INSECT SCIENCE, Issue 1 2004
Li-rong Shen
Abstract, The venomous phospholipase A2 (AcPLA2) coding reading region of the Chinese honeybee (Apis cerana cerana), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15kD. Detection of western blot using ant-European honeybee (Apis mellifera) phospholipase A2 (AmPLA2) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA2. The result demonstrated that the AcPLA2 peptide had been expressed in E. coli and the AcPLA2 has the similar antigenicity as the AmPLA2. [source]

THERMAL DEATH TIMES OF ESCHERICHIA COLI IN YOUNG COCONUT ENDOSPERM BEVERAGE

JOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 2009
ALONZO A. GABRIEL
ABSTRACT The decimal reduction times (D values) of Escherichia coli (American Type Culture Collection 25922) were established in a young coconut endosperm beverage, a famous local drink in the Philippines and in many tropical countries. Artificially inoculated cells were heated to 60, 70 and 80C at various heating times prior to survivor enumeration by surface plating onto pre-solidified Eosine Methylene Blue Agar. Results showed that the surviving populations significantly (P < 0.05) decreased with increasing exposure time and temperature. The calculated D values ranged from 0.26 ± 0.01 to 0.56 ± 0.08 min. Validation of the results by establishing the thermal resistance of other E. coli isolates in the coconut beverage medium was recommended. PRACTICAL APPLICATION The study established the thermal inactivation rates of Escherichia coli (American Type Culture Collection 25922) in a young coconut endosperm beverage medium in various heating temperatures. The results obtained from this study may be used in the calculations of appropriate thermal process schedules for the test beverage against the test organism. [source]

HIGH PREVALENCE OF EXTENDED-SPECTRUM ,-LACTAMASES ESCHERICHIA COLI AND VANCOMYCIN-RESISTANT ENTEROCOCCI ISOLATES FROM CHICKEN PRODUCTS.

JOURNAL OF FOOD SAFETY, Issue 1 2010
A PROBLEM OF PUBLIC HEALTH
ABSTRACT Twenty-nine chicken products were acquired from different supermarkets in Portugal during September to December 2007 and were analyzed for extended-spectrum ,-lactamases (ESBL) Escherichia coli and vancomycin-resistant enterococci (VRE). Cefotaxime-resistant E. coli isolates were recovered in 27 of 29 chicken samples representing 93% of the analyzed samples. The highest percentages of resistance (more than 50% of the isolates) were detected for ampicillin, nalidixic acid and tetracycline. VRE isolates were detected in 17 of 29 samples of chicken origin (59%) and were identified as Enterococcus durans (n = 15) and E. faecium (n = 2) with the highest percentages of resistance being detected for erythromycin, tetracycline, ampicillin and ciprofloxacin. Seven E. durans and the two E. faecium isolates recovered from chicken wings, gizzard and skin show gelatinase activity. The high rate of colonization of chicken products by these bacteria supports other studies suggesting that the food chain could be a source of ESBL and VRE colonization in humans representing a public health problem. PRACTICAL APPLICATIONS The data indicate that chicken products may be contaminated with a high prevalence of extended-spectrum ,-lactamases Escherichia coli and vancomycin-resistant enterococci (VRE). It is important to mention that the isolates present a diversity of phenotypes of antimicrobial resistance, and half of the VRE isolates show also gelatinase activity, indicating that these animals may be a reservoir of bacteria showing virulence and increased resistance to antimicrobial agents, raising special concerns about their transmission to humans through the food chain. [source]

REDUCTIONS OF ESCHERICHIA COLI, COLIFORMS, AEROBIC PLATE COUNTS AND CAMPYLOBACTER JEJUNI BY A SMALL-SCALE, HIGH-PRESSURE SYSTEM DEVISED TO CLEAN A MINIATURIZED POULTRY GIBLETS TRANSPORT SYSTEM

