Survival differences among freeze-dried genetically engineered and wild-type bacteria.

Because the death mechanisms of freeze-dried and air-dried bacteria are thought to be similar, freeze-drying was used to investigate the survival differences between potentially airborne genetically engineered microorganisms and their wild types. To this end, engineered strains of Escherichia coli and Pseudomonas syringae were freeze-dried and exposed to air, visible light, or both. The death rates of all engineered strains were significantly higher than those of their parental strains. Light and air exposure were found to increase the death rates of all strains. Application of death rate models to freeze-dried engineered bacteria to be released into the environment is discussed.     

Novel method for monitoring genetically engineered microorganisms in the environment.

A method has been devised for directly detecting and monitoring genetically engineered microorganisms (GEMs) by using in vitro amplification of the target DNAs by a polymerase chain reaction and then hybridizing the DNAs with a specific oligonucleotide or DNA probe. A cloned 0.3-kilobase napier grass (Pennisetum purpureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments was inserted into a derivative of a 2,4-dichlorophenoxyacetic acid-degradative plasmid, pRC10, and transferred into Escherichia coli. This genetically altered microorganism, seeded into filter-sterilized lake and sewage water samples (10(4)/ml), was detected by a plate count method in decreasing numbers for 6 and 10 days of sample incubation, respectively. The new method detected the amplified unique marker (0.3-kilobase DNA) of the GEM even after 10 to 14 days of incubation. This method is highly sensitive (it requires only picogram amounts of DNA) and has an advantage over the plate count technique, which can detect only culturable microorganisms. The method may be useful for monitoring GEMs in complex environments, where discrimination between GEMs and indigenous microorganisms is either difficult or requires time-consuming tests.     

Release of genetically engineered micro-organisms into the environment

Summary Genetically engineered micro-organisms (GEM) at present are the subject of much public attention. They are being considered for biological control, frost protection of plants, and other applications. There is a need to test such organisms before release to the environment. Examples of GEM includePseudomonas fluorescens into which have been cloned -toxin genes ofBacillus thuringiensis, ice-minus bacteria, and other organisms modified by addition, rearrangement and/or deletion of genetic material. Prior to release, the survival, fate, and effects of GEM in the environment must be established. Because organisms, once released, cannot be recalled or always controlled, it is imperative that a full understanding of the risks be known. Predictive ecology must include the new sub-discipline of molecular microbial ecology, if the need for information prior to release of GEM is to be met. One of the most important aspects of deliberate release which must be considered is the ability to detect and monitor GEM in the environment. It has been discovered that micro-organisms can undergo dormancy, i.e. enter a viable but non-recoverable stage in the natural environment. New techniques have been developed, employing immunofluorescent/epifluorescent microscopy, coupled with 5S rRNA sequencing, which allow accurate non-genetic detection of GEM. These techniques have been employed in aquatic systems. Charateristics of GEM important in release to the environment include ability to colonize surfaces, transfer genetic material and persist in specific environments. Clearly, the effects of GEM on the environment cannot be precisely predicted, unless the organisms have been so debilitated that they cannot persist in any natural habitat and cannot exchange genetic material with any other organism. It must be recognized that micro-organisms are extremely diverse and versatile. Uniformly applied, standard regulations governing deliberate release of GEM to the environment cannot be applied in the same way as for regulation of chemicals or medical devices. Case-by-case regulation appears to be the best approach for the immediate future. The implications of each organism, in terms of its own biology, will have to be considered.

