; Sacco, Olga; Lopes, Wilton Silva; Sannino, Diana

    2016-06-01

    The release of antibiotics into the environment can result in antibiotic resistance (AR) spread, which in turn can seriously affect human health. Antibiotic resistant bacteria have been detected in different aquatic environments used as drinking water source. Water disinfection may be a possible solution to minimize AR spread but conventional processes, such as chlorination, result in the formation of dangerous disinfection by-products. In this study advanced oxidation processes (AOPs), namely H2O2/UV, TiO2/UV and N-TiO2/UV, have been compared with chlorination in the inactivation of an AR Escherichia coli (E. coli) strain in surface water. TiO2 P25 and nitrogen doped TiO2 (N-TiO2), prepared by sol-gel method at two different synthesis temperatures (0 and -20°C), were investigated in heterogeneous photocatalysis experiments. Under the investigated conditions, chlorination (1.0 mg L(-1)) was the faster process (2.5 min) to achieve total inactivation (6 Log). Among AOPs, H2O2/UV resulted in the best inactivation rate: total inactivation (6 Log) was achieved in 45 min treatment. Total inactivation was not observed (4.5 Log), also after 120 min treatment, only for N-doped TiO2 synthesized at 0°C. Moreover, H2O2/UV and chlorination processes were evaluated in terms of cytotoxicity potential by means of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenylte-trazolium colorimetric test on a human-derived cell line and they similarly affected HepG2 cells viability.

  9. Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy.

    PubMed

    Joseph, Benesh; Sikora, Arthur; Bordignon, Enrica; Jeschke, Gunnar; Cafiso, David S; Prisner, Thomas F

    2015-05-18

    Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin-labeled in whole cells and outer membranes and interspin distances were measured to a spin-labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.

  10. Survival of verocytotoxigenic Escherichia coli O157 in soil, water and on surfaces.

    PubMed

    Maule, A

    2000-01-01

    Cattle and sheep are major reservoirs of Escherichia coli O157 and consequently these and certain other farm animals can pass out large numbers of this organism in their faeces. Thus the ability of the organism to survive in faeces, on pastureland and in associated water systems has important implications for its spread to crops by direct application of manure, by irrigation with infected water or directly to man by contact with animals or contaminated soil. Model systems were used to determine the persistence of the organism in river water, cattle faeces, soil cores and on stainless steel work surfaces. Survival of the organism was found to be greatest in soil cores containing rooted grass. Under these conditions viable numbers were shown to decline from approximately 10(8) g(-1) soil to between 10(6) and 10(7) g(-1) soil after 130 d. When the organism was inoculated into cattle faeces it remained detectable at high levels for more than 50 d. In contrast the organism survived much less readily in cattle slurry and river water where it fell in numbers from more than 10(6) ml(-1) to undetectable levels in 10 and 27 d, respectively. The survival of E. coli O157 was also investigated on stainless steel surfaces, where as air-dried deposits, it was shown to survive for periods in excess of 60 d. It was most stable at chill temperatures (4 degrees C) and viability was only partially reduced at 18 degrees C. In addition to stainless steel, the organism was shown to survive for extended periods on domestic (plastic) cutting boards, both at room and chill temperatures. Sanitizing agents, such as hypochlorites and a compound comprising both cationic and anionic-based active ingredients were found to be effective in killing various VTEC on stainless steel surfaces.

  11. Changes in Escherichia coli cells starved in seawater or grown in seawater-wastewater mixtures.

    PubMed Central

    Munro, P M; Gauthier, M J; Laumond, F M

    1987-01-01

    Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater. PMID:3116927

  12. Ruminant Gastrointestinal Cell Proliferation and Clearance of Escherichia coli O157:H7

    PubMed Central

    Magnuson, Bernadene A.; Davis, Margaret; Hubele, Suzanna; Austin, Paula R.; Kudva, Indira T.; Williams, Christopher J.; Hunt, Carl W.; Hovde, Carolyn J.

    2000-01-01

    Human infections with Escherichia coli O157:H7 cause hemorrhagic colitis that can progress to a life-threatening sequelae. The most common mode of disease transmission is ingestion of contaminated bovine food products, and it is well established that E. coli O157:H7 is a transient member of the bovine microbiota. However, the conditions that induce acquisition and subsequent clearance of this bacterium from the ruminant gastrointestinal tract (GIT) are not understood. Evidence that the rates of epithelial cell proliferation in the lower GIT of cattle are associated with the duration animals remained E. coli O157:H7 culture positive is presented. Cattle with slower rates of intestinal cell proliferation in the cecum and the distal colon were culture positive significantly longer than cohort cattle with faster cell proliferation rates. Cell death rates (apoptotic indices) between the short- and long-term culture-positive animals were not different. Typical grain-based finishing diets and forage-based growing diets did not effect GIT cell proliferation or the duration animals remained E. coli O157:H7 culture positive. To identify a dietary intervention that would effect GIT cell proliferation, we used sheep as a model ruminant. A fasting-refeeding regime that increased the rate of GIT cell proliferation was developed. The fasting-refeeding protocol was used in cattle to test the hypothesis that feeding interventions that increase the rate of GIT cell proliferation induce the clearance of E. coli O157:H7 from the bovine GIT. PMID:10858188

  13. Invasion of epithelial cells by locus of enterocyte effacement-negative enterohemorrhagic Escherichia coli.

    PubMed

    Luck, Shelley N; Bennett-Wood, Vicki; Poon, Rachael; Robins-Browne, Roy M; Hartland, Elizabeth L

    2005-05-01

    The majority of enterohemorrhagic Escherichia coli (EHEC) strains associated with severe disease carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the ability to induce attaching and effacing lesions on the host intestinal mucosa. While LEE is essential for colonization of the host in these pathogens, strains of EHEC that do not carry LEE are regularly isolated from patients with severe disease, although little is known about the way these organisms interact with the host epithelium. In this study, we compared the adherence properties of clinical isolates of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. Transmission electron microscopy revealed that LEE-negative EHEC O113:H21 was internalized by Chinese hamster ovary (CHO-K1) epithelial cells and that intracellular bacteria were located within a membrane-bound vacuole. In contrast, EHEC O157:H7 remained extracellular and intimately attached to the epithelial cell surface. Quantitative gentamicin protection assays confirmed that EHEC O113:H21 was invasive and also showed that several other serogroups of LEE-negative EHEC were internalized by CHO-K1 cells. Invasion by EHEC O113:H21 was significantly reduced in the presence of the cytoskeletal inhibitors cytochalasin D and colchicine and the pan-Rho GTPase inhibitor compactin, whereas the tyrosine kinase inhibitor genistein had no significant impact on bacterial invasion. In addition, we found that EHEC O113:H21 was invasive for the human colonic cell lines HCT-8 and Caco-2. Overall these studies suggest that isolates of LEE-negative EHEC may employ a mechanism of host cell invasion to colonize the intestinal mucosa.

  14. Comparative Analysis of UV Irradiation Effects on Escherichia coli and Pseudomonas aeruginosa Bacterial Cells Utilizing Biological and Computational Approaches.

    PubMed

    Margaryan, A; Badalyan, H; Trchounian, A

    2016-09-01

    Microorganisms have a large number of tools to withstand different, and sometimes strong, environmental stresses, including irradiation, but this ability should be further evaluated for certain applications. Growth inhibition and morphological alterations of Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild-type cells caused by UV-A irradiation have been detected in the present study. Comparative analysis was carried out using well-established microbiological methods (determination of specific growth rate, growth lag phase duration, and colony-forming unit number-CFU) and computational approaches, employing light microscopy and digital image analysis to evaluate bacterial cell morphology. Decreases in the specific growth rate, prolonged lag-phases, and lowered CFUs were observed after 5 and 10 min of UV irradiation (approx. 40 Gy) compared to the control (nonirradiated) cells. Accordingly, two computational parameters-the average bacterial cell surface area and the bacterial cell perimeter (i.e., of the 2D projection of bacterial cells in microscopy image)-were reduced. The ratio of bacterial cell surface area (S) to the square of the perimeter (p (2) ) was reduced after 5 min of irradiation, but after 10 min of irradiation the studied bacterial cells became flat cylinders. The revealed findings are concluded to be highly useful in developing new, rapid analysis methods to monitor environmental and UV irradiation effects on bacteria and to detect bacterial cell morphology alterations. PMID:27334536

  15. DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

    PubMed Central

    Khandige, Surabhi; Asferg, Cecilie Antoinette; Rasmussen, Karina Juhl; Larsen, Martin Jakob; Overgaard, Martin

    2016-01-01

    ABSTRACT The ability to change cell morphology is an advantageous characteristic adopted by multiple pathogenic bacteria in order to evade host immune detection and assault during infection. Uropathogenic Escherichia coli (UPEC) exhibits such cellular dynamics and has been shown to transition through a series of distinct morphological phenotypes during a urinary tract infection. Here, we report the first systematic spatio-temporal gene expression analysis of the UPEC transition through these phenotypes by using a flow chamber-based in vitro infection model that simulates conditions in the bladder. This analysis revealed a novel association between the cell division gene damX and reversible UPEC filamentation. We demonstrate a lack of reversible bacterial filamentation in a damX deletion mutant in vitro and absence of a filamentous response by this mutant in a murine model of cystitis. While deletion of damX abrogated UPEC filamentation and secondary surface colonization in tissue culture and in mouse infections, transient overexpression of damX resulted in reversible UPEC filamentation. In this study, we identify a hitherto-unknown damX-mediated mechanism underlying UPEC morphotypical switching. Murine infection studies showed that DamX is essential for establishment of a robust urinary tract infection, thus emphasizing its role as a mediator of virulence. Our study demonstrates the value of an in vitro methodology, in which uroepithelium infection is closely simulated, when undertaking targeted investigations that are challenging to perform in animal infection models. PMID:27486187

  16. Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose employing Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuo...

  17. Quantifying potential sources of surface water contamination with Campylobacter jejuni and Campylobacter coli.

    PubMed

    Mughini-Gras, Lapo; Penny, Christian; Ragimbeau, Catherine; Schets, Franciska M; Blaak, Hetty; Duim, Birgitta; Wagenaar, Jaap A; de Boer, Albert; Cauchie, Henry-Michel; Mossong, Joel; van Pelt, Wilfrid

    2016-09-15

    Campylobacter is the most common causative agent of human bacterial gastroenteritis and is frequently found in surface water, where it indicates recent contamination with animal faeces, sewage effluent, and agricultural run-off. The contribution of different animal reservoirs to surface water contamination with Campylobacter is largely unknown. In the Netherlands, the massive poultry culling to control the 2003 avian influenza epidemic coincided with a 44-50% reduction in human campylobacteriosis cases in the culling areas, suggesting substantial environment-mediated spread of poultry-borne Campylobacter. We inferred the origin of surface water Campylobacter jejuni and Campylobacter coli strains in Luxembourg and the Netherlands, as defined by multilocus sequence typing, by comparison to strains from poultry, pigs, ruminants, and wild birds, using the asymmetric island model for source attribution. Most Luxembourgish water strains were attributed to wild birds (61.0%), followed by poultry (18.8%), ruminants (15.9%), and pigs (4.3%); whereas the Dutch water strains were mainly attributed to poultry (51.7%), wild birds (37.3%), ruminants (9.8%), and pigs (1.2%). Attributions varied over seasons and surface water types, and geographical variation in the relative contribution of poultry correlated with the magnitude of poultry production at either the national or provincial level, suggesting that environmental dissemination of Campylobacter from poultry farms and slaughterhouses can be substantial in poultry-rich regions. PMID:27244295

  18. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles. PMID:25184988

  19. Inactivation of Escherichia coli Cells in Aqueous Solution by Atmospheric-Pressure N2, He, Air, and O2 Microplasmas

    PubMed Central

    Zhou, Renwu; Zhang, Xianhui; Bi, Zhenhua; Zong, Zichao; Niu, Jinhai; Song, Ying; Yang, Size

    2015-01-01

    Atmospheric-pressure N2, He, air, and O2 microplasma arrays have been used to inactivate Escherichia coli cells suspended in aqueous solution. Measurements show that the efficiency of inactivation of E. coli cells is strongly dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2 and He microplasma arrays, air and O2 microplasma arrays may be utilized to more efficiently kill E. coli cells in aqueous solution. The efficiencies of inactivation of E. coli cells in water can be well described by using the chemical reaction rate model, where reactive oxygen species play a crucial role in the inactivation process. Analysis indicates that plasma-generated reactive species can react with E. coli cells in water by direct or indirect interactions. PMID:26025895

  20. Inactivation of Escherichia coli Cells in Aqueous Solution by Atmospheric-Pressure N2, He, Air, and O2 Microplasmas.

    PubMed

    Zhou, Renwu; Zhang, Xianhui; Bi, Zhenhua; Zong, Zichao; Niu, Jinhai; Song, Ying; Liu, Dongping; Yang, Size

    2015-08-01

    Atmospheric-pressure N2, He, air, and O2 microplasma arrays have been used to inactivate Escherichia coli cells suspended in aqueous solution. Measurements show that the efficiency of inactivation of E. coli cells is strongly dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N2 and He microplasma arrays, air and O2 microplasma arrays may be utilized to more efficiently kill E. coli cells in aqueous solution. The efficiencies of inactivation of E. coli cells in water can be well described by using the chemical reaction rate model, where reactive oxygen species play a crucial role in the inactivation process. Analysis indicates that plasma-generated reactive species can react with E. coli cells in water by direct or indirect interactions.

  1. Interaction of Gram-negative bacteria with cationic proteins: Dependence on the surface characteristics of the bacterial cell

    PubMed Central

    Prokhorenko, Isabella R; Zubova, Svetlana V; Ivanov, Alexandr Yu; Grachev, Sergey V

    2009-01-01

    Gram-negative bacteria can enter the bloodstream and interact with serum cationic proteins. The character of interaction will depend on the surface characteristics of bacterial cells, which are determined by bacterial chemotype and density of lipopolysaccharide (LPS) packing in the cell wall. It was shown that the lysozyme treatment resulted in the increase sensitivity to hypotonic shock. Significant differences to this effect were found between Escherichia coli strain D21 and D21f2 under treatment with physiological protein concentration. On the basis of electrokinetic measurements and studies of the interaction of cells with lysozyme, the hypothesis was formed that the cell wall of the E. coli strain D21f2 contains more LPS and has a higher density of their packing than the cell wall of the E. coli D21 cells. The effect of lysozyme and lactoferrin on the viability of E. coli cells of two different strains was examined. Lysozyme was found to more effectively inhibit the growth of the E. coli D21 bacteria, and lactoferrin suppressed mainly the growth of the E. coli D21f2 bacteria. These results indicate that the differences in LPS core structure of bacterial R-chemotype, which determines surface charge and density of LPS packing, plays an essential role in the mechanisms of interaction of the cationic proteins with the cell wall. PMID:20360884

  2. Integrating artificial with natural cells to translate chemical messages that direct E. coli behaviour.

    PubMed

    Lentini, Roberta; Santero, Silvia Perez; Chizzolini, Fabio; Cecchi, Dario; Fontana, Jason; Marchioretto, Marta; Del Bianco, Cristina; Terrell, Jessica L; Spencer, Amy C; Martini, Laura; Forlin, Michele; Assfalg, Michael; Dalla Serra, Mauro; Bentley, William E; Mansy, Sheref S

    2014-01-01

    Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms. PMID:24874202

  3. Influenza A Virus Infection of Intestinal Epithelial Cells Enhances the Adhesion Ability of Crohn’s Disease Associated Escherichia coli Strains

    PubMed Central

    Aleandri, Marta; Conte, Maria Pia; Simonetti, Giovanna; Panella, Simona; Celestino, Ignacio; Checconi, Paola; Marazzato, Massimiliano; Longhi, Catia; Goldoni, Paola; Nicoletti, Mauro; Barnich, Nicolas; Palamara, Anna Teresa; Schippa, Serena; Nencioni, Lucia

    2015-01-01

    Modifications of intestinal glycoreceptors expression, in particular CEACAM6, typically found in ileal Crohn's disease (CD), favor, among the commensal species of microbiota, the enrichment in Escherichia coli. Removal of protein glycosidic residues by neuraminidase, a sialidase typical of influenza virus, increases adhesion ability of Escherichia coli to Caco-2 intestinal cells. In this study we investigated whether influenza virus infection of human intestinal epithelial cells could influence the adhesiveness of different Escherichia coli strains isolated from CD patients by altering surface glycoreceptors. Influenza virus infection of intestinal cells increased exposure of galactose and mannose residues on the cell surface. In particular, glycoreceptors Thomsen-Friedenreich and CEACAM6 were over-expressed in influenza virus infected cells. In the same experimental conditions, a significant increase in bacterial adhesiveness was observed, independently of their own adhesive ability. The increase was reverted by treatment with anti-TF and anti-CEACAM6 antibodies. Interestingly, influenza virus was able to efficiently replicate in human primary intestinal cells leading to TF exposure. Finally, intestinal infected cells produced high levels of pro-inflammatory cytokines compared to control. Overall these data suggest that influenza virus infection, could constitute an additional risk factor in CD patients. PMID:25706391

  4. Processing of complex heteroduplexes in Escherichia coli and Cos-1 monkey cells.

    PubMed Central

    Abastado, J P; Cami, B; Dinh, T H; Igolen, J; Kourilsky, P

    1984-01-01

    Upon transformation into Escherichia coli or Cos-1 monkey cells, heteroduplex DNA made of two sequences containing many nucleotide mismatches yields a wide array of different molecules, some with a patchwork structure. Thus, complex heteroduplexes can be processed to generate many genetic variants. Images PMID:6091108

  5. Problem-Solving Test: RNA and Protein Synthesis in Bacteriophage-Infected "E. coli" Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    The classic experiment presented in this problem-solving test was designed to identify the template molecules of translation by analyzing the synthesis of phage proteins in "Escherichia coli" cells infected with bacteriophage T4. The work described in this test led to one of the most seminal discoveries of early molecular biology: it dealt a…

  6. Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles.

    PubMed

    Ngwenya, Bryne T; Curry, Philip; Kapetas, Leon

    2015-08-01

    A mechanistic understanding of processes controlling the transport and viability of bacteria in porous media is critical for designing in situ bioremediation and microbiological water decontamination programs. We investigated the combined influence of coating sand with iron oxide and silver nanoparticles on the transport and viability of Escherichia coli cells under saturated conditions. Results showed that iron oxide coatings increase cell deposition which was generally reversed by silver nanoparticle coatings in the early stages of injection. These observations are consistent with short-term, particle surface charge controls on bacteria transport, where a negatively charged surface induced by silver nanoparticles reverses the positive charge due to iron oxide coatings, but columns eventually recovered irreversible cell deposition. Silver nanoparticle coatings significantly increased cell inactivation during transit through the columns. However, when viability data is normalised to volume throughput, only a small improvement in cell inactivation is observed for silver nanoparticle coated sands relative to iron oxide coating alone. This counterintuitive result underscores the importance of net surface charge in controlling cell transport and inactivation and implies that the extra cost for implementing silver nanoparticle coatings on porous beds coated with iron oxides may not be justified in designing point of use water filters in low income countries. PMID:26042624

  7. Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles.

    PubMed

    Ngwenya, Bryne T; Curry, Philip; Kapetas, Leon

    2015-08-01

    A mechanistic understanding of processes controlling the transport and viability of bacteria in porous media is critical for designing in situ bioremediation and microbiological water decontamination programs. We investigated the combined influence of coating sand with iron oxide and silver nanoparticles on the transport and viability of Escherichia coli cells under saturated conditions. Results showed that iron oxide coatings increase cell deposition which was generally reversed by silver nanoparticle coatings in the early stages of injection. These observations are consistent with short-term, particle surface charge controls on bacteria transport, where a negatively charged surface induced by silver nanoparticles reverses the positive charge due to iron oxide coatings, but columns eventually recovered irreversible cell deposition. Silver nanoparticle coatings significantly increased cell inactivation during transit through the columns. However, when viability data is normalised to volume throughput, only a small improvement in cell inactivation is observed for silver nanoparticle coated sands relative to iron oxide coating alone. This counterintuitive result underscores the importance of net surface charge in controlling cell transport and inactivation and implies that the extra cost for implementing silver nanoparticle coatings on porous beds coated with iron oxides may not be justified in designing point of use water filters in low income countries.

  8. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro.

    PubMed

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-10-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46(+)/CD3(-)) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis. PMID:27638120

  9. Ultraviolet-C light inactivation of Escherichia coli O157:H7 and Listeria monocytogenes on organic fruit surfaces.

    PubMed

    Adhikari, Achyut; Syamaladevi, Roopesh M; Killinger, Karen; Sablani, Shyam S

    2015-10-01

    This study investigated UV-C light inactivation of Escherichia coli O157:H7 and Listeria monocytogenes on the surface of organic apples, pears, strawberries, red raspberries and cantaloupes. Fruit surfaces spot inoculated with cocktail strains of E. coli O157:H7 and L. monocytogenes were exposed to UV-C doses up to 11.9 kJ/m(2) at 23 °C. Fruit surface roughness, contact angle, and surface energy were determined and correlated with UV-C inactivation kinetics. Results demonstrate that bacterial pathogens on fruit surfaces respond differently to UV-C light exposure. E. coli O157:H7 on apple and pear surfaces was reduced by 2.9 and 2.1 log CFU/g, respectively when treated with UV-C light at 0.92 kJ/m(2) (60s). For berries, the reduction of E. coli O157:H7 was lower with 2.0 (strawberry) and 1.1 log CFU/g (raspberry) achieved after UV-C treatment at 7.2 kJ/m(2) (8 min) and at 10.5 kJ/m(2) (12 min), respectively. Similarly, a higher reduction of L. monocytogenes was observed on apple (1.6 log CFU/g at 3.75 kJ/m(2)) and pear (1.7 log CFU/g at 11.9 kJ/m(2)) surfaces compared to cantaloupe and strawberry surfaces (both achieved 1.0 log CFU/g at 11.9 kJ/m(2)). L. monocytogenes shows more resistance than E. coli O157:H7. Inactivation rates were higher for less hydrophobic fruits with smoother surfaces (apples and pears) as compared to fruits with rougher surfaces (cantaloupe, strawberry and raspberry). Findings indicate that UV-C light can effectively reduce E. coli O157:H7 and L. monocytogenes populations on fruit and berry surfaces. However, surface characteristics influence the efficacy of UV-C light.

  10. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

    PubMed

    Montanaro, Jacqueline; Inic-Kanada, Aleksandra; Ladurner, Angela; Stein, Elisabeth; Belij, Sandra; Bintner, Nora; Schlacher, Simone; Schuerer, Nadine; Mayr, Ulrike Beate; Lubitz, Werner; Leisch, Nikolaus; Barisani-Asenbauer, Talin

    2015-01-01

    To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery. PMID:26229437

  11. Highly selective and rapid arsenic removal by metabolically engineered Escherichia coli cells expressing Fucus vesiculosus metallothionein.

    PubMed

    Singh, Shailendra; Mulchandani, Ashok; Chen, Wilfred

    2008-05-01

    An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus was expressed in Escherichia coli, resulting in 30- and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting E. coli cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA.

  12. Beta-glucanase productivity improvement via cell immobilization of recombinant Escherichia coli cells in different matrices.

    PubMed

    Beshay, Usama; El-Enshasy, Hesham; Ismail, I M K; Moawad, Hassan; Abd-El-Ghany, Sawsan

    2011-01-01

    The studies have been performed to analyze the production of beta-glucanase by a recombinant strain of Escherichia coli immobilized in different matrices. Porous sintered glass SIRAN, Ceramic supporting matrices and Broken Pumice stone as well as SIRAN Raschig-rings were examined for the immobilization of whole bacterial cells. The beta-glucanase activity of bacteria immobilized in CeramTec PST 5 (4-5 mm) was very low. CeramTec PST 5 (1.5-2.5 mm) was found to be the best carrier compared to all other matrices regarding glucanase production (630 U/ml) and compared to enzyme activity produced by free cells (500 U/ml). Different doses of matrices were applied (2, 5, 7, 10 g/lask) in the form of "matrix weight". Using 2 g/flask of CeramTec PST 5 (1.5-2.5 mm) yielded enzyme activity of 630 U/ml). CeramTec gives highest operational stability of beta-glucanase by repeated batch fermentation to 5 cycles, and activity reached 660 U/ml. Scanning electron microscopy observations showed a high number of vegetative cells that continued growth inside the matrices, indicating that beta-glucanase activity improvement was due to the immobilization of the cells.

  13. Entire sequence of the colonization factor coli surface antigen 6-encoding plasmid pCss165 from an enterotoxigenic Escherichia coli clinical isolate.

    PubMed

    Wajima, Takeaki; Sabui, Subrata; Kano, Shigeyuki; Ramamurthy, Thandavarayan; Chatterjee, Nabendu Sekhar; Hamabata, Takashi

    2013-11-01

    Coli surface antigen 6 (CS6) is one of the most prevalent colonization factors among enterotoxigenic Escherichia coli (ETEC) isolated in developing countries. Although it is known that CS6 is encoded by a plasmid, there are no reports on the sequence analysis of the CS6-encoding plasmid or genes exhibiting similar behavior to CS6. Here, we report the isolation of the CS6-encoding plasmid, pCss165Kan, from 4266 ΔcssB::kanamycin (Km) and its complete nucleotide sequence. This plasmid consisted of 165,311bp and 222 predicted coding sequences. Remarkably, there were many insertion sequence (IS) elements, which comprised 24.4% of the entire sequence. Virulence-associated genes such as heat-stable enterotoxin, homologues of ATP-binding cassette transporter in enteroaggregative E. coli (EAEC), and ETEC autotransporter A were also present, although the ETEC autotransporter A gene was disrupted by the integration of IS629. We found that 2 transcriptional regulators belonging to the AraC family were not involved in CS6 expression. Interestingly, pCss165 had conjugative transfer genes, as well as 3 toxin-antitoxin systems that potentially exclude other plasmid-free host bacteria. These genes might be involved in the prevalence of CS6 among ETEC isolates. PMID:23933356

  14. A novel putrescine importer required for type 1 pili-driven surface motility induced by extracellular putrescine in Escherichia coli K-12.

    PubMed

    Kurihara, Shin; Suzuki, Hideyuki; Oshida, Mayu; Benno, Yoshimi

    2011-03-25

    Recently, many studies have reported that polyamines play a role in bacterial cell-to-cell signaling processes. The present study describes a novel putrescine importer required for induction of type 1 pili-driven surface motility. The surface motility of the Escherichia coli ΔspeAB ΔspeC ΔpotABCD strain, which cannot produce putrescine and cannot import spermidine from the medium, was induced by extracellular putrescine. Introduction of the gene deletions for known polyamine importers (ΔpotE, ΔpotFGHI, and ΔpuuP) or a putative polyamine importer (ΔydcSTUV) into the ΔspeAB ΔspeC ΔpotABCD strain did not affect putrescine-induced surface motility. The deletion of yeeF, an annotated putative putrescine importer, in the ΔspeAB ΔspeC ΔpotABCD ΔydcSTUV strain abolished surface motility in putrescine-supplemented medium. Complementation of yeeF by a plasmid vector restored surface motility. The surface motility observed in the present study was abolished by the deletion of fimA, suggesting that the surface motility is type 1 pili-driven. A transport assay using the yeeF(+) or ΔyeeF strains revealed that YeeF is a novel putrescine importer. The K(m) of YeeF (155 μM) is 40 to 300 times higher than that of other importers reported previously. On the other hand, the V(max) of YeeF (9.3 nmol/min/mg) is comparable to that of PotABCD, PotFGHI, and PuuP. The low affinity of YeeF for putrescine may allow E. coli to sense the cell density depending on the concentration of extracellular putrescine.

