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At least 631 records · Page 35Linked to original sources
PubMed2002

Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates.

This paper deals with the microbial production of polyhydroxyalkanoates (PHAs), biodegradable thermoplastics which perform excellently as a material, from inexpensive renewable carbon sources. To date, with the help of genetic engineering techniques, it has become possible to design several types of PHAs with different compositions and to enhance the productivities of PHAs. In addition, molecular breeding of PHA biosynthesis enzymes has been demonstrated to improve polymer production. Mutant PHA synthases generated by an in vitro evolution technique have allowed the enhanced production and quality alteration of PHAs. Furthermore, use of inexpensive renewable carbon sources, such as plant oils, waste materials, and carbon dioxide, would be a key for a reduction in PHA production cost.

Journal Article↗
PubMed2005

Chemoenzymatic synthesis of cryptophycin/arenastatin natural products.

Microbially derived modular polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways are a rich source of novel natural products. Development of these systems for the engineered biosynthesis of diverse secondary metabolites continues to progress as a robust source of chemical diversity. Recent efforts that employ individual enzymes and catalytic domains for the production or modification of small molecules have met with growing success. In this study, the thioesterase domain from the cryptophycin biosynthetic pathway was isolated and its function evaluated with a series of linear chain elongation intermediates in developing a novel chemoenzymatic synthesis of the cryptophycin/arenastatin class of antitumor agents. The results show the high efficiency of the thioesterase in generating the 16-membered depsipeptide ring of this important natural product system. Moreover, analysis of selected substrates revealed considerable tolerance for structural variation within the seco-cryptophycin unit C beta-alanine residue, but strict structural requirements at the phenyl group position of the unit A delta-hydroxy octadienoate chain elongation intermediates.

Amino Acid Sequence↗
PubMed1994

The roles of magnesium in biotechnology.

This review highlights the important roles played by magnesium in the growth and metabolic functions of microbial and animal cells, and therefore assigns a key role for magnesium ions in biotechnology. The fundamental biochemical and physiological actions of magnesium as a regulatory cation are outlined. Such actions are deemed to be relevant in an applied sense, because Mg2+ availability in cell culture and fermentation media can dramatically influence growth and metabolism of cells. Manipulation of extracellular and intracellular magnesium ions can thus be envisaged as a relatively simplistic, but nevertheless versatile, means of physiological cell engineering. In addition, biological antagonism between calcium and magnesium at the molecular level may have profound consequences for the optimization of biotechnological processes that exploit cells. In fermentation, for example, it is argued that the efficiency of microbial conversion of substrate to product may be improved by altering Mg:Ca concentration ratios in industrial feedstocks in a way that makes more magnesium available to the cells. With particular respect to yeast-based biotechnologies, magnesium availability is seen as being crucially important in governing central pathways of carbohydrate catabolism, especially ethanolic fermentation. It is proposed that such influences of magnesium ions are expressed at the combined levels of key enzyme activation and cell membrane stabilization. The former ensures optimum flow of substrate to ethanol and the latter acts to protect yeasts from physical and chemical stress.

Animals↗
PubMed2003

Rapid selection of phage-resistant mutants in Streptococcus thermophilus by immunoselection and cell sorting.

Immunoselection and flow cytometry allowed the isolation from Streptococcus thermophilus strain Str31 of double mutants displaying resistance to the phage phi31 and good acid production. Strain Str31 is very sensitive to phage phi31. This phage-host system seemed therefore particularly suitable to test the validity of the selection method adopted in this study. Mutants were stable with respect to both characters. The isolation of the double mutants required 4 to 5 days. The approach does not involve genetic manipulations and can therefore be an alternative to genetic engineering when this technology cannot be applied.

Adsorption↗
PubMed1990

Luminescence-based nonextractive technique for in situ detection of Escherichia coli in soil.

Measurement of light output by luminometry was used to estimate quantitatively the cell concentrations of luminescent strains of Escherichia coli in liquid culture and inoculated into soil. Strains were constructed in which luciferase production was autoinducible or constitutive. In the former, light output per cell varied considerably during growth but was constant in constitutive strains. In liquid culture, the lower detection limit was in the order of 10(2) cells ml-1. Sensitivity was reduced by approximately 1 order of magnitude for cells inoculated into soil, when 2 x 10(2) to 6 x 10(3) cells g of soil-1 could be detected. Light output measurements were obtained within 5 min of sampling, and luminometry therefore potentially offers a rapid and sensitive detection technique for genetically engineered microorganisms.

Bacteriological Techniques↗
PubMed1992

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Benzoates↗
PubMed1965

Procedures necessary for the prevention of planetary contamination.