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Resumen En el campo de la biotecnología, en rápida expansión, causa preocupación la liberación deliberada al medio ambiente de microorganismos manipulados genéticamente. Se estan llevando a cabo estudios para determinar el destino y el efecto de tales microorganismos en el medio ambiente y especialmente su impacto ambiental. Estos estudios indican que la detección directa de dichos microorganismos, no cultivables pero que permanecen viables en un entorno natural, se puede realizar empleando microscopía de inmunofluorescencia/epifluorescencia junto con secuencias 5S rARN utilizadas como etiquetas, lo que proporciona un sistema eficaz para rastrear estos organismos en muestras del medio ambiente. La utilización de microcosmos y los estudios realizados hasta el momento indican que, efectivamente, los microorganismos sufren una transformación que los mantiene viables aunque no recuperables, en un estado similar a la dormancia, por lo que se requieren métodos de detección directa para controlar la liberación al medio ambiente de microorganismos producidos mediante ingeniería genética. Este fenómeno ocurre también frecuentemente en otros microorganismos liberados al ambiente, entre ellos posibles patógenos. Los estudios realizados sobreVibrio cholerae proporcionan un ejemplo interesante tanto de los métodos empleados como de los resultados obtenidos. Las conclusiones obtenidas indican que el organismo esta ampliamente distribuido en el entorno natural, pero que solo puede ser detectado mediante microscopía directa debido al estado viable, aunque no vultivable, en que se mantiene en el ambiente. Ensayos en animales muentran que se pueden recolectar células deV. cholerae a partir de muestras del medio ambiente y recuperarse como células viables despues de haber sido inyectadas en el íleo de conejos, habiendose demostrado una respuesta ileal positiva para aquellas cepas que son enterotóxicas. Obviamente las ideas actuales acerca de la persistencia y función de los microorganismos en el ambiente necesitaran ser modificadas a medida que se vayan desarrollando nuevos métodos y nuevos conceptos en ecología microbiana.

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Résumé Le développement rapide de la biotechnologie a fait naître des préoccupations concernant le relâchement délibéré dans l'environment de micro-organismes génétiquement manipulés. Des études sont en cours pour déterminer le sort et les effects de ces micro-organismes dans la nature. Les travaux entrepris montrent que la microscopie d'immuno-fluorescence et d'épifluorescence, associée à la détérmination de séquences-marqueurs de r-ARN 5S, permet de détecter des micro-organismes non-cultivables, mais demeurés viables dans les milieux naturels. Les études déjà réalisées sur divers microcosmes indiquent que des micro-organismes peuvent effectivement se trouver dans un état viable, mais non-cultivable, qui ressemble à une dormance, ce qui exige, pour contrôler le relâchement dans la nature d'organismes génétiquement manipulés, l'emploi de méthodes de détection directe. Ce phénomène se produit fréquemment aussi dans le cas de beaucoup d'autres organismes, y compris des pathogènes potentiels, qui sont relâchés dans l'environnement. Des études réalisées avecVibrio cholereae fournissent un exemple de méthodes utiles et de résultats intéressants. Il résulte de ces études queV. cholereae est largement distribué dans l'environnement naturel et ne peut souvent y être détecté que par microscopie directe, à cause de l'état viable, mais non-cultivable, sous lequel il se maintient dans la nature. Les études sur l'animal montrent qu'on peut collecter à partir d'échantillons de milieu naturel des cellules deV. cholereae viables mais noncultivables, après avoir donné, dans le cas des souches entéro-toxigènes, une réponse intestinale positive. Il est clair que les idées courantes sur la persistance et la fonction des micro-organismes dans l'environnement demandent à être modifiées en fonction de l'émergence de nouvelles méthodes et de nouveaux concepts en écologie microbienne.

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Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki: 12–16.8.1985. Session 4     

Use of bioluminescence for detection of genetically engineered microorganisms released into the environment.

The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.     