  15. A moonlighting enzyme links Escherichia coli cell size with central metabolism.

    PubMed

    Hill, Norbert S; Buske, Paul J; Shi, Yue; Levin, Petra Anne

    2013-01-01

    Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.

  16. Effect of Antimicrobial Agents on MinD Protein Oscillations in E. coli Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Kelly, Corey; Murphy, Megan; Giuliani, Maximiliano; Dutcher, John

    2011-03-01

    The pole-to-pole oscillation of the MinD proteins in E. coli determines the location of the division septum, and is integral to healthy cell division. It has been shown previously that the MinD oscillation period is approximately 40 s for healthy cells but is strongly dependant on environmental factors such as temperature, which may place stress on the cell [2,3]. We use a strain of E. coli in which the MinD proteins are tagged with green fluorescent protein (GFP), allowing fluorescence visualization of the MinD oscillation. We use high resolution total internal reflection fluorescence (TIRF) microscopy to observe the effect of exposure to antimicrobial agents on the MinD oscillation period and, more generally, to analyze the time variation of the spatial distribution of the MinD proteins within the cells. These measurements provide insight into the mechanism of antimicrobial action.

  17. Localization microscopy study of FtsZ structures in E. coli cells during SOS-response

    NASA Astrophysics Data System (ADS)

    Vedyaykin, A. D.; Sabantsev, A. V.; Vishnyakov, I. E.; Borchsenius, S. N.; Fedorova, Y. V.; Melnikov, A. S.; Serdobintsev, P. Yu; Khodorkovskii, M. A.

    2014-10-01

    Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. This is achieved by temporal separation of single fluorophore molecules emission and subsequent localization of them with the precision of few tens of nanometers. This method was previously successfully used to obtain images of FtsZ structures in Escherichia coli cells using FtsZ fusion with fluorescent protein mEos2. In this work we obtained superresolution images of FtsZ structures in fixed E. coli cells using immunocytochemical labeling. Comparison of superresolution FtsZ structures in cells undergoing SOS-response and "healthy" cells shows that FtsZ structures are partially disassembled during SOS-response, but still retain some periodicity.

  18. [Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method].

    PubMed

    Iusupov, M M; Spirin, A S

    1986-11-01

    A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure. PMID:3542056

  19. Following cell-fate in E. coli after infection by phage lambda.

    PubMed

    Zeng, Lanying; Golding, Ido

    2011-01-01

    The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we describe the full procedure for performing the infection experiments described in our earlier work. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome

  20. Correlation of Surface Toll-Like Receptor 9 Expression with IL-17 Production in Neutrophils during Septic Peritonitis in Mice Induced by E. coli

    PubMed Central

    Ren, Yunjia; Hao, Xu; Zhao, Peiyan; Yu, Yongli; Wang, Liying

    2016-01-01

    IL-17 is a proinflammatory cytokine produced by various immune cells. Polymorphonuclear neutrophils (PMNs) are the first line of defense in bacterial infection and express surface Toll-like receptor 9 (sTLR9). To study the relationship of sTLR9 and IL-17 in PMNs during bacterial infection, we infected mice with E. coli intraperitoneally to establish a septic peritonitis model for studying the PMNs response in peritoneal cavity. We found that PMNs and some of “giant cells” were massively accumulated in the peritoneal cavity of mice with fatal septic peritonitis induced by E. coli. Kinetically, the CD11b+ PMNs were increased from 20–40% at 18 hours to >80% at 72 hours after infection. After E. coli infection, sTLR9 expression on CD11b+ and CD11b− PMNs and macrophages in the PLCs were increased at early stage and deceased at late stage; IL-17 expression was also increased in CD11b+ PMNs, CD11b− PMNs, macrophages, and CD3+ T cells. Using experiments of in vitro blockage, qRT-PCR and cell sorting, we confirmed that PMNs in the PLCs did increase their IL-17 expression during E. coli infection. Interestingly, sTLR9−CD11b+Ly6G+ PMNs, not sTLR9+CD11b+Ly6G+ PMNs, were found to be able to increase their IL-17 expression. Together, the data may help understand novel roles of PMNs in septic peritonitis. PMID:27057095

  1. Biofilm Formation on Biotic and Abiotic Surfaces in the Presence of Antimicrobials by Escherichia coli Isolates from Cases of Bovine Mastitis

    PubMed Central

    Silva, Vitor O.; Soares, Larissa O.; Silva Júnior, Abelardo; Mantovani, Hilário C.; Chang, Yung-Fu

    2014-01-01

    Escherichia coli is a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of four E. coli isolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genes fliC, csgA, fimA, and luxS in the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion of E. coli isolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occur in vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds. PMID:25063668

  2. Unbalanced fermentation of glycerol in Escherichia coli via heterologous production of an electron transport chain and electrode interaction in microbial electrochemical cells.

    PubMed

    Sturm-Richter, Katrin; Golitsch, Frederik; Sturm, Gunnar; Kipf, Elena; Dittrich, André; Beblawy, Sebastian; Kerzenmacher, Sven; Gescher, Johannes

    2015-06-01

    Microbial electrochemical cells are an emerging technology for achieving unbalanced fermentations. However, organisms that can serve as potential biocatalysts for this application are limited by their narrow substrate spectrum. This study describes the reprogramming of Escherichia coli for the efficient use of anodes as electron acceptors. Electron transfer into the periplasm was accelerated by 183% via heterologous expression of the c-type cytochromes CymA, MtrA and STC from Shewanella oneidensis. STC was identified as a target for heterologous expression via a two-stage screening approach. First, mass spectroscopic analysis revealed natively expressed cytochromes in S. oneidensis. Thereafter, the corresponding genes were cloned and expressed in E. coli to quantify periplasmic electron transfer activity using methylene blue. This redox dye was further used to expand electron transfer to carbon electrode surfaces. The results demonstrate that E. coli can be reprogrammed from glycerol fermentation to respiration upon production of the new electron transport chain.

  3. Tetracycline rapidly reaches all the constituent cells of uropathogenic Escherichia coli biofilms

    NASA Technical Reports Server (NTRS)

    Stone, G.; Wood, P.; Dixon, L.; Keyhan, M.; Matin, A.; Demain, A. L. (Principal Investigator)

    2002-01-01

    We have developed a method for visualizing Escherichia coli cells that are exposed to tetracycline in a biofilm, based on a previous report that liposomes containing the E. coli TetR(B) protein fluoresce when exposed to this antibiotic. By our method, cells devoid of TetR(B) also exhibited tetracycline-dependent fluorescence. At 50 microg of tetracycline ml(-1), planktonic cells of a uropathogenic E. coli (UPEC) strain developed maximal fluorescence after 7.5 to 10 min of exposure. A similar behavior was exhibited by cells in a 24- or 48-h UPEC biofilm, as examined by confocal laser microscopy, regardless of whether they lined empty spaces or occupied densely packed regions. Further, a comparison of phase-contrast and fluorescent images of corresponding biofilm zones showed that all the cells fluoresced. Thus, all the biofilm cells were exposed to tetracycline and there were no pockets within the biofilm where the antibiotic failed to reach. It also appeared unlikely that niches of reduced exposure to the antibiotic existed within the biofilms.

  4. Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model.

    PubMed Central

    Miliotis, M D; Koornhof, H J; Phillips, J I

    1989-01-01

    The invasive capacity of 13 enteropathogenic Escherichia coli strains was assessed in vitro in Henle 407 cell culture. Both fluorescent microscopy of infected monolayers stained with acridine orange and electron microscopy revealed the presence of intracellular bacteria. As shown by acridine orange-stained infected monolayers, the number of internalized bacteria increased with time. Monolayers infected for 3 h were treated with antibiotics and either [14C]glutamine or [3H]leucine and incubated for various time intervals, after which the amount of radioactivity present in the washed monolayers was measured. A significant (P less than 0.005) increase in uptake was evident for up to 4 h after the addition of radiolabeled amino acid. This finding was confirmed by an increase in bacterial number in cultured cells and in protein concentration of infected cells with time. None of the South African enteropathogenic E. coli isolates used in these studies produced Vero cytotoxin. These findings demonstrate that, in addition to adherence, cell penetration and intracellular multiplication take place in epithelial cell-derived tissue culture cells infected by enteropathogenic E. coli. Images PMID:2659527

  5. CONTRIBUTION OF NUTRIENTS AND E. COLI TO SURFACE WATER CONDITION IN THE OZARKS I. USING PARTIAL LEAST SQUARES PREDICTIONS WHEN STANDARD REGRESSION ASSUMPTIONS ARE VIOLATED

    EPA Science Inventory

    We present here the application of PLS regression to predicting surface water total phosphorous, total ammonia and Escherichia coli from landscape metrics. The amount of variability in surface water constituents explained by each model reflects the composition of the contributi...

  6. Stress response of Escherichia coli induced by surface streamer discharge in humid air

    NASA Astrophysics Data System (ADS)

    Doležalová, Eva; Prukner, Václav; Lukeš, Petr; Šimek, Milan

    2016-02-01

    Inactivation of Escherichia coli by means of surface streamer discharge has been investigated to obtain new insights into the key mechanisms involved, with a particular emphasis placed on the microbial response to plasma-induced stress. The surface streamer discharge was produced in coplanar dielectric barrier discharge electrode geometry, and was driven by an amplitude-modulated ac high voltage in humid synthetic air at atmospheric pressure. The response to plasma-induced stress was evaluated by using conventional cultivation, sublethal injury and resazurin assay and the LIVE/DEAD® BacLight™ Bacterial Viability kit. Compared to conventional cultivation, the LIVE/DEAD® test labels bacteria with damaged membranes, while resazurin assay tracks their metabolic activity. Our results clearly demonstrate that the treated bacteria partly lost their ability to grow properly, i.e. they became injured and culturable, or even viable but nonculturable (VBNC). The ability to develop colonies could have been lost due to damage of the bacterial membrane. Damage of the membranes was mainly caused by the lipid peroxidation, evidencing the key role of oxygen reactive species, in particular ozone. We conclude that the conventional cultivation method overestimates the decontamination efficiency of various plasma sources, and must therefore be complemented by alternative techniques capable of resolving viable but nonculturable bacteria.

  7. Thermal inactivation of Escherichia coli O157:H7 and non-O157 shiga toxin-producing Escherichia coli cells in mechanically tenderized veal.

    PubMed

    Luchansky, John B; Porto-Fett, Anna C S; Shoyer, Bradley A; Thippareddi, Harshavardhan; Amaya, Jesus R; Lemler, Michael

    2014-07-01

    Preflattened veal cutlets (ca. 71.5 g, ca. 0.32 cm thick) were surface inoculated with ca. 6.8 log CFU/g of a multistrain cocktail of Escherichia coli O157:H7 (ECOH) or a cocktail made of single strains of serogroups O26, O45, O103, O104, O111, O121, and O145 of Shiga toxin-producing E. coli (STEC) cells and then were mechanically tenderized by passing once through a "Sir Steak" tenderizer. For each cooking time, in each of at least three trials, three inoculated and tenderized cutlets, with and without breading, were individually cooked in 15 or 30 ml of canola oil for 0.0, 0.75, 1.0, 1.25, 1.5, 1.75, or 2.25 min per side on an electric skillet set at 191.5°C. The temperatures of the meat and of the skillet were monitored and recorded using a type J thermocouple. Regardless of the breading or volume of oil used to cook the meat, the longer the cooking times, the higher was the internal temperature of the meat, along with a greater reduction of both ECOH and STEC. The average final internal temperature of the meat at the approximate geometric center ranged from 56.8 to 93.1°C. Microbial reductions of ca. 2.0 to 6.7 log CFU/g and ca. 2.6 to 6.2 log CFU/g were achieved for ECOH and STEC, respectively. Our data also revealed no differences in thermal inactivation of ECOH relative to the volume of oil used to cook nonbreaded cutlets. However, when cooking breaded cutlets, the use of more (30 ml) compared with less (15 ml) cooking oil resulted in greater reductions in pathogen numbers. To deliver about a 5.0-log reduction of ECOH and STEC, and to achieve the recommended internal temperature of 71.1°C, it was necessary to cook mechanically tenderized veal cutlets for at least 1.5 min per side on a preheated electric skillet set at 191.5°C and containing 15 ml of cooking oil. These data also established that cooking times and temperatures effective for inactivating serotype O157:H7 strains of E. coli in tenderized veal are equally effective against the additional six

  8. Enterotoxigenic Escherichia coli TibA Glycoprotein Adheres to Human Intestine Epithelial Cells

    PubMed Central

    Lindenthal, Christoph; Elsinghorst, Eric A.

    2001-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis. PMID:11119488

  9. Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells.

    PubMed

    Lindenthal, C; Elsinghorst, E A

    2001-01-01

    Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis. PMID:11119488

  10. Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

    PubMed

    Hotchkiss, Arland T; Nuñez, Alberto; Strahan, Gary D; Chau, Hoa K; White, André K; Marais, Jannie P J; Hom, Kellie; Vakkalanka, Malathi S; Di, Rong; Yam, Kit L; Khoo, Christina

    2015-06-17

    Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) β-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae. PMID:25973733

  11. Quantitative risk analysis for potentially resistant E. coli in surface waters caused by antibiotic use in agricultural systems.

    PubMed

    Limayem, Alya; Martin, Elizabeth M

    2014-01-01

    Antibiotics are frequently used in agricultural systems to promote livestock health and to control bacterial contaminants. Given the upsurge of the resistant fecal indicator bacteria (FIB) in the surface waters, a novel statistical method namely, microbial risk assessment (MRA) was performed, to evaluate the probability of infection by resistant FIB on populations exposed to recreational waters. Diarrheagenic Escherichia coli, except E. coli O157:H7, were selected for their prevalence in aquatic ecosystem. A comparative study between a typical E. coli pathway and a case scenario aggravated by antibiotic use has been performed via Crystal Ball® software in an effort to analyze a set of available inputs provided by the US institutions including E. coli concentrations in US Great Lakes through using random sampling and probability distributions. Results from forecasting a possible worst-case scenario dose-response, accounted for an approximate 50% chance for 20% of the exposed human populations to be infected by recreational water in the U.S. However, in a typical scenario, there is a 50% chance of infection for only 1% of the exposed human populations. The uncertain variable, E. coli concentration accounted for approximately 92.1% in a typical scenario as the major contributing factor of the dose-response model. Resistant FIB in recreational waters that are exacerbated by a low dose of antibiotic pollutants would increase the adverse health effects in exposed human populations by 10 fold.

  12. An efficient bacterial surface display system based on a novel outer membrane anchoring element from the Escherichia coli protein YiaT.

    PubMed

    Han, Mee-Jung; Lee, Seung Hwan

    2015-01-01

    In a bacterial surface display system, the display of a successful recombinant protein is highly dependent on the choice of anchoring motif. In this study, we developed an efficient Escherichia coli display system using novel anchoring motifs derived from the protein YiaT. To determine the best surface-anchoring motif, full-length YiaT and two of its C-terminal truncated forms, cut at the R181 and R232 sites, were evaluated. Two industrial enzymes, a lipase from Pseudomonas fluorescens SIK W1 and an α-amylase from Bacillus subtilis, were used as the target proteins for display. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, immunofluorescence microscopy and whole-cell enzyme activity measurements confirmed the expression of the fusion proteins on the E. coli surface. Using YiaTR181 or YiaTR232 as the anchoring motif, the fusion proteins showed very high enzyme activities and did not exert any adverse effects on either cell growth or the outer membrane integrity. Additionally, these fusion proteins were suitable for displaying proteins of large molecular size in an active form. Compared with the previous anchoring motifs FadL and OprF, YiaTR181 and YiaTR232 had approximately 10-fold and 20-fold higher enzyme activities, respectively. These results suggest that YiaT can be used as an E. coli anchoring motif to efficiently display various enzymes; hence, this system could be employed in a variety of biotechnological and industrial applications.

  13. Model development and verification for mass transport to Escherichia coli cells in a turbulent flow

    NASA Astrophysics Data System (ADS)

    Hondzo, Miki; Al-Homoud, Amer

    2007-08-01

    Theoretical studies imply that fluid motion does not significantly increase the molecular diffusive mass flux toward and away from microscopic organisms. This study presents experimental and theoretical evidence that small-scale turbulence modulates enhanced mass transport to Escherichia coli cells in a turbulent flow. Using the technique of inner region and outer region expansions, a model for dissolved oxygen and glucose uptake by E. coli was developed. The mass transport to the E. coli was modeled by the Sherwood (Sh)-Péclet (Pe) number relationship with redefined characteristic length and velocity scales. The model Sh = (1 + Pe1/2 + Pe) agreed with the laboratory measurements well. The Péclet number that quantifies the role and function of small-scale turbulence on E. coli metabolism is defined by Pe = (?) where Ezz is the root mean square of fluid extension in the direction of local vorticity, ηK is the Kolmogorov length scale, Lc is the length scale of E. coli, and D is the molecular diffusion coefficient. An alternative formulation for the redefined Pe is given by Pe = (?) where ? = 0.5(ɛν)1/4 is the Kolmogorov velocity averaged over the Kolmogorov length scale, ɛ is dissipation of turbulent kinetic energy, and ν is the kinematic viscosity of fluid. The dissipation of turbulent kinetic energy was estimated directly from measured velocity gradients and was within the reported range in engineered and natural aquatic ecosytems. The specific growth of E. coli was up to 5 times larger in a turbulent flow in comparison to the still water controls. Dissolved oxygen and glucose uptake were enhanced with increased ɛ in the turbulent flow.

  14. Quantitative phase imaging of cell division in yeast cells and E.coli using digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Pandiyan, Vimal Prabhu; John, Renu

    2015-12-01

    Digital holographic microscope (DHM) is an emerging quantitative phase imaging technique with unique imaging scales and resolutions leading to multitude of applications. DHM is promising as a novel investigational and applied tool for cell imaging, studying the morphology and real time dynamics of cells and a number of related applications. The use of numerical propagation and computational digital optics offer unique flexibility to tune the depth of focus, and compensate for image aberrations. In this work, we report imaging the dynamics of cell division in E.coli and yeast cells using a DHM platform. We demonstrate 3-D and depth imaging as well as reconstruction of phase profiles of E.coli and yeast cells using the system. We record a digital hologram of E.coli and yeast cells and reconstruct the image using Fresnel propagation algorithm. We also use aberration compensation algorithms for correcting the aberrations that are introduced by the microscope objective in the object path using linear least square fitting techniques. This work demonstrates the strong potential of a DHM platform in 3-D live cell imaging, fast clinical quantifications and pathological applications.

  15. Efficient Surface Display of Diisopropylfluorophosphatase (DFPase) in E. coli for Biodegradation of Toxic Organophosphorus Compounds (DFP and Cp).

    PubMed

    Latifi, Ali Mohammad; Karami, Ali; Khodi, Samaneh

    2015-10-01

    Compounds including organophosphorus pesticides (OPs) and chemical nerve agents are toxic compounds synthesized recently which disrupt the mechanisms of neural transmission. Therefore, a critical requirement is the development of a bio-refining technology to facilitate the biodegradation of organophosphorus pollutants. The diisopropylfluorophosphatase (DFPase, EC 3.1.8.2) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some OPs. Intracellular production of OPs-degrading enzymes or the use of native bacteria and fungi leads to a low degradation rate of OPs due to a mass transfer issue which reduces the overall catalytic efficiency. To overcome this challenge, we expressed DFPase on the surface of E. coli for the first time by employing the N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif. Tracking the recombinant protein confirmed that DFPase is successfully located on the outer membrane. Further studies on its activity to degrade diisopropylfluorophosphate (DFP) showed its significant ability for the biodegradation of diisopropylfluorophosphate (DFP) with a specific activity of 500 U/mg of wet cell weight. Recombinant cells could also degrade chlorpyrifos (Cp) with an activity equivalent to a maximum value of 381.44 U/ml with a specific activity of 476.75 U/mg of cell, analyzed using HPLC technique. The optimum activity of purified DFPase was found at 30 °C. A more increased activity was also obtained in the presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 mg/L Co(2+) ion. These results highlight the high potential of the InaV-N anchoring domain to produce an engineered bacterium that can be used in the bioremediation of pesticide-contaminated environments.

  16. Efficient Surface Display of Diisopropylfluorophosphatase (DFPase) in E. coli for Biodegradation of Toxic Organophosphorus Compounds (DFP and Cp).

    PubMed

    Latifi, Ali Mohammad; Karami, Ali; Khodi, Samaneh

    2015-10-01

    Compounds including organophosphorus pesticides (OPs) and chemical nerve agents are toxic compounds synthesized recently which disrupt the mechanisms of neural transmission. Therefore, a critical requirement is the development of a bio-refining technology to facilitate the biodegradation of organophosphorus pollutants. The diisopropylfluorophosphatase (DFPase, EC 3.1.8.2) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some OPs. Intracellular production of OPs-degrading enzymes or the use of native bacteria and fungi leads to a low degradation rate of OPs due to a mass transfer issue which reduces the overall catalytic efficiency. To overcome this challenge, we expressed DFPase on the surface of E. coli for the first time by employing the N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif. Tracking the recombinant protein confirmed that DFPase is successfully located on the outer membrane. Further studies on its activity to degrade diisopropylfluorophosphate (DFP) showed its significant ability for the biodegradation of diisopropylfluorophosphate (DFP) with a specific activity of 500 U/mg of wet cell weight. Recombinant cells could also degrade chlorpyrifos (Cp) with an activity equivalent to a maximum value of 381.44 U/ml with a specific activity of 476.75 U/mg of cell, analyzed using HPLC technique. The optimum activity of purified DFPase was found at 30 °C. A more increased activity was also obtained in the presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 mg/L Co(2+) ion. These results highlight the high potential of the InaV-N anchoring domain to produce an engineered bacterium that can be used in the bioremediation of pesticide-contaminated environments. PMID:26239441

  17. Strategies for recovering of planktonic and sessile cells of Escherichia coli O157:H7 from freshwater environment.

    PubMed

    Marucci, Patricia L; Cubitto, María A

    2016-07-01

    The experiments were performed with Escherichia coli O157:H7 EDL 933 in freshwater microcosms at 12 °C. At 35, 45, and 70 days, samples were taken and filtered through 0.45 μm membrane filters. The following alternatives were tested to evaluate the recovery percentage of injured cells: (1) selective media CHROMagar(™)O157 and chromID(™)O157:H7 agar, at 37 °C for 24 h; (2) tryptic soy agar supplemented with yeast extract (TSAE), incubated at 25 °C for 2 or 4 h, then transferred to CHROMagar(™)O157 or chromID(™)O157:H7 agar at 37 °C (TSAE2h-CHROM, TSAE4h-CHROM and TSAE2h-ID, TSAE4h-ID); (3) thin agar layer (TAL) method, TSAE was overlaid on CHROMagar(™)O157 or chromID(™)O157:H7 agar (TALCHROM and TALID, respectively) and incubated at 37 °C for 24 h; and (4) TALCHROM at 25 °C for 4 h, then continued up to complete 24 h at 37 °C (TALCHROM4h). Furthermore, the recovery of E. coli O157:H7 cells adhering to glass coverslips were evaluated to mimic biofilm conditions. The recovery percentages obtained from each alternative were calculated relative to TSAE counts. After 70 days, TSAE4h-CHROM and TALCHROM4h showed the highest recovery percentage (>90 %) from water microcosms. Despite the improved recovery of cell adhering to glass surfaces, the percentages obtained with TSAE4h-CHROM were low. Further studies for the recovery of biofilm-forming E. coli O157:H7 are required. Pre-incubation on TSAE at 25 °C for 4 h, combined with CHROMagar(™)O157, or by thin agar layer method (TALCHROM) enhanced significantly the recovery of viable cells of E. coli O157:H7 after prolonged stay in water microcosms. PMID:27344557

  18. Single-Cell E. coli Response to an Instantaneously Applied Chemotactic Signal

    PubMed Central

    Sagawa, Takashi; Kikuchi, Yu; Inoue, Yuichi; Takahashi, Hiroto; Muraoka, Takahiro; Kinbara, Kazushi; Ishijima, Akihiko; Fukuoka, Hajime

    2014-01-01

    In response to an attractant or repellant, an Escherichia coli cell controls the rotational direction of its flagellar motor by a chemotaxis system. When an E. coli cell senses an attractant, a reduction in the intracellular concentration of a chemotaxis protein, phosphorylated CheY (CheY-P), induces counterclockwise (CCW) rotation of the flagellar motor, and this cellular response is thought to occur in several hundred milliseconds. Here, to measure the signaling process occurring inside a single E. coli cell, including the recognition of an attractant by a receptor cluster, the inactivation of histidine kinase CheA, and the diffusion of CheY and CheY-P molecules, we applied a serine stimulus by instantaneous photorelease from a caged compound and examined the cellular response at a temporal resolution of several hundred microseconds. We quantified the clockwise (CW) and CCW durations immediately after the photorelease of serine as the response time and the duration of the response, respectively. The results showed that the response time depended on the distance between the receptor and motor, indicating that the decreased CheY-P concentration induced by serine propagates through the cytoplasm from the receptor-kinase cluster toward the motor with a timing that is explained by the diffusion of CheY and CheY-P molecules. The response time included 240 ms for enzymatic reactions in addition to the time required for diffusion of the signaling molecule. The measured response time and duration of the response also revealed that the E. coli cell senses a similar serine concentration regardless of whether the serine concentration is increasing or decreasing. These detailed quantitative findings increase our understanding of the signal transduction process that occurs inside cells during bacterial chemotaxis. PMID:25099812

  19. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

    PubMed Central

    Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  20. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation.

