Methods for detecting recombinant DNA in the environment

The successful introduction of genetically modified and genetically engineered microorganisms into the environment requires a quantitative evaluation of the survival and dispersion of the microorganisms and specific gene(s) in the environment. The objective of this article is to examine the applicability, suitability, and significance of existing and new methods for detecting and monitoring the recombinant genes or organisms introduced into the environment. Conventional microbiological method(s) involving the selective and differential growth of microorganism(s) adn other quantitative approaches such as the most-probable-number (MPN) method and direct microscopic observation (e.g., acridine orange direct count analysis) have drawbacks and are not specific or universally applicable. Direct enumeration by immunofluorescence by the use of fluorescent dye seems more sensitive although still not perfect. However, the molecular methodologies such as the use of gene probes, plasmid epidemiology, antibiotic resistant marker strains, and protein electrophoresis and bacteriophage sensitivity are receiving more attention. As yet, the technology of DNA:DNA hybridization appears to be very useful, sensitive, and accurate for detecting and monitoring the microorganisms in the environment, although improvements are required. New approaches can be developed which may include biochemical signature compounds as well as gene cassettes to be used in a complementary fashion with conventional and molecular techniques for quantifying specific genotypes and genes in the environment.     

Leaching of Pseudomonas aeruginosa, and transconjugants containing pR68.45 through unsaturated, intact soil columns

Abstract Leaching of genetically engineered microbes (GEMs) through soil is a significant concern related to groundwater quality. The objective of this study was to examine the leaching, survival and gene transfer of a genetically engineered microbe and indigenous recipients of pR68.45 in nonsterile, undisturbed soil columns. Pseudomonas aeruginosa PAO25, containing the plasmid R68.45, was added to the surface of undisturbed soil columns (10 cm diameter × 80 cm length). Unsaturated flow conditions were maintained by 100 ml daily additions of 2 mM CaCl₂ for a period of 70 days. The population of the GEM exhibited a significant ( P = 0.05) linear decline with time. The GEM leached only to a depth of 30–40 cm in 70 days. Transfer of pR68.45 was shown to occur from P. aeruginosa into the indigenous bacterial population although relatively low numbers of transconjugants were observed (log 2 cfu g⁻¹ dry soil). The number of transconjugants also decreased with depth and time. Leaching of transconjugants, however, occured more readily than that of the GEM, probably as a result of plasmid transfer into smaller, more mobile bacteria. At 70 days incubation, no GEMs were detected in the columns, while transconjugants were observed at several depths. These results demonstrate the importance of examining both the survival and movement of GEMs and transconjugants in 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.     

The fate of a genetically modified Pseudomonas strain and its transgene during the composting of poultry manure

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 x 1010 CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 degrees C or above for one or more days. In contrast, in the compost microcosms incubated at 23 degrees C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 degrees C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.     

基因工程微生物的环境监测及生物防御体系研究进展

随着生物技术的发展, 研究人员构建出了大量具有特定功能的基因工程微生物, 这些基因工程微生物在实际应用时常受到限制, 因为它们释放到环境中有可能带来新的污染。为了减少或消除其对环境的潜在危害, 有必要采取措施对这些基因工程微生物进行监测和安全控制。通常要求这类基因工程微生物带有便于监测的检测标记以及能进行自消亡的主动生物防御体系。对基因工程微生物的检测标记以及主动生物防御体系的研究现状进行了综述。     

Field applications of genetically engineered microorganisms for bioremediation processes

Genetically engineered microorganisms (GEMs) have shown potential for bioremediation applications in soil, groundwater, and activated sludge environments, exhibiting enhanced degradative capabilities encompassing a wide range of chemical contaminants. However, the vast majority of studies pertaining to genetically engineered microbial bioremediation are supported by laboratory-based experimental data. In general, relatively few examples of GEM applications in environmental ecosystems exist. Unfortunately, the only manner in which to fully address the competence of GEMs in bioremediation efforts is through long-term field release studies. It is therefore essential that field studies be performed to acquire the requisite information for determining the overall effectiveness and risks associated with GEM introduction into natural ecosystems.     