JOURNAL OF FOOD SAFETY, Issue 4 2009
OMAR A. OYARZABAL
ABSTRACT The efficacy of using direct high-pressure hot water (60C, 140F) and a quaternary ammonium compound to clean the inside of stainless steel pipe used to transport chicken giblets was evaluated. The giblets were collected from a commercial processing plant and were inoculated with Campylobacter jejuni. The cleaning system was effective in reducing the numbers of inoculated C. jejuni and naturally occurring mesotrophic bacteria (aerobic plate counts) on the inside surface of the stainless steel pipe used to transport the giblets. However, the decreases in naturally occurring Escherichia coli and coliforms were not significant. These results suggest that additional improvements are needed to better disinfect the piping system used to transport giblets to reduce the potential for cross-contamination with C. jejuni and E. coli. The devised cleaning system could be optimized to reduce the use of chemical agents, the cleaning time and the cost of cleaning pipes in poultry processing facilities. PRACTICAL APPLICATIONS These experiments suggest that the traditional use of hot water and quaternary ammonium compounds to clean the inside of the piping system used to transport chicken giblets may not be sufficient to reduce the contamination with Campylobacter jejuni and mesotrophic bacteria (aerobic plate count). Poultry processors should be aware of the limitations of cleaning closed piping systems and develop and test high-pressure systems to thoroughly clean the pipes used to transport giblets after processing to avoid potential sources of cross-contamination with C. jejuni and mesotrophic bacteria. [source]

ACID TOLERANCE OF ESCHERICHIA COLI FOLLOWING COLD SHOCK TREATMENT

JOURNAL OF FOOD SAFETY, Issue 2 2003
GREG BLANK
ABSTRACT The effect of an initial cold shock treatment (2 h at 10C), following an abrupt downshift in temperature from 37 to 10C, on the subsequent growth and survival of Escherichia coli strains O157:H7 and MY20 (Biotype 1) in acidified Trypticase soy broth (TSB) and fruit juices (orange, apple) was investigated. Overall, no difference in growth at 37C was observed between each cold shocked and noncold shocked E. coli strain when cultured in TSB adjusted with either acetic acid (pH 6.0)or malic, citric and tartaric acid (each adjusted to: pH 4.5, 5.0, 5.5, 6.0). However, significant (P ± 0.05) differences in survival were observed between cold shocked and noncold shocked populations in TSB acidified with acetic acid (pH 5.0) or citric, malic and tartaric acid (pH 4.0). For strain MY20, survivor levels for cold shocked cells in TSB acidified with acetic acid citric, malic and tartaric acid at 8C were significantly (P ± 0.05) higher than in noncold shocked populations. Also, at 37C survival levels for cold shocked cells were significantly (P ± 0.05) higher than noncold shocked cells in TSB acidified with either malic or tartaric acid (pH 4.0). For the O157:H7 strain, survivor levels were higher (P ± 0.05) for cold shocked cells when maintained in TSB at 37C regardless of acid type. At 8C, cold shock treatment only increased (P ± 0.05) the survival of the O157:H7 strain in TSB adjusted with acetic acid (pH 6.0). Acid cross protection induced by cold shocking, as evidenced by enhanced survival, was not apparent for either E. coil strain in apple (pH 3.5) or orange juice (pH 3.8) maintained at 8C. [source]

Contact-Killing Polyelectrolyte Microcapsules Based on Chitosan Derivatives

ADVANCED FUNCTIONAL MATERIALS, Issue 19 2010
Di Cui
Abstract Polyelectrolyte-multilayer microcapsules are made by layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes onto sacrificial colloidal particles, followed by core removal. In this paper, contact-killing polyelectrolyte microcapsules are prepared based solely on polysaccharides. To this end, water-soluble quaternized chitosan (QCHI) with varying degrees of substitution (DS) and hyaluronic acid (HA) are assembled into thin films. The quaternary ammonium groups are selectively grafted on the primary amine group of chitosan by exploiting its reaction with glycidyltrimethylammonium chloride (GTMAC) under homogeneous aqueous acidic conditions. The morphology of the capsules is closely dependent on the DS of the quaternized chitosan derivatives, which suggests differences in their complexation with HA. The DS is also a key parameter to control the antibacterial activity of QCHI against Escherichia Coli (E. coli). Thus, capsules containing the QCHI derivative with the highest DS are shown to be the most efficient to kill E. coli while retaining their biocompatibility toward myoblast cells, which suggests their potential as drug carriers able to combat bacterial infections. [source]