    PubMed

    Binder, Dennis; Probst, Christopher; Grünberger, Alexander; Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  1. Effects of Copper-phenanthroline on Pentachlorophenol-induced Adaptation and Cell Death of Escherichia coli1

    PubMed Central

    Zhang, Xue-Wen; Li, Rong-Gui; Wang, Xin; Zhou, Shuan-Hu

    2009-01-01

    Objective To evaluate the effects of copper-phenanthroline (CuOP) on pentachlorophenol (PCP)-induced adaptation and cell death of Escherichia coli. Methods Bacterial growth and adaptation to PCP were monitored spectrophotometrically at 600 nm. Inactivation of bacterial cells was determined from colony count on agar dishes. Cellular ATP content and accumulation of PCP were assessed by chemiluminescence and HPLC analysis respectively. The formation of PCP-Cu-OP complex was shown by UV-visible spectra. Results Escherichia coli (E. coli) could adapt to PCP, a wood preservative and insecticide used in agriculture. The adaptation of E. coli to PCP prevented its death to the synergistic cytotoxicity of CuOP plus PCP and declined cellular accumulation and uncoupling of oxidative phosphorylation of PCP. Furthermore, CuOP and PCP neither produced reactive oxygen species (ROS) nor had a synergistic effect on uncoupling of oxidative phosphorylation in E. coli. The synergistic cytotoxicity of CuOP and PCP in E. coli might be due to the formation of lipophilic PCP-Cu-OP complex. Conclusion Our data suggested that adaptation of E. coli to PCP decreased the synergistic effects of CuOP and PCP on prokaryotic cell death due to the formation of lipophilic PCP-Cu-OP complex, but it had no effect on the uncoupling of oxidative phosphorylation and production of reactive oxygen species in E. coli. PMID:17624183

  2. Engineering Escherichia coli into a Protein Delivery System for Mammalian Cells

    PubMed Central

    2015-01-01

    Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications. PMID:25853840

  3. Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome

    PubMed Central

    Díaz-Mejía, Juan Javier; Babu, Mohan; Emili, Andrew

    2009-01-01

    The bacterial cell-envelope consists of a complex arrangement of lipids, proteins and carbohydrates that serves as the interface between a microorganism and its environment or, with pathogens, a human host. Escherichia coli has long been investigated as a leading model system to elucidate the fundamental mechanisms underlying microbial cell-envelope biology. This includes extensive descriptions of the molecular identities, biochemical activities and evolutionary trajectories of integral transmembrane proteins, many of which play critical roles in infectious disease and antibiotic resistance. Strikingly, however, only half of the c. 1200 putative cell-envelope-related proteins of E. coli currently have experimentally attributed functions, indicating an opportunity for discovery. In this review, we summarize the state of the art of computational and proteomic approaches for determining the components of the E. coli cell-envelope proteome, as well as exploring the physical and functional interactions that underlie its biogenesis and functionality. We also provide a comprehensive comparative benchmarking analysis on the performance of different bioinformatic and proteomic methods commonly used to determine the subcellular localization of bacterial proteins. PMID:19054114

  4. Engineering Escherichia coli into a protein delivery system for mammalian cells.

    PubMed

    Reeves, Analise Z; Spears, William E; Du, Juan; Tan, Kah Yong; Wagers, Amy J; Lesser, Cammie F

    2015-05-15

    Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications. PMID:25853840

  5. Enteropathogenic Escherichia coli inhibits intestinal vitamin B1 (thiamin) uptake: studies with human-derived intestinal epithelial Caco-2 cells.

    PubMed

    Ashokkumar, Balasubramaniem; Kumar, Jeyan S; Hecht, Gail A; Said, Hamid M

    2009-10-01

    Infection with the gram-negative enteropathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. Whereas diarrhea is a major consequence of this infection, malnutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with nonpathogenic E. coli, killed EPEC, or filtered supernatant) leads to a significant (P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 and -2 proteins, (determined by the biotinylation method) showed a significantly (P < 0.01) lower expression in EPEC-treated cells compared with controls. EPEC infection also affected the steady-state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS) or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 and -2. Furthermore, the inhibition appears to be dependent on a functional TTSS of EPEC.

  6. Quantifying transfer rates of Salmonella and Escherichia coli O157:H7 between fresh-cut produce and common kitchen surfaces.

    PubMed

    Jensen, Dane A; Friedrich, Loretta M; Harris, Linda J; Danyluk, Michelle D; Schaffner, Donald W

    2013-09-01

    Cross-contamination between foods and surfaces in food processing environments and home kitchens may play a significant role in foodborne disease transmission. This study quantifies the cross-contamination rates between a variety of fresh-cut produce and common kitchen surfaces (ceramic, stainless steel, glass, and plastic) using scenarios that differ by cross-contamination direction, surface type, produce type, and drying time/moisture level. A five-strain cocktail of rifampin-resistant Salmonella was used in transfer scenarios involving celery, carrot, and watermelon, and a five-strain cocktail of rifampin-resistant Escherichia coli O157:H7 was used in transfer scenarios involving lettuce. Produce or surface coupons were placed in buffer-filled filter bags and homogenized or massaged, respectively, to recover cells. The resulting solutions were serially diluted in 0.1% peptone and surface plated onto tryptic soy agar with 80 μg/ml rifampin and bismuth sulfite agar with 80 μg/ml rifampin for Salmonella or sorbitol MacConkey agar with 80 μg/ml rifampin for E. coli O157:H7. When the food contact surface was freshly inoculated, a high amount (>90%) of the inoculum was almost always transferred to the cut produce item. If the inoculated food contact surfaces were allowed to dry for 1 h, median transfer was generally >90% for carrots and watermelon but ranged from <1 to ∼70% for celery and lettuce. Freshly inoculated celery or lettuce transferred more bacteria (<2 to ∼25% of the inoculum) compared with freshly inoculated carrots or watermelon (approximately <1 to 8%). After 1 h of drying, the rate of transfer from inoculated celery, carrot, and lettuce was <0.01 to ∼5% and <1 to ∼5% for watermelon. Surface moisture and direction of transfer have the greatest influence on microbial transfer rates. PMID:23992497

  7. Diffusing colloidal probes of cell surfaces.

    PubMed

    Duncan, Gregg A; Fairbrother, D Howard; Bevan, Michael A

    2016-05-25

    Measurements and analyses are reported to quantify dynamic and equilibrium interactions between colloidal particles and live cell surfaces using dark field video microscopy. Two-dimensional trajectories of micron-sized polyethylene glycol (PEG)-coated silica colloids relative to adherent epithelial breast cancer cell perimeters are determined allowing measurement of position dependent diffusivities and interaction potentials. PEG was chosen as the material system of interest to assess non-specific interactions with cell surfaces and establishes a basis for investigation of specific interactions in future studies. Analysis of measured potential energies on cell surfaces reveals the spatial dependence in cell topography. With the measured cell topography and models for particle-cell surface hydrodynamic interactions, excellent agreement is obtained between theoretical and measured colloidal transport on cell surfaces. Quantitative analyses of association lifetimes showed that PEG coatings act to stabilize colloids above the cell surface through net repulsive, steric interactions. Our results demonstrate a self-consistent analysis of diffusing colloidal probe interactions due to conservative and non-conservative forces to characterize biophysical cell surface properties. PMID:27117575

  8. Diffusing colloidal probes of cell surfaces.

    PubMed

    Duncan, Gregg A; Fairbrother, D Howard; Bevan, Michael A

    2016-05-25

    Measurements and analyses are reported to quantify dynamic and equilibrium interactions between colloidal particles and live cell surfaces using dark field video microscopy. Two-dimensional trajectories of micron-sized polyethylene glycol (PEG)-coated silica colloids relative to adherent epithelial breast cancer cell perimeters are determined allowing measurement of position dependent diffusivities and interaction potentials. PEG was chosen as the material system of interest to assess non-specific interactions with cell surfaces and establishes a basis for investigation of specific interactions in future studies. Analysis of measured potential energies on cell surfaces reveals the spatial dependence in cell topography. With the measured cell topography and models for particle-cell surface hydrodynamic interactions, excellent agreement is obtained between theoretical and measured colloidal transport on cell surfaces. Quantitative analyses of association lifetimes showed that PEG coatings act to stabilize colloids above the cell surface through net repulsive, steric interactions. Our results demonstrate a self-consistent analysis of diffusing colloidal probe interactions due to conservative and non-conservative forces to characterize biophysical cell surface properties.

  9. Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

    PubMed

    Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

    2016-06-01

    Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed. PMID:26967458

  10. Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

    PubMed

    Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

    2016-06-01

    Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

  11. Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology

    PubMed Central

    2012-01-01

    Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture. PMID:22336426

  12. A portable, shock-proof, surface-heated droplet PCR system for Escherichia coli detection.

    PubMed

    Angus, Scott V; Cho, Soohee; Harshman, Dustin K; Song, Jae-Young; Yoon, Jeong-Yeol

    2015-12-15

    A novel polymerase chain reaction (PCR) device was developed that uses wire-guided droplet manipulation (WDM) to guide a droplet over three different heating chambers. After PCR amplification, end-point detection is achieved using a smartphone-based fluorescence microscope. The device was tested for identification of the 16S rRNA gene V3 hypervariable region from Escherichia coli genomic DNA. The lower limit of detection was 10(3) genome copies per sample. The device is portable with smartphone-based end-point detection and provides the assay results quickly (15 min for a 30-cycle amplification) and accurately. The system is also shock and vibration resistant, due to the multiple points of contact between the droplet and the thermocouple and the Teflon film on the heater surfaces. The thermocouple also provides real-time droplet temperature feedback to ensure it reaches the set temperature before moving to the next chamber/step in PCR. The device is equipped to use either silicone oil or coconut oil. Coconut oil provides additional portability and ease of transportation by eliminating spilling because its high melting temperature means it is solid at room temperature. PMID:26164008

  13. Controlled surface chemistries and quantitative cell response

    NASA Astrophysics Data System (ADS)

    Plant, Anne L.

    2002-03-01

    Living cells experience a large number of signaling cues from their extracellular matrix. As a result of these inputs, a variety of intracellular signaling pathways are apparently initiated simultaneously. The vast array of alternative responses that result from the integration of these inputs suggests that it may be reasonable to look for cellular response not as an 'on' or 'off' condition but as a distribution of responses. A difficult challenge is to determine whether variations in responses from individual cells arise from the complexity of intracellular signals or are due to variations in the cell culture environment. By controlling surface chemistry so that every cell 'sees' the same chemical and physical environment, we can begin to assess how the distribution of cell response is affected strictly by changes in the chemistry of the cell culture surface. Using the gene for green fluorescent protein linked to the gene for the promoter of the extracellular matrix protein, tenascin, we can easily probe the end product in a signaling pathway that is purported to be linked to surface protein chemistry and to cell shape. Cell response to well-controlled, well-characterized, and highly reproducible surfaces prepared using soft lithography techniques are compared with more conventional ways of preparing extracellular matrix proteins for cell culture. Using fluorescence microscopy and image analysis of populations of cells on these surfaces, we probe quantitatively the relationship between surface chemistry, cell shape and variations in gene expression endpoint.

  14. Plasmid-encoded production of coli surface-associated antigen 1 (CS1) in a strain of Escherichia coli serotype O139.H28.

    PubMed

    Willshaw, G A; Smith, H R; McConnell, M M; Gaastra, W; Thomas, A; Hibberd, M; Rowe, B

    1990-07-01

    Production of coli surface-associated antigen 1 (CS1) by Escherichia coli strain E24377 of serotype O139.H28 was controlled by a plasmid that also encoded heat stable and heat labile enterotoxins and CS3. The presence of a regulatory sequence was detected on this plasmid by hybridization with the cfaD gene that regulates expression of colonization factor antigen I fimbriae and is at least 96% homologous with the rns sequence controlling production of CS1 or CS2 fimbriae by strains of serotype O6.H16 of appropriate biotype. A separate plasmid, pDEP20, carrying the structural genes for CS1 synthesis was identified and transformed into E. coli strain HB101 or a derivative of strain E24377 without large plasmids. Transformants carrying pDEP20 did not produce CS1 fimbrial antigen, but antigen expression was obtained when a cloned cfaD gene or a wild-type plasmid carrying the rns sequence was introduced. Transposon mutagenesis with Tn1000 identified a 3.7 kbp region of pDEP20 essential for production of CS1 fimbriae. Genes encoding production of CS1 fimbriae were cloned on a 9.9 kbp BamHI fragment and were expressed in the presence of the cfaD sequence. A strain producing both CS1 and CS2 antigens was constructed by introduction of the cloned cfaD gene into a strain of serotype O6.H16 biotype C carrying plasmid pDEP20.

  15. Real-Time, Nonlinear Optical Probe of Molecular Transport across Living Escherichia coli Cell Membranes

    NASA Astrophysics Data System (ADS)

    Zeng, Jia; Eckenrode, Heather; Dai, Hai-Lung

    2006-03-01

    We will demonstrate for the first time that a nonlinear optical technique- Second Harmonic Generation- can be used to monitor, with real time resolution, the transport of a molecule across the membranes of a living cell. The transport of the hydrophobic ionic dye molecule malachite green (MG) through both membranes of the gram-negative bacteria Escherichia coli, the outer membrane and the cytoplasmic membrane, has been studied. A kinetic model, assuming that the MG molecules penetrate the bacteria outer membrane through classic porin channels while transport across the cytoplasmic membrane is by diffusion through the phospholipid bilayer, is proposed to account for experimental observations. Analysis of the SHG data enables quantitative determination of the transport rate constants and the adsorption equilibrium constants for the Escherichia coli cells living in different environments.

  16. Participation of Integrin α5β1 in the Fibronectin-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

    PubMed Central

    Izquierdo, Mariana; Nataro, James P.; Ruiz-Perez, Fernando; Farfan, Mauricio J.

    2014-01-01

    Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells. PMID:25177698

  17. Participation of integrin α5β1 in the fibronectin-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Alvestegui, Alejandra; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-01-01

    Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells.

  18. Participation of integrin α5β1 in the fibronectin-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

    PubMed

    Izquierdo, Mariana; Alvestegui, Alejandra; Nataro, James P; Ruiz-Perez, Fernando; Farfan, Mauricio J

    2014-01-01

    Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells. PMID:25177698

  19. Relevance of nontoxigenic strains as surrogates for Escherichia coli O157:H7 in groundwater contamination potential: role of temperature and cell acclimation time.

    PubMed

    Castro, Felipe D; Tufenkji, Nathalie

    2007-06-15

    Nontoxigenic bacteria are commonly used as indicators for predicting the contamination potential of pathogens in natural or engineered aqueous environments. In this study, column transport experiments were used to examine the relevance of two nontoxigenic strains of Escherichia coli O157:H7 as potential surrogates for the well-known pathogen. Experiments conducted at 11 degrees C indicate that only one of the nontoxigenic strains may be an appropriate surrogate for predicting the migration potential of the pathogen at low solution ionic strengths. Results of various bacterial characterization methods indicate that differences in cell attachment could qualitatively, but convincingly, be related to differences in cell surface charge. Additional experiments conducted at 22 degrees C reveal the influence of temperature on bacterial cell surface charge and cell attachmentto sand. The role of cell acclimation time to an artificial groundwater solution is also examined, showing little change in the degree of cell attachment over a period of several weeks.

  20. Analysis of cell surface antigens by Surface Plasmon Resonance imaging.

    PubMed

    Stojanović, Ivan; Schasfoort, Richard B M; Terstappen, Leon W M M

    2014-02-15

    Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells.

  1. Spatial coordination between chromosomes and cell division proteins in Escherichia coli.

    PubMed

    Männik, Jaan; Bailey, Matthew W

    2015-01-01

    To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness. PMID:25926826

  2. Spatial coordination between chromosomes and cell division proteins in Escherichia coli

    PubMed Central

    Männik, Jaan; Bailey, Matthew W.

    2015-01-01

    To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness. PMID:25926826

  3. Development of a fluorometric microplate antiadhesion assay using uropathogenic Escherichia coli and human uroepithelial cells.

    PubMed

    Kimble, Lindsey L; Mathison, Bridget D; Kaspar, Kerrie L; Khoo, Christina; Chew, Boon P

    2014-05-23

    A fluorometric microplate assay has been developed to determine Escherichia (E.) coli adhesion to uroepithelial cells (UEC). P-fimbriated E. coli were labeled with BacLight Green and preincubated 30 min with human urine or standard. Fluorescent-E. coli were added to UEC in mircoplates at a 400:1 ratio, incubated 1 h, and washed, and the fluorescence intensity was measured. Specific labeling and adherence were confirmed by flow cytometry. A myricetin (1) standard curve (0-30 μg/mL) was developed; the lower limit of detection was 0.1 μg/mL, and half-maximal inhibitory concentration was 0.88 μg/mL (intra- and interassay coefficients of variance were <10% and <15%, respectively). Vaccinium macrocarpon (cranberry) extracts, quercetin (2), and procyanidins B1 (3), B2 (4), and C1 (5) showed similar inhibition. Antiadhesion activity of urine samples from subjects (n = 12) consuming placebo or V. macrocarpon beverage determined using this assay was positively correlated (R(2) = 0.78; p < 0.01) with a radiolabeled-E. coli assay.

  4. Chemotaxis for enhanced immobilization of Escherichia coli and Legionella pneumophila on biofunctionalized surfaces of GaAs.

    PubMed

    Hassen, Walid M; Sanyal, Hashimita; Hammood, Manar; Moumanis, Khalid; Frost, Eric H; Dubowski, Jan J

    2016-06-20

    The authors have investigated the effect of chemotaxis on immobilization of bacteria on the surface of biofunctionalized GaAs (001) samples. Escherichia coli K12 bacteria were employed to provide a proof-of-concept of chemotaxis-enhanced bacterial immobilization, and then, these results were confirmed using Legionella pneumophila. The recognition layer was based on a self-assembled monolayer of thiol functionalized with specific antibodies directed toward E. coli or L. pneumophila, together with the enzyme beta-galactosidase (β-gal). The authors hypothesized that this enzyme together with its substrate lactose would produce a gradient of glucose which would attract bacteria toward the biochip surface. The chemotaxis effect was monitored by comparing the number of bacteria bound to the biochip surface with and without attractant. The authors have observed that β-gal plus lactose enhanced the immobilization of bacteria on our biochips with a higher effect at low bacterial concentrations. At 100 and 10 bacteria/ml, respectively, for E. coli and L. pneumophila, the authors observed up to 11 and 8 times more bacteria bound to biochip surfaces assisted with the chemotaxis effect in comparison to biochips without chemotaxis. At 10(4) bacteria/ml, the immobilization enhancement rate did not exceed two times.

  5. Dynamics of SOS-Response in UVR-Mutants of Escherichia Coli Cells under Ultraviolet Irradiation

    NASA Astrophysics Data System (ADS)

    Tuchina, M. A.; Parkhomenko, A. Yu.; Belov, O. V.; Bugai, A. N.

    2010-01-01

    A mathematical model of the genetic regulatory system of the SOS-response induced by ultraviolet radiation in excision repair-deficient mutants of E. coli bacterial cells is developed. On the basis of the model, the dynamics of the SOS system regulatory proteins is analyzed. The influence of excision repair on the induction of the key gene products during the SOS-response is studied.

  6. Release of cell wall peptides into culture medium by exponentially growing Escherichia coli.

    PubMed Central

    Goodell, E W; Schwarz, U

    1985-01-01

    Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation. PMID:2858468

  7. Acetate metabolism by Escherichia coli in high-cell-density fermentation.

    PubMed Central

    Kleman, G L; Strohl, W R

    1994-01-01

    Little is known about the cellular physiology of Escherichia coli at high cell densities (e.g., greater than 50 g [dry cell weight] per liter), particularly in relation to the cellular response to different growth conditions. E. coli W3100 cultures were grown under identical physical and nutritional conditions, by using a computer-controlled fermentation system which maintains the glucose concentration at 0.5 g/liter, to high cell densities at pH values of 6.0, 6.5, 7.0, and 7.5. The data suggest a relationship between the pH of the environment and the amount of acetate excreted by the organism during growth. At pH values of 6.0 and 6.5, the acetate reached a concentration of 6 g/liter, whereas at pH 7.5, the acetate reached a concentration of 12 g/liter. Furthermore, at pH values of 6.0 to 7.0, the E. coli culture undergoes a dramatic metabolic switch in which oxygen and glucose consumption and CO2 evolution all temporarily decreased by 50 to 80%, with a concomitant initiation of acetate utilization. After a 30-min pause in which approximately 50% of the available acetate is consumed, the culture recovers and resumes consuming glucose and oxygen and producing acetate and CO2 at preswitch levels. During the switch period, the specific activity of isocitrate lyase typically increases approximately fourfold. PMID:7993084

  8. Translocation of surface-inoculated Escherichia coli into whole muscle nonintact beef striploins following blade tenderization.

    PubMed

    Johns, Daniel F; Bratcher, Christy L; Kerth, Chris R; McCaskey, Thomas

    2011-08-01

    Translocation of Escherichia coli among beef loins processed with a mechanical tenderizer was evaluated. Two beef striploins were inoculated on the lean side with 6.4 to 7.2 ml of a nalidixic acid-resistant E. coli at 8.2 to 10.1 log CFU/ml. Total E. coli inoculated onto striploins ranged from 1.12 × 10(9) to 9.10 × 10(10) CFU. Striploins were passed once (lean side up, anterior end first) through a mechanical blade tenderizer. After the inoculated striploins had been tenderized, uninoculated beef striploins (n = 5) were passed once (lean side up, anterior end first) through the same mechanical tenderizer. This procedure was repeated twice for a total of 12 striploins. Six core samples were taken from each striploin starting with the anterior end. Each core was cut into six sections; sections 1 through 4 represented the top 4 cm of the core sample, and sections 5 and 6 represented the remaining part of the core split in half. After tenderization, E. coli levels were highest (P < 0.05) in loin 1. Loin 2 had higher levels (P < 0.05) than did loins 4, 5, and 6. No differences in E. coli levels (P > 0.05) were found among loins 3, 4, 5, and 6, for which levels were below the limit of detection. Levels of E. coli from section 1 were higher than those for all other sections. Section 2 had higher levels (P < 0.05) than did sections 3, 4, 5, and 6. E. coli recovery from section 6 was higher (P < 0.05) than that from sections 3, 4, and 5. No differences in E. coli recovery (P > 0.05) were found among sections 3, 4, and 5. Data indicate that even after inoculation of E. coli a high initial levels, contamination from one loin to another is quickly reduced to < 10 CFU/g. PMID:21819662

  9. Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: Concept of a leading edge

    SciTech Connect

    Wientjes, F.B.; Nanninga, N. )

    1989-06-01

    The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with (meso-{sup 3}H)diaminopimelic acid (({sup 3}H)Dap). The second method was autoradiography of cells pulse-labeled with ({sup 3}H)Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surface components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of ({sup 3}H)Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific {beta}-lactam antibiotic furazlocillin did not affect ({sup 3}H)Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.

  10. Hot air treatment for surface decontamination of table eggs experimentally infected with Salmonella, Listeria, and Escherichia coli.

    PubMed

    Manfreda, G; Cevoli, C; Lucchi, A; Pasquali, F; Fabbri, A; Franchini, A

    2010-06-01

    Hot-air pasteurization was investigated in the EU-funded project "Reducing Egg Susceptibility to Contaminations in Avian Production in Europe (RESCAPE)" as an innovative treatment for surface bacterial decontamination of table eggs. Possible side effects of the treatment on egg quality traits were also studied. The decontamination power of hot air was evaluated over 1 month on shell eggs that were experimentally inoculated with Salmonella enteritidis, Escherichia coli, or Listeria monocytogenes. The S. Enteritidis and L. monocytogenes populations on the surfaces of treated eggs showed a significant reduction compared with untreated eggs. No statistically significant results were obtained comparing E. coli loads on treated and untreated eggs. No detrimental effects on quality traits either immediately after treatment or after 28 days of storage at 20 degrees C were recorded.

  11. Adhesion of cells to polystyrene surfaces

    PubMed Central

    1983-01-01

    The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin. PMID:6355120

  12. Cell-surface remodelling during mammalian erythropoiesis.

    PubMed

    Wraith, D C; Chesterton, C J

    1982-10-15

    Current evidence suggests that the major cell-surface modification occurring during mammalian erythropoiesis could be generated by two separate mechanisms: either selective loss of membrane proteins during enucleation or endocytosis at the subsequent reticulocyte and erythrocyte stages. The former idea was tested by collecting developing rabbit erythroid cells before and after the enucleation step and comparing their cell-surface protein composition via radiolabelling and electrophoresis. Few changes were observed. Our data thus lend support to the endocytosis mechanism.

  13. Chemical modification of the surfaces of bacterial cell walls.

    PubMed

    Neihof, R A; Echols, W H

    1978-01-01

    The surfaces of the isolated cell walls of four bacterial species were studied by microelectrophoresis following chemical treatments intended to remove specific charged groups. Acid-base titrations of the walls were used to assess specificity and extent of the modifications. Carboxyl groups were specifically and completely modified by activation with a water-soluble carbodiimide and subsequent reaction with a nucleophile, such as glycinamide, to give an uncharged pH-stable product. Aqueous media and mild reaction conditions make the method suitable for modifying carboxyl groups on cell surfaces too labile to withstand the harsh conditions required for conventional esterification reactions. Use of the carbodiimide-mediated reaction for discharging carboxyl groups, along with fluorodinitrobenzene for discharging amino groups and extraction procedures for removing constituents carrying phosphoester groups (teichoic acids), made it possible to obtain information about the spatial arrangement of charged groups on the wall surfaces. Removal of the exterior negative charge dominating wall surfaces allowed underlying amino groups to become electrokinetically effective and, in the case of E. coli, also revealed a lipophilic region with an affinity for a cationic surfactant.

  14. Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli.

    PubMed

    Koh, Seung Y; George, Sajan; Brözel, Volker; Moxley, Rodney; Francis, David; Kaushik, Radhey S

    2008-07-27

    Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.

  15. Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces

    PubMed Central

    Wang, Liyun; Fan, Daming; Chen, Wei; Terentjev, Eugene M.

    2015-01-01

    In medicine and food industry, bacterial colonisation on surfaces is a common cause of infections and severe illnesses. However, the detailed quantitative information about the dynamics and the mechanisms involved in bacterial proliferation on solid substrates is still lacking. In this study we investigated the adhesion and detachment, the individual growth and colonisation, and the cell size control of Escherichia coli (E. coli) MG1655 on polyethylene terephthalate (PET) surfaces. The results show that the bacterial growth curve on PET exhibits the distinct lag and log phases, but the generation time is more than twice longer than in bulk medium. Single cells in the lag phase are more likely to detach than clustered ones in the log phase; clustered bacteria in micro-colonies have stronger adhesive bonds with surfaces and their neighbours with the progressing colonisation. We show that the cell size is under the density-dependent pathway control: when the adherent cells are at low density, the culture medium is responsible for coordinating cell division and cell size; when the clustered cells are at high population density, we demonstrate that the effect of quorum sensing causes the cell size decrease as the cell density on surfaces increases. PMID:26464114

  16. Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces.

    PubMed

    Wang, Liyun; Fan, Daming; Chen, Wei; Terentjev, Eugene M

    2015-10-14

    In medicine and food industry, bacterial colonisation on surfaces is a common cause of infections and severe illnesses. However, the detailed quantitative information about the dynamics and the mechanisms involved in bacterial proliferation on solid substrates is still lacking. In this study we investigated the adhesion and detachment, the individual growth and colonisation, and the cell size control of Escherichia coli (E. coli) MG1655 on polyethylene terephthalate (PET) surfaces. The results show that the bacterial growth curve on PET exhibits the distinct lag and log phases, but the generation time is more than twice longer than in bulk medium. Single cells in the lag phase are more likely to detach than clustered ones in the log phase; clustered bacteria in micro-colonies have stronger adhesive bonds with surfaces and their neighbours with the progressing colonisation. We show that the cell size is under the density-dependent pathway control: when the adherent cells are at low density, the culture medium is responsible for coordinating cell division and cell size; when the clustered cells are at high population density, we demonstrate that the effect of quorum sensing causes the cell size decrease as the cell density on surfaces increases.