基因工程微生物的环境监测及生物防御体系研究进展

随着生物技术的发展, 研究人员构建出了大量具有特定功能的基因工程微生物, 这些基因工程微生物在实际应用时常受到限制, 因为它们释放到环境中有可能带来新的污染。为了减少或消除其对环境的潜在危害, 有必要采取措施对这些基因工程微生物进行监测和安全控制。通常要求这类基因工程微生物带有便于监测的检测标记以及能进行自消亡的主动生物防御体系。对基因工程微生物的检测标记以及主动生物防御体系的研究现状进行了综述。     

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)     

同源重组法构建多功能农药降解基因工程菌研究

构建遗传稳定的多功能农药降解基因工程菌可以为农药污染的生物修复提供良好的菌种资源,然而,构建遗传稳定且不带入外源抗性的基因工程菌是一个难点。通过以受体菌的16S rDNA为同源重组指导序列、sacB基因为双交换正筛选标记构建同源重组载体,二亲结合的方法将甲基对硫磷水解酶基因（mpd）整合到呋喃丹降解菌Sphingomonas sp.CDS1染色体的16S rDNA位点,分别成功构建了含1个和2个mpd基因插入到rDNA位点且不带入外源抗性的基因工程菌株CDSmpd和CDS-2mpd。同源重组单交换的效率为3.7×10^－7～6.8×10^－7。通过PCR和Southern杂交的方法验证了同源重组事件。基因工程菌遗传稳定,能同时降解甲基对硫磷和呋喃丹。甲基对硫磷水解酶（MPH）的比活在各生长时期均高于原始出发菌株,比活最高达6.22 mu/μg。     

Conceptualizing "suicidal genetically engineered microorganisms" for bioremediation applications

Use of genetically modified microorganisms (GEMs) for pollution abatement has been limited because of risks associated with their release in the environment. Recent developments in the area of recombinant DNA technologies have paved the way for conceptualizing "suicidal genetically engineered microorganisms" (S-GEMS) to minimize such anticipated hazards and to achieve efficient and safer bioremediation of contaminated sites. Our strategy of designing a novel S-GEM is based on the knowledge of killer-anti-killer gene(s) that would be susceptible to programmed cell death after detoxification of any given contaminated site(s).     

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.     

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)     

A review of available systems to investigate transfer of DNA to indigenous soil bacteria

The deliberate or accidental release of genetically engineered microorganisms (GEMs) in the environment has led to some questions concerning microbial survival, transfer of DNA to the indigenous microflora and environmental consequences. Amongst horizontal gene transfer mechanisms, conjugation is probably the most frequent in the environment. With the aim of evaluating risks associated with environmental release of GEMs and their engineered DNA, studies of conjugative gene transfer between a donor strain and indigenous microflora have been conducted. Such studies required the development of a donor counterselection system to prevent growth of donor cells on transconjugant selective plates. This review summarizes the known and potential donor counterselection systems.     

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.     

Evaluation of methods for detecting ecological effects from genetically engineered microorganisms and microbial pest control agents in terrestrial systems

This report summarizes and evaluates research from several laboratories that deals with the detection of ecological effects induced through exposure of microbes or plants to genetically engineered microorganisms (GEMs) and microbial pest control agents (MPCAs). Some 27 potential endpoints for measuring effects have been studied. Perturbations induced by GEMs have been detected in about one-half of these endpoints. Detectable effects have been recorded for over half of the 16 species of bacteria and fungi studied. The effects caused by GEMs and MPCAs include inhibition of beneficial mycorrhizal fungi growing on Douglas fir seedling roots, depression in plant root and shoot growth, inhibition of predatory soil protozoa, accumulation of a toxic metabolite during biodegradation that inhibits soil fungi, increased microbial community respiration due to rapid lignin breakdown in soil, and the displacement of a broad group of gram-negative bacteria that inhabit the root surface of cereal crops. These effects were usually, but not always, of short duration. However, some of the changes were irreversible during the observation time of days, weeks, or in one case, months.     

A new medium for the enumeration and subculture of bacteria from potable water.

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new medium for the enumeration and subculture of bacteria from potable water

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Detection of genetically engineered traits among bacteria in the environment.

The release of genetically engineered microorganisms (GEMs) into the environment has, as its main aims, the benefits of improved agricultural yield and control of environmental pollution. However, effective and safe release programmes necessitate the development of sensitive, selective detection methods to monitor the environmental impact of released organisms.     

Suicidal genetically engineered microorganisms for bioremediation: need and perspectives

In the past few decades, increased awareness of environmental pollution has led to the exploitation of microbial metabolic potential in the construction of several genetically engineered microorganisms (GEMs) for bioremediation purposes. At the same time, environmental concerns and regulatory constraints have limited the in situ application of GEMs, the ultimate objective behind their development. In order to address the anticipated risks due to the uncontrolled survival/dispersal of GEMs or recombinant plasmids into the environment, some attempts have been made to construct systems that would contain the released organisms. This article discusses the designing of safer genetically engineered organisms for environmental release with specific emphasis on the use of bacterial plasmid addiction systems to limit their survival thus minimizing the anticipated risk. We also conceptualize a novel strategy to construct "Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring/combining the knowledge of different plasmid addiction systems (such as antisense RNA-regulated plasmid addiction, proteic plasmid addiction etc.) and inducible degradative operons of bacteria.