Etiologic spectrum and pattern of antimicrobial drug susceptibility in bacterial meningitis in Sokoto, Nigeria

ACTA PAEDIATRICA, Issue 8 2000
FE EmeleArticle first published online: 2 JAN 200
Etiologic agents of meningitis were prospectively investigated among patients admitted to Usman Danfodio University Teaching Hospital, Sokoto. Of 1097 cerebrospinal fluid (CSF) samples submitted to the microbiology laboratory from various wards of the hospital, 289 (26%) were microscopically, culturally and/or serologically proven to be bacterial meningitis. The etiologic spectrum was as follows: Neisseria meningitidis (61%), Streptococcus pneumoniae (18%), Haemophilus influenzae (10%), Staphylococcus aureus (6%), Coliform bacilli (3%), Escherichia coli (0.7%), Mycobacterium tuberculosis (0.7%), Listeria monocytogenes (0.4%), Flavobacterium meningosepticum (0.4%) and Pseudomonas putrifasciens (0.4%). Bacterial meningitis was most prevalent (195 or 68%) among children aged 1-9 y, while adults and neonates were least affected. Coliform bacilli caused five of eight neonatal cases. Males were more frequently affected than females (x2=12.50;p < 0.05). Culture and microscopy were comparatively less efficient than the search for bacterial antigens, especially in the diagnosis of Haemophilus meningitis. Antimicrobial susceptibility of N. meningitidis to ampicillin and benzyl penicillin reduced progressively over the years (F = 406.98;p < 0.001). Nineteen (11%) of the isolates (5 Meningococci, 7 Staph. aureus, 1 Haem. influenza and 6 others) showed simultaneous resistance to chloramphenicol, ampicillin and benzyl penicillin. [source]

Gastrointestinal Bacterial Transmission among Humans, Mountain Gorillas, and Livestock in Bwindi Impenetrable National Park, Uganda