  17. Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters.

    PubMed

    Aldea, M; Garrido, T; Pla, J; Vicente, M

    1990-11-01

    The cell division ftsQAZ cluster and the ftsZ-dependent bolA morphogene of Escherichia coli are found to be driven by gearboxes, a distinct class of promoters characterized by showing an activity that is inversely dependent on growth rate. These promoters contain specific sequences upstream from the mRNA start point, and their -10 region is essential for the inverse growth rate dependence. Gearbox promoters are essential for driving ftsQAZ and bolA gene expression so that the encoded products are synthesized at constant amounts per cell independently of cell size. This mode of regulation would be expected for the expression of proteins that either play a regulatory role in cell division or form a stoichiometric component of the septum, a structure that, independently of cell size and growth rate, is produced once per cell cycle.

  18. Study of mechanical properties of DNA in E. coli cells by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kafle, Rudra; Liebeskind, Molly; Meiners, Jens-Christian

    Mechanical quantities like the elasticity of cells are conventionally measured by directly probing them mechanically. Measurements of these quantities for subcellular structures in living cells are almost impossible this way. We use fluorescence correlation spectroscopy (FCS) to measure such mechanical quantities in chromosomal DNA in E. coli cells. We present methods to address complexities of live-cell FCS such as photobleaching, and calculate the viscoelastic moduli from the FCS data. We compare the measured viscoelastic moduli of live cells with those that are ATP-depleted to stop all molecular motor action and find substantial differences. Active processes are stopped in ATP-depleted cells and hence the bacterial DNA appears to become stiffer and the surrounding intracellular medium more viscous. We also compare our results with the FCS data obtained from the lambda DNA solution in various concentrations to mimic the cellular environment.

  19. Surface modification for interaction study with bacteria and preosteoblast cells

    NASA Astrophysics Data System (ADS)

    Song, Qing

    Surface modification plays a pivotal role in bioengineering. Polymer coatings can provide biocompatibility and biofunctionalities to biomaterials through surface modification. In this dissertation, initiated chemical vapor deposition (iCVD) was utilized to coat two-dimensional (2D) and three-dimensional (3D) substrates with differently charged polyelectrolytes in order to generate antimicrobial and osteocompatible biomaterials. ICVD is a modified CVD technique that enables surface modification in an all-dry condition without substrate damage and solvent contamination. The free-radical polymerization allows the vinyl polymers to conformally coat on various micro- and nano-structured substrates and maintains the delicate structure of the functional groups. The vapor deposition of polycations provided antimicrobial activity to planar and porous substrates through destroying the negatively charged bacterial membrane and brought about high contact-killing efficiency (99.99%) against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Additionally, the polyampholytes synthesized by iCVD exhibited excellent antifouling performance against the adhesion of Gram-positive Listeria innocua and Gram-negative E. coli in phosphate buffered saline (PBS). Their antifouling activities were attributed to the electrostatic interaction and hydration layers that served as physical and energetic barriers to prevent bacterial adhesion. The contact-killing and antifouling polymers synthesized by iCVD can be applied to surface modification of food processing equipment and medical devices with the aim of reducing foodborne diseases and medical infections. Moreover, the charged polyelectrolyte modified 2D polystyrene surfaces displayed good osteocompatibility and enhanced osteogenesis of preosteoblast cells than the un-modified polystyrene surface. In order to promote osteoinduction of hydroxyapatite (HA) scaffolds, bioinspired polymer-controlled mineralization was conducted

  20. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

    SciTech Connect

    Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R; Huser, T

    2007-01-09

    Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in

  1. Origins of Escherichia coli growth rate and cell shape changes at high external osmolality.

    PubMed

    Pilizota, Teuta; Shaevitz, Joshua W

    2014-10-21

    In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.

  2. Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

    PubMed Central

    Pilizota, Teuta; Shaevitz, Joshua W.

    2014-01-01

    In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis. PMID:25418177

  3. In vitro adhesion of enterotoxigenic Escherichia coli to human intestinal epithelial cells from mucosal biopsies.

    PubMed

    Knutton, S; Lloyd, D R; Candy, D C; McNeish, A S

    1984-05-01

    An adhesion assay with isolated human enterocytes prepared from duodenal biopsies has been developed and tested by using human enterotoxigenic Escherichia coli expressing colonization factor antigens I and II (CFA/I and CFA/II) and type 1 fimbriae. Enterotoxigenic E. coli strains H10407 (CFA/I) and B2C (CFA/II) bound to duodenal enterocytes to a much greater extent (mean of 4.6 and 4.0 bacteria per brush border) than did strain H10407P, a CFA/I- mutant of H10407 (mean of 0.1 bacteria per brush border). Type 1 fimbriae also promoted adhesion of strain H10407P to duodenal enterocytes but attachment was to basolateral rather than brush border surfaces. CFA/I and CFA/II, on the other hand, promoted adhesion only to human enterocyte brush borders.

  4. Prevalence of multi-antimicrobial-agent resistant, shiga toxin and enterotoxin producing Escherichia coli in surface waters of river Ganga.

    PubMed

    Ram, Siya; Vajpayee, Poornima; Shanker, Rishi

    2007-11-01

    The consumption of polluted surface water for domestic and recreational purposes by large populations in developing nations is a major cause of diarrheal disease related mortality. The river Ganga and its tributaries meet 40% of the water requirement for drinking and irrigation in India. In this study, Escherichia coli isolates (n=75) of the river Ganga water were investigated for resistance to antimicrobial agents (n=15) and virulence genes specific to shiga toxin (STEC) and enterotoxin producing E. coli (ETEC). E. coli isolates from the river Ganga water exhibit resistance to multiple antimicrobial agents. The distribution of antimicrobial agent resistance in E. colivaries significantly (chi2: 81.28 at df = 24, p < 0.001) between the sites. Both stx1 and stx2 genes were present in 82.3% of STEC (n=17) while remaining isolates possess either stxl (11.8%) or stx2 (5.9%). The presence of eaeA, hlyA, and chuA genes was observed in 70.6, 88.2, and 58.8% of STEC, respectively. Both LT1 and ST1 genes were positive in 66.7% of ETEC (n=15) while 33.3% of isolates harbor only LT1 gene. The prevalence of multi-antimicrobial-agent resistant E. coli in the river Ganga water poses increased risk of infections in the human population.

  5. Prevalence of multi-antimicrobial-agent resistant, shiga toxin and enterotoxin producing Escherichia coli in surface waters of river Ganga.

    PubMed

    Ram, Siya; Vajpayee, Poornima; Shanker, Rishi

    2007-11-01

    The consumption of polluted surface water for domestic and recreational purposes by large populations in developing nations is a major cause of diarrheal disease related mortality. The river Ganga and its tributaries meet 40% of the water requirement for drinking and irrigation in India. In this study, Escherichia coli isolates (n=75) of the river Ganga water were investigated for resistance to antimicrobial agents (n=15) and virulence genes specific to shiga toxin (STEC) and enterotoxin producing E. coli (ETEC). E. coli isolates from the river Ganga water exhibit resistance to multiple antimicrobial agents. The distribution of antimicrobial agent resistance in E. colivaries significantly (chi2: 81.28 at df = 24, p < 0.001) between the sites. Both stx1 and stx2 genes were present in 82.3% of STEC (n=17) while remaining isolates possess either stxl (11.8%) or stx2 (5.9%). The presence of eaeA, hlyA, and chuA genes was observed in 70.6, 88.2, and 58.8% of STEC, respectively. Both LT1 and ST1 genes were positive in 66.7% of ETEC (n=15) while 33.3% of isolates harbor only LT1 gene. The prevalence of multi-antimicrobial-agent resistant E. coli in the river Ganga water poses increased risk of infections in the human population. PMID:18044515

  6. Effect of Antimicrobial Agents on MinD Protein Oscillations in E. coli Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Kelly, Corey; Giuliani, Maximiliano; Dutcher, John

    2012-02-01

    The pole-to-pole oscillation of MinD proteins in E. coli cells determines the location of the division septum, and is integral to healthy cell division. It has been shown previously that the MinD oscillation period is approximately 40 s for healthy cells [1] but is strongly dependant on environmental factors such as temperature, which may place stress on the cell [2,3]. We use a strain of E. coli in which the MinD proteins are tagged with green fluorescent protein (GFP), allowing fluorescence visualization of the MinD oscillation. We use high-resolution total internal reflection fluorescence (TIRF) microscopy and a custom, temperature controlled flow cell to observe the effect of exposure to antimicrobial agents on the MinD oscillation period and, more generally, to analyze the time variation of the spatial distribution of the MinD proteins within the cells. These measurements provide insight into the mechanism of antimicrobial action. [1] Raskin, D.M.; de Boer, P. (1999) Proc. Natl. Acad. Sci. 96: 4971-4976. [2] Touhami, A.; Jericho, M; Rutenberg, A. (2006) J. Bacteriol. 188: 7661-7667. [3] Downing, B.; Rutenberg, A.; Touhami, A.; Jericho, M. (2009) PLoS ONE 4: e7285.

  7. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Shiga toxigenic Escherichia coli O104:H4 bares the characteristics of both enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga...

  8. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications

    PubMed Central

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods. PMID:26157424

  9. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

    PubMed

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

  10. Profiling of the cell surface proteome.

    PubMed

    Jang, Jun Ho; Hanash, Samir

    2003-10-01

    The in depth-mining of the proteome necessitates the comprehensive analysis of proteins in individual subcellular compartments to uncover interesting patterns of protein expression that include assessment of protein location, trafficking and of post-translational modifications that are location specific. One of the compartments of substantial interest from a diagnostic and therapeutic point of view is the plasma membrane which contains intrinsic membrane proteins and other proteins expressed on the cell surface. Technologies are currently available for the comprehensive profiling of the cell surface proteome that rely on protein tagging of intact cells. Studies are emerging that point to unexpected patterns of expression of specific proteins on the cell surface, with a common occurrence of proteins previously considered to occur predominantly in other compartments, notably the endoplasmic reticulum. The profiling of the cell surface and plasma membrane proteomes will likely provide novel insights and uncover disease related alterations. PMID:14625857

  11. Biocatalytic Formation of Gold Nanoparticles Decorated with Functional Proteins inside Recombinant Escherichia coli Cells.

    PubMed

    Hosomomi, Yukiho; Niide, Teppei; Wakabayashi, Rie; Goto, Masahiro; Kamiya, Noriho

    2016-01-01

    A novel strategy for the preparation of protein-decorated gold nanoparticles (Au NPs) was developed inside Escherichia coli cells, where an artificial oxidoreductase, composed of antibody-binding protein (pG), Bacillus stearothermophilus glycerol dehydrogenase (BsGLD) and a peptide tag with gold-binding affinity (H6C), was overexpressed in the cytoplasm. In situ formation of Au NPs was promoted by a natural electron-donating cofactor, nicotinamide adenine dinucleotide (NAD), which was regenerated to the reduced form of NADH by the catalytic activity of the fusion protein (pG-BsGLD-H6C) overexpressed in the cytoplasm of E. coli, with the concomitant addition of exogenous glycerol to the reaction system. The fusion protein was self-immobilized on Au NPs inside the E. coli cells, which was confirmed by SDS-PAGE and western blotting analyses of the resultant Au NPs. Finally, the IgG binding ability of the pG moiety displayed on Au NPs was evaluated by an enzyme-linked immunosorbent assay. PMID:26960608

  12. [Mutagenic and DNA-damaging effect of potassium bichromate in Escherichia coli cells].

    PubMed

    Kalinina, L M; Minseitova, S R

    1983-12-01

    Mutagenic effects and DNA degradation in Escherichia coli cells treated with potassium bichromate were studied in this work. It is estimated that events taking place in cells treated with the mutagen are controlled by recB recC and sbcB gene products. The method for sedimentation analysis has revealed DNA degradation. The use of the impulse label of 3H-thymidine made it possible to detect DNA breaks in the daughter strands. This effect of potassium bichromate on DNA degradation is, possibly, connected with mutagenic repair.

  13. FACTORS LIMITING BACTERIAL GROWTH : III. CELL SIZE AND "PHYSIOLOGIC YOUTH" IN BACTERIUM COLI CULTURES.

    PubMed

    Hershey, A D; Bronfenbrenner, J

    1938-07-20

    1. Measurements of the rate of oxygen uptake per cell in transplants of Bacterium coli from cultures of this organism in different phases of growth have given results in essential agreement with the observations of others. 2. Correlations of viable count, centrifugable nitrogen, and turbidity, with oxygen consumption, indicate that the increased metabolism during the early portion of the growth period is quantitatively referable to increased average size of cells. 3. Indirect evidence has suggested that the initial rate of growth of transplants is not related to the phase of growth of the parent culture.

  14. In-cell NMR in E. coli to Monitor Maturation Steps of hSOD1

    PubMed Central

    Banci, Lucia; Barbieri, Letizia; Bertini, Ivano; Cantini, Francesca; Luchinat, Enrico

    2011-01-01

    In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes. PMID:21887272

  15. Group V Phospholipase A2 in Bone Marrow-derived Myeloid Cells and Bronchial Epithelial Cells Promotes Bacterial Clearance after Escherichia coli Pneumonia*

    PubMed Central

    Degousee, Norbert; Kelvin, David J.; Geisslinger, Gerd; Hwang, David M.; Stefanski, Eva; Wang, Xing-Hua; Danesh, Ali; Angioni, Carlo; Schmidt, Helmut; Lindsay, Thomas F.; Gelb, Michael H.; Bollinger, James; Payré, Christine; Lambeau, Gérard; Arm, Jonathan P.; Keating, Armand; Rubin, Barry B.

    2011-01-01

    Group V-secreted phospholipase A2 (GV sPLA2) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA2 in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA2 after pulmonary E. coli infection. GV−/− mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV+/+ mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV+/+ and GV−/− mice after lung E. coli infection. In contrast, GV+/+ mice have higher levels of prostaglandin D2 (PGD2), PGF2α, and 15-keto-PGE2 in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV−/− mice after lung infection with E. coli. Selective deletion of GV sPLA2 in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA2 in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA2 in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli. PMID:21849511

  16. Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

    PubMed Central

    ALBIERO, Mayra Laino; AMORIM, Bruna Rabelo; MARTINS, Luciane; CASATI, Márcio Zaffalon; SALLUM, Enilson Antonio; NOCITI, Francisco Humberto; SILVÉRIO, Karina Gonzales

    2015-01-01

    Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities. PMID:26018305

  17. Solving a four-destination traveling salesman problem using Escherichia coli cells as biocomputers.

    PubMed

    Esau, Michael; Rozema, Mark; Zhang, Tuo Huang; Zeng, Dawson; Chiu, Stephanie; Kwan, Rachel; Moorhouse, Cadence; Murray, Cameron; Tseng, Nien-Tsu; Ridgway, Doug; Sauvageau, Dominic; Ellison, Michael

    2014-12-19

    The Traveling Salesman Problem involves finding the shortest possible route visiting all destinations on a map only once before returning to the point of origin. The present study demonstrates a strategy for solving Traveling Salesman Problems using modified E. coli cells as processors for massively parallel computing. Sequential, combinatorial DNA assembly was used to generate routes, in the form of plasmids made up of marker genes, each representing a path between destinations, and short connecting linkers, each representing a given destination. Upon growth of the population of modified E. coli, phenotypic selection was used to eliminate invalid routes, and statistical analysis was performed to successfully identify the optimal solution. The strategy was successfully employed to solve a four-destination test problem. PMID:25524102

  18. Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis.

    PubMed

    Ferreira Antunes, Márcia; Eggimann, Fabian Kurt; Kittelmann, Matthias; Lütz, Stephan; Hanlon, Steven P; Wirz, Beat; Bachler, Thorsten; Winkler, Margit

    2016-10-10

    Human xanthine oxidoreductase (XOR), which is responsible for the final steps of the purine metabolism pathway and involved in oxidative drug metabolism, was successfully expressed in Escherichia coli BL21(DE3) Gold. Recombinant human (rh) XOR yielded higher productivity with the gene sequence optimized for expression in E.coli than with the native gene sequence. Induction of XOR expression with lactose or IPTG resulted in complete loss of activity whereas shake flasks cultures using media rather poor in nutrients resulted in functional XOR expression in the stationary phase. LB medium was used for a 25L fermentation in fed-batch mode, which led to a 5 fold increase of the enzyme productivity when compared to cultivation in shake flasks. Quinazoline was used as a substrate on the semi-preparative scale using an optimized whole cell biotransformation protocol, yielding 73mg of the isolated product, 4-quinazolinone, from 104mg of starting material.

  19. High Cell Density Production of Europium-Labeled Escherichia coli for Tracing of Bacteria in Mantled Karst of Northwest Arkansas

    NASA Astrophysics Data System (ADS)

    Ting, T.; Thoma, G. J.; Beitle, R. B.; Davis, R. K.; Brahana, J. V.; Liu, H.

    2004-12-01

    The preparation of europium-labeled E. coli as a bacterial tracer in our study is separated into two major steps: the production of large quantities of cells, and the labeling of the cells at high density. Indigenous E. coli isolated from a natural spring at the University of Arkansas's Savoy Experimental Watershed (SEW), Savoy, Arkansas was fermented in BIOFLO II (New Brunswick Scientific, Edison, NJ) bioreactor using a fed-batch technique. Either a concentrated glucose solution or an ammonium hydroxide solution was pulsed into the reactor automatically using closed-loop pH control in a reactor feeding strategy designed to optimize cell growth. E. coli cells were harvested at the stationary phase of the bacterial growth profile, washed and centrifuged prior to the europium labeling step. A concentrated europium chloride solution was prepared by dissolving europium (III) chloride in 1-L of deionized water; the salt solution was chilled at 6oC overnight. A batch of 100-g wet weight of the washed E. coli was suspended in the chilled europium salt solution, and the cells were incubated at 6oC for 2 hours with stirring. After the cold incubation, the cells were washed with chilled deionized water and centrifuged repeatedly to remove excess europium. We have successfully prepared 760-g wet weight of labeled E. coli using the high cell density fermentation and europium labeling technique in a 9-day period. Preparation of large quantities of viable europium-tagged bacteria is critical for use as an environmental tracer. The europium uptake by the E. coli was found to be 15-mg europium per gram of labeled cell (wet weight). A field injection of multiple tracers along with the europium-tagged E. coli was successfully performed during the summer of 2004 at SEW to elucidate the transport, storage and viability of fecal contaminants in a karst basin. Prior investigations suggest that, unlike conservative tracers, E. coli become deposited along the flow path in the aquifer, and

  20. A comparative study on cell disruption methods for release of aspartase from E. coli K-12.

    PubMed

    Singh, R S

    2013-11-01

    Applicability of different mechanical cell disruption techniques namely sonication, bead milling and French press for the release of aspartase from E. coli K-12 was compared. Various operating parameters of each technique were optimized to obtain maximum aspartase release. The efficiency of aspartase release and cell disruption by all the methods was also compared under optimal conditions. The maximum release of aspartase (98.22%) and maximum cell breakage (84.25%) was observed using French press, while 92% of aspartase release was obtained by both sonication and bead milling. The order of cell disruption constant (k) for aspartase release by these methods was French press > bead milling > sonication. Disruption of cells using French press also demonstrated maximum protein release (14.12 mg/mL). The crude enzyme preparations can be further used for purification and its applications.

  1. Escherichia coli persister cells suppress translation by selectively disassembling and degrading their ribosomes.

    PubMed

    Cho, Junho; Rogers, Janet; Kearns, Mark; Leslie, Macall; Hartson, Steven D; Wilson, Kevin S

    2015-01-01

    Bacterial persisters are rare, phenotypically distinct cells that survive exposure to multiple antibiotics. Previous studies indicated that formation and maintenance of the persister phenotype are regulated by suppressing translation. To examine the mechanism of this translational suppression, we developed novel methodology to rapidly purify ribosome complexes from persister cells. We purified His-tagged ribosomes from Escherichia coli cells that over-expressed HipA protein, which induces persister formation, and were treated with ampicillin to remove antibiotic-sensitive cells. We profiled ribosome complexes and analyzed the ribosomal RNA and protein components from these persister cells. Our results show that (i) ribosomes in persisters exist largely as inactive ribosomal subunits, (ii) rRNAs and tRNAs are mostly degraded and (iii) a small fraction of the ribosomes remain mostly intact, except for reduced amounts of seven ribosomal proteins. Our findings explain the basis for translational suppression in persisters and suggest how persisters survive exposure to multiple antibiotics.

  2. Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells.

    PubMed

    Berlec, Aleš; Strukelj, Borut

    2013-04-01

    Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.

  3. Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide.

    PubMed

    Ueda, Takafumi; Akuta, Teruo; Kikuchi-Ueda, Takane; Imaizumi, Keitaro; Ono, Yasuo

    2016-04-01

    We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production. PMID:26724416

  4. A Single-Amino-Acid Substitution in Obg Activates a New Programmed Cell Death Pathway in Escherichia coli

    PubMed Central

    Dewachter, Liselot; Verstraeten, Natalie; Monteyne, Daniel; Kint, Cyrielle Ines; Versées, Wim; Pérez-Morga, David; Fauvart, Maarten

    2015-01-01

    ABSTRACT Programmed cell death (PCD) is an important hallmark of multicellular organisms. Cells self-destruct through a regulated series of events for the benefit of the organism as a whole. The existence of PCD in bacteria has long been controversial due to the widely held belief that only multicellular organisms would profit from this kind of altruistic behavior at the cellular level. However, over the past decade, compelling experimental evidence has established the existence of such pathways in bacteria. Here, we report that expression of a mutant isoform of the essential GTPase ObgE causes rapid loss of viability in Escherichia coli. The physiological changes that occur upon expression of this mutant protein—including loss of membrane potential, chromosome condensation and fragmentation, exposure of phosphatidylserine on the cell surface, and membrane blebbing—point to a PCD mechanism. Importantly, key regulators and executioners of known bacterial PCD pathways were shown not to influence this cell death program. Collectively, our results suggest that the cell death pathway described in this work constitutes a new mode of bacterial PCD. PMID:26695632

  5. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy.

  6. Co-Detection of Virulent Escherichia coli Genes in Surface Water Sources

    PubMed Central

    Ndlovu, Thando; Le Roux, Marcellous; Khan, Wesaal; Khan, Sehaam

    2015-01-01

    McNemar’s test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR) assays confirmed the presence of the aggR gene (69%), ipaH gene (23%) and the stx gene (15%) carried by Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Enterohermorrhagic E. coli (EHEC), respectively, in river water samples collected from the Berg River (Paarl, South Africa). Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa). In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC) in 69%, stx (EHEC) in 15%, ipaH (EIEC) in 31% and eae (EPEC) in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC) was detected in 46% of the samples, while eae (EPEC) was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05) between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar’s test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health. PMID:25659126

  7. Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss.

    PubMed

    Newton, Joseph M; Schofield, Desmond; Vlahopoulou, Joanna; Zhou, Yuhong

    2016-07-01

    Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction-point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069-1076, 2016.

  8. Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

    PubMed Central

    Newton, Joseph M.; Schofield, Desmond; Vlahopoulou, Joanna

    2016-01-01

    Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016 PMID:27111912

  9. High vacuum cells for classical surface techniques

    SciTech Connect

    Martinez, Imee Su; Baldelli, Steven

    2010-04-15

    Novel glass cells were designed and built to be able to perform surface potential and surface tension measurements in a contained environment. The cells can withstand pressures of approximately 1x10{sup -6} Torr, providing a reasonable level of control in terms of the amounts of volatile contaminants during experimentation. The measurements can take several hours; thus the cells help maintain the integrity of the sample in the course of the experiment. To test for the feasibility of the cell design, calibration measurements were performed. For the surface potential cell, the modified TREK 6000B-7C probe exhibited performance comparable to its unmodified counterpart. The correlation measurements between applied potential on the test surface and the measured potential showed R-values very close to 1 as well as standard deviation values of less than 1. Results also demonstrate improved measurement values for experiments performed in vacuum. The surface tension cell, on the other hand, which was used to perform the pendant drop method, was tested on common liquids and showed percentage errors of 0.5% when compared to literature values. The fabricated cells redefine measurements using classical surface techniques, providing unique and novel methods of sample preparation, premeasurement preparation, and sample analysis at highly beneficial expenditure cost.

  10. Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions.

    PubMed

    Mancusso, Romina; Cruz, Eduardo; Cataldi, Marcela; Mendoza, Carla; Fuchs, James; Wang, Hsin; Yang, Xiaomin; Tasayco, María Luisa

    2004-04-01

    Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to

  11. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

  12. Mouse Paneth Cell Secretory Responses to Cell Surface Glycolipids of Virulent and Attenuated Pathogenic Bacteria

    PubMed Central

    Tanabe, Hiroki; Ayabe, Tokiyoshi; Bainbridge, Brian; Guina, Tina; Ernst, Robert K.; Darveau, Richard P.; Miller, Samuel I.; Ouellette, Andre J.

    2005-01-01

    Mouse Paneth cells respond to bacteria and bacterial cell surface antigens by discharging secretory granules into the lumen of small intestinal crypts (T. Ayabe et al., Nat. Immunol. 1:113-118, 2000). To investigate mechanisms regulating these responses, purified surface glycolipid molecules with known acyl chain modifications and attenuated properties were tested for the ability to stimulate Paneth cell secretion. The antigens included lipopolysaccharide (LPS) from wild-type and msbB-null Escherichia coli and phoP-null and phoP-constitutive Salmonella enterica serovar Typhimurium strains, as well as LPS, lipid A, and lipoteichoic acid from Pseudomonas aeruginosa and Listeria monocytogenes grown in Mg2+-limited media. Measurements of total secreted protein, secreted lysozyme, and the bactericidal peptide activities of collected secretions showed that the purified antigens elicited similar secretory responses from Paneth cells in mouse crypts ex vivo, regardless of glycolipid acyl chain modification. Despite their impaired Tlr4 pathway, Paneth cells in ex vivo C3H/HeJ mouse crypts released equivalent amounts of bactericidal peptide activity in response to purified bacterial antigens, including lipid A. Thus, mouse Paneth cells respond equivalently to purified bacterial cell envelope glycolipids, regardless of functional Tlr4, the structural properties of glycolipid acyl chains, or their association with virulence in humans. PMID:15784576

  13. MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation

    PubMed Central

    Kadoya, Ryosuke; Matsumoto, Ken’ichiro; Ooi, Toshihiko; Taguchi, Seiichi

    2015-01-01

    Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly(lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers. PMID:26039058

  14. MreB Orientation Correlates with Cell Diameter in Escherichia coli.

    PubMed

    Ouzounov, Nikolay; Nguyen, Jeffrey P; Bratton, Benjamin P; Jacobowitz, David; Gitai, Zemer; Shaevitz, Joshua W

    2016-09-01

    Bacteria have remarkably robust cell shape control mechanisms. For example, cell diameter only varies by a few percent across a given population. The bacterial actin homolog, MreB, is necessary for establishment and maintenance of rod shape although the detailed properties of MreB that are important for shape control remained unknown. In this study, we perturb MreB in two ways: by treating cells with the polymerization-inhibiting drug A22 and by creating point mutants in mreB. These perturbations modify the steady-state diameter of cells over a wide range, from 790 ± 30 nm to 1700 ± 20 nm. To determine which properties of MreB are important for diameter control, we correlated structural characteristics of fluorescently tagged MreB polymers with cell diameter by simultaneously analyzing three-dimensional images of MreB and cell shape. Our results indicate that the helical pitch angle of MreB inversely correlates with the cell diameter of Escherichia coli. Other correlations between MreB and cell diameter are not found to be significant. These results demonstrate that the physical properties of MreB filaments are important for shape control and support a model in which MreB organizes the cell wall growth machinery to produce a chiral cell wall structure and dictate cell diameter. PMID:27602731

  15. Low-Reflectance Surfaces For Solar Cells

    NASA Technical Reports Server (NTRS)

    Bailey, Sheila G.; Landis, Geoffrey A.; Fatemi, Navid; Jenkins, Phillip P.