The release of genetically engineered micro-organisms and viruses into the environment

This review considers the reasons for, and research governing, the regulation and monitoring of genetically engineered micro-organisms and viruses (GEMs) released into the environment. The hazards associated with releasing GEMs into the environment are the creation and evolution of new pests and diseases, and damage to the ecosystem and non target species. The similarities and differences between GEMs and conventional micro-organisms are discussed in relation to risk assessment. Other issues covered include the persistence of micro-organisms in the environment, transgene dispersal to non-engineered microbes and other organisms, the effects of transgenes and transformation on fitness, and the evolution of pests and pathogens that are given or acquire transgenes. Areas requiring further research are identified and recommendations for risk assessment made.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

A method to evaluate survival of genetically engineered bacteria in soil extracts

A technique of potential use to the biotechnology industry was developed for studying the survival of bacteria in aqueous extracts of soil. The aqueous extracts of soil were placed into test tubes, amended as desired, inoculated with bacteria containing recombinant DNA, and incubated. Most bacteria introduced into filter-sterilized soil extracts were capable of multiplying and maintained populations of 10 E6 to 10 E8 cfu/ml over 13 days. However, bacteria introduced into nonsterile soil extracts at 10 E5 cfu/ml were found to decrease by 2–3 logs over a 13-day period. The soil extract method revealed that recombinant DNA plasmids had no significant effect on survival of thePseudomonas spp. andEscherichia coli strains examined. Extracts from soil provide a convenient and homogeneous milieu for estimating relative competitiveness and documenting survival characteristics of genetically engineered microorganisms. The use of aqueous extracts of soil offer convenience, a means of obtaining homogeneous cell suspensions, and ease of experimental replication over the inoculation of bacteria uniformly into soil.     

Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments.

Many methods for detecting model genetically engineered microorganisms (GEMs) in experimental ecosystems rely on cultivation of introduced cells. In this study, survival of Escherichia coli was monitored with the green fluorescent protein (GFP) gene. This approach allowed enumeration of GEMs by both plating and microscopy. Use of the GFP-marked GEMs revealed that E. coli persisted in stream water at higher densities as determined microscopically than as determined by CFU enumeration. The GFP gene did not negatively impact the fitness of the host strain.     

Survival and catabolic activity of natural and genetically engineered bacteria in a laboratory-scale activated-sludge unit.

The survival of selected naturally occurring and genetically engineered bacteria in a fully functional laboratory-scale activated-sludge unit (ASU) was investigated. The effect of the presence of 3-chlorobenzoate (3CB) on the survival of Pseudomonas putida UWC1, with or without a chimeric plasmid, pD10, which encodes 3CB catabolism, was determined. P. putida UWC1(pD10) did not enhance 3CB breakdown in the ASU, even following inoculation at a high concentration (3 x 10(8) CFU/ml). The emergence of a natural, 3CB-degrading population appeared to have a detrimental effect on the survival of strain UWC1 in the ASU. The fate of two 3CB-utilizing bacteria, derived from activated-sludge microflora, was studied in experiments in which these strains were inoculated into the ASU. Both strains, AS2, an unmanipulated natural isolate which flocculated readily in liquid media, and P. putida ASR2.8, a transconjugant containing the recombinant plasmid pD10, survived for long periods in the ASU and enhanced 3CB breakdown at 15 degrees C. The results reported in this paper illustrate the importance of choosing strains which are well adapted to environmental conditions if the use of microbial inoculants for the breakdown of target pollutants is to be successful.     

Interpretation of phenotype in genetically engineered mice.

BACKGROUND AND PURPOSE: In mice, genetic engineering involves two general approaches-addition of an exogenous gene, resulting in transgenic mice, and use of knockout mice, which have a targeted mutation of an endogenous gene. The advantages of these approaches is that questions can be asked about the function of a particular gene in a living mammalian organism, taking into account interactions among cells, tissues, and organs under normal, disease, injury, and stress situations. METHODS: Review of the literature concentrating principally on knockout mice and questions of unexpected phenotypes, lack of phenotype, redundancy, and effect of genetic background on phenotype will be discussed. CONCLUSION: There is little gene redundancy in mammals; knockout phenotypes exist even if none are immediately apparent; and investigating phenotypes in colonies of mixed genetic background may reveal not only more phenotypes, but also may lead to better understanding of the molecular or cellular mechanism underlying the phenotype and to discovery of modifier gene(s).     