CONSERVATION BIOLOGY, Issue 6 2008
INNOCENT B. RWEGO
ecología de enfermedades; Escherichia coli; primates; salud del ecosistema; zoonosis Abstract:,Habitat overlap can increase the risks of anthroponotic and zoonotic pathogen transmission between humans, livestock, and wild apes. We collected Escherichia coli bacteria from humans, livestock, and mountain gorillas (Gorilla gorilla beringei) in Bwindi Impenetrable National Park, Uganda, from May to August 2005 to examine whether habitat overlap influences rates and patterns of pathogen transmission between humans and apes and whether livestock might facilitate transmission. We genotyped 496 E. coli isolates with repetitive extragenic palindromic polymerase chain reaction fingerprinting and measured susceptibility to 11 antibiotics with the disc-diffusion method. We conducted population genetic analyses to examine genetic differences among populations of bacteria from different hosts and locations. Gorilla populations that overlapped in their use of habitat at high rates with people and livestock harbored E. coli that were genetically similar to E. coli from those people and livestock, whereas E. coli from gorillas that did not overlap in their use of habitats with people and livestock were more distantly related to human or livestock bacteria. Thirty-five percent of isolates from humans, 27% of isolates from livestock, and 17% of isolates from gorillas were clinically resistant to at least one antibiotic used by local people, and the proportion of individual gorillas harboring resistant isolates declined across populations in proportion to decreasing degrees of habitat overlap with humans. These patterns of genetic similarity and antibiotic resistance among E. coli from populations of apes, humans, and livestock indicate that habitat overlap between species affects the dynamics of gastrointestinal bacterial transmission, perhaps through domestic animal intermediates and the physical environment. Limiting such transmission would benefit human and domestic animal health and ape conservation. Resumen:,El traslape de hábitats puede incrementar los riesgos de transmisión de patógenos antroponótica y zoonótica entre humanos, ganado y simios silvestres. Recolectamos bacterias Escherichia coli de humanos, ganado y gorilas de montaña (Gorilla gorilla beringei) en el Parque Nacional Bwindi Impenetrable, Uganda, de mayo a agosto 2005 para examinar sí el traslape de hábitat influye en las tasas y patrones de transmisión de patógenos entre humanos y simios y sí el ganado facilita esa transmisión. Determinamos el genotipo de 496 aislados de E. coli con marcaje de reacción en cadena de polimerasa palindrómica extragénica (rep-PCR) y medimos la susceptibilidad a 11 antibióticos con el método de difusión de disco. Realizamos análisis de genética poblacional para examinar las diferencias genéticas entre poblaciones de bacterias de huéspedes y localidades diferentes. Las poblaciones de gorilas con alto grado de traslape en el uso de hábitat con humanos y ganado presentaron E. coli genéticamente similar a E. coli de humanos y ganado, mientras que E. coli de gorilas sin traslape en el uso hábitat con humanos y ganado tuvo relación lejana con las bacterias de humanos y ganado. Treinta y cinco porciento de los aislados de humanos, 27% de los aislados de ganado y 17% de los aislados de gorilas fueron clínicamente resistentes a por lo menos un antibiótico utilizado por habitantes locales, y la proporción de gorilas individuales con presencia de aislados resistentes declinó en las poblaciones proporcionalmente con la disminución en el grado de traslape con humanos. Estos de patrones de similitud genética y resistencia a antibióticos entre E. coli de poblaciones de simios, humanos y ganado indican que el traslape de hábitat entre especies afecta la dinámica de transmisión de bacterias gastrointestinales, probablemente a través de animales domésticos intermediarios y el ambiente físico. La limitación de esa transmisión beneficiaría a la salud de humanos y animales domésticos y a la conservación de simios. [source]

Effect of ,-trinositol on secretion induced by Escherichia coli ST-toxin in rat jejunum

ACTA PHYSIOLOGICA, Issue 4 2003
A.-M. Lahti
Abstract Aim:,d -myo-inositol-1,2,6-trisphosphate (, -trinositol, PP56), is a synthetic isomer of the intracellular second messenger, d -myo-inositol-1,4,5-trisphospahate. The pharmacological actions of , -trinositol include potent anti-inflammatory properties and inhibition of the secretion induced by cholera toxin and obstructive ileus. In the present study, we investigated whether , -trinositol was able to influence the secretion induced by heat-stable ST-toxin from Escherichia coli in the rat jejunum. Methods:, A midline abdominal incision was performed in anaesthetized male Sprague,Dawley rats and a 6,7 cm long jejunal segment was isolated with intact vascular supply and placed in a chamber suspended from a force displacement transducer connected to a Grass® polygraph. Intestinal net fluid transport was continuously monitored gravimetrically. Crystalline ST-toxin (120 mouse units) was introduced into the intestinal lumen and left there for the rest of the experiment. When a stable secretion was observed, , -trinositol (60 mg kg,1 h,1) or saline were infused during 2 h, followed by a 2-h control period. Results:, , -Trinositol induced a significant (P < 0.001) inhibition of ST-toxin secretion within 30 min, lasting until 2 h after infusion had stopped. The agent also moderately increased (P < 0.05) net fluid absorption in normal jejunum. Mean arterial pressure (P < 0.001) and heart rate (P < 0.001) were reduced by , -trinositol. Conclusion:, The inhibition by , -trinositol of ST-toxin induced intestinal secretion is primarily secondary to inhibition of secretory mechanisms and only to lesser extent due to increased absorption. The detailed mechanisms of action have not been clarified but may involve suppression of inflammation possibly by means of cellular signal transduction. [source]