    1994-01-01

    Improved method for increasing solar cell efficiency has potential application for space-based and terrestrial solar power systems and optoelectronic devices. Etched low-angle grooves help recover reflected light. Light reflected from v-grooved surface trapped in cover glass and adhesive by total internal reflection. Reflected light redirected onto surface, and greater fraction of incident light absorbed, producing more electrical energy in InP solar photovoltaic cell.

  16. Enzyme Evolution by Yeast Cell Surface Engineering.

    PubMed

    Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Artificial evolution of proteins with the aim of acquiring novel or improved functionality is important for practical applications of the proteins. We have developed yeast cell surface engineering methods (or arming technology) for evolving enzymes. Here, we have described yeast cell surface engineering coupled with in vivo homologous recombination and library screening as a method for the artificial evolution of enzymes such as firefly luciferases. Using this method, novel luciferases with improved substrate specificity and substrate reactivity were engineered. PMID:26060078

  17. Increase in UV mutagenesis by heat stress on UV-irradiated E. coli cells.

    PubMed

    Saha, Swati; Basu, Tarakdas

    2012-06-01

    When leu- auxotrophs of Escherichia coli, after UV irradiation, were grown at temperatures between 30 and 47°C, the frequency of UV-induced mutation from leu- to leu+ revertant increased as the UV dose and the temperature increased. For cells exposed to a UV dose of 45 J/m2, the mutation frequency at 47°C was 1.9 times that at 30°C; for a dose of 90 J/m2, it was 3.25 times; and for 135 J/m2, it was 4.8 times. Similar enhancement of reversion frequency was observed when the irradiated cells were grown at 30°C in the presence of a heat shock inducer, ethanol (8% v/v). Heat shock-mediated enhancement of UV mutagenesis did not occur in an E. coli mutant sigma 32 (heat shock regulator protein), but sigma 32 overexpression in the mutant strain (transformed with a sigma 32-bearing plasmid) increased the UV-induced mutation frequency. These results suggest that heat stress alone has no mutagenic property, but when applied to UV-damaged cells, it enhances the UV-induced frequency of cell mutation.

  18. Role of major surface structures of Escherichia coli O157:H7 in initial attachment to biotic and abiotic surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection by human pathogens through fresh, minimally processed produce and solid plant-derived foods is a major concern of U.S. and global food industry and public health services. The enterohemorrhagic Escherichia coli O157:H7 is a frequent and potent food borne pathogen that causes severe disease...

  19. Essential oils--their antimicrobial activity against Escherichia coli and effect on intestinal cell viability.

    PubMed

    Fabian, Dusan; Dusan, Fabian; Sabol, Marián; Marián, Sabol; Domaracká, Katarína; Katarína, Domaracká; Bujnáková, Dobroslava; Dobroslava, Bujnáková

    2006-12-01

    Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria. The main objective of this study was to evaluate possible harmful effects of four commonly used essential oils and their major components on intestinal cells. Antimicrobial activity of selected plant extracts against enteroinvasive Escherichia coli was dose dependent. However, doses of essential oils with the ability to completely inhibit bacterial growth (0.05%) showed also relatively high cytotoxicity to intestinal-like cells cultured in vitro. Lower doses of essential oils (0.01%) had only partial antimicrobial activity and their damaging effect on Caco-2 cells was only modest. Cell death assessment based on morphological and viability staining followed by fluorescence microscopy showed that essential oils of cinnamon and clove and their major component eugenol had almost no cytotoxic effect at lower doses. Although essential oil of oregano and its component carvacrol slightly increased the incidence of apoptotic cell death, they showed extensive antimicrobial activity even at lower concentrations. Relatively high cytotoxicity was demonstrated by thyme oil, which increased both apoptotic and necrotic cell death incidence. In contrast, its component thymol showed no cytotoxic effect as well as greatly-reduced ability to inhibit visible growth of the chosen pathogen in the doses used. On the other hand, the addition of all essential oils and their components at lower doses, with the exception of thyme oil, to bacterial suspension significantly reduced the cytotoxic effect of E. coli on Caco-2 cells after 1h culture. In conclusion, it is possible to find appropriate doses of essential oils showing both antimicrobial activity and very low detrimental effect on intestinal cells. PMID:16919909

  20. Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water

    SciTech Connect

    Barcina, I.; Gonzalez, J.M.; Iriberri, J.; Egea, L.

    1989-01-01

    Some effects of visible light on the survival of Escherichia coli in waters of the Butron river were studied by comparing illuminated and nonilluminated systems. The following count methods were used: CFU on a selective medium (eosin-methylene blue agar), CFU on a medium of recuperation (Trypticase soy agar with yeast extract and glucose), number of metabolically active cells by reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, and total number of E. coli cells as determined by the acridine orange direct-count method. In the illuminated systems, decreases in CFU of E. coli and in the number of metabolically active cells were observed. However, no decline of the total number of E. coli cells was observed. By count methods, different stages of progressive dormancy of E. coli cells were determined to exist in illuminated systems. Culturable and recoverable cells were defined as viable cells, and metabolically active cells and morphologically intact cells were defined as somnicells. Indirect activity measurements were also done by using (14C)glucose. In illuminated systems, a decrease of glucose uptake by E. coli cells was observed throughout the experiments. The assimilated fraction of (14C)glucose decreased faster than the respired fraction in illuminated systems. The percentage of respired (14C)glucose (14CO2 production) with respect to the total glucose uptake increased throughout the experiments, and the percentage of assimilated glucose decreased. Therefore, the visible light was also responsible for an additional inhibition of biosynthetic processes.

  1. Reactivation of cell surface transport in Reticulomyxa.

    PubMed

    Orokos, D D; Bowser, S S; Travis, J L

    1997-01-01

    Granuloreticulosean protists transport particles (e.g., bacteria, algae, and sand grains) along the outer surfaces of their pseudopodia. This cell surface transport plays a vital role in feeding, reproduction, shell construction, and locomotion and can be visualized by the movements of extracellularly adherent polystyrene microspheres (i.e., latex beads). Our videomicroscopic analyses of transport associated with the pseudopodia of Reticulomyxa filosa revealed two distinct types of both intracellular and cell surface transport: (1) saltatory, bidirectional transport of individual or clustered organelles and/or surface-attached particles, and (2) continuous, unidirectional bulk or "resolute" motion of aggregated organelles and/or surface-bound particles. Organelles and surface-attached polystyrene microspheres remained firmly attached to the microtubule cytoskeletons of detergent-extracted pseudopodia. Both saltatory and resolute organelle and surface transport reactivated upon the addition of 0.01-1.0 mM ATP. At 1 mM ATP, the velocities of reactivated saltatory transport were indistinguishable from those observed in vivo. The reactivated transport was microtubule-dependent and was not inhibited by incubation with Ca(2+)-gelsolin under conditions that abolish rhodamine-phalloidin detection of actin filaments. These findings provide further support that both intracellular organelle and membrane surface transport are mediated by a common mechanism, and establish Reticulomyxa as a unique model system to further study the mechanochemistry of cell surface transport in vitro.

  2. Transcription of ftsZ oscillates during the cell cycle of Escherichia coli.

    PubMed

    Garrido, T; Sánchez, M; Palacios, P; Aldea, M; Vicente, M

    1993-10-01

    The FtsZ protein is a key element controlling cell division in Escherichia coli. A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells. The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates. This cell cycle dependency is specifically due to the two proximal ftsZ promoters. A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer. In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate-limiting role of this protein in cell division. Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated. This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles.

  3. Structure and functions of fungal cell surfaces

    NASA Technical Reports Server (NTRS)

    Nozawa, Y.

    1984-01-01

    A review with 24 references on the biochemistry, molecular structure, and function of cell surfaces of fungi, especially dermatophytes: the chemistry and structure of the cell wall, the effect of polyene antibiotics on the morphology and function of cytoplasmic membranes, and the chemical structure and function of pigments produced by various fungi are discussed.

  4. Probes for anionic cell surface detection

    DOEpatents

    Smith, Bradley D.

    2013-03-05

    Embodiments of the present invention are generally directed to compositions comprising a class of molecular probes for detecting the presence of anionic cell surfaces. Embodiments include compositions that are enriched for these compositions and preparations, particularly preparations suitable for use as laboratory/clinical reagents and diagnostic indicators, either alone or as part of a kit. An embodiment of the invention provides for a highly selective agent useful in the discernment and identification of dead or dying cells, such as apoptotic cells, in a relatively calcium-free environment. An embodiment of the invention provides a selective agent for the identification of bacteria in a mixed population of bacterial cells and nonbacterial cells.

  5. [Hepatitis E virus capsid protein production by high cell density culture of recombinant Escherichia coli].

    PubMed

    Liu, Ru-Shi; He, Zhi-Qiang; Li, Shao-Wei; Yang, Kun-Yu; Xian, Yang-Ling; Pang, Shu-Qiang; Zhang, Jun; Li, Yi-Min; Xia, Ning-Shao

    2004-05-01

    Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility. PMID:15971623

  6. The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli

    PubMed Central

    Peters, Katharina; Kannan, Suresh; Rao, Vincenzo A.; Biboy, Jacob; Vollmer, Daniela; Erickson, Stephen W.; Lewis, Richard J.

    2016-01-01

    ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. PMID:27329754

  7. Effect of Micro- and Nanoscale Topography on the Adhesion of Bacterial Cells to Solid Surfaces

    PubMed Central

    Hsu, Lillian C.; Fang, Jean; Borca-Tasciuc, Diana A.; Worobo, Randy W.

    2013-01-01

    Attachment and biofilm formation by bacterial pathogens on surfaces in natural, industrial, and hospital settings lead to infections and illnesses and even death. Minimizing bacterial attachment to surfaces using controlled topography could reduce the spreading of pathogens and, thus, the incidence of illnesses and subsequent human and financial losses. In this context, the attachment of key microorganisms, including Escherichia coli, Listeria innocua, and Pseudomonas fluorescens, to silica and alumina surfaces with micron and nanoscale topography was investigated. The results suggest that orientation of the attached cells occurs preferentially such as to maximize their contact area with the surface. Moreover, the bacterial cells exhibited different morphologies, including different number and size of cellular appendages, depending on the topographical details of the surface to which they attached. This suggests that bacteria may utilize different mechanisms of attachment in response to surface topography. These results are important for the design of novel microbe-repellant materials. PMID:23416997

  8. Cell Adhesion on Surface-Functionalized Magnesium.

    PubMed

    Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

    2016-05-18

    The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance. PMID:27089250

  9. [Effect of Stress on Emergence of Antibiotic-resistant Escherichia coli Cells].

    PubMed

    Loiko, N G; Kozlova, A N; Nikolaev, Yu A; Gaponov, A M; Tutel'yan, A V; El'-Registan, G I

    2015-01-01

    Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45 degrees C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli 12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10(-4) to 10(-2)% of the intial CFU number) surviving lethal doses of an antibiotic (a mpicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term persisters, and the cells of slowly growing SCV variants with small colonies (d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete. PMID:27169240

  10. MioC and GidA proteins promote cell division in E. coli.

    PubMed

    Lies, Mark; Visser, Bryan J; Joshi, Mohan C; Magnan, David; Bates, David

    2015-01-01

    The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.

  11. Characterization of MreB polymers in E. coli and their correlations to cell shape

    NASA Astrophysics Data System (ADS)

    Nguyen, Jeffrey; Ouzonov, Nikolay; Gitai, Zemer; Shaevitz, Joshua

    2015-03-01

    Shape influences all facets of how bacteria interact with their environment. The size of E. coli is determined by the peptidoglycan cell wall and internal turgor pressure. The cell wall is patterned by MreB, an actin homolog that forms short polymers on the cytoplasmic membrane. MreB coordinates the breaking of old material and the insertion of new material for growth, but it is currently unknown what mechanism sets the absolute diameter of the cell. Using new techniques in fluorescence microscopy and image processing, we are able to quantify cell shape in 3- dimensions and access previously unattainable data on the conformation of MreB polymers. To study how MreB affects the diameter of bacteria, we analyzed the shapes and polymers of cells that have had MreB perturbed by one of two methods. We first treated cells with the MreB polymerization-inhibiting drug A22. Secondly, we created point mutants in MreB that change MreB polymer conformation and the cell shape. By analyzing the correlations between different shape and polymer metrics, we find that under both treatments, the average helical pitch angle of the polymers correlates strongly with the cell diameter. This observation links the micron scale shape of the cell to the nanometer scale MreB cytoskeleton.

  12. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes

    PubMed Central

    1989-01-01

    The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli. PMID:2538544

  13. Protection against Escherichia coli infection by antibody to the Staphylococcus aureus poly-N-acetylglucosamine surface polysaccharide

    PubMed Central

    Cerca, Nuno; Maira-Litrán, Tomás; Jefferson, Kimberly K.; Grout, Martha; Goldmann, Donald A.; Pier, Gerald B.

    2007-01-01

    Poly-N-acetylglucosamine (PNAG) is a surface polysaccharide produced by Staphylococcus aureus and Staphyloccus epidermidis and is an effective target for opsonic and protective Ab for these two organisms. Recently, it has been found that Escherichia coli produces an exo-polysaccharide, designated polyglucosamine, that is biochemically indistinguishable from PNAG. We analyzed 30 E. coli strains isolated from urinary tract and neonatal bloodstream infections for the pga locus, PNAG antigen production, and susceptibility to opsonic killing and protection from lethal infection by Ab to PNAG. Twenty-six of 30 strains carried the pga locus, 25 of 30 expressed immunologically detectable PNAG, and 21 of 30 could be killed by rabbit IgG specific for the deacetylated form of the staphylococcal PNAG. Ab to staphylococcal PNAG protected mice against lethality from five different E. coli strains expressing PNAG. PNAG expression by both Gram-negative and Gram-positive organisms could make this antigen a conserved vaccine target for multiple pathogenic species of bacteria. PMID:17446272

  14. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

    PubMed Central

    Canuto, K.S.; Sergio, L.P.S.; Guimarães, O.R.; Geller, M.; Paoli, F.; Fonseca, A.S.

    2015-01-01

    Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia colicells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficientE. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out. PMID:26445338

  15. Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells.

    PubMed

    Canuto, K S; Sergio, L P S; Guimarães, O R; Geller, M; Paoli, F; Fonseca, A S

    2015-10-01

    Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia colicells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficientE. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out.

  16. Surface Structures Involved in Plant Stomata and Leaf Colonization by Shiga-Toxigenic Escherichia Coli O157:H7

    PubMed Central

    Saldaña, Zeus; Sánchez, Ethel; Xicohtencatl-Cortes, Juan; Puente, Jose Luis; Girón, Jorge A.

    2011-01-01

    Shiga-toxigenic Escherichia coli (STEC) O157:H7 uses a myriad of surface adhesive appendages including pili, flagella, and the type 3 secretion system (T3SS) to adhere to and inflict damage to the human gut mucosa. Consumption of contaminated ground beef, milk, juices, water, or leafy greens has been associated with outbreaks of diarrheal disease in humans due to STEC. The aim of this study was to investigate which of the known STEC O157:H7 adherence factors mediate colonization of baby spinach leaves and where the bacteria reside within tainted leaves. We found that STEC O157:H7 colonizes baby spinach leaves through the coordinated production of curli, the E. coli common pilus, hemorrhagic coli type 4 pilus, flagella, and T3SS. Electron microscopy analysis of tainted leaves revealed STEC bacteria in the internal cavity of the stomata, in intercellular spaces, and within vascular tissue (xylem and phloem), where the bacteria were protected from the bactericidal effect of gentamicin, sodium hypochlorite or ozonated water treatments. We confirmed that the T3S escN mutant showed a reduced number of bacteria within the stomata suggesting that T3S is required for the successful colonization of leaves. In agreement, non-pathogenic E. coli K-12 strain DH5α transformed with a plasmid carrying the locus of enterocyte effacement (LEE) pathogenicity island, harboring the T3SS and effector genes, internalized into stomata more efficiently than without the LEE. This study highlights a role for pili, flagella, and T3SS in the interaction of STEC with spinach leaves. Colonization of plant stomata and internal tissues may constitute a strategy by which STEC survives in a nutrient-rich microenvironment protected from external foes and may be a potential source for human infection. PMID:21887151

  17. The Synchronization of Replication and Division Cycles in Individual E. coli Cells.

    PubMed

    Wallden, Mats; Fange, David; Lundius, Ebba Gregorsson; Baltekin, Özden; Elf, Johan

    2016-07-28

    Isogenic E. coli cells growing in a constant environment display significant variability in growth rates, division sizes, and generation times. The guiding principle appears to be that each cell, during one generation, adds a size increment that is uncorrelated to its birth size. Here, we investigate the mechanisms underlying this "adder" behavior by mapping the chromosome replication cycle to the division cycle of individual cells using fluorescence microscopy. We have found that initiation of chromosome replication is triggered at a fixed volume per chromosome independent of a cell's birth volume and growth rate. Each initiation event is coupled to a division event after a growth-rate-dependent time. We formalize our findings in a model showing that cell-to-cell variation in division timing and cell size is mainly driven by variations in growth rate. The model also explains why fast-growing cells display adder behavior and correctly predict deviations from the adder behavior at slow growth. PMID:27471967

  18. Human milk oligosaccharides protect bladder epithelial cells against uropathogenic Escherichia coli invasion and cytotoxicity.

    PubMed

    Lin, Ann E; Autran, Chloe A; Espanola, Sophia D; Bode, Lars; Nizet, Victor

    2014-02-01

    The invasive pathogen uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). Recurrent infection that can progress to life-threatening renal failure has remained as a serious global health concern in infants. UPEC adheres to and invades bladder epithelial cells to establish infection. Studies have detected the presence of human milk oligosaccharides (HMOs) in urine of breast-fed, but not formula-fed, neonates. We investigated the mechanisms HMOs deploy to elicit protection in human bladder epithelial cells infected with UPEC CFT073, a prototypic urosepsis-associated strain. We found a significant reduction in UPEC internalization into HMO-pretreated epithelial cells without observing any significant effect in UPEC binding to these cells. This event coincides with a rapid decrease in host cell cytotoxicity, recognized by LIVE/DEAD staining and cell detachment, but independent of caspase-mediated or mitochondrial-mediated programmed cell death pathways. Further investigation revealed HMOs, and particularly the sialic acid-containing fraction, reduced UPEC-mediated MAPK and NF-κB activation. Collectively, our results indicate that HMOs can protect bladder epithelial cells from deleterious cytotoxic and proinflammatory effects of UPEC infection, and may be one contributing mechanism underlying the epidemiological evidence of reduced UTI incidence in breast-fed infants.

  19. Residence and transit times of MinD in E. coli bacterial cells

    NASA Astrophysics Data System (ADS)

    Giuliani, Maximiliano; Kelly, Corey; Dutcher, John

    2012-02-01

    A key step in the life of a bacterial cell is its division into two daughters cells of equal size. This process is carefully controlled and regulated so that an equal partitioning of the main cell components is obtained, which is critical for the viability of the daughter cells. In E. coli this regulation is accomplished in part by the Min protein system, that determines the localization of the division machinery. Of particular interest is the MinD protein that exhibits an oscillation between the poles in the rod shaped bacteria. The oscillation relies on a ATP mediated dimerization of the MinD protein that allows its insertion into the inner membrane at one of the poles of the cell, followed by an interaction with the MinE protein, which releases the MinD from the membrane, allowing it to travel to the other pole of the cell where the cycle is repeated. We have studied the spatio-temporal characteristics of the MinD oscillation from which we extract the average times for the two main processes that determine the oscillation period: the residence time in the membrane and the transit time to travel the length of the cell. Additionally, we explore how these two timescales are affected by stresses on the bacterial cells due to unfavorable physiological conditions.

  20. Deformation of Filamentous Escherichia coli Cells in a Microfluidic Device: A New Technique to Study Cell Mechanics

    PubMed Central

    Caspi, Yaron

    2014-01-01

    The mechanical properties of bacterial cells are determined by their stress-bearing elements. The size of typical bacterial cells, and the fact that different time and length scales govern their behavior, necessitate special experimental techniques in order to probe their mechanical properties under various spatiotemporal conditions. Here, we present such an experimental technique to study cell mechanics using hydrodynamic forces in a microfluidic device. We demonstrate the application of this technique by calculating the flexural rigidity of non-growing Escherichia coli cells. In addition, we compare the deformation of filamentous cells under growing and non-growing conditions during the deformation process. We show that, at low forces, the force needed to deform growing cells to the same extent as non-growing cells is approximately two times smaller. Following previous works, we interpret these results as the outcome of the difference between the elastic response of non-growing cells and the plastic-elastic response of growing cells. Finally, we observe some heterogeneity in the response of individual cells to the applied force. We suggest that this results from the individuality of different bacterial cells. PMID:24392095

  1. Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli.

    PubMed

    Clark, Michelle W; Yie, Anna M; Eder, Elizabeth K; Dennis, Richard G; Basting, Preston J; Martinez, Keith A; Jones, Brian D; Slonczewski, Joan L

    2015-01-01

    Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2-7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.

  2. Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli

    PubMed Central

    Clark, Michelle W.; Yie, Anna M.; Eder, Elizabeth K.; Dennis, Richard G.; Basting, Preston J.; Martinez, Keith A.; Jones, Brian D.; Slonczewski, Joan L.

    2015-01-01

    Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2–7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress. PMID:26713733

  3. Progenitor cells for ocular surface regenerative therapy.

    PubMed

    Casaroli-Marano, Ricardo P; Nieto-Nicolau, Nuria; Martínez-Conesa, Eva M

    2013-01-01

    The integrity and normal function of the corneal epithelium are essential for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio-replacement, such as cultured limbal epithelial transplantation and cultured oral mucosal epithelial transplantation, present very encouraging clinical results for treating limbal stem cell deficiencies. Another emerging therapeutic strategy consists of obtaining and implementing human progenitor cells of different origins using tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal stromal cells, represents a significant breakthrough in the treatment of certain eye diseases and also offers a more rational, less invasive and more physiological approach to ocular surface regeneration. PMID:23257987

  4. Progenitor cells for ocular surface regenerative therapy.

    PubMed

    Casaroli-Marano, Ricardo P; Nieto-Nicolau, Nuria; Martínez-Conesa, Eva M

    2013-01-01

    The integrity and normal function of the corneal epithelium are essential for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio-replacement, such as cultured limbal epithelial transplantation and cultured oral mucosal epithelial transplantation, present very encouraging clinical results for treating limbal stem cell deficiencies. Another emerging therapeutic strategy consists of obtaining and implementing human progenitor cells of different origins using tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal stromal cells, represents a significant breakthrough in the treatment of certain eye diseases and also offers a more rational, less invasive and more physiological approach to ocular surface regeneration.

  5. Data in support of Gallium (Ga(3+)) antibacterial activities to counteract E. coli and S. epidermidis biofilm formation onto pro-osteointegrative titanium surfaces.

    PubMed

    Cochis, A; Azzimonti, B; Sorrentino, R; Della Valle, C; De Giglio, E; Bloise, N; Visai, L; Bruni, G; Cometa, S; Pezzoli, D; Candiani, G; Rimondini, L; Chiesa, R

    2016-03-01

    This paper contains original data supporting the antibacterial activities of Gallium (Ga(3+))-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in "The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii" (Cochis et al. 2016) [1]. In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga(3+) enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga(3+) activity were compared to Silver.

  6. Data in support of Gallium (Ga3+) antibacterial activities to counteract E. coli and S. epidermidis biofilm formation onto pro-osteointegrative titanium surfaces

    PubMed Central

    Cochis, A.; Azzimonti, B.; Sorrentino, R.; Della Valle, C.; De Giglio, E.; Bloise, N.; Visai, L.; Bruni, G.; Cometa, S.; Pezzoli, D.; Candiani, G.; Rimondini, L.; Chiesa, R.

    2016-01-01

    This paper contains original data supporting the antibacterial activities of Gallium (Ga3+)-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in “The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii” (Cochis et al. 2016) [1]. In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga3+ enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga3+ activity were compared to Silver. PMID:26909385

  7. Data in support of Gallium (Ga(3+)) antibacterial activities to counteract E. coli and S. epidermidis biofilm formation onto pro-osteointegrative titanium surfaces.

    PubMed

    Cochis, A; Azzimonti, B; Sorrentino, R; Della Valle, C; De Giglio, E; Bloise, N; Visai, L; Bruni, G; Cometa, S; Pezzoli, D; Candiani, G; Rimondini, L; Chiesa, R

    2016-03-01

    This paper contains original data supporting the antibacterial activities of Gallium (Ga(3+))-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in "The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii" (Cochis et al. 2016) [1]. In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga(3+) enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga(3+) activity were compared to Silver. PMID:26909385

  8. Development of a rapid capture-cum-detection method for Escherichia coli O157 from apple juice comprising nano-immunomagnetic separation in tandem with surface enhanced Raman scattering.

    PubMed

    Najafi, Roya; Mukherjee, Shubhasish; Hudson, Jim; Sharma, Anup; Banerjee, Pratik

    2014-10-17

    A combined capture and detection method comprising of nano-immunomagnetic separation (NIMS) and surface enhanced Raman spectroscopy (SERS) was developed to detect Escherichia coli O157 from liquid media including apple juice. The capture antibodies (cAbs) were immobilized on magnetite-gold (Fe3O4/Au) magnetic nanoparticles (MNPs) which were used for separation and concentration of the E. coli O157 cells from model liquid food matrix. The capture efficiency (CE) for E. coli O157 using MNP was found to be approximately 84-94%. No cross reactivity was observed with background non-target organisms. There was a significant difference in the mean CE of bacteria captured by MNP and commercially sourced immunomagnetic microbeads (p<0.05). For the detection of target pathogen, SERS labels were prepared by conjugating gold nanoparticles with Raman reporter molecules and the detector antibody (dAb). Au-Raman label-dAb was interacted with gold coated MNP-cAb-E. coli O157 complex. The ability of this immunoassay to detect E. coli O157 in apple juice was investigated. We have successfully applied the synthesized Fe3O4/Au nanoclusters to E. coli O157 detection in apple juice using the SERS method. The lowest detectable bacterial cell concentration in apple juice was 10(2)CFU/mL with a total analysis time of less than an hour. This method presents a convenient way of preconcentration, separation, and detection of low levels of target pathogen from liquid food matrix.