Allele-specific activation of genetically engineered receptors.

The binding of agonists and antagonists to the beta-adrenergic receptor (beta AR) is postulated to involve an ionic interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor. To explore the importance of this interaction in the binding of ligands to the beta AR, a Ser residue was substituted for Asp113, and the ability of this mutant receptor to respond to compounds which could potentially interact with the hydroxyl side chain of the Ser residue was assessed. The mutant receptor was fully activated by catechol-containing esters and ketones, compounds which did not activate the wild-type beta AR. The demonstration that the molecular substitution of a single amino acid residue can alter the ligand binding specificity of the beta AR provides evidence that the chemical nature of this residue is a critical determinant in the recognition site of the receptor. Further, the ability to modify the specificity of a receptor by the replacement of amino acids at the binding site demonstrates the potential for the rational design of drugs which function specifically at genetically engineered receptors.     

Advancing ecological risk assessment on genetically engineered breeding stacks with combined insect-resistance traits

Transgenic Research - To inform the ecological risk assessment (ERA) of a transgenic crop with multiple insecticidal traits combined by conventional breeding (breeding stack), a comparative field...     

The release into the environment of genetically engineered viruses, vaccines and viral pesticides

Viruses are being developed by genetic engineering procedures for two purposes: as improved vaccines or vectors for inserting foreign genes into a vaccinated hosts, and as improved viral pesticides. Both uses raise environmental issues.     

A microcosm for measuring survival and conjugation of genetically engineered bacteria in rhizosphere environments

A microcosm is described to evaluate and measure bacterial conjugation in the rhizosphere of barley and radish with strains ofPseudomonas cepacia. The purpose was to describe a standard method useful for evaluating the propensity of genetically engineered microorganisms (GEMs) to transfer DNA to recipient bacteria. Results demonstrated the formation of transconjugants from the rhizosphere of each plant 24 h after inoculation. Transconjugant populations peaked at 1.8 × 10² colony forming units (CFU)/g root and associated soil in barley and 2.0×10² CFU/g root and associated soil in radish; they then declined over the next five days of the experiment. No significant differences were found in the survival of transconjugant populations monitored from the two plant species. The microcosm was also used to document the formation of false positive transconjugants, which resulted from donor and recipientP. cepacia mating on the surface of selective agar plates instead of in microcosms. Transconjugants resulting from such plate mating occurred in substantial numbers during the first 5 days of the experiment but declined to undetectable numbers by day 7. The use of nalidixic acid was investigated to determine the magnitude of plate mating. The number of transconjugants detected from radish rhizosphere was reduced by two orders of magnitude by including nalidixic acid in the plating medium; this indicated that 99% of the transconjugants were a result of plate mating.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

Survival and catabolic activity of natural and genetically engineered bacteria in a laboratory-scale activated-sludge unit

The survival of selected naturally occurring and genetically engineered bacteria in a fully functional laboratory-scale activated-sludge unit (ASU) was investigated. The effect of the presence of 3-chlorobenzoate (3CB) on the survival of Pseudomonas putida UWC1, with or without a chimeric plasmid, pD10, which encodes 3CB catabolism, was determined. P. putida UWC1(pD10) did not enhance 3CB breakdown in the ASU, even following inoculation at a high concentration (3 x 10(8) CFU/ml). The emergence of a natural, 3CB-degrading population appeared to have a detrimental effect on the survival of strain UWC1 in the ASU. The fate of two 3CB-utilizing bacteria, derived from activated-sludge microflora, was studied in experiments in which these strains were inoculated into the ASU. Both strains, AS2, an unmanipulated natural isolate which flocculated readily in liquid media, and P. putida ASR2.8, a transconjugant containing the recombinant plasmid pD10, survived for long periods in the ASU and enhanced 3CB breakdown at 15 degrees C. The results reported in this paper illustrate the importance of choosing strains which are well adapted to environmental conditions if the use of microbial inoculants for the breakdown of target pollutants is to be successful.