Quantification of Shigella IcsA required for bacterial actin polymerization

CYTOSKELETON, Issue 4 2002
Juana Magdalena
Abstract Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500,2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella -infected cells. Cell Motil. Cytoskeleton 51:187,196, 2002. © 2002 Wiley-Liss, Inc. [source]

Gram-negative meningitis and infections in individuals treated with intrathecal baclofen for spasticity: a retrospective study

DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 6 2006
Colleen A Wunderlich MD MSc
The aim of this retrospective study was to describe signs, symptoms, and clinical outcomes of individuals undergoing intrathecal baclofen (ITB) therapy who experienced pumprelated Gram-negative infections including meningitis. Participants included 12 individuals (nine males, three females) aged 10 to 32 years (mean 17y 9mo), nine of whom had quadriplegic CP. A total of 571 baclofen pump surgeries were performed with 45 total infections. Of the 45 infections, 12 were by Gram-negative organisms, two resulting in meningitis. Ten of 12 Gram-negative infections (21 site encounters) occurred within 60 days of surgery. Eleven of 12 pumps were explanted. By site encounters, Pseudomonas aeruginosa accounted for eight Gram-negative infections, Escherichia coli for five, Proteus for three, Enterobacter cloacae for two, and Klebsiella, Enterobacter aerogenes, and Enterobacter vulnaris for one each. Two individuals with Gram-negative meningitis were admitted 72 to 96 hours after hospital discharge following pump replacement. Both patients had rapid deterioration requiring transfer to the pediatric intensive care unit, and developed coagulopathy and decrease in responsiveness. Both have improved and have elected not to replace the ITB pump. In Gram-negative infections in ITB therapy, the progression of signs and symptoms can be swift and devastating. Identification of the infectious agent in such cases is imperative; these infections can quickly become life threatening. [source]

Factors influencing the challenges of modelling and treating fecal indicator bacteria in surface waters

ECOHYDROLOGY, Issue 4 2009
Cristiane Q. Surbeck
Abstract In the United States, thousands of creeks, rivers, and coastal zones are listed as impaired in the Clean Water Act's 303(d) list. The number one general cause of impairments is denoted as ,pathogens', which can include known pathogenic organisms or, more commonly, fecal indicator bacteria (FIB), such as fecal coliform bacteria, Escherichia coli, and enterococci bacteria. Despite efforts by water quality managers to reduce FIB in surface waters via treatment, successful and significant reduction of FIB has been difficult to achieve to meet water quality standards. In addition, current efforts to numerically model FIB concentrations in surface waters do not consider many complexities associated with FIB as a pollutant. Reasons for the challenge of treating and modelling FIB are their varied sources and mechanisms of survival and decay in the environment. This technical note addresses this challenge by discussing the nature of FIB, their sources, and their fate and transport mechanisms. Sources of FIB to surface waters include wastewater, stormwater and dry-weather runoff, and animals. Mechanisms of pathogen indicator occurrence in surface waters are transport in stormwater, ecological proliferation, and interaction with sediments. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Amperometric Biosensors for Detection of Sugars Based on the Electrical Wiring of Different Pyranose Oxidases and Pyranose Dehydrogenases with Osmium Redox Polymer on Graphite Electrodes

ELECTROANALYSIS, Issue 2-3 2007
Federico Tasca
Abstract Electrical wiring of different types of pyranose oxidase (P2O) (fungal wild type, recombinant wild type with a hexa-histidine tag, mutant form E542K with a hexa-histidine tag) from Trametes multicolor, and recombinant P2O from Coriolus sp. overexpressed in Escherichia coli as well as of pyranose dehydrogenase (PDH) from Agaricus meleagris and Agaricus xanthoderma with an osmium redox polymer (poly(1-vinylimidazole)12 -[Os(4,4,-dimethyl-2,2,-dipyridyl)2Cl2]2+/+) on graphite electrodes was carried out. After optimization studies using glucose as substrate, the biosensors, which showed the best characteristics in terms of linear range, detection limit and sensitivity were selected, viz. wild type P2O from T. multicolor and PDH from A. meleagris. These two enzymes were used and investigated for their selectivity for a number of different sugars. [source]

