Genetically Engineered Microorganisms Research Articles

Development of Detection Methods for Monitoring of Genetically Engineered Microorganisms Released into Environment

Application of genetically engineered microorganisms (GEMs) to solve many kinds of environmental pollution problems such as removing environmental hazardous contaminants has been studied. Before releasing GEMs into environment, the behavior of these newly developed microorganisms must be checked sufficiently. The fundamental checking points for GEMs are their survival and propagative possibilities in the environment and their capability of transferring or mobilizing their genetically engineered genes. In this study, the authors investigated and developed highly sensitive methodology for monitoring the GEMs' genes, to establish the effective analytical methods for the GEMs' behavior in water pollution control systems. DNA polymerase chain reaction (PCR) method was adopted to amplify the detecting sensitivity against target genes of GEMs. Nucleic acids hybridization technology is usually used to detect specific genes or DNA segments. In this study, the combined methodology of PCR amplification of target DNA and detection by nucleic acid hybridization with specific DNA probes was developed. The other important factor of the detection is recovering target genes or DNAs from environmental samples. Therefore, we also established the recovering methods of GEMs' nucleic acids from water or soil to which GEMs were released.

Persistence of a xylE marker gene in Pseudomonas putida introduced into soils of differing texture

Summary: The growth and survival of two genetically engineered micro-organisms (GEMs), Pseudomonas putida strain PaW8 containing either marker plasmid pLV1013 (IncQ) or pLV1016 (IncP), both of which contain the xylE gene, were investigated in soils ranging in texture from a sandy loam to a peat. Viable GEMs were enumerated over a period of 89 d. Analysis of the decay kinetics indicated that GEM survival, as monitored by the persistence of the xylE gene, was biphasic in the sandy and clay loams. Both organisms showed evidence of initial growth in the peat soil prior to a rapid death phase. The results indicated that soil texture has an effect on the survival of genetically engineered P. putida, death decreasing with increasing clay content.

Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms.

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-Döbler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.

Evaluation of aquatic sediment microcosms and their use in assessing possible effects of introduced microorganisms on ecosystem parameters.

In this paper we describe a sediment microcosm system consisting of 20 undisturbed, layered sediment cores with overlying site water which are incubated under identical conditions of temperature, light, stirring rate of overlying water, and water exchange rate. Ecosystem parameters (nutrient level, photosynthetic potential, community structure of heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients) of the laboratory microcosms and the source ecosystem were compared and shown to be indistinguishable for the first 2 weeks. In weeks 3 and 4, small differences were detectable in the nutrient level, community structure of heterotrophic bacteria, and thymidine incorporation rate. However, the photosynthetic potential, depth profiles of heterotrophic bacterial community structure, and oxygen microgradients were maintained throughout the incubation period and did not differ between laboratory microcosms and the source ecosystem. The microcosm system described here would thus appear to be a valid model of aquatic sediments for up to 4 weeks; the actual period would depend on the sediment source and incubation temperature. The validated systems were used with Rhine river sediment to assess possible effects on ecosystem parameters of Pseudomonas sp. strain B13 FR1(pFRC20P), a genetically engineered microorganism (GEM) that had been constructed to degrade mixtures of halo- and alkylbenzoates and -phenols. The GEM survived in the surface sediment at densities of 5 x 10(4) to 5 x 10(5)/g (dry weight) for 4 weeks and degraded added chloro- and methylaromatics. The GEM did not measurably influence ecosystem parameters such as photosynthesis, densities of selected heterotrophic bacteria, thymidine incorporation rate, and oxygen microgradients. Thus, the microcosm system described here would seem to be useful for the study of the ecology of biodegradation and the fate and effect of microorganisms introduced into the environment.

A DNA probe suitable for monitoring genetically engineered microorganisms in the environment.

A bacterial haloacetate dehalogenase (H-1 type enzyme, EC 3.8.1.3) was proved to be one of the useful probes for detecting a genetically engineered microorganism (GEM) from environmental samples. A mono-fluoroacetate assimilating bacterium (Pseudomonas sp.), characteristically encoding H-1 gene on its plasmid pUO12, was applied to two sorts of intact paddy soil and was detected by colony hybridization probing 32P-labeled H-1 DNA fragment (1.6kb). Target bacteria could be quantitatively counted by autoradiography with the samples of no less than 10-3-fold soil dilution while using nutrient agar plates for colonization. Thus, the detection limit of the target bacteria was regarded as 400 CFU (colony-forming unit) per gram of dry weight of soil introducing the principle of most-probable-number method. When the same experiment was carried out using minimal agar containing monofluoroacetate as a sole source of carbon, target colonies were clearly detected even in the 10-1 soil dilution and thus, the detection limit was improved to as low as 4 CFU per gram of dry weight of soil. These redults suggested that H-1 gene probe was very valuable for tracing a GEM in the environment because of its unique nucleotide sequence and high sensitivity.

