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At least 919 records · Page 51Linked to original sources
PubMed1992

Production of recombinant human interferon-alpha 1 by Escherichia coli using a computer-controlled cultivation process.

Genetically engineered E. coli K12 BMH-71-18 with plasmid PBV-867 was used for constitutive expression of human interferon-alpha 1 (IFN) with a defined medium. A manual, time-based, fed-batch cultivation process produced a cell density of 26.3 g l-1 (OD550 89), an IFN activity of 1.55 x 10(8) IU l-1 and a specific IFN productivity of 0.65 x 10(6) IU g-1. An analysis was conducted to characterize the problems involved in the high density microbial processes of recombinant protein production. The strategy suggested by the analysis is to establish a nutrient feeding profile that improves both the plasmid stability and the overall productivity of IFN. The nutrient feeding procedure developed here was based on the growth dynamics and a glucose consumption model. By using this procedure to continuously supply nutrients during cultivations, cell density reached 58 to 80 g l-1 and the specific IFN productivities of these runs were increased over that of the manual process. Nutrient feeding rates were found to affect the specific IFN productivity substantially. The optimized process achieved an IFN activity of 1.26 x 10(9) IU l-1, a cell density of 58 g l-1 and a specific IFN productivity of 2.2 x 10(7) IU g-1. More significantly, the overall productivity IU l-1 h-1 of the optimized, computer-controlled cultivation process was increased 12.9-fold over that of the manual cultivation process.

Cloning, Molecular↗
PubMed1993

Use of DNA probes and plasmid capture in a search for new interesting environmental genes.

Adaptation to a stressed environment leads to organisms bearing DNA, encoding defense mechanisms. These mechanisms can be heavy metal resistance, catabolism of organic xenobiotics or stress reactions. Genes responsible for these mechanisms can be used for monitoring changing environments and therefore it can be important to store such bacteria in a bank. DNA-probing will be presented by the use of DNA fragments (of Alcaligenes eutrophus) coding for heavy metal resistance or xenobiotic degradation. Some strains do not grow on petri dishes and accordingly cannot be isolated from soils. In order to isolate plasmids from such strains, coding for heavy metal resistances or xenobiotic degradations, an exogenous plasmid isolation method was developed. In this method, the endogenous population is conjugated with Pseudomonas or Alcaligenes strains bearing a retrotransfer plasmid like RP4. In that way new plasmids from various sources including non-culturable strains could be obtained. With these methods, a large number of specimens adapted to stressed situations can be isolated or constructed (in the case of the exogenous plasmid isolation method). They form a source of interesting genetic material that can be used to restore polluted areas in natural areas, if necessary with the aid of genetic engineering (in vitro or in vivo techniques). Full knowledge of such bacteria and their resistance mechanisms or degradation pathways, can lead to new constructions able to attack recalcitrant mixtures of different organics and to resist heavy metals.

Alcaligenes↗
PubMed2002

Antiinfective and encrustation-inhibiting materials--myth and facts.

Catheters, urethral and ureteral stents and other urological implants are frequently affected by encrustration and infection due to their permanent contact with urine. Indwelling urinary catheters provide a haven for microorganisms and thus require extensive monitoring. Several surface modification techniques have been proposed to improve the performance of devices including the immobilization of biomolecules, the incorporation of hydrophilic grafts to reduce protein adsorption, the creation of hydrophobic surfaces, the creation of microdomains to regulate cellular and protein adhesion, new polymers and antimicrobial coatings. Physico-chemical explanation to elucidate the mechanism of such encrustation or infection inhibiting materials is still not available. Our series of experiments showed a marked decrease of silver-activity in biological fluids which corresponds with the controversial clinical results obtained with silver coated urinary catheters. Rifampicin/minocycline coated catheters had very low activity against Gram-negative rods, enterococci and Candida spp., the main causing organisms of urinary catheter infection. Surface engineered materials and antimicrobial drug delivery systems will be the next generation of sophisticated urinary catheters and stents, if both efficacy as well as efficiency has been proved clinically.

Anti-Bacterial Agents↗
PubMed1994

[Susceptibility to antimicrobial drugs of strains of bacilli used as a basis for various probiotics].

Five strains of the genus Bacillus used as components of biological preparations i.e. bacterin SL, biosporin, bactisubtil and subalin were tested for their susceptibility to 38 antimicrobial agents, The strains were simultaneously resistant to 5 antibiotics: astreonam, colistin, penicillin, ceftizoxime and cefuroxime. High susceptibility of the strains to some penicillins, cephalosporins and aminoglycosides was recorded. The gene engineered strain B. subtilis 2335(105) marked by the kanamycin resistance gene showed cross resistance to amikacin and tobramycin. Trimetoprim, sulfotrimetoprim and norfloxacin proved to be active against the strains.

Anti-Bacterial Agents↗
PubMed2003

The threonine story.