Cationic and anionic lipid-based nanoparticles in CEC for protein separation

ELECTROPHORESIS, Issue 11 2010
Christian Nilsson
Abstract The development of new separation techniques is an important task in protein science. Herein, we describe how anionic and cationic lipid-based liquid crystalline nanoparticles can be used for protein separation. The potential of the suggested separation methods is demonstrated on green fluorescent protein (GFP) samples for future use on more complex samples. Three different CEC-LIF approaches for protein separation are described. (i) GFP and GFP N212Y, which are equally charged, were separated with high resolution by using anionic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (ii) High efficiency (800,000 plates/m) and peak capacity were demonstrated separating GFP samples from Escherichia coli with cationic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (iii) Three single amino-acid-substituted GFP variants were separated with high resolution using an approach based on a physical attached double-layer coating of cationic and anionic nanoparticles combined with anionic lipid nanoparticles suspended in the electrolyte. The soft and porous lipid-based nanoparticles were synthesized by a one-step procedure based on the self-assembly of lipids, and were biocompatible with a large surface-to-volume ratio. The methodology is still under development and the optimization of the nanoparticle chemistry and separation conditions can further improve the separation system. In contrast to conventional LC, a new interaction phase is introduced for every analysis, which minimizes carry-over and time-consuming column regeneration. [source]

Bacteria concentration using a membrane type insulator-based dielectrophoresis in a plastic chip

ELECTROPHORESIS, Issue 18 2009
Yoon-Kyoung Cho
Abstract We report an insulator-based (or, electrodeless) dielectrophoresis utilizing microfabricated plastic membranes. The membranes with honeycomb-type pores have been fabricated by patterning the SU-8 layer on a substrate which was pretreated with self-assembled monolayer of octadecyltrichlorosilane for the easy release. The fabricated membrane was positioned between two electrodes and alternating current field was applied for the particle trap experiments. The particle could be trapped due to the dielectrophoresis force generated by the non-uniformities of the electric fields applied through the membranes with pores. Simulations using CFD-ACE+(CFD Research, Huntsville, Alabama) suggested that the dielectrophoresis force is stronger in the edge of the pores where the field gradient is highest. The bacteria could be captured on the near edge of the pores when the electric field was turned on and the trapped bacteria could be released when the field was turned off with the release efficiency of more than 93±7%. The maximal trapping efficiency of 66±7% was obtained under the electric fields (E=128,V/mm and f=300,kHz) when the dilute bacteria solution (Escherichia coli: 9.3×103,cell/mL, 0.5,mS/m) flowed with a flow rate of 100,,L/min. [source]

Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresis

ELECTROPHORESIS, Issue 14 2009
Jingdan Liang
Abstract Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps. [source]

The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis

ELECTROPHORESIS, Issue 15 2008
Chi-Hui Lin
Abstract The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity. [source]

Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression

ELECTROPHORESIS, Issue 21 2007
Divya Kapoor
Abstract The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE. [source]

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

ELECTROPHORESIS, Issue 9 2006
Peng Gao
Abstract The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S.,aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S.,aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S.,aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0×105 colony forming unit/mL. We have also utilized this technology to identify S.,aureus in a stool sample coming from a healthy volunteer spiked successfully with S.,aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S.,aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available. [source]

Improved 2-DE of microorganisms after acidic extraction

ELECTROPHORESIS, Issue 8 2006
Ben R. Herbert Professor
Abstract 2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100,kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH,3 by 80,mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. [source]

Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations

ELECTROPHORESIS, Issue 7-8 2004
Yi Zhu
Abstract Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains. [source]

Separation of Escherichia coli 055:B5 lipopolysaccharide and detoxified lipopolysaccharide by high-performance capillary electrophoresis