Field calibration of soil-core microcosms: Fate of a genetically altered rhizobacterium

Microcosms containing intact soil-cores are a potential tool for assessing the risks of the release of genetically engineered microorganisms (GEMs) to the environment. Before microcosms become a standard assessment tool, however, they must first be calibrated to ensure that they adequately simulate key parameters in the field. Four systems were compared: intact soil-core microcosms located in the laboratory at ambient temperature and in a growth chamber with temperature fluctuations that simulated average conditions in the field, field lysimeters, and field plots. These four systems were inoculated with rifampicin-resistantPseudomonas sp. and planted to winter wheat. Populations of thePseudomonas sp. in soil decreased more rapidly at ambient temperature, but population size at the three-leaf stage of wheat growth was the same in all four systems. Populations of thePseudomonas sp. on the rhizoplane of wheat were the same at the three-leaf stage in all four systems, and colonization with depth at the final boot stage-sampling was also similar. In general, microcosms incubated at ambient temperature in the laboratory or in the growth chamber were similar to those in the field with respect to survival of and colonization of the rhizoplane by the introducedPseudomonas sp.

GENE TRANSFER BETWEEN DISTANTLY RELATED BACTERIA

'[;:��:��:� i ��.::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: : Transduction . EVIDENCE FOR GENE EXCHANGE BETWEEN DIFFERENT SPECIES . Phenotypic Evidence for Gene Exchange ..... . Experimental Demonstrations of [ntergeneric Gene Transfers . . Effective Conjugation between Distantly-Related Microbes . Naturally occurring Plasmids and their Tran�fer . Retrotransfer . IMPLICATIONS OF GENE TRANSFER . The Role of Gene Transfer in Speciation . . Gene Transfer in Nature and the Question of Risk Assessment during the Release of Genetically-Engineered Microorganisms (GEM) . Industrial Applications . CONCLUSION . Facilitating Transfer between Microbial Species in the Laboratory

Effects of Genetically Engineered Microorganisms on Nitrogen Transformations and Nitrogen-Transforming Microbial Populations in Soil

The principal concern about releasing genetically engineered microorganisms (GEMs) into the environment is their potential adverse effects on the environment, whether caused directly or indirectly by the GEMs. The effects of five GEMs on ammonification, nitrification, and denitrification in soil were studied. With the possible exception of a strain of Enterobacter cloacae carrying a plasmid, no consistent statistically or ecologically significant differences in effects on these processes or on the population dynamics of the microorganisms responsible for the processes were observed between soils inoculated with the GEMs or their homologous plasmidless hosts and those that were not inoculated. Increasing the concentration of montmorillonite in the soil enhanced the rate of nitrification, regardless of the inoculum, indicating that the perfusion technique used was sensitive enough to detect changes in nitrification rates when they occurred.

Monitoring a genetically engineered bacterium in a freshwater environment by rapid enzymatic amplification of a synthetic DNA "number-plate".

In order to set up a sensitive and reliable detection method to monitor environmentally released genetically engineered microorganisms (GEMs) a 72-bp, double-stranded DNA fragment has been built by annealing and ligating four synthetic oligonucleotides. Binding sites for two 20-mer oligonucleotides are situated inside the DNA fragment, flanking the centre. Into the central part of the construction a 30-nucleotide identification sequence has been fitted. Thanks to the presence of the two oligonucleotide binding sites, the synthetic construction ("number-plate") can be submitted to enzymatic amplification using the polymerase chain reaction (PCR), thus enabling the identification system to take advantage of the outstanding sensitivity of this technique. When released into a freshwater microcosm, cells of Pseudomonas putida carrying a "number-plated" chromosome could be easily and rapidly detected merely by submitting boiled cell sediments to PCR amplification.

Risk assessment in genetic engineering

This book is a practical guide to assessing the risks involved in introducing genetically-engineered microorganisms and plants into the environment. Five methodologies are presented for integrating the diverse data essential for accurate risk assessment. The social and economic impacts of biotechnology and related ethical issues are presented as essential components of risk-benefit assessment.

Monitoring survival and gene transfer in soil microcosms of recombinant Escherichia coli designed to represent an industrial production strain.