  9. Surface and internalized Escherichia coli O157:H7 on field-grown spinach and lettuce treated with spray-contaminated irrigation water.

    PubMed

    Erickson, Marilyn C; Webb, Cathy C; Diaz-Perez, Juan Carlos; Phatak, Sharad C; Silvoy, John J; Davey, Lindsey; Payton, Alison S; Liao, Jean; Ma, Li; Doyle, Michael P

    2010-06-01

    Numerous field studies have revealed that irrigation water can contaminate the surface of plants; however, the occurrence of pathogen internalization is unclear. This study was conducted to determine the sites of Escherichia coli O157:H7 contamination and its survival when the bacteria were applied through spray irrigation water to either field-grown spinach or lettuce. To differentiate internalized and surface populations, leaves were treated with a surface disinfectant wash before the tissue was ground for analysis of E. coli O157:H7 by direct plate count or enrichment culture. Irrigation water containing E. coli O157:H7 at 10(2), 10(4), or 10(6) CFU/ml was applied to spinach 48 and 69 days after transplantation of seedlings into fields. E. coli O157:H7 was initially detected after application on the surface of plants dosed at 10(4) CFU/ml (4 of 20 samples) and both on the surface (17 of 20 samples) and internally (5 of 20 samples) of plants dosed at 10(6) CFU/ml. Seven days postspraying, all spinach leaves tested negative for surface or internal contamination. In a subsequent study, irrigation water containing E. coli O157:H7 at 10(8) CFU/ml was sprayed onto either the abaxial (lower) or adaxial (upper) side of leaves of field-grown lettuce under sunny or shaded conditions. E. coli O157:H7 was detectable on the leaf surface 27 days postspraying, but survival was higher on leaves sprayed on the abaxial side than on leaves sprayed on the adaxial side. Internalization of E. coli O157:H7 into lettuce leaves also occurred with greater persistence in leaves sprayed on the abaxial side (up to 14 days) than in leaves sprayed on the adaxial side (2 days).

  10. Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

    PubMed

    San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

    2015-10-01

    This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain.

  11. Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis

    PubMed Central

    San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

    2015-01-01

    This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. PMID:26185055

  12. The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion.

    PubMed

    Shimizu, Takeshi; Ichimura, Kimitoshi; Noda, Masatoshi

    2016-02-01

    Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection. PMID:26644384

  13. The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion

    PubMed Central

    Ichimura, Kimitoshi; Noda, Masatoshi

    2015-01-01

    Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection. PMID:26644384

  14. Polysaccharide-based silver nanoparticles synthesized by Klebsiella oxytoca DSM 29614 cause DNA fragmentation in E. coli cells.

    PubMed

    Baldi, Franco; Daniele, Salvatore; Gallo, Michele; Paganelli, Stefano; Battistel, Dario; Piccolo, Oreste; Faleri, Claudia; Puglia, Anna Maria; Gallo, Giuseppe

    2016-04-01

    Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO3 to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS(aer) and the AgNPs-EPS(anaer), were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS(aer), in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS(aer). In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS(aer) exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS(aer). The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag(+) release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations. PMID:26886276

  15. Recent advances in adherence and invasion of pathogenic Escherichia coli

    PubMed Central

    Kalita, Anjana; Hu, Jia; Torres, Alfredo G.

    2014-01-01

    Purpose of review Colonization of the host epithelia by pathogenic Escherichia coli is influenced by the ability of the bacteria to interact with host surfaces. Because the initial step of an E. coli infection is to adhere, invade, and persist within host cells, some strategies used by intestinal and extra-intestinal E. coli to infect host cell are presented. Recent findings This review highlights recent progress understanding how extra-intestinal pathogenic E. coli strains express specific adhesins/invasins that allow colonization of the urinary tract or the meninges, while intestinal E. coli strains are able to colonize different regions of the intestinal tract using other specialized adhesins/invasins. Finally, evaluation of, different diets and environmental conditions regulating the colonization of these pathogens is discussed. Summary Discovery of new interactions between pathogenic E. coli and the host epithelial cells unravels the need of more mechanistic studies that can provide new clues in how to combat these infections. PMID:25023740

  16. Real-time observation of Escherichia coli cells under irradiation with a 2-MeV H{sup +} microbeam

    SciTech Connect

    Kato, Mikio; Meissl, Walter; Ikeda, Tokihiro; Yamazaki, Yasunori; Umezawa, Kenji

    2012-05-07

    A high-energy H{sup +} microbeam generated by tapered glass capillary optics was applied to a single Escherichia coli cell, in order to evaluate the effects of irradiation on the activity of the flagellar motor and cell growth in real time. The flagellar motor of the tethered cells was stopped by irradiation with an average ion fluence of 2.0 x 10{sup 12} protons/cm{sup 2}. When a lower dose was applied to the cells attached to the substrate, an elongated cell, which seemed ready to divide, divided into two daughter cells; however, the daughter cells did not elongate, neither did further cell division occur.

  17. A model for chromosome organization during the cell cycle in live E. coli

    PubMed Central

    Liu, Yuru; Xie, Ping; Wang, Pengye; Li, Ming; Li, Hui; Li, Wei; Dou, Shuoxing

    2015-01-01

    Bacterial chromosomal DNA is a highly compact nucleoid. The organization of this nucleoid is poorly understood due to limitations in the methods used to monitor the complexities of DNA organization in live bacteria. Here, we report that circular plasmid DNA is auto-packaged into a uniform dual-toroidal-spool conformation in response to mechanical stress stemming from sharp bending and un-winding by atomic force microscopic analysis. The mechanism underlying this phenomenon was deduced with basic physical principles to explain the auto-packaging behaviour of circular DNA. Based on our observations and previous studies, we propose a dynamic model of how chromosomal DNA in E. coli may be organized during a cell division cycle. Next, we test the model by monitoring the development of HNS clusters in live E. coli during a cell cycle. The results were in close agreement with the model. Furthermore, the model accommodates a majority of the thus-far-discovered remarkable features of nucleoids in vivo. PMID:26597953

  18. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli.

    PubMed

    Zheng, Zhaojuan; Xu, Ying; Sun, Ye; Mei, Wending; Ouyang, Jia

    2015-01-01

    Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h). No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose. PMID:26462117

  19. Biocatalytic Production of Trehalose from Maltose by Using Whole Cells of Permeabilized Recombinant Escherichia coli

    PubMed Central

    Sun, Ye; Mei, Wending; Ouyang, Jia

    2015-01-01

    Trehalose is a non-reducing disaccharide, which can protect proteins, lipid membranes, and cells from desiccation, refrigeration, dehydration, and other harsh environments. Trehalose can be produced by different pathways and trehalose synthase pathway is a convenient, practical, and low-cost pathway for the industrial production of trehalose. In this study, 3 candidate treS genes were screened from genomic databases of Pseudomonas and expressed in Escherichia coli. One of them from P. stutzeri A1501 exhibited the best transformation ability from maltose into trehalose and the least byproduct. Thus, whole cells of this recombinant E. coli were used as biocatalyst for trehalose production. In order to improve the conversion rate of maltose to trehalose, optimization of the permeabilization and biotransformation were carried out. Under optimal conditions, 92.2 g/l trehalose was produced with a high productivity of 23.1 g/(l h). No increase of glucose was detected during the whole course. The biocatalytic process developed in this study might serve as a candidate for the large scale production of trehalose. PMID:26462117

  20. Inactivation of E. Coli cell viability and DNA Photo-breakage by Pulsed Nitrogen Laser Radiation

    SciTech Connect

    Cheba, Ben Amar; Alzaag, Ali; Tilfah, Nafie A.

    2005-03-17

    The mutagenic and lethal effect of nitrogen laser radiation: 337.1 nm wave length, 1.5 millijoul pulse energy, 10 nanosecond pulse with and pulse repetition rate range from 1 to 50 Pulse/ second was evaluated on E. Coli cells. Results indicated that irradiation of E. coli JMP39 with pulse repetition of 8 , 16 , 32 pulse/sec, for 1, 5 , 10, 25 min respectively led to a significant decrease in cell count proportional to irradiation dose with significant increase in lacmutation frequency accompanied with some mutations in pattern of antibiotic resistance. The effect of nitrogen laser on the genomic content of the strain JMP39 was also studied by irradiating the total DNA with 30 pulse/second for 1 ,5, 15 , 30 min then subjected to both agarose gel electrophoresis and scanning spectrophotometry. The first technique revealed to DNA photo breakage and significant decrease in DNA absorbency was noticed by scanning spectrophotometry. This could be attributed to photo-decomposition resulted from multi-photo-excitation of UV-Laser pulses.

  1. The Cell Surface Proteome of Human Mesenchymal Stromal Cells

    PubMed Central

    Pursche, Theresia; Bornhäuser, Martin; Corbeil, Denis; Hoflack, Bernard

    2011-01-01

    Background Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers. Methodology/Principal Findings To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316) were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously. Conclusions/Significance Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention. PMID:21637820

  2. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  3. Escherichia coli cell division mutation ftsM1 is in serU

    SciTech Connect

    Leclerc, G.; Sirard, C.; Drapeau, G.R.

    1989-04-01

    The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.

  4. Organization of ribosomes and nucleoids in Escherichia coli cells during growth and in quiescence.

    PubMed

    Chai, Qian; Singh, Bhupender; Peisker, Kristin; Metzendorf, Nicole; Ge, Xueliang; Dasgupta, Santanu; Sanyal, Suparna

    2014-04-18

    We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

  5. Polar Location of the Chemoreceptor Complex in the Escherichia coli Cell

    NASA Astrophysics Data System (ADS)

    Maddock, Janine R.; Shapiro, Lucille

    1993-03-01

    The eukaryotic cell exhibits compartmentalization of functions to various membrane-bound organelles and to specific domains within each membrane. The spatial distribution of the membrane chemoreceptors and associated cytoplasmic chemotaxis proteins in Escherichia coli were examined as a prototypic functional aggregate in bacterial cells. Bacterial chemotaxis involves a phospho-relay system brought about by ligand association with a membrane receptor, culminating in a switch in the direction of flagellar rotation. The transduction of the chemotaxis signal is initiated by a chemoreceptor-CheW-CheA ternary complex at the inner membrane. These ternary complexes aggregate predominantly at the cell poles. Polar localization of the cytoplasmic CheA and CheW proteins is dependent on membrane-bound chemoreceptor. Chemoreceptors are not confined to the cell poles in strains lacking both CheA and CheW. The chemoreceptor-CheW binary complex is polarly localized in the absence of CheA, whereas the chemoreceptor-CheA binary complex is not confined to the cell poles in strains lacking CheW. The subcellular localization of the chemotaxis proteins may reflect a general mechanism by which the bacterial cell sequesters different regions of the cell for specialized functions.

  6. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    PubMed

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA.

  7. Enterotoxigenic Escherichia coli CS21 pilus contributes to adhesion to intestinal cells and to pathogenesis under in vivo conditions

    PubMed Central

    Guevara, C. P.; Luiz, W. B.; Sierra, A.; Cruz, C.; Qadri, F.; Kaushik, R. S.; Ferreira, L. C. S.

    2013-01-01

    Colonization surface antigens (CSs) represent key virulence-associated factors of enterotoxigenic Escherichia coli (ETEC) strains. They are required for gut colonization, the first step of the diarrhoeal disease process induced by these bacteria. One of the most prevalent CSs is CS21, or longus, a type IV pili associated with bacterial self-aggregation, protection against environmental stresses, biofilm formation and adherence to epithelial cell lines. The objectives of this study were to assess the role of CS21 in adherence to primary intestinal epithelial cells and to determine if CS21 contributes to the pathogenesis of ETEC infection in vivo. We evaluated adherence of a CS21-expressing wild-type ETEC strain and an isogenic CS21-mutant strain to pig-derived intestinal cell lines. To determine the role of CS21 in pathogenesis we used the above ETEC strains in a neonatal mice challenge infection model to assess mortality. Quantitative adherence assays confirmed that ETEC adheres to primary intestinal epithelial cells lines in a CS21-dependent manner. In addition, the CS21-mediated ETEC adherence to cells was specific as purified LngA protein, the CS21 major subunit, competed for binding with the CS21-expressing ETEC while specific anti-LngA antibodies blocked adhesion to intestinal cells. Neonatal DBA/2 mice died after intra-stomach administration of CS21-expressing strains while lack of CS21 expression drastically reduced the virulence of the wild-type ETEC strain in this animal model. Collectively these results further support the role of CS21 during ETEC infection and add new evidence on its in vivo relevance in pathogenesis. PMID:23760820

  8. Enterotoxigenic Escherichia coli CS21 pilus contributes to adhesion to intestinal cells and to pathogenesis under in vivo conditions.

    PubMed

    Guevara, C P; Luiz, W B; Sierra, A; Cruz, C; Qadri, F; Kaushik, R S; Ferreira, L C S; Gómez-Duarte, O G

    2013-08-01

    Colonization surface antigens (CSs) represent key virulence-associated factors of enterotoxigenic Escherichia coli (ETEC) strains. They are required for gut colonization, the first step of the diarrhoeal disease process induced by these bacteria. One of the most prevalent CSs is CS21, or longus, a type IV pili associated with bacterial self-aggregation, protection against environmental stresses, biofilm formation and adherence to epithelial cell lines. The objectives of this study were to assess the role of CS21 in adherence to primary intestinal epithelial cells and to determine if CS21 contributes to the pathogenesis of ETEC infection in vivo. We evaluated adherence of a CS21-expressing wild-type ETEC strain and an isogenic CS21-mutant strain to pig-derived intestinal cell lines. To determine the role of CS21 in pathogenesis we used the above ETEC strains in a neonatal mice challenge infection model to assess mortality. Quantitative adherence assays confirmed that ETEC adheres to primary intestinal epithelial cells lines in a CS21-dependent manner. In addition, the CS21-mediated ETEC adherence to cells was specific as purified LngA protein, the CS21 major subunit, competed for binding with the CS21-expressing ETEC while specific anti-LngA antibodies blocked adhesion to intestinal cells. Neonatal DBA/2 mice died after intra-stomach administration of CS21-expressing strains while lack of CS21 expression drastically reduced the virulence of the wild-type ETEC strain in this animal model. Collectively these results further support the role of CS21 during ETEC infection and add new evidence on its in vivo relevance in pathogenesis. PMID:23760820

  9. Surface texturing and patterning in solar cells

    SciTech Connect

    Green, M.A.

    1993-11-01

    Surface texture can perform a number of functions in modern solar cell design. The most obvious function is in control of reflection from surfaces on which sunlight is incident. However, texture can also be used to influence the fate of light that is refracted into the cell. Light steering by surface texture can ensure this refracted light is absorbed in regions of the cell which are most responsive. When used with rear reflectors, surface texture can help trap weakly absorbed light into the cell, increasing the effective path length or optical thickness of the cell by factors of 30--60. Two general types of texture are considered. One involves macroscopic features of controlled shape designed to control the direction of interacting light. The other is based on the use of irregular features of size comparable to wavelength of the light. These can be very effective in scattering light into a wide range of directions. Non-optical uses of texture are also briefly described. 62 refs., 22 figs.

  10. Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

    PubMed Central

    Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J.; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F.; Emili, Andrew

    2011-01-01

    As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. PMID:22125496

  11. Effects of chromosomal inversion on cell fitness in Escherichia coli K-12.

    PubMed

    Hill, C W; Gray, J A

    1988-08-01

    In an effort to learn what factors might mitigate the establishment of Escherichia coli variants bearing major chromosomal rearrangements, we have examined the effects on cell growth of two inversions between rRNA operons. One of these inversions, IN(rrnD-rrnE), had been propagated in a commonly used subline of E. coli K-12 for approximately 30 yr before its discovery, a fact that illustrates the absence of obvious detrimental effects associated with the inversion. We found that culturing under conditions requiring repeated transition from stationary phase to rapid growth led to the replacement of IN(rrnD-rrnE) cells by cells that had undergone either of two types of additional chromosomal inversion: one type fully restored the wild-type order, while the other partially restored it. The partial reinversion was also between rrn operons, but it left a small transposition. The tendency for overgrowth by these revertants persisted through several rounds of periodic selection. In contrast, the other inversion, IN(rrnG-rrnE), was associated with severe, detrimental effects. The effects of IN(rrnG-rrnE) were also alleviated by full or partial reinversion. The probable relationship between the severity of the effects caused by the inversions and the degree of displacement of the replication origin is discussed. Spontaneous inversion events between rrn operons separated by 18% of the chromosome were estimated to occur at a frequency of roughly 10(-5). If extended to natural situations, the growth disadvantage together with the relatively high frequency of reinversion suggest that clones of cells with an inversion between these rrn operons would be readily overgrown by revertants.

  12. Lipopolysaccharide (LPS)-induced autophagy is involved in the restriction of Escherichia coli in peritoneal mesothelial cells

    PubMed Central

    2013-01-01

    Background Host cell autophagy is implicated in the control of intracellular pathogen. Escherichia coli (E.coli) is the most common organism caused single-germ enterobacterial peritonitis during peritoneal dialysis. In this study, we investigated autophagy of peritoneal mesothelial cells and its role in defense against E.coli. Results Autophagy in human peritoneal mesothelial cell line (HMrSV5) was induced by lipopolysaccharide (LPS) in a dose-dependent and time-dependent way, which was demonstrated by increased expression of Beclin-1 and light chain 3 (LC3)-II, the accumulation of punctate green fluorescent protein-LC3, and a higher number of monodansylcadaverine-labeled autophagic vacuoles. After incubation of HMrSV5 cells with E.coli following LPS stimulation, both the intracellular bactericidal activity and the co-localization of E.coli (K12-strain) with autophagosomes were enhanced. Conversely, blockade of autophagy with 3-methyladenine, wortmannin or Beclin-1 small-interfering RNA (siRNA) led to a significant reduction in autophagy-associated protein expression, attenuation of intracellular bactericidal activity, and reduced co-localization of E.coli with monodansylcadaverine-labeled autophagosomes. In addition, treatment of HMrSV5 cells with LPS caused a dose-dependent and time-dependent increase in Toll-like receptor 4 (TLR4) expression. Both knockdown of TLR4 with siRNA and pharmacological inhibition of TLR4 with Polymyxin B significantly decreased LPS-induced autophagy. Furthermore, TLR4 siRNA attenuated remarkably LPS-induced intracellular bactericidal activity. Conclusions Our findings demonstrated for the first time that LPS-induced autophagy in peritoneal mesothelial cells could enhance the intracellular bactericidal activity and the co-localization of E.coli with autophagosomes. The activation of TLR4 signaling was involved in this process. These results indicate that LPS-induced autophagy may be a cell-autonomous defense mechanism triggered in

  13. Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells.

    PubMed

    Eaves-Pyles, Tonyia; Allen, Christopher A; Taormina, Joanna; Swidsinski, Alexander; Tutt, Christopher B; Jezek, G Eric; Islas-Islas, Martha; Torres, Alfredo G

    2008-07-01

    Inflammatory diseases of the intestinal tract are a major health concern both in the United States and around the world. Evidence now suggests that a new category of Escherichia coli, designated Adherent Invasive E. coli (AIEC) is highly prevalent in Crohn's Disease (CD) patients. AIEC strains have been shown to colonize and adhere to intestinal epithelial cells (IEC). However, the role AIEC strains play in the induction of an inflammatory response is not known. Therefore, we examined several E. coli strains (designated LF82, O83:H1, 6604 and 6655) that were isolated from CD patients for their ability to induce inflammation in two IEC, Caco-2BBe and T-84 cells. Results showed that each strain had varying abilities to adhere to and invade IEC as well as induced cytokine secretion from polarized IEC. However, E. coli O83:H1 displayed the best characteristics of AIEC strains as compared to the prototype AIEC strain LF82, inducing cytokine secretion from IEC and promoting immune cell migration through IEC. Upon further analysis, E. coli O83:H1 did not harbor virulence genes present in known pathogenic intestinal organisms. Further characterization of E. coli O83:H1 virulence determinants showed that a non-flagellated O83:H1 strain significantly decreased the organism's ability to adhere to and invade both IEC and elicit IEC cytokine secretion compared to the wild type and complemented strains. These findings demonstrate that E. coli O83:H1 possesses the characteristics of the AIEC LF82 strain that may contribute to the low-grade, chronic inflammation observed in Crohn's disease. PMID:17900983

  14. Simulation of E. coli Gene Regulation including Overlapping Cell Cycles, Growth, Division, Time Delays and Noise

    PubMed Central

    Luo, Ruoyu; Ye, Lin; Tao, Chenyang; Wang, Kankan

    2013-01-01

    Due to the complexity of biological systems, simulation of biological networks is necessary but sometimes complicated. The classic stochastic simulation algorithm (SSA) by Gillespie and its modified versions are widely used to simulate the stochastic dynamics of biochemical reaction systems. However, it has remained a challenge to implement accurate and efficient simulation algorithms for general reaction schemes in growing cells. Here, we present a modeling and simulation tool, called ‘GeneCircuits’, which is specifically developed to simulate gene-regulation in exponentially growing bacterial cells (such as E. coli) with overlapping cell cycles. Our tool integrates three specific features of these cells that are not generally included in SSA tools: 1) the time delay between the regulation and synthesis of proteins that is due to transcription and translation processes; 2) cell cycle-dependent periodic changes of gene dosage; and 3) variations in the propensities of chemical reactions that have time-dependent reaction rates as a consequence of volume expansion and cell division. We give three biologically relevant examples to illustrate the use of our simulation tool in quantitative studies of systems biology and synthetic biology. PMID:23638057

  15. Assessment of the manufacturability of Escherichia coli high cell density fermentations.

    PubMed

    Perez-Pardo, M A; Ali, S; Balasundaram, B; Mannall, G J; Baganz, F; Bracewell, D G

    2011-01-01

    The physical and biological conditions of the host cell obtained at the end of fermentation influences subsequent downstream processing unit operations. The ability to monitor these characteristics is central to the improvement of biopharmaceutical manufacture. In this study, we have used a combination of techniques such as adaptive focus acoustics (AFA) and ultra scale-down (USD) centrifugation that utilize milliliter quantities of sample to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. This is achieved primarily through an assessment of cell strength and its impact on large-scale disc stack centrifugation performance, measuring critical attributes such as viscosity and particle size distribution. An Escherichia coli fed-batch fermentation expressing antibody fragments in the periplasm was used as a model system representative of current manufacturing challenges. The weakening of cell strength during cultivation time, detected through increased micronization and viscosity, resulted in a 2.6-fold increase in product release rates from the cell (as measured by AFA) and approximately fourfold decrease in clarification performance (as measured by USD centrifugation). The information obtained allows for informed harvest point decisions accounting for both product leakages during fermentation and potential losses during primary recovery. The clarification performance results were verified at pilot scale. The use of these technologies forms a route to the process understanding needed to tailor the host cell and upstream process to the product and downstream process, critical to the implementation of quality-by-design principles.

  16. Sat, the Secreted Autotransporter Toxin of Uropathogenic Escherichia coli, Is a Vacuolating Cytotoxin for Bladder and Kidney Epithelial Cells

    PubMed Central

    Guyer, Debra M.; Radulovic, Suzana; Jones, Faye-Ellen; Mobley, Harry L. T.

    2002-01-01

    The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection. PMID:12117966

  17. Sat, the secreted autotransporter toxin of uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells.

    PubMed

    Guyer, Debra M; Radulovic, Suzana; Jones, Faye-Ellen; Mobley, Harry L T

    2002-08-01

    The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.

  18. Production of cell surface and secreted glycoproteins in mammalian cells.

    PubMed

    Seiradake, Elena; Zhao, Yuguang; Lu, Weixian; Aricescu, A Radu; Jones, E Yvonne

    2015-01-01

    Mammalian protein expression systems are becoming increasingly popular for the production of eukaryotic secreted and cell surface proteins. Here we describe methods to produce recombinant proteins in adherent or suspension human embryonic kidney cell cultures, using transient transfection or stable cell lines. The protocols are easy to scale up and cost-efficient, making them suitable for protein crystallization projects and other applications that require high protein yields. PMID:25502196

  19. Escherichia coli Common Pilus (ECP) Targets Arabinosyl Residues in Plant Cell Walls to Mediate Adhesion to Fresh Produce Plants*

    PubMed Central

    Rossez, Yannick; Holmes, Ashleigh; Lodberg-Pedersen, Henriette; Birse, Louise; Marshall, Jacqueline; Willats, William G. T.; Toth, Ian K.; Holden, Nicola J.

    2014-01-01

    Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR–GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells. PMID:25320086

  20. Escherichia coli common pilus (ECP) targets arabinosyl residues in plant cell walls to mediate adhesion to fresh produce plants.

    PubMed

    Rossez, Yannick; Holmes, Ashleigh; Lodberg-Pedersen, Henriette; Birse, Louise; Marshall, Jacqueline; Willats, William G T; Toth, Ian K; Holden, Nicola J

    2014-12-01

    Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.

  1. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  2. Escherichia coli as a cell factory for heterologous production of nonribosomal peptides and polyketides.

    PubMed

    Li, Jian; Neubauer, Peter

    2014-12-25

    Nonribosomal peptides (NRPs) and polyketides (PKs) are two classes of natural products with numerous bioactivities such as antiviral, antimicrobial and anticancer activity. However, pharmaceutical applications of these products are often impeded because many native producers are difficult to cultivate or show a low productivity. Over the last decade, with the development of synthetic biology and metabolic engineering, more and more bioactive natural products including NRPs and PKs have been heterologously produced using easy-to-handle surrogate microbes. In this process, the full biosynthetic pathway for the production of a target compound is first identified and isolated from the native producer, and then reconstituted in a well-characterized and easily culturable heterologous producer like Escherichia coli. Thereafter, the productivity could be rationally improved through multiple strategies from strain to bioprocess optimization. This review summarizes the endeavors and progresses made in the heterologous production of NRPs, PKs and NRP/PK hybrids using E. coli as a robust whole-cell factory in recent years.

  3. Toxicity of ZnO and TiO2 to Escherichia coli cells

    NASA Astrophysics Data System (ADS)

    Leung, Yu Hang; Xu, Xiaoying; Ma, Angel P. Y.; Liu, Fangzhou; Ng, Alan M. C.; Shen, Zhiyong; Gethings, Lee A.; Guo, Mu Yao; Djurišić, Aleksandra B.; Lee, Patrick K. H.; Lee, Hung Kay; Chan, Wai Kin; Leung, Frederick C. C.