ELECTROPHORESIS, Issue 17 2003
Nicola Volpi
Abstract A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the lipopolysaccharide (LPS) and detoxified LPS (D-LPS), produced by both alkaline treatment in anhydrous conditions and mild acid hydrolysis, from Escherichia coli 055:B5 bacteria. LPS and D-LPS are separated and readily determined within 25 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship (correlation coefficient greater than about 0.97) was found for the LPS and the two D-LPS species over a wide range of concentrations, from approximately 120 to 360 ng, with a detection sensitivity less than about 100 ng. Furthermore, HPCE was able to separate several molecular species mainly due to the presence of populations with O -specific polysaccharides of distinct and increasing mean chain lengths. This approach could be of great importance for the quantitative determination of LPS and D-LPS during the purification and preparation processes, also considering the importance of D-LPS in the preparation of human vaccines, and for the qualitative evaluation of the heterogeneity of LPS and the O -polysaccharide components. [source]

Applications of PAT-Process Analytical Technology in Recombinant Protein Processes with Escherichia coli

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2008
C. Kaiser
Abstract Monitoring of bioprocesses and thus observation and identification of such processes is one of the main aims of bioprocess engineering. It is of vital importance in bioprocess development to improve the overall productivity by avoiding unintentional limitations to ensure not only optimal process conditions but also the observation of established production processes. Furthermore, reproducibility needs to be improved and final product quality and quantity be guaranteed. Therefore, an advanced monitoring and control system has been developed, which is based on different in-line, on-line and at-line measurements for substrates and products. Observation of cell viability applying in-line radio frequency impedance measurement and on-line determination of intracellular recombinant target protein using the reporter protein T-Sapphire GFP based on in-line fluorescence measurement show the ability for the detection of critical process states. In this way, the possibility for the on-line recognition of optimal harvest times arises and disturbances in the scheduled process route can be perceived. [source]

Enhancement of the NAD(P)(H) Pool in Escherichia coli for Biotransformation

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007
F. Heuser
Abstract In pyridine nucleotide-dependent, reductive whole cell biotransformation with resting cells of Escherichia coli, the availability of intracellular NAD(P)(H) is a pivotal point for an efficient and highly productive substrate conversion. The question whether an increase of the intracellular NAD(P)(H) concentration could increase the productivity was discussed controversially in the past. This is the first report on an E. coli strain with an increased NAD(P)(H) pool which was tested in a reductive biotransformation system for an increased productivity. Biotransformation was performed with a strain overexpressing a gene encoding an (R)-specific alcohol dehydrogenase for the stereospecific, NADPH-dependent reduction of methyl acetoacetate (MAA) to (R)-methyl-3-hydroxybutanoate (MHB). Cofactor regeneration was implemented via glucose oxidation by coexpression of a gene encoding glucose dehydrogenase. The specific MHB productivity (mmol mg,1 cell dry weight,1h,1) enabled a comparison between the E. coli,BL21(DE3) wild-type and a genetically modified strain. The enhancement of the NAD(P)(H) pool was achieved by genetic manipulation of the NAD(H) biosynthetic pathways. After simultaneous overexpression of the pncB and nadE genes, encoding nicotinic acid phosphoribosyltransferase and NAD synthetase, measurements of the total NAD(P)(H) pool, sizes showed a 7-fold and 2-fold increased intracellular concentration of NAD(H) and NADP(H), respectively. However, the implementation of an E.,coli strain carrying a genomically integrated pncB gene with an upstream T7,promoter for biotransformation did not result in reproducible increased specific cell productivity. [source]

Optimization and Control of Industrial Microbial Cultivation Processes

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2006
M. Jenzsch
Abstract Compared to the immense achievements in fundamental molecular biological sciences, the improvements in the fermentation and downstream processing technologies used in industry have been less spectacular over the last decade. Hence, there is a misbalance between new cellular systems and production technologies, resulting in a decreasing annual rate of approved production processes. In its PAT initiative the U.S. Food and Drug Administration identifies the potential for continuous improvement and makes concrete suggestions how this can be achieved. Here, some of these suggestions were applied to recombinant protein production with Escherichia coli and Pichia pastoris cultures. Concretely, the development of process operational procedures is discussed that allow a more tight supervision of the processes and the automatic control in cases where processes deviate from their set-point profiles. [source]