L-Threonine is an essential amino acid which has recently been brought into agricultural industry for balancing the livestock feed. L-Threonine is produced by microbial synthesis using glucose or sucrose as substrates. For the process to be cost-effective, the microbial strain must be capable of threonine overproduction. This paper reviews the biochemical pathways of L-threonine synthesis in bacteria and the regulation of these pathways, the principles and the techniques of constructing high-producing strains, and the most efficient strains thus developed.

Alcohols↗
PubMed2006

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe.

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4+/-0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.

Anaerobiosis↗
PubMed1992

Emerging strategies for enhancing crop resistance to microbial pathogens.

There are marked differences in the pattern of host gene expression in incompatible plant:microbial pathogen interactions compared with compatible interactions, associated with the elaboration of inducible defenses. Constitutive expression of genes encoding a chitinase or a ribosome-inactivating protein in transgenic plants confers partial protection against fungal attack, and a large repertoire of such antimicrobial genes has been identified for further manipulation. In addition, strategies are emerging for the manipulation of multigenic defenses such as lignin deposition and synthesis of phytoalexin antibiotics by overexpression of genes encoding rate determining steps, modification of transcription factors or other regulatory genes, and engineering production of novel phytoalexins by interspecies transfer of biosynthetic genes. The imminent cloning of disease resistance genes, further molecular dissection of stress signal perception and transduction mechanisms, and identification of genes that affect symptom development will provide attractive new opportunities for enhancing crop protection. Combinatorial integration of these novel strategies into ongoing breeding programs should make an important contribution to effective, durable field resistance.

Amino Acid Sequence↗
PubMed1986

Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains.

The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (5----10 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced overproduction of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.

Biodegradation, Environmental↗
PubMed2003

Fungal biotechnology.

Fungi are used in many industrial processes, such as the production of enzymes, vitamins, polysaccharides, polyhydric alcohols, pigments, lipids, and glycolipids. Some of these products are produced commercially while others are potentially valuable in biotechnology. Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. In addition to the multiple reaction sequences of fermentations, fungi are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine-chemical industry in the production of single-isomer intermediates. Recombinant DNA technology, which includes yeasts and other fungi as hosts, has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as a means of increasing titers and yields of microbial processes and in the discovery of new drugs. Today, fungal biology is a major participant in global industry. Moreover, the best is yet to come as genomes of additional species are sequenced at some level (cDNA, complete genomes, expressed sequence tags) and gene and protein arrays become available.

Biotechnology↗
PubMed1998

Engineering protein-based machines to emulate key steps of metabolism (biological energy conversion)

Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill. This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions. Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity. Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed2003

Detection and quantitative estimation of Dehalococcoides spp. in a dechlorinating bioreactor by a combination of fluorescent in situ hybridisation (FISH) and kinetic analysis.

The unique capacity of Dehalococcoides ethenogenes of completely dechlorinating the common groundwater pollutant tetrachloroethene (PCE) to the harmless ethene makes this microorganism very attractive for application in natural or engineered bioremediation systems. In this study, the qualitative and quantitative determination of Dehalococcoides spp. in a lab-scale bioreactor was performed based on the combination of fluorescent in situ hybridisation (FISH) for specific detection, and kinetic batch tests at non-limiting hydrogen and PCE concentration for quantitative determination. The dechlorinating bioreactor was operated at a high and constant PCE loading rate of 255 micromol PCE [g volatile suspended solids (VSS)](-1) day(-1). Pale coccoid cells resembling the distinctive morphotype of D. ethenogenes were present in the microbial culture. These cocci hybridised with both eubacterial probes and the Dhe1259t probe recently designed for detecting Dehalococcoides spp. Positive hybridisation was also observed when the DHC1377 reverse primer was used as a specific probe and applied to the dechlorinating microbial consortium. The maximum dechlorination rate obtained under non-limiting hydrogen and PCE concentrations was 3.22 +/- 0.08 mmol Cl(-) l(-1 )day(-1). From the specific activity of D. ethenogenes [i.e. 0.055 +/- 0.008 mmol Cl(-) (mg VSS)(-1) day(-1)], as reported from pure culture study, this observed maximum rate corresponded to a concentration of this bacterium in the mixed liquor of the bioreactor of 59.0+/-10.4 mg VSS.l(-1) (41.5+/-11.2% of overall VSS). This calculated relative abundance of D. ethenogenes was in agreement with the percentage of methanol (in terms of reducing equivalents) channeled to reductive dechlorination (approximately 30%) supporting the assumption that most reductive dechlorination was actually due to this microorganism.