Studies of microbial survival in simulated deep-space conditions have established that these conditions will not sterilize contaminated spacecraft. Likewise, data presented at this and previous meetings of COSPAR have shown that many terrestrial microbes, particularly anaerobic sporeformers, readily tolerate simulated Martian environments. Any spacecraft landing on Mars must have a low probability of harboring any terrestrial organisms, especially sporeformers, if the goals of exobiology are not to be compromised. Before the planning, production and monitoring of sterile spacecraft can be undertaken, the theory and principles of sterilization and their application to the special problem of spacecraft sterilization must be understood. The response of a given population of microorganisms to sterilizing agents is affected by several factors; these factors in turn regulate the kinetics of the killing process. The only processes that achieve both surface and interior sterilization are heat and radiation. Of these, dry heat is the agent of choice. If all parts of the spacecraft are assembled under the most rigorous conditions of cleanliness, the capsule can be brought to terminal sterilization containing a total of not more than 10(5) viable organisms. These can be killed by exposure to 135 degrees for 24 hours with a confidence of greater than 10(4). Spacecraft must be specifically designed to withstand such heat sterilization without reduction of reliability. The number of labile parts must be kept to a minimum, sterilized by methods other than heat, and inserted into the sterilized spacecraft by sterile techniques. The entire process must be under very tight control with tests and records of every detail. The sterilized spacecraft must be encapsulated and remain therein during final testing and launch until the capsule is opened in deep space. The design and production engineer must understand how all these procedures will affect the spacecraft, and the biologist must constantly monitor and educate the engineers on the scope and limitations of these various sterilizing procedures.

Colony Count, Microbial↗
PubMed2005

Direct determination of p-nitrophenyl substituent organophosphorus nerve agents using a recombinant Pseudomonas putida JS444-modified Clark oxygen electrode.

A microbial biosensor for rapid, sensitive, selective, and cost-effective determination of the total content of organophosphorus nerve agents with p-nitrophenyl substituent is reported. The biosensor consisted of genetically engineered PNP-degrader Pseudomonas putida JS444 expressing organophosphorus hydrolase (OPH) on its cell surface immobilized on a dissolved oxygen electrode. Surface-expressed OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, methyl parathion, and parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen. The oxygen consumption was measured and correlated to the concentration of organophosphates. The sensor signal and response time were optimized with 0.086 mg dry weight of cell and operating in 50 mM pH 7.5 citrate-phosphate buffer with 50 microM CoCl(2) at room temperature. When operated at optimized conditions, the biosensor measured as low as 55 ppb of paraoxon, 53 ppb of methyl parathion, and 58 ppb of parathion without interference from most phenolic compounds and other commonly used pesticides, such as atrazine, coumaphos, sutan, sevin, and diazinon. The operational life of the microbial biosensor was approximately 5 days when stored in the operating buffer at 4 degrees C.

Aryldialkylphosphatase↗
PubMed1999

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.

Amino Acid Motifs↗
PubMed1993

Monitoring of hospital water supplies for Legionella.

In order to determine the value of regular surveillance for Legionella in the prevention of hospital-acquired (nosocomial) legionellosis, water samples were obtained over a three-year period from 17 hospitals located in England and Scotland. Prior to the study, all of the hospitals had in operation defined protocols and maintenance schedules which followed national guidelines for the prevention of legionellosis in health care premises. Six samples, from key locations in the water system of each hospital, were taken at six-monthly intervals. Total viable bacterial count (TVC), coliform count and legionella cultures were performed on all the samples. No coliforms were detected in any of the samples, whereas the TVC was variable. Legionella pneumophila was isolated from both the hot and cold water supplies of two hospitals. The TVC was not related to the isolation of Legionella. Confirmation of the presence of Legionella was subsequently attributed to defects in the equipment and water maintenance programmes. It was concluded that the microbiological examination of water is an effective approach to the audit of the maintenance of hospital water systems in order to prevent legionellosis.

Colony Count, Microbial↗
PubMed2002

Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system.

An increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. To facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. We made two large pools of independent transposon mutants in Escherichia coli using modified Tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated loxP recombination, we obtained E. coli strains in which large genomic fragments (59-117 kilobases) were deleted. Some of these individual deletions were then combined into a single "cumulative deletion strain" that lacked 287 open reading frames (313.1 kilobases) but that nevertheless exhibited normal growth under standard laboratory conditions.

Bacteriophage P1↗
PubMed2001

Clinical research results with dl1520 (Onyx-015), a replication-selective adenovirus for the treatment of cancer: what have we learned?