Process Development in Biotechnology , A Re-Evaluation

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2005
K. Schügerl
Abstract This review considers some process development problems in biotechnology and presents examples of solutions, which were developed in cooperation with industrial partners. These processes include the production of restriction endonuclease EcoRI by recombinant Escherichia coli, which is toxic to the cell, penicillin V by Penicillium chrysogenum, xylanase by Aspergillus awamori, cephalosporin C by Acremonium chrysogenum, erythritol by Moniliella tomentosa var pollinis, and alkaline serine protease by Bacillus licheniformis. Special attention is given to the practical aspects of product development. [source]

Frameshift mutations induced by four isomeric nitroacridines and their des-nitro counterpart in the lacZ reversion assay in Escherichia coli

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2006
George R. Hoffmann
Abstract Acridines are well-known as compounds that intercalate noncovalently between DNA base pairs and induce ±1 frameshift mutations at sites of monotonous repeats of a single base. Reactive derivatives of acridines, including acridine mustards and nitroacridines, form covalent adducts in DNA and exhibit mutagenic properties different from the simple intercalators. We compared the frameshift mutagenicity of the cancer chemotherapy drug nitracrine (1-nitro-9-(3,-dimethylaminopropylamino)-acridine), its des-nitro counterpart 9-(3,-dimethylaminopropylamino)-acridine (DAPA), and its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA) in the lacZ reversion assay in Escherichia coli. DAPA is a simple intercalator, much like the widely studied 9-aminoacridine. It most strongly induced ±1 frameshift mutations in runs of guanine residues and more weakly induced ,1 frameshifts in a run of adenine residues. A nitro group in the 1, 3, or 4 position of DAPA reduced the yield of ±1 frameshift mutations. DAPA weakly induced ,2 frameshifts in an alternating CG sequence. In contrast, nitracrine and its 3-nitro isomer resembled the 3-nitroacridine Entozon in effectively inducing ,2 frameshift mutations. The 2- and 4-nitro isomers were less effective than the 1- and 3-nitro compounds in ,2 frameshift mutagenesis. The results are interpreted with respect to intercalation, steric interactions, effects of base strength on DNA binding, enzymatic processing, and a slipped mispairing model of frameshift mutagenesis. Environ. Mol. Mutagen., 2006. © 2005 Wiley-Liss, Inc. [source]

Ion transport and osmotic adjustment in Escherichia coli in response to ionic and non-ionic osmotica

ENVIRONMENTAL MICROBIOLOGY, Issue 1 2009
Lana Shabala
Summary Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using Escherichia coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above ,1.0 Os kg,1 causes a dose-dependent K+ leak from the cell, resulting in a substantial decrease in cytosolic K+ content and a concurrent accumulation of Na+ in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K+ uptake. Ion flux data are consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K+ uptake to efflux. Microarray experiments reveal that about 40% of upregulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic and non-ionic osmotica. The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K+ transporters for bacterial adaptation to hyperosmotic stress. [source]

Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex method

ENVIRONMENTAL MICROBIOLOGY, Issue 10 2008
David M. Gordon
Summary It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85,90% of E. coli strains can be assigned to a phylo-group and that 80,85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group. [source]

The multicopper oxidase (CueO) and cell aggregation in Escherichia coli

ENVIRONMENTAL MICROBIOLOGY, Issue 8 2007
Jai J. Tree
Summary cueO encodes a periplasmic multicopper oxidase, which is known to be involved in copper homeostasis and protection against oxidative stress in Escherichia coli K12. Transcriptional profiling showed that expression of genes associated with motility was lowered in a cueO mutant while expression of genes associated with autoaggregation was elevated. Increased aggregation was correlated with increased expression of cell surface proteins antigen 43 and curli. Changes in gene expression caused by the deletion of cueO were essentially independent of SoxR and OxyR, the global regulators of oxidative stress response. [source]