Biodegradation, Environmental↗
PubMed2002

Viral binding proteins as antibody surrogates in immunoassays of cytokines.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative approach explored here is the use of pathogen-encoded cytokine-binding proteins, which have Kd in the nanomolar range. It is anticipated that pathogens have evolved binding proteins, antagonists, and/or specific neutralizing phenotypes directed against key signaling and effector molecules involved in the multifaceted host defense system. Thus, by screening the genomes of a wide range of microbial agents, we would expect to find coding sequences for binding proteins for the most important cytokines. Consistent with this view is the identification of poxvirus genes encoding binding activities for TNF type I and type II interferons, interleukin (IL)-1beta, IL-18, and beta-chemokines. These high-affinity receptors have the potential to act as surrogate antibodies in a number of applications in cytokine quantification and purification and could be potentially useful reagents to complement the existing panel of anti-cytokine, monoclonal, polyclonal, or engineered antibodies that are currently available.

Antibodies↗
PubMed1993

Construction and detection of bioluminescent strains of Bacillus subtilis.

Bioluminescence (lux) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.

Bacillus subtilis↗
PubMed1991

Molecular biology: new tools for studying microbial ecology.

The general aim of studies in microbial ecology is to determine the activities of particular groups of organisms in the natural environment. Current developments in molecular biology are providing methods that can be adopted for investigations in microbial ecology. Presently, molecular techniques are being used to determine population and community structures in samples. Following on from this, methods are needed to determine the activities of these organisms in their natural state. This review describes the application of molecular techniques to the study of micro-organisms in environmental samples.

Bacteria↗
PubMed1971

Changes in the microflora of man during long-term confinement.

Extended space missions may change the human normal microflora, including the intestinal flora. The bacterial composition of the intestinal microflora is an important factor in natural resistance to infection. During the year-long medico-engineering experiment human intestinal microflora was thoroughly studied. It was necessary to collect more detailed information concerning the composition in healthy people. 83 microbiological tests of faeces of 27 healthy male subjects were carried out. It was found that spore-less anaerobic bacteria predominated in the fecal microflora, reaching 90% of total micro-organisms. Aerobic micro-organisms were less than 6% of total micro-organisms. Long-term enclosure of men was shown to induce changes in the intestinal microflora which involved a sharp reduction of different microbial groups, and occasionally complete elimination of certain representatives of the intestinal flora. The composition tended to be simplified. A study of the isolated cultures of Cl. perfringens demonstrated an increased amount of strains with toxicogenic properties. Staphylococci were isolated from the test subjects during their long-term enclosure. The pharynx and nose of the three test subjects were examined and 1445 staphylococcal strains were isolated, of which 745 strains (51.5%) were coagulase producers. In 745 strains phage-type, coagulase, toxicity, hemolysins, lecitinase, fibrinolysin and DNA-ase were determined. Phage-typing of the isolated staphylococci revealed a microbial exchange between the men. Staphylococcal strains of the same phage-type (29/52) isolated during the experiment showed an increase of the toxin titre (from 1:40-1:80 to 1:640-1:1280) and development of additional pathogenic properties that were not found previously. An increase in beta-hemolysin and fibrinolysin production and an acceleration of DNA-ase activity. Experiments on mice demonstrated increased virulence of the staphylococcal strains isolated by the end of the experiment. The data are discussed from the point of view of the immunological responses of the human body.

Aerospace Medicine↗
PubMed2000

Anhydrobiotic engineering of gram-negative bacteria.

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.

Biotechnology↗
PubMed1995

Flux analysis of microbial metabolic pathways using a visual programming environment.

This paper describes the use of a visual programming environment (LabVIEW) for the flux analysis of metabolic pathways. Representations of metabolic pathways are constructed in software from individual reaction elements (icons) which are linked together to indicate potential flux routes. Off-line bioprocess data are then used to supply the inputs and outputs to the metabolic pathway and the pathway fluxes are calculated. The metabolic system can be modelled at different levels of complexity and new pathways can be inserted into existing models. To illustrate this, flux analyses are performed on three Escherichia coli mutants with metabolic pathway deletions and insertions. The first analysis looks at organic acid production and the second at the effect of the presence in E. coli of an engineered pathway for toluene degradation.

Acetates↗
PubMed2004

A DNA vaccine producing LcrV antigen in oligomers is effective in protecting mice from lethal mucosal challenge of plague.

There is an urgent need to develop effective vaccines against pneumonic plague, a highly lethal and contagious disease caused by the Gram-negative bacterium Yersinia pestis. Here we demonstrate that a novel DNA vaccine expressing a modified V antigen (LcrV) of Y. pestis, with a human tissue plasminogen activator (tPA) signal sequence, elicited strong V-specific antibody responses in BALB/c mice. This tPA-V DNA vaccine protected mice from intranasal challenge with lethal doses of Y. pestis. In comparison, a DNA vaccine expressing the wild type V antigen was much less effective. Only tPA-V formed oligomers spontaneously, and elicited a higher IgG2a anti-V antibody response in immunized mice, suggesting increased TH1 type cellular immune response. Our data indicate that antigen engineering is effective in inducing high quality protective immune responses against conformationally sensitive antigens. These results support that optimized DNA vaccines have the potential to protect against bacterial pathogens than is generally recognized.

Animals↗