A genetically engineered microorganism (GEM) was designed to exemplify bacterial strains used for the production of biological material in industry. The recombinant DNA was located on a safety plasmid (pUC19). Survival and persistence of the GEM and its recombinant DNA (rDNA) was determined in soil microcosms by using different monitoring methods, including the polymerase-chain reaction, to amplify and detect the specific rDNA. Depending on nutritional status, both the GEM and its rDNA had disappeared within 16 (amended soil) or 28 days (non-amended soil) with a limit of detection of 5 cells/g soil and 20 fg DNA/g soil.

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.

A selective plating method to enumerate target microorganisms in an environment.

A sensitive method using selective plating media to detect a genetically engineered microorganism (GEM) released into the environment was developed. In experiments, a strain of Pseudomonas sp. capable of assimilating monofluoroacetate (FA) as a sole carbon source was chosen as the GEM model. Two sorts of Japanese paddy soil were used as a microcosm. In a trial when serial 10-fold soil dilutions were spread on minimal FA agar plates and incubated, native oligotrophic microorganisms (105-106CFU/g of dry weight of soil) grew on the plates. So, it was difficult to pick out a few populations of target bacteria from such a high number of background microbial communities. A further examination showed that those indigenous microorganisms colonized even on plates containing only water and agar. For this reason, we tested silica-gel instead of agar as an agent to solidify the selective media. On minimal FA silica-gel plates, the number of indigenous oligotrophic colonies was greatly decreased. When a definite number of target Pseudomonas cells was mixed with the soil dilutions and cultured on minimal FA silica-gel plates, only the target colonies were appeared. We applied the most-probable-number technique to the plate-counting method to attain as low as 4CFU/g of dry weight of soil as the statistical limit of detecting FA-assimilating bacteria. This selective plating method is suitable for environmental monitoring of GEMs which assimilate FA aerobically, as it is on highly sensitive, specific and feasible.

Construction of tracer plasmids for Bacillus sphaericus 1593 utilizing the xylE gene from Pseudomonas putida

Genetically engineered microorganisms (GEMS) released into the environment must be traceable in order to accurately assess their impact on the area of release. Tracer genes other than those that introduce antibiotic resistance are preferred for use in identifying genetically engineered strains. In this study, we describe the construction of a series of tracer plasmids for use in Bacillus sphaericus using the xylE gene from the Pseudomonas putida TOL plasmid. This gene codes for the enzyme catechol 2,3-dioxygenase which converts the colorless substance catechol to 2-hydroxymuconic semialdehyde, a yellow product which is easily detected. Colonies of cells which express the xylE gene turn yellow shortly after being exposed with a solution of catechol.

The release of genetically-engineered microorganisms. London: Academic Press Limited, 1988. 306 pp., £ 15.95 ISBN 0-12-677521-4 (c), ISBN 0-12-677522-2 (p/b)

Acta BiotechnologicaVolume 11, Issue 1 p. 56-56 Book Review The release of genetically-engineered microorganisms. London: Academic Press Limited, 1988. 306 pp., £ 15.95 ISBN 0-12-677521-4 (c), ISBN 0-12-677522-2 (p/b) P. Dobrowolski, P. DobrowolskiSearch for more papers by this author P. Dobrowolski, P. DobrowolskiSearch for more papers by this author First published: 1991 https://doi.org/10.1002/abio.370110116AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume11, Issue11991Pages 56-56 RelatedInformation

Persistence of genetically engineeredErwinia carotovora in perturbed and unperturbed aquatic microcosms and effect on recovery of indigenous bacteria

Genetically engineeredErwinia carotovora persisted significantly longer in thermally perturbed microcosms (35 days) than in nonstressed microcosms (5 days). Decreased pressure of competitors and predators and increased nutrient availability were examined as the most probable reasons for greater vulnerability of perturbed microcosms to colonization by genetically engineered microorganisms (GEMs). Indigenous bacteria that competed with GEMs for the same nutrient sources (protein, cellulose, pectate) were present immediately after perturbation in densities one to two orders of magnitude lower than in unperturbed microcosms, but their populations increased to densities significantly higher than in unperturbed microscosms 10 to 15 days after inoculation. Predators of bacteria (protozoans, cladocerans, nematodes, and rotifers) were present during the experiment in unperturbed microcosms, while dense populations of bacteriovorous nanoflagellates developed in perturbed microcosms. Preemptive inoculation of perturbed microcosms with GEMs did not have a longlasting effect on the recovery of total, proteolytic, cellulolytic, and pectolytic bacteria in perturbed microscosms, indicating the absence of competitive exclusion.