Replication-selective microbial agents hold promise as a novel cancer treatment platform. dl1520 (Onyx-015), an E1B-55 kD gene-deleted adenovirus, was the first such genetically engineered agent to be tested in humans. Over 200 cancer patients have been treated to date on over 10 clinical trials (phases I-III). The virus was generally well-tolerated at doses of up to 2 x 10(12) particles by intratumoral, intraperitoneal, hepatic arterial and intravenous administration; no maximally tolerated doses were identified by any route of administration. Viral replication was tumor-selective, and was documented after administration by all routes; replication was generally transient (<10 days), however, and was variable depending on tumor histology. single agent efficacy has been limited to date (0-14% local tumor regression rates). in combination with chemotherapy, however, encouraging antitumoral activity has been demonstrated. these clinical research results demonstrate the potential of this novel treatment platform, as well as the hurdles to be overcome. novel replication-selective agents with improved potency are needed.

Adenoviridae↗
PubMed2001

Bacteriological validation of a new apparatus for disinfection of hospital waste at the point of disposal.

OBJECTIVES: To evaluate the ability of a new apparatus (Dipsys 25, Société SGN, Bagnols sur Cèze, France) to disinfect biomedical waste, including both potentially infectious agents and the normal saprophytic flora of the waste. METHODS: Disinfection was assessed using standard methods (reference strains were fixed on reference carriers according to the French AFNOR methods) and nonstandard assays. Assays in conditions of hospital use, evaluations of bacterial survival during storage, sporicidal effect, and spore survival during storage were performed in parallel. Finally, bactericidal effect in extreme conditions (association of high contamination and high bacterial protection conditions) was tested with normal fecal flora. Bacterial counts were performed after treatment by the apparatus and without treatment (controls). All tests were carried out in triplicate. RESULTS: In all treated carriers, a bacterial population decrease of at least 5 log10 was obtained. Assays performed in hospital-use conditions did not show any bacterial growth. Concerning the evaluation of sporicidal effect and spore revival during conservation, a minimum reduction of 5 log10 was observed in all assays performed, without survival. Finally, concerning assays in extreme conditions, the decrease of bacterial population was between 5 log10 and 10 log10 for vegetative anaerobes of normal fecal flora. CONCLUSION: Under our study conditions, the study apparatus reduced the tested microbial populations by a minimal factor of 5 log10. The main advantage of the apparatus is the opportunity to treat contaminated waste inside hospital wards, at the point of initial collection, without pulverization, by nonspecialized staff.

Bacteria, Aerobic↗
PubMed2006

Efficient production of L-lactic acid from xylose by Pichia stipitis.

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to approximately 15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.

Biotechnology↗
PubMed1994

Eubacteria show their true colors: genetics of carotenoid pigment biosynthesis from microbes to plants.

The opportunities to understand eubacterial carotenoid biosynthesis and apply the lessons learned in this field to eukaryotes have improved dramatically in the last several years. On the other hand, many questions remain. Although the pigments illustrated in Fig. 2 represent only a small fraction of the carotenoids found in nature, the characterization of eubacterial genes required for their biosynthesis has not yet been completed. Identifying those eukaryotic carotenoid biosynthetic mutants, genes, and enzymes that have no eubacterial counterparts will also prove essential for a full description of the biochemical pathways (81). Eubacterial crt gene regulation has not been studied in detail, with the notable exceptions of M. xanthus and R. capsulatus (5, 33, 39, 45, 46, 84). Determination of the rate-limiting reaction(s) in carotenoid biosynthesis has thus far yielded species-specific results (12, 27, 47, 69), and the mechanisms of many of the biochemical conversions remain obscure. Predicted characteristics of some carotenoid biosynthesis gene products await confirmation by studying the purified proteins. Despite these challenges, (over)expression of eubacterial or eukaryotic carotenoid genes in heterologous hosts has already created exciting possibilities for the directed manipulation of carotenoid levels and content. Such efforts could, for example, enhance the nutritional value of crop plants or yield microbial production of novel and desirable pigments. In the future, the functional compatibility of enzymes from different organisms will form a central theme in the genetic engineering of carotenoid pigment biosynthetic pathways.

Bacteria↗
PubMed1997

A simple method for the eradication of Legionella pneumophila from potable water systems.

In this paper we describe a simple method, noncorrosive to pipes, for the eradication of Legionella pneumophila from potable water systems. This method is based on the systematic purging of the pipe networks with cold water containing 1-1.5 mg residual chlorine/L. In the hot water system, a new pipe bypassing the water heater was installed, whereas in the air conditioning system, the circuit is purged with water from the tap water system. The feasibility of this method was studied in two hotels in which the presence of Legionella was detected despite treatment of the water by the hyperchlorination method. The evolution of the presence of Legionella was studied by culture and polymerase chain reaction. Eighty samples from hotel A and sixty-seven samples from hotel B were analyzed during the time that the eradication method was applied. Our results showed that this method permitted the effective elimination of L. pneumophila after 5 months in hotel A and 7 months in hotel B.

Chlorine↗