    2016-10-01

    We performed a comprehensive investigation of the toxicity of ZnO and TiO2 nanoparticles using Escherichia coli as a model organism. Both materials are wide band gap n-type semiconductors and they can interact with lipopolysaccharide molecules present in the outer membrane of E. coli, as well as produce reactive oxygen species (ROS) under UV illumination. Despite the similarities in their properties, the response of the bacteria to the two nanomaterials was fundamentally different. When the ROS generation is observed, the toxicity of nanomaterial is commonly attributed to oxidative stress and cell membrane damage caused by lipid peroxidation. However, we found that significant toxicity does not necessarily correlate with up-regulation of ROS-related proteins. TiO2 exhibited significant antibacterial activity, but the protein expression profile of bacteria exposed to TiO2 was different compared to H2O2 and the ROS-related proteins were not strongly expressed. On the other hand, ZnO exhibited lower antibacterial activity compared to TiO2, and the bacterial response involved up-regulating ROS-related proteins similar to the bacterial response to the exposure to H2O2. Reasons for the observed differences in toxicity and bacterial response to the two metal oxides are discussed.

  4. Femtosecond fabricated surfaces for cell biology

    NASA Astrophysics Data System (ADS)

    Day, Daniel; Gu, Min

    2010-08-01

    Microfabrication using femtosecond pulse lasers is enabling access to a range of structures, surfaces and materials that was not previously available for scientific and engineering applications. The ability to produce micrometre sized features directly in polymer and metal substrates is demonstrated with applications in cell biology. The size, shape and aspect ratio of the etched features can be precisely controlled through the manipulation of the fluence of the laser etching process with respect to the properties of the target material. Femtosecond laser etching of poly(methyl methacrylate) and aluminium substrates has enabled the production of micrometre resolution moulds that can be accurately replicated using soft lithography. The moulded surfaces are used in the imaging of T cells and demonstrate the improved ability to observe biological events over time periods greater than 10 h. These results indicate the great potential femtosecond pulse lasers may have in the future manufacturing of microstructured surfaces and devices.

  5. Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures.

    PubMed

    Choi, Okkyoung; Yu, Chang-Ping; Esteban Fernández, G; Hu, Zhiqiang

    2010-12-01

    Biofilms are often more resistant to toxic chemicals such as heavy metals and antimicrobial agents than planktonic cells. Nanosilver has a broad range of applications with strong antimicrobial activity. However, biofilm susceptibility to nanosilver toxicity is not well understood. We studied the bacterial activity in planktonic or biofilm cultures after nanosilver exposure using oxygen quenching fluorescence-based microrespirometry. We also determined the aggregation behavior and the spatial distribution of nanosilver having red fluorescence in biofilms of Escherichia coli expressing green fluorescent protein. At the same bacterial concentrations (3 × 10(8) CFU/mL), biofilms were about four times more resistant to nanosilver inhibition than planktonic cells. The minimum bactericidal concentrations (MBCs) of nanosilver (size from 15 to 21 nm), defined as the lowest concentration that kills at least 99.9% of a planktonic or biofilm bacterial population, were 38 and 10 mg/L Ag, respectively. For comparison, silver ions were more toxic to E. coli than nanosilver with MBCs of 2.4 and 1.2 mg/L Ag for planktonic and biofilm cultures, respectively. Nanosilver was aggregated in the presence of planktonic or biofilm-forming cells resulting in an increase of average particle size by a factor of 15 and 40, respectively. The nanosilver particles were able to penetrate to approximately 40 μm in a thick biofilm after 1-h exposure. These findings suggested that biofilm resistance to nanosilver could be at least partially due to nanoparticle aggregation and retarded silver ion/particle diffusion.

  6. Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef.

    PubMed

    Fitzmaurice, J; Duffy, G; Kilbride, B; Sheridan, J J; Carroll, C; Maher, M

    2004-11-01

    In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.

  7. Living Toroids - Cells on Toroidal Surfaces

    NASA Astrophysics Data System (ADS)

    Chang, Ya-Wen; Angelini, Thomas; Marquez, Samantha; Kim, Harold; Fernandez-Nieves, Alberto

    2014-03-01

    Cellular environment influences a multitude of cellular functions by providing chemical and physical signals that modulate cell behavior, dynamics, development, and eventually survival. Substrate mechanics has been recognized as one of the important physical cues that governs cell behavior at single cell level as well as in collective cell motion. Past research has suggested several contact-guided behaviors to be the result of surface curvature. However, studies on the effect of curvature are relatively scarce likely due to the difficulty in generating substrates with well-defined curvature. Here we describe the generation of toroidal droplets, which unlike spherical droplets, have regions of both positive and negative Gaussian curvature. Additionally, the range of curvatures can be controlled by varying the size and aspect ratio of the torus. Cells are either encapsulated inside toroidal droplets or located on toroidal hydrogel surfaces. Preliminary studies use B. Subtilis to study the organization of bacteria biofilms. When confined in droplets surrounded by yield-stress fluid, bacteria self-organize into heterogeneous biofilm at fluid- substrate interface. It is found that the surface curvature in the sub-millimeter scale has little effect on biofilm architecture.

  8. Solar cell having improved front surface metallization

    SciTech Connect

    Lillington, D.R.; Mardesich, N.; Dill, H.G.; Garlick, G.F.J.

    1987-09-15

    This patent describes a solar cell comprising: a first layer of gallium arsenide semiconductor material of an N+ conductivity; a second layer of gallium arsenide semiconductor material of an N conductivity overlying the first layer; a third layer of gallium arsenide semiconductor material of a P conductivity overlying the N conductivity layer and forming a P-N junction therebetween. A layer of aluminium gallium arsenide semiconductor material of a p conductivity overlying the front major surface of the P conductivity third layer and having an exposed surface essentially parallel to the front major surface and at least one edge; a plurality of metallic contact lines made of a first metal alloy composition and being spaced apart by a first predetermined distance traversing the exposed surface and extending through the aluminium gallium arsenide layer to the front major surface and making electrical contact to the third layer; a plurality of longitudinally disposed metallic grid lines made of a second metal alloy composition and being spaced apart by a second predetermined distance located on the exposed surface of the aluminium gallium arsenide layer and which cross the metallic contact lines and make electrical contact to the metallic lines; a flat metallic strip disposed on the aluminium gallium arsenide layer exposed surface near the edge, the strip electrically coupling the metallic grid lines to one another; and a back contact located on the back major surface.

  9. Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells.

    PubMed

    Hartland, E L; Batchelor, M; Delahay, R M; Hale, C; Matthews, S; Dougan, G; Knutton, S; Connerton, I; Frankel, G

    1999-04-01

    Enteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells. This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study, we have localized the intimin-binding domain of Tir to a central 107-amino-acid region, designated Tir-M. We provide evidence that both the amino- and carboxy-termini of Tir are located within the host cell. In addition, using immunogold labelling electron microscopy, we have confirmed that intimin can bind independently to host cells even in the absence of Tir. This Tir-independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectin-like module residing at the carboxy-terminus of the intimin polypeptide. Using the yeast two-hybrid system and gel overlays, we show that intimin can bind both Tir and Tir-M even when the lectin-like domain is disrupted. These data provide strong evidence that intimin interacts not only with Tir but also in a lectin-like manner with a host cell intimin receptor.

  10. Effect of the Min System on Timing of Cell Division in Escherichia coli

    PubMed Central

    Jia, Shuxin; Keilberg, Daniela; Hot, Edina; Thanbichler, Martin; Søgaard-Andersen, Lotte; Lenz, Peter

    2014-01-01

    In Escherichia coli the Min protein system plays an important role in positioning the division site. We show that this system also has an effect on timing of cell division. We do this in a quantitative way by measuring the cell division waiting time (defined as time difference between appearance of a division site and the division event) and the Z-ring existence time. Both quantities are found to be different in WT and cells without functional Min system. We develop a series of theoretical models whose predictions are compared with the experimental findings. Continuous improvement leads to a final model that is able to explain all relevant experimental observations. In particular, it shows that the chromosome segregation defect caused by the absence of Min proteins has an important influence on timing of cell division. Our results indicate that the Min system affects the septum formation rate. In the absence of the Min proteins this rate is reduced, leading to the observed strongly randomized cell division events and the longer division waiting times. PMID:25090009

  11. [Change of Electrophysical Properties of Escherichia coli Cells Due to Levomycetin and Tetracycline Action].

    PubMed

    Guliy, O I; Bunin, V D; Larionova, O S; Zhnichkova, E G; Balkos, A B; Ignatov, O V

    2016-01-01

    The effect of chloramphenicol and tetracycline, as inhibitors of protein synthesis, on electrophysical properties of Escherichia coli K-12 cells was investigated. Significant changes in the orientation spectra (OS) of the cell suspensions incubated with various concentrations of chloramphenicol were observed only at the first five frequencies of the electric field (10-1000 kHz). When the cells were exposed to chloramphenicol (1.5 mcg/ml) or tetracycline (1.7 mcg/ml), no changes in the OS were recorded. Significant changes in the electrooptic signal were observed, when the K-12 cells were simultaneously incubated with chloramphenicol (1.5 mcg/ml) and tetracycline (1.7 mcg/ml), that could be due to the synergistic action of the antibiotics. Therefore, the electrooptic analysis provided registration of higher antibacterial effect with the simultaneous use of chloramphenicol and tetracycline. Additional control experiments with the cell culture on the LB nutrient medium containing chloramphenicol and tetracycline were performed. The results suggested that the use of electrophysical methods for investigation of antibiotics effect on microorganisms was rather efficient. PMID:27337862

  12. Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli

    NASA Astrophysics Data System (ADS)

    Buryakina, Tatyana Yu.; Su, Pin-Tzu; Syu, Wan-Jr; Allen Chang, C.; Fan, Hsiu-Fang; Kao, Fu-Jen

    2012-10-01

    Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

  13. A comparative toxicity evaluation of Escherichia coli-targeted ssDNA and chlorine in HepG2 cells.

    PubMed

    Kaushik, Rajni; Balasubramanian, Rajasekhar

    2014-01-01

    In this study, a comparative assessment of the effectiveness of ssDNA and chlorine as disinfectants for treating water contaminated with Escherichia coli (E. coli) was investigated on the basis of cytotoxicity and genotoxicity. The gene targets addressed for the ssDNA based inhibition method were marA (multiple antibiotic resistance) and groL (essential gene Hsp60) in E. coli. Based on the maximum log reduction in E. coli cell numbers when compared to no ssDNA control, groL-1 was chosen as the optimized ssDNA for gene silencing-based inactivation. For toxicity assessment, HepG2 cells were exposed to extracts corresponding to concentrations of 0.2, 1, 5, 25 and 50 mL water/mL medium of chlorine doped water and 1, 10, 100, 300 nM of ssDNA. Compared with ssDNA, HepG2 cells exposed to extracts of chlorine doped water for 24 h showed higher cytotoxicity, caspase 3/7 levels, DNA damage, micronuclei frequency, and decreased cell viability. Water doped with chlorine was found to be more toxic than that by ssDNA when exposed to HepG2 cells. The results of this study provide a scientific basis for comparative evaluation of new and conventional disinfection methods by taking into consideration the outcome of cytotoxicity and genotoxicity assessments.

  14. Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone production

    PubMed Central

    2013-01-01

    Background Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them. Results In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process. Conclusions High NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was

  15. Properties of pili from Escherichia coli SS142 that mediate mannose-resistant adhesion to mammalian cells.

    PubMed Central

    Mett, H; Kloetzlen, L; Vosbeck, K

    1983-01-01

    We isolated pili from Escherichia coli SS142. These pili had a diameter of 6 nm and an average length of 400 nm. They were composed of subunits with a molecular weight of 18,000. Their amino acid composition was determined; methionine and proline were not detected. The isolated pili retained mannose-resistant hemagglutinating activity. Proteolytic digestion and glutaraldehyde fixation led to partial or complete loss of the hemagglutinating activity of the pili without causing any detectable damage to their supramolecular structure, which was only disintegrated by treatment with hot sodium dodecyl sulfate. The hemagglutinating activity of E. coli SS142 was inhibited by the glycoproteins fetuin and Tamm-Horsfall protein, as well as by the glycolipids phytyl lactoside, dansyl-sphingosine lactoside, and digalactosyl diglyceride. Isolated pili inhibited the adhesion of the homologous strain E. coli SS142 to Intestine 407 cell monolayers, but did not inhibit the adhesion of E. coli strain B-413, B-506, or 2699. This indicates that E. coli SS142 binds to a receptor different from those recognized by the other strains and that mannose-resistant adhesion to tissue culture cells can be classified into different subtypes. Images PMID:6130060

  16. Process of Cellular Division in Escherichia coli: Physiological Study on Thermosensitive Mutants Defective in Cell Division

    PubMed Central

    Ricard, Matthieu; Hirota, Yukinori

    1973-01-01

    Thermosensitive fts mutants of Escherichia coli belonging to seven previously identified genetic classes (ftsA to ftsG) were studied from a physiological standpoint. These mutants immediately stopped dividing and formed multinucleated filaments when the temperature was shifted to 41 C. Macromolecular syntheses (deoxyribonucleic acid), ribonucleic acid, cell mass, and murein) continued exponentially for at least 40 to 120 min. The number of surviving bacteria remained constant during the time of incubation, and this number began to decrease exponentially, as the rate of cell mass increase leveled off from the initial rate. The recovery of cell division at 30 C in these filamentous cells was studied after 60 min of incubation at 41 C. The existence of three types of mutants was shown. The ftsA and ftsE mutants resumed cell division without new protein synthesis; ftsD mutants resumed cell division only if new protein synthesis occured, while ftsB, C, F and G mutants did not resume cell division at all. No alteration in the cell envelope was detected by the method used here, although the ftsA, B, D, F and G mutations, in contrast with ftsC and E, caused an increased resistance to penicillin G. It was also shown that the recA mutation did not suppress the effect of the fts mutations and that none of the lysogenic fts mutants induced prophage multiplication while forming filaments. The effects of osmotic pressure and salts which rescue the mutant phenotype is described. PMID:4583216

  17. Effects of sub-minimum inhibitory concentrations of ciprofloxacin on enteroaggregative Escherichia coli and the role of the surface protein dispersin

    SciTech Connect

    Fowlkes, Jason Davidson; Doktycz, Mitchel John; Allison, David Post

    2011-01-01

    Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

  18. The effect of sub-minimum inhibitory concentration of ciprofloxacin concentrations on enteroaggregative Escherichia coli and the role of the surface protein dispersin

    SciTech Connect

    Mortensen, Ninell P; Fowlkes, Jason Davidson; Trevino-Dopatka, Sonia; Maggart, Michael J; Boisen, Nadia; Doktycz, Mitchel John; Nataro, James; Allison, David P

    2011-01-01

    Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

  19. Comparison of the Transport of Tetracycline-Resistant and Tetracycline-Susceptible Escherichia coli Isolated from Lake Michigan

    PubMed Central

    Walczak, Jacob J.; Bardy, Sonia L.; Feriancikova, Lucia

    2011-01-01

    It was recently reported that tetracycline could enhance the mobility of manure-derived Escherichia coli within saturated porous media (Walczak et al. (Water Research 45:1681–1690, 2011)). It was also shown, however, that E. coli from various sources could display marked variation in their mobility (Bolster et al. (Journal of Environmental Quality 35:1018–1025, 2009)). The focus of this research was to examine if the observed difference in the mobility of manure-derived tetracycline-resistant (tetR) and tetracycline-susceptible (tetS) E. coli strains was source-dependent. Specifically, E. coli were isolated from Lake Michigan, and the influence of tetracycline resistance on Lake Michigan-derived E. coli was investigated through column transport experiments. Additionally, a variety of cell morphology and surface properties were determined and related to the observed bacterial transport behavior. Our experimental results showed that, consistent with previous observations, the deposition rate coefficients of the tetR E. coli strain was ~20–100% higher than those of the tetS E. coli strain. The zeta potential of the tetR E. coli cells was ~25 mV more negative than the tetS E. coli cells. Because the surfaces of the E. coli cells and the quartz sands were negatively charged, the repulsive electrostatic double-layer interaction between the tetR E. coli cells and the quartz sands was stronger, and the mobility of the tetR E. coli cells in the sand packs was thus higher. The tetR E. coli cells were also more hydrophilic than the tetS E. coli cells. Results from migration to hydrocarbon phase (MATH) tests showed that about ~35% more tetS E. coli cells partitioned to the hydrocarbon phase. As it was previously shown that cell hydrophobicity could enhance the attachment of bacterial cells to quartz sand, the difference in cell hydrophobicity could also have contributed to the observed higher mobility of the tetR E. coli cells. The size of the tetR and tetS E. coli cells

  20. Engineering novel cell surface chemistry for selective tumor cell targeting

    SciTech Connect

    Bertozzi, C.R. |

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  1. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli.

    PubMed

    Sohoni, Sujata Vijay; Nelapati, Dhanaraj; Sathe, Sneha; Javadekar-Subhedar, Vaishali; Gaikaiwari, Raghavendra P; Wangikar, Pramod P

    2015-01-01

    Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl β-D-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32 °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process. PMID:25739996

  2. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  3. Identification and quantitation of elongation factor EF-P in Escherichia coli cell-free extracts.

    PubMed

    An, G; Glick, B R; Friesen, J D; Ganoza, M C

    1980-11-01

    EF-P, an elongation factor that stimulates peptide bond synthesis in vitro with some aminoacyl-tRNAs, has been identified by two-dimensional gel electrophoresis and the cellular content at three points in the growth curve has been measured. The molecular weight of EF-P is estimated to be 21 000. EF-P is a slightly acidic protein whose isoelectric point is close to RNA polymerase subunit alpha. The amount of EF-P present in Escherichia coli is about 1/10th that of EF-G and the level is independent of the stage of cell growth; there is about one EF-P per 10 ribosomes. It is also shown that a highly purified preparation of EF-P is free of all known protein synthesis factors and ribosomal proteins. PMID:7011506

  4. [Bacteriophage lambda:lux: design and expression of bioluminescence in E. coli cells].

    PubMed

    Duzhiĭ, D E; Zavil'gel'skiĭ, G B

    1994-01-01

    The bacteriophages lambda:lux and lambda:luxAB have been constructed by ligation of phage arms generated by BamHI or SalGI restriction endonucleases digestion of EMBL4 to BamHI digested plasmid pF1 lux+ or to SalGI digested plasmid pF2 lambda:luxA+B+. Cells of Escherichia coli prototrophic strain Cs were infected with lambda:lux or lambda:luxAB and intensity of bioluminiscence of the samples registered at different time intervals determined. The signal of bioluminiscence was first detected 15 min after infection and its level increased exponentially thereafter demonstrating replication of the lambda:lux bacteriophages. We have used the recombinant lambda:luxAB bacteriophage to detect the enteric indicator bacteria without enrichment in 15 min, provided that they are present at levels higher than 10(4).

  5. Enzymatic synthesis of 2-phenoxyethanol galactoside by whole cells of β-galactosidase-containing Escherichia coli.

    PubMed

    Lee, Hyang-Yeol; Jung, Kyung-Hwan

    2014-09-01

    We investigated whether β-galactosidase (β-gal)-containing Escherichia coli cells could transfer a galactose to 2-phenoxyethanol, resulting in 2-phenoxyethanol galactoside (PE-Gal). PE-Gal was confirmed by liquid chromatography-mass spectrometry. In addition, we also confirmed that a galactose molecule was covalently bonded with PE during thin-layer chromatography analysis of the β-gal hydrolysate of PE-Gal. The yield for PE-Gal synthesis was about 37.5% (weight basis), which was about 7-8 times greater than that of a previous report. In addition, the concentration of β-gal (0.96 U/ml) used in this PE-Gal synthesis was about 20 times less than that in a previous report.

  6. The occurrence of heritable Mu excisions in starving cells of Escherichia coli.

    PubMed

    Foster, P L; Cairns, J

    1994-11-01

    A strain of Escherichia coli constructed by Shapiro has a segment of Mu bacteriophage DNA inserted between the araC and lacZ genes. Excision events that produce an in-frame fusion of lacZ to araB result in a cell (here designated Ara-Lac+) that can grow on lactose if arabinose is present as an inducer. Whether or not these excision events occur in the absence of selection for the Ara-Lac+ phenotype has figured prominently in the debate of the phenomenon known as 'directed' or 'adaptive' mutation. In an attempt to settle the issue, we have used classic fluctuation tests to show that cells capable of producing a clone of descendants that are phenotypically Ara-Lac+ do, indeed, arise in stationary phase cultures kept starving in depleted minimal medium. We found that Ara-Lac+ progenitors arise rapidly under these conditions, in contrast to the delayed appearance of Ara-Lac+ mutants when cells are incubated on lactose-arabinose minimal plates. Similar results are reported in the accompanying paper by Maenhaut-Michel and Shapiro, who used indirect selection to isolate Ara-Lac+ cells in the absence of selection. However, their sequencing data have introduced a new unexpected complication to the interpretation of all such experiments, and it is no longer clear exactly when the fusions arise.

  7. Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli.

    PubMed Central

    Driehuis, F; Wouters, J T

    1987-01-01

    The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall. Images PMID:3539928

  8. Human intestinal cells modulate conjugational transfer of multidrug resistance plasmids between clinical Escherichia coli isolates.

    PubMed

    Machado, Ana Manuel Dantas; Sommer, Morten O A

    2014-01-01

    Bacterial conjugation in the human gut microbiota is believed to play a major role in the dissemination of antibiotic resistance genes and virulence plasmids. However, the modulation of bacterial conjugation by the human host remains poorly understood and there is a need for controlled systems to study this process. We established an in vitro co-culture system to study the interaction between human intestinal cells and bacteria. We show that the conjugation efficiency of a plasmid encoding an extended spectrum beta-lactamase is reduced when clinical isolates of Escherichia coli are co-cultured with human intestinal cells. We show that filtered media from co-cultures contain a factor that reduces conjugation efficiency. Protease treatment of the filtered media eliminates this inhibition of conjugation. This data suggests that a peptide or protein based factor is secreted on the apical side of the intestinal cells exposed to bacteria leading to a two-fold reduction in conjugation efficiency. These results show that human gut epithelial cells can modulate bacterial conjugation and may have relevance to gene exchange in the gut.

  9. High cell density cultivation and high recombinant protein production of Escherichia coli strain expressing uricase.

    PubMed

    Nakagawa, S; Oda, H; Anazawa, H

    1995-12-01

    Uricase from Cellulomonas flavigena SK-4 is an industrially useful enzyme for commercial formulations of hair coloring. The uricase production by recombinant Escherichia coli strain with a high cell density cultivation technique was described. Of three kinds of media, synthetic media with the feeding of a high concentration of glucose solution were suitable for high cell density cultivation. As for feeding, both biomass concentration and uricase productivity were increased by about two (61.2 g dry cell weight (DCW)/liter) and three times (1037 U/ml broth), respectively, in 24 h by continuous supply. In the case of feeding by a DO-stat method, however, cell concentration was comparable to continuous glucose supply but uricase activity was reduced. By supplying pure oxygen to compensate for oxygen limitation during cultivation, the highest values of 77.4g DCW/liter and 1113 U/ml broth of the uricase activity were achieved with the total cultivation time of 15 h.

  10. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

    PubMed

    Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

    2015-09-18

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells. PMID:26017572

  11. Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

    PubMed

    Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

    2015-09-18

    Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells.

  12. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose. PMID:23300051

  13. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.

  14. The Core Lipopolysaccharide of Escherichia coli Is a Ligand for the Dendritic-Cell-Specific Intercellular Adhesion Molecule Nonintegrin CD209 Receptor

    PubMed Central

    Klena, John; Zhang, Pei; Schwartz, Olivier; Hull, Sheila; Chen, Tie

    2005-01-01

    The dendritic-cell-specific intercellular adhesion molecule nonintegrin (DC-SIGN) CD209 is a receptor for Escherichia coli K-12 that promotes bacterial adherence and phagocytosis. However, the ligand of E. coli for DC-SIGN has not yet been identified. In this study, we found that DC-SIGN did not mediate the phagocytosis of several pathogenic strains of E. coli, including enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, and uropathogenic E. coli, in dendritic cells or HeLa cells expressing human DC-SIGN antigen. However, we showed that an outer core lipopolysaccharide (LPS) (rough) mutant, unlike an inner core LPS (deep rough) mutant or O-antigen-expressing recombinant of E. coli K-12 was phagocytosed. These results demonstrate that the host cells expressing DC-SIGN can phagocytose E. coli in part by interacting with the complete core region of the LPS molecule. These results provide a mechanism for how O antigen acts as an antiphagocytic factor. PMID:15716442

  15. Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water.

    PubMed

    Canal, Natália; Meneghetti, Karine Lena; de Almeida, Clara Ponzi; da Rosa Bastos, Marina; Otton, Letícia Muner; Corção, Gertrudes

    2016-01-01

    Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacEΔ1 gene at the 3' conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans. PMID:26991286

  16. Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water.

    PubMed

    Canal, Natália; Meneghetti, Karine Lena; de Almeida, Clara Ponzi; da Rosa Bastos, Marina; Otton, Letícia Muner; Corção, Gertrudes

    2016-01-01

    Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacEΔ1 gene at the 3' conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans.

  17. Hydrogen peroxide signals E. coli phagocytosis by human polymorphonuclear cells; up-stream and down-stream pathway.

    PubMed

    Petropoulos, Michalis; Karamolegkou, Georgia; Rosmaraki, Eleftheria; Tsakas, Sotiris

    2015-12-01

    Hydrogen peroxide (Η2Ο2) is produced during a variety of cellular procedures. In this paper, the regulatory role of Η2Ο2, in Escherichia coli phagocytosis by the human polymorphonuclears, was investigated. White blood cells were incubated with dihydrorhodamine (DHR) in order to study H2O2 synthesis and E. coli-FITC to study phagocytosis. Flow cytometry revealed increased synthesis of H2O2 in polymorphonuclears which incorporated E. coli-FITC. The blocking of H2O2 synthesis by specific inhibitors, N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for superoxide dismutase (SOD), decreased E. coli phagocytosis, as well. Immunoblot analysis of white blood cell protein extracts revealed that the blocking of NADPH oxidase and SOD decreased ERK-1/2 phosphorylation, while it had no effect on JNK and p38. Confocal microscopy showed that phosphorylation of MAPKs and phagocytosis solely occur in the polymorphonuclear and not in mononuclear cells. The use of specific MAPKs inhibitors showed that all of them are necessary for phagocytosis, but only phospho-p38 affects H2O2 synthesis. The blocking of JNK phosphorylation, in the presence of E. coli, evoked a further decrease of cytoplasmic p47 thus increasing its translocation onto the plasma membrane for the assembly of NADPH oxidase. It appears that newly synthesised H2O2 invigorates the phosphorylation and action of ERK-1/2 in E. coli phagocytosis, while phospho-JNK and phospho-p38 appear to regulate H2O2 production.

  18. [Enzymatic control of homologous recombination in Escherichia coli cells and hyper-recombination].