Accurate and sensitive method of enumeration of a specific microorganism,Pseudomonus paucimobilisSS86, in nonsterile soil

Abstract Selective, sensitive, and accurate counting of a specific microorganism in nonsterile soil is a prerequisite for the study of auteeology of microorganisms, including genetically engineered microorganisms (GEMs) introduced into soil. Pseudomonas paucimobilis SS86, which has the rare ability to decompose γ-HCH (γ-BHC, Lindane) rapidly and completely, is considered to be a useful microorganism for such investigations. The P. paucimobilis SS86 bacteria in nonsterile soil could be counted selectively and with a high sensitivity (100-107 cells/g soil) by using a simple and low cost MPN method. In addition, the population level of P. paucimobilis SS86 in nonsterile soil could be selectively controlled by the addition of an appropriate amount of y-HCH. These results indicated that a detailed autecological study of P. paucimobilis SS86 was possible and that the information obtained from the autecological study could be useful for the environmental release of GEMs.

Small-scale field test of the genetically engineered lacZY marker

Commercial genetic engineering is advancing into areas that require the small-scale introduction of genetically engineered microorganisms (GEMs) to better quantify variables that affect microorganism distribution and survival and to document potential long-term consequences. A recombinant DNA marker system, the lacZY marker, developed by the Monsanto Agricultural Co., enables the distribution and fate of marked fluorescent pseudomonad organisms to be monitored under actual field conditions. Critical evaluation of GEMs under field conditions is imperative if plant-beneficial effects are to be correlated with organism release. This paper evaluates the effectiveness of this marker system and its ability to facilitate the assessment of risks associated with deliberate environmental introductions of genetically engineered microorganisms. Results of prerelease contained growth chamber and field experiments demonstrated that: (1) the scientific risk assessment methodology adopted by Monsanto and approved by the U.S. Environmental Protection Agency was appropriate and comprehensive; (2) the deliberate introduction of a GEM did not pose unaccepatable or unforeseen risks to human health or the environment; (3) the lacZY marker is an effective environmental tracking tool; and (4) regulatory oversight should reflect the expected risk and not be excessively burdensome for all GEMs.

Evaluation of intact soil-core microcosms for determining potential impacts on nutrient dynamics by genetically engineered microorganisms

Nutrient export from intact soil-core microcosms in leachate or by plant uptake was evaluated as a means to assess the ecosystem impacts from the environmental release of genetically modified root-colonizing bacteria. Intact cores of two soil types, a Burbank sandy loam and a Palouse silt loam, were excavated for use as microcosms, seeded with corn and wheat, respectively, and inoculated with Azospirillum lipoferum transposon Tn5 mutants. Microcosms were leached at 33 d and 67 d after seeding and the leachate analyzed for sulfate, phosphate, ammonium, nitrite, nitrate, dissolved organic carbon and inorganic carbon. Statistically significant differences in leachate nutrient concentrations between treatments were due to differences in soil types, rather than inoculation with the bacteria. The high variation in the concentrations of nutrients in the leachates from replicate microcosms, attributed to real field variation, suggest that the measurement of nutrient export in leachate from microcosms may not be a sensitive indicator of ecosystem impacts. One exception was dissolved organic carbon (and total carbon). A statistically significant difference (p ≥ 0.05) was observed between the microcosms receiving live inoculum and those receiving heat-killed cells for leachate organic carbon concentration. Plant uptake of N, P, S, B, Cu, Fe, Mg, Mn and Zn was not affected by inoculation but the coefficients of variation were considerably lower than for the leachate analyses. It is suggested that, although the leaching of organic carbon from microcosms may be used as a holistic indicator of ecosystem impacts from the release of genetically engineered microorganisms (GEMs), measurements of plant assimilation of nutrients and specific rates of microbial transformation of nutrients may provide more sensitive effects end points.

Plasmid mobilization from genetically engineered bacteria to members of the indigenous soil microflora in situ

For study of gene transfer and expression in a large variety of soil bacteria in situ, a gene-promoter cassette was constructed and inserted into an expression vector that allowed expression and maintenance in a wide host range. The hybrid replicon was transformed into a mobilizer strain that transferred its DNA to other Gram-negative bacteria at very high rates. This genetically engineered microorganism (GEM) was introduced into nonsterile soil microcosms. Plasmid transfer from the introduced GEM to members of the native soil flora was observed in situ.