    PubMed

    Bakhlanova, I V; Dudkina, A V; Baĭtin, D M

    2013-01-01

    The RecA protein is a major enzyme of homologous recombination in bacterial cell. Forming a right-handed helical filament on ssDNA, it provides a homology search between two DNA molecules and homologous strand exchange. The RecA protein not only defends the cell from exposure to ionizing radiation and UV-irradiation, but also ensures the recombination process in the course of normal cell growth. A number of wild-type or mutant RecA proteins demonstrate increased recombinogenic properties in vitro and in vivo as compared with the wild-type RecA protein from Escherichia coli, which leads to hyper-recombination. The hyper-rec activity of RecA proteins during the recombination process in many depends on the filamentation dynamics on ssDNA and DNA-transferase properties. Changes in filamentation and DNA-transferase abilities of RecA protein may be the result of not only specific amino-acid substitutions, but also the functioning of the cell enzymatic apparatus, including such proteins as RecO, RecR, RecF, RecX, DinI, SSB, PsiB. To date, the function of each of these proteins is identified at the molecular level. However, the role of some of them in the cell metabolism remains to be seen. Increase in recombination in vivo is not always useful for a cell and faces various limitations. Moreover, in the bacterial cell some mechanisms are activated, that cause genomic reorganization, directed to suppress the expression of hyper-active RecA protein. The ways of hyper-active RecA protein regulation are very interesting, and they are studied in different model systems. PMID:23808153

  19. Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

    PubMed Central

    1986-01-01

    The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets. PMID:3025223

  20. Mechanism of cell integration on biomaterial implant surfaces in the presence of bacterial contamination.

    PubMed

    Yue, Chongxia; van der Mei, Henny C; Kuijer, Roel; Busscher, Henk J; Rochford, Edward T J

    2015-11-01

    Bacterial contamination during biomaterial implantation is often unavoidable, yielding a combat between cells and bacteria. Here we aim to determine the modulatory function of bacterial components on stem-cell, fibroblast, and osteoblast adhesion to a titanium alloy, including the role of toll-like-receptors (TLRs). Presence of heat-sacrificed Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa induced dose and cell-type dependent responses. Stem-cells were most sensitive to bacterial presence, demonstrating decreased adhesion number yet increased adhesion effort with a relatively large focal adhesion contact area. Blocking TLRs had no effect on stem-cell adhesion in presence of S. aureus, but blocking both TLR2 and TLR4 induced an increased adhesion effort in presence of E. coli. Neither lipopolysaccharide, lipoteichoic acid, nor bacterial DNA provoked the same cell response as did whole bacteria. Herewith we suggest a new mechanism as to how biomaterials are integrated by cells despite the unavoidable presence of bacterial contamination. Stimulation of host cell integration of implant surfaces may open a new window to design new biomaterials with enhanced healing, thereby reducing the risk of biomaterial-associated infection of both "hardware-based" implants as well as of tissue-engineered constructs, known to suffer from similarly high infection risks as currently prevailing in "hardware-based" implants. PMID:25966819

  1. Enhanced Bioaccumulation of Heavy Metal Ions by Bacterial Cells Due to Surface Display of Short Metal Binding Peptides

    PubMed Central

    Kotrba, Pavel; Dolečková, Lucie; de Lorenzo, Víctor; Ruml, Tomas

    1999-01-01

    Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal. PMID:10049868

  2. Persistence of Salmonella and E. coli on the surface of restaurant menus.

    PubMed

    Sirsat, Sujata A; Choi, Jin-Kyung K; Almanza, Barbara A; Neal, Jack A

    2013-03-01

    To the authors' knowledge, the role of restaurant menus as a vehicle for pathogens has not been explored. Menus, however, can pose as a vector for bacterial contamination and transfer. Sampling menus from two restaurants in the Houston, Texas, area showed the presence of up to 100 CFU/cm2 aerobic bacteria. Follow-up studies designed to investigate the ability of Salmonella and E. coli to persist on paper and laminated menus at various time points (0, 6, 24, 48, and 72 hours) demonstrated that bacteria persist more efficiently on laminated menus as compared to paper menus. Transfer studies performed to quantitatively determine the ability of bacteria to transfer from menus to fingertips and from fingertips to clean menus showed that bacteria can be transferred for up to 24 hours. The study described here showed that restaurant menus may serve as vehicles for pathogens and hence present a public health issue within the retail food environment. PMID:23505769

  3. An immobilized biotin ligase: surface display of Escherichia coli BirA on Saccharomyces cerevisiae.

    PubMed

    Parthasarathy, Ranganath; Bajaj, Jitin; Boder, Eric T

    2005-01-01

    The Escherichia coli biotin ligase enzyme BirA has been extensively used in recent years to generate site-specifically biotinylated proteins via a biotin acceptor peptide tag. In the present study, BirA was displayed for the first time on the yeast Saccharomyces cerevisiae using the Aga1p-Aga2p platform and assayed using a peptide-tagged protein as the substrate. The enzyme is fully functional and resembles the soluble form in many of its properties, but the yeast-displayed enzyme demonstrates stability and reusability on the time scale of weeks. Thus, the yeast-displayed BirA system represents a facile and highly economical alternative for producing site-specifically biotinylated proteins.

  4. Rapid culture-independent quantitative detection of enterotoxigenic Escherichia coli in surface waters by real-time PCR with molecular beacon.

    PubMed

    Ram, Siya; Vajpayee, Poornima; Shanker, Rishi

    2008-06-15

    Rapid and reliable detection of enterotoxigenic Escherichia coli (ETEC) is critical for the management of the waterborne diseases threatening human lives worldwide. In this study, a culture-independent real-time PCR assay, in molecular beacon format was designed and validated for detection and quantitative enumeration of ETEC harboring LT1 gene (encoding heat labile toxin) in surface waters contaminated by fecal pollutants of human and animal origin. It was observed that the assay was able to detect 2 CFU/mL of ETEC (r = 0.997; PCR efficiency = 99.8%) from water samples spiked by a reference organism (E. coli MTCC 723). In the presence of 10(6) CFU/mL of nonpathogenic E. coli(E. coli DH5alpha), the lowest detection limit from spiked water samples was 4 CFU/mL. The assay was 500 times more sensitive than conventional PCR using the same oligomers (Student's t test p < 0.05). The assay could specifically detect and quantify ETEC (1.2 x 10(3) to 1.4 x 10(6) CFU/100 mL) in polluted surface waters of river Gomti. The rapid culture-independent assay developed in this study for detection and quantitative enumeration of ETEC can be used for preliminary monitoring of surface waters to prevent waterborne outbreaks.

  5. Alginate immobilization of Escherichia coli MTCC 1652 whole cells for bioconversion of glycyrrhizinic acid and into 18-beta glycyrrhetinic acid.

    PubMed

    Ahmad, M; Panda, B P

    2013-12-15

    Microbial biotransformation of Glycyrrhizinic acid (GL) into 18-beta Glycyrrhetinic Acid (GA) was achieved using Escherichia coli MTCC 1652 whole cell. The E. coli whole cell was immobilized by entrapment method within calcium alginate beads using cell suspension of equal volume with sodium alginate 8%. The pH of solution, reaction volume and % of GL were optimized during the immobilization procedure and optimum pH 6.5, reaction volume of 4 mL and at 3% GL concentration for 12 h of incubation time showed highest concentration of GA (72.649 microg mL(-1)) with 76% bioconversion of GL to GA. Under optimized condition the immobilized cell produces 58.663 microg per mL of GA in licorice root extract containing 95.118 microg of GL per mL of the extract with 61% conversion at 12 h.

  6. Maintaining replication fork integrity in UV-irradiated Escherichia coli cells.

    PubMed

    Rudolph, Christian J; Upton, Amy L; Lloyd, Robert G

    2008-09-01

    In dividing cells, the stalling of replication fork complexes by impediments to DNA unwinding or by template imperfections that block synthesis by the polymerase subunits is a serious threat to genomic integrity and cell viability. What happens to stalled forks depends on the nature of the offending obstacle. In UV-irradiated Escherichia coli cells DNA synthesis is delayed for a considerable period, during which forks undergo extensive processing before replication can resume. Thus, restart depends on factors needed to load the replicative helicase, indicating that the replisome may have dissociated. It also requires the RecFOR proteins, which are known to load RecA recombinase on single-stranded DNA, implying that template strands are exposed. To gain a further understanding of how UV irradiation affects replication and how replication resumes after a block, we used fluorescence microscopy and BrdU or radioisotope labelling to examine chromosome replication and cell cycle progression. Our studies confirm that RecFOR promote efficient reactivation of stalled forks and demonstrate that they are also needed for productive replication initiated at the origin, or triggered elsewhere by damage to the DNA. Although delayed, all modes of replication do recover in the absence of these proteins, but nascent DNA strands are degraded more extensively by RecJ exonuclease. However, these strands are also degraded in the presence of RecFOR when restart is blocked by other means, indicating that RecA loading is not sufficient to stabilise and protect the fork. This is consistent with the idea that RecA actively promotes restart. Thus, in contrast to eukaryotic cells, there may be no factor in bacterial cells acting specifically to stabilise stalled forks. Instead, nascent strands may be protected by the simple expedient of promoting restart. We also report that the efficiency of fork reactivation is not affected in polB mutants. PMID:18644471

  7. Segregation of chromosome arms in growing and non-growing Escherichia coli cells

    PubMed Central

    Woldringh, Conrad L.; Hansen, Flemming G.; Vischer, Norbert O. E.; Atlung, Tove

    2015-01-01

    In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model. PMID:26029188

  8. Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid.

    PubMed

    Szymańska, Grazyna; Sobierajski, Bogusław; Chmiel, Aleksander

    2011-01-01

    For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour. PMID:21905626

  9. Decolorization of acid and basic dyes: understanding the metabolic degradation and cell-induced adsorption/precipitation by Escherichia coli.

    PubMed

    Cerboneschi, Matteo; Corsi, Massimo; Bianchini, Roberto; Bonanni, Marco; Tegli, Stefania

    2015-10-01

    Escherichia coli strain DH5α was successfully employed in the decolorization of commercial anthraquinone and azo dyes, belonging to the general classes of acid or basic dyes. The bacteria showed an aptitude to survive at different pH values on any dye solution tested, and a rapid decolorization was obtained under aerobic conditions for the whole collection of dyes. A deep investigation about the mode of action of E. coli was carried out to demonstrate that dye decolorization mainly occurred via three different pathways, specifically bacterial induced precipitation, cell wall adsorption, and metabolism, whose weight was correlated with the chemical nature of the dye. In the case of basic azo dyes, an unexpected fast decolorization was observed after just 2-h postinoculation under aerobic conditions, suggesting that metabolism was the main mechanism involved in basic azo dye degradation, as unequivocally demonstrated by mass spectrometric analysis. The reductive cleavage of the azo group by E. coli on basic azo dyes was also further demonstrated by the inhibition of decolorization occurring when glucose was added to the dye solution. Moreover, no residual toxicity was found in the E. coli-treated basic azo dye solutions by performing Daphnia magna acute toxicity assays. The results of the present study demonstrated that E. coli can be simply exploited for its natural metabolic pathways, without applying any recombinant technology. The high versatility and adaptability of this bacterium could encourage its involvement in industrial bioremediation of textile and leather dyeing wastewaters.

  10. Inactivation of Escherichia coli in broth and sausage by combined high pressure and Lactobacillus casei cell extract.

    PubMed

    Chung, Hyun-Jung; Yousef, Ahmed E

    2010-10-01

    The purpose of this study was to investigate the effect of combined high pressure and Lactobacillus casei cell extract (CE) on Escherichia coli O157 strains with variation in pressure resistance in broth and sausage. Pressure-resistant (O157:H7 and O157:H12) and -sensitive (O157-M1 and O157-M2) E. coli strains were used. Pressure treatment at 350 MPa for 20 min in broth caused 1.1-1.2 logs reduction in O157:H12 and O157:H7 and 4.1-5.5 logs reduction in the O157-M1 and O157-M2. When high pressure was treated in the presence of CE (32 CEAU/mL), the combination treatment caused a significant inactivation in the pressure-resistant O157:H7 strains resulting in the viability loss of 4.3-4.6 logs and the synergistic effect increased with increase in treatment time (p < 0.05). Similar result was observed in sausage. Differential scanning calorimetry thermogram showed that the presence of Lb. casei CE may cause considerable damage to cellular components of E. coli during the high pressure treatment. The synergy between high pressure processing and Lb. casei OSY-LB6A CE against pressure-resistant E. coli O157 strains suggests the feasibility of using this combination to minimize the risk of transmission of E. coli O157 by food.

  11. Water disinfection using silver nanoparticle impregnated activated carbon: Escherichia coli cell-killing in batch and continuous packed column operation over a long duration.

    PubMed

    Biswas, Pritam; Bandyopadhyaya, Rajdip

    2016-09-01

    Silver nanoparticles (Ag-NP) were selectively impregnated on the external surface of plasma treated activated carbon (AC) granules (referred to as Ag-AC hybrid, having 0.8 wt% of Ag), for achieving continuous disinfection of water in a single flow-column set-up. First, Ag-NPs (28 nm mean size) were synthesized by UV reduction. Subsequently, Escherichia coli cell-killing experiments were performed in both shake flask (i. e. batch-mode) and flow-column (i. e. continuous-mode) operations, using E. coli K12 (MTCC 1302) as a model organism. Batch results using 8 mg Ag-AC hybrid/ml of cell suspension showed that, 10(4) CFU/ml of cells were killed within 25 min contact time, with cell concentration decaying exponentially in time. Maintaining almost the same contact time as in the batch experiments, three columns packed with Ag-AC (all having a height of 25 cm but increasing diameters of 1, 5 and 8 cm, respectively) were used for monitoring cell-killing performance over a long duration. For all columns, inlet water having 10(4) CFU/ml E. coli could be completely disinfected to produce treated, outlet water having zero cell count. Specifically for the 8 cm diameter column, a maximum throughput of treating 1.62 L of contaminated water per hour could be maintained for at least up to 16 days. Moreover, the Ag concentration in the outlet water was only up to 29.8 μg/L at steady state, which is well within the recommended limit of 100 μg/L for drinking water. Hence, water disinfection for potable quality water (zero E. coli count and <100 μg/L Ag) can be achieved in a continuous manner over a long duration, with our packed Ag-AC column. PMID:27179597

  12. Water disinfection using silver nanoparticle impregnated activated carbon: Escherichia coli cell-killing in batch and continuous packed column operation over a long duration.

    PubMed

    Biswas, Pritam; Bandyopadhyaya, Rajdip

    2016-09-01

    Silver nanoparticles (Ag-NP) were selectively impregnated on the external surface of plasma treated activated carbon (AC) granules (referred to as Ag-AC hybrid, having 0.8 wt% of Ag), for achieving continuous disinfection of water in a single flow-column set-up. First, Ag-NPs (28 nm mean size) were synthesized by UV reduction. Subsequently, Escherichia coli cell-killing experiments were performed in both shake flask (i. e. batch-mode) and flow-column (i. e. continuous-mode) operations, using E. coli K12 (MTCC 1302) as a model organism. Batch results using 8 mg Ag-AC hybrid/ml of cell suspension showed that, 10(4) CFU/ml of cells were killed within 25 min contact time, with cell concentration decaying exponentially in time. Maintaining almost the same contact time as in the batch experiments, three columns packed with Ag-AC (all having a height of 25 cm but increasing diameters of 1, 5 and 8 cm, respectively) were used for monitoring cell-killing performance over a long duration. For all columns, inlet water having 10(4) CFU/ml E. coli could be completely disinfected to produce treated, outlet water having zero cell count. Specifically for the 8 cm diameter column, a maximum throughput of treating 1.62 L of contaminated water per hour could be maintained for at least up to 16 days. Moreover, the Ag concentration in the outlet water was only up to 29.8 μg/L at steady state, which is well within the recommended limit of 100 μg/L for drinking water. Hence, water disinfection for potable quality water (zero E. coli count and <100 μg/L Ag) can be achieved in a continuous manner over a long duration, with our packed Ag-AC column.

  13. Plasmolysis and cell shape depend on solute outer-membrane permeability during hyperosmotic shock in E. coli.

    PubMed

    Pilizota, Teuta; Shaevitz, Joshua W

    2013-06-18

    The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.

  14. Plasmolysis and cell shape depend on solute outer-membrane permeability during hyperosmotic shock in E. coli.

    PubMed

    Pilizota, Teuta; Shaevitz, Joshua W

    2013-06-18

    The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously. PMID:23790382

  15. Plasmolysis and Cell Shape Depend on Solute Outer-Membrane Permeability during Hyperosmotic Shock in E. coli

    PubMed Central

    Pilizota, Teuta; Shaevitz, Joshua W.

    2013-01-01

    The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously. PMID:23790382

  16. Cell surface receptors for CCN proteins.

    PubMed

    Lau, Lester F

    2016-06-01

    The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvβ3, αvβ5, α5β1, α6β1, αIIbβ3, αMβ2, and αDβ2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities.

  17. Hydrogen Peroxide Induced Cell Death: The Major Defences Relative Roles and Consequences in E. coli.

    PubMed

    Uhl, Lionel; Dukan, Sam

    2016-01-01

    We recently developed a mathematical model for predicting reactive oxygen species (ROS) concentration and macromolecules oxidation in vivo. We constructed such a model using Escherichia coli as a model organism and a set of ordinary differential equations. In order to evaluate the major defences relative roles against hydrogen peroxide (H2 O2), we investigated the relative contributions of the various reactions to the dynamic system and searched for approximate analytical solutions for the explicit expression of changes in H2 O2 internal or external concentrations. Although the key actors in cell defence are enzymes and membrane, a detailed analysis shows that their involvement depends on the H2 O2 concentration level. Actually, the impact of the membrane upon the H2 O2 stress felt by the cell is greater when micromolar H2 O2 is present (9-fold less H2 O2 in the cell than out of the cell) than when millimolar H2 O2 is present (about 2-fold less H2 O2 in the cell than out of the cell). The ratio between maximal external H2 O2 and internal H2 O2 concentration also changes, reducing from 8 to 2 while external H2 O2 concentration increases from micromolar to millimolar. This non-linear behaviour mainly occurs because of the switch in the predominant scavenger from Ahp (Alkyl Hydroperoxide Reductase) to Cat (catalase). The phenomenon changes the internal H2 O2 maximal concentration, which surprisingly does not depend on cell density. The external H2 O2 half-life and the cumulative internal H2 O2 exposure do depend upon cell density. Based on these analyses and in order to introduce a concept of dose response relationship for H2 O2-induced cell death, we developed the concepts of "maximal internal H2 O2 concentration" and "cumulative internal H2 O2 concentration" (e.g. the total amount of H2 O2). We predict that cumulative internal H2 O2 concentration is responsible for the H2 O2-mediated death of bacterial cells. PMID:27494019

  18. Hydrogen Peroxide Induced Cell Death: The Major Defences Relative Roles and Consequences in E. coli

    PubMed Central

    Uhl, Lionel; Dukan, Sam

    2016-01-01

    We recently developed a mathematical model for predicting reactive oxygen species (ROS) concentration and macromolecules oxidation in vivo. We constructed such a model using Escherichia coli as a model organism and a set of ordinary differential equations. In order to evaluate the major defences relative roles against hydrogen peroxide (H2 O2), we investigated the relative contributions of the various reactions to the dynamic system and searched for approximate analytical solutions for the explicit expression of changes in H2 O2 internal or external concentrations. Although the key actors in cell defence are enzymes and membrane, a detailed analysis shows that their involvement depends on the H2 O2 concentration level. Actually, the impact of the membrane upon the H2 O2 stress felt by the cell is greater when micromolar H2 O2 is present (9-fold less H2 O2 in the cell than out of the cell) than when millimolar H2 O2 is present (about 2-fold less H2 O2 in the cell than out of the cell). The ratio between maximal external H2 O2 and internal H2 O2 concentration also changes, reducing from 8 to 2 while external H2 O2 concentration increases from micromolar to millimolar. This non-linear behaviour mainly occurs because of the switch in the predominant scavenger from Ahp (Alkyl Hydroperoxide Reductase) to Cat (catalase). The phenomenon changes the internal H2 O2 maximal concentration, which surprisingly does not depend on cell density. The external H2 O2 half-life and the cumulative internal H2 O2 exposure do depend upon cell density. Based on these analyses and in order to introduce a concept of dose response relationship for H2 O2-induced cell death, we developed the concepts of “maximal internal H2 O2 concentration” and “cumulative internal H2 O2 concentration” (e.g. the total amount of H2 O2). We predict that cumulative internal H2 O2 concentration is responsible for the H2 O2-mediated death of bacterial cells. PMID:27494019

  19. Topoisomerase IIα Binding Domains of Adenomatous Polyposis Coli Influence Cell Cycle Progression and Aneuploidy

    PubMed Central

    Wang, Yang; Coffey, Robert J.; Osheroff, Neil; Neufeld, Kristi L.

    2010-01-01

    Background Truncating mutations in the tumor suppressor gene APC (Adenomatous Polyposis Coli) are thought to initiate the majority of colorectal cancers. The 15- and 20-amino acid repeat regions of APC bind β-catenin and have been widely studied for their role in the negative regulation of canonical Wnt signaling. However, functions of APC in other important cellular processes, such as cell cycle control or aneuploidy, are only beginning to be studied. Our previous investigation implicated the 15-amino acid repeat region of APC (M2-APC) in the regulation of the G2/M cell cycle transition through interaction with topoisomerase IIα (topo IIα). Methodology/Principal Findings We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo IIα in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo IIα isoform and lacking topo IIβ did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo IIα. Of note, although M3-APC can bind β-catenin, the G2 arrest did not correlate with β-catenin expression or activity, similar to what was seen with M2-APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2-APC region. Conclusions/Significance Together, our data establish that the 20-amino acid repeat region of APC interacts with topo IIα to enhance its activity in vitro, and leads to G2 cell cycle accumulation and aneuploidy when expressed in cells containing full-length APC. These findings provide an additional explanation for the aneuploidy associated with many colon cancers that possess truncated APC. PMID:20368985

  20. Effect of gold nanoparticles on thermal gradient generation and thermotaxis of E. coli cells in microfluidic device.

    PubMed

    Murugesan, Nithya; Panda, Tapobrata; Das, Sarit K

    2016-08-01

    Bacteria responds to changing chemical and thermal environment by moving towards or away from a particular location. In this report, we looked into thermal gradient generation and response of E. coli DH5α cells to thermal gradient in the presence and in the absence of spherical gold nanoparticles (size: 15 to 22 nm) in a static microfluidic environment using a polydimethylsiloxane (PDMS) made microfluidic device. A PDMS-agarose based microfluidic device for generating thermal gradient has been developed and the thermal gradient generation in the device has been validated with the numerical simulation. Our studies revealed that the presence of gold nanoparticles, AuNPs (0.649 μg/mL) has no effect on the thermal gradient generation. The E. coli DH5α cells have been treated with AuNPs of two different concentrations (0.649 μg/mL and 0.008 μg/mL). The thermotaxis behavior of cells in the presence of AuNPs has been studied and compared to the thermotaxis of E.coli DH5α cells in the absence of AuNPs. In case of thermotaxis, in the absence of the AuNPs, the E. coli DH5α cells showed better thermotaxis towards lower temperature range, whereas in the presence of AuNPs (0.649 μg/mL and 0.008 μg/mL) thermotaxis of the E. coli DH5α cells has been inhibited. The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration. The reason for the failure in thermotaxis response mechanism may be due to decreased F-type ATP synthase activity and collapse of membrane potential by AuNPs, which, in turn, leads to decreased ATP levels. This has been hypothesized since both thermotaxis and chemotaxis follows the same response mechanism for migration in which ATP plays critical role.

  1. Nanoparticle energy transfer on the cell surface.

    PubMed

    Bene, László; Szentesi, Gergely; Mátyus, László; Gáspár, Rezso; Damjanovich, Sándor

    2005-01-01

    Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2-10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal-fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle-fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range essentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore-nanoparticle distance. The intensity of 90 degrees scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90 degrees light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging

  2. Internalization of Escherichia Coli O157:H7 by Bovine Rectal Epithelial Cells

    PubMed Central

    Sheng, Haiqing; Wang, Jing; Lim, Ji Youn; Davitt, Christine; Minnich, Scott A.; Hovde, Carolyn J.

    2011-01-01

    Escherichia coli O157:H7 (O157) causes human diarrheal disease and healthy cattle are its primary reservoir. O157 colonize the bovine epithelial mucosa at the recto-anal junction (RAJ). Previous studies show that O157 at this site are not eliminated by aggressive interventions including applications of O157-specific lytic bacteriophages and other bactericidal agents. We hypothesize that some O157 at the RAJ mucosa are protected from these killing agents by host cell internalization. To test this hypothesis, rectal biopsies from O157 culture positive and negative cattle were analyzed by fluorescent microscopy and subjected to gentamicin protection assays. GFP-labeled bacteria were found located deep within the tissue crypts and a small number of O157 were recovered from rectal biopsies after gentamicin treatment. Primary bovine rectal epithelial (PBRE) cell cultures were incubated with O157 and subjected to gentamicin protection assays. Strains ATCC 43895, 43894, Sakai, and WSU180 entered the PBRE cells with different levels of efficiency ranging from 0.18 to 19.38% of the inocula. Intracellular bacteria were confirmed to be within membrane-bounded vacuoles by electron microscopy. Cytochalasin D curtailed internalization of O157 indicating internalization was dependent on eukaryotic microfilament assembly. Strain ATCC 43895 exhibited the highest efficiency of internalization and survived for at least 24 h within PBRE cells. Deletion mutation of intimin or its receptor in ATCC 43895 did not reduce bacterial internalization. This strain produced more biofilm than the others tested. Retrospective analysis of cattle challenged with two O157 strains, showed ATCC 43895, the most efficient at host cell internalization, was most persistent. PMID:21687423

  3. Individuality and universality in the growth-division laws of single E. coli cells.

    PubMed

    Kennard, Andrew S; Osella, Matteo; Javer, Avelino; Grilli, Jacopo; Nghe, Philippe; Tans, Sander J; Cicuta, Pietro; Cosentino Lagomarsino, Marco

    2016-01-01

    The mean size of exponentially dividing Escherichia coli cells in different nutrient conditions is known to depend on the mean growth rate only. However, the joint fluctuations relating cell size, doubling time, and individual growth rate are only starting to be characterized. Recent studies in bacteria reported a universal trend where the spread in both size and doubling times is a linear function of the population means of these variables. Here we combine experiments and theory and use scaling concepts to elucidate the constraints posed by the second observation on the division control mechanism and on the joint fluctuations of sizes and doubling times. We found that scaling relations based on the means collapse both size and doubling-time distributions across different conditions and explain how the shape of their joint fluctuations deviates from the means. Our data on these joint fluctuations highlight the importance of cell individuality: Single cells do not follow the dependence observed for the means between size and either growth rate or inverse doubling time. Our calculations show that these results emerge from a broad class of division control mechanisms requiring a certain scaling form of the "division hazard rate function," which defines t