Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.     

Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis

A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10⁴Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences.     

遗传工程微生物细胞间发生的自然遗传转化

将两株具有不同遗传标记的枯草芽孢杆菌在基本培养基中分别培养至对数生长后期后进行短时间混合静置培养,经选择平板筛选、DNaseI敏感性试验、质粒检测和产蛋白酶活性检测,发现两菌株之间可通过自然遗传转化进行染色体DNA和质粒DNA的交换。研究结果表明,自然遗传转化可在细胞间进行,这对揭示微生物群居的自然环境中可能存在的细胞间的DNA转移,以及正确评估遗传工程微生物(GEMs)的安全使用具有重要意义。 Abstract：The culture fluids of two genetically distinct Bacillus subtilis strains were mixed and coincubated for a short time after they reached post-exponentially growth phase in minimal media.The steadily bidirectional gene transfer involving chromosomal DNA and plasmid DNA by natural genetic transformation between these two strains has been demonstrated by the methods of selective medium screening,DNaseI sensitivity test,plasmid detection and the detection of the capability of producing protease.This result indicates that natural genetic transformation occurs not only between“naked”DNA and cells but also between cells.This conclusion is significant in the assessment of both the possibility of intercelluar DNA transfer in natural habitats of microorganisms and the risk of the application of genetically engineered microorganisms (GEMs).     

遗传工程微生物细胞间发生的自然遗传转化

将两株具有不同遗传标记的枯草芽孢杆菌在基本培养基中分别培养至对数生长后期后进行短时间混合静置培养 ,经选择平板筛选、DNaseI敏感性试验、质粒检测和产蛋白酶活性检测 ,发现两菌株之间可通过自然遗传转化进行染色体DNA和质粒DNA的交换。研究结果表明 ,自然遗传转化可在细胞间进行 ,这对揭示微生物群居的自然环境中可能存在的细胞间的DNA转移 ,以及正确评估遗传工程微生物(GEMs)的安全使用具有重要意义。     

基因工程菌在生物降解中的应用及其发展

在简要阐述基因工程菌构建方法的基础上,系统综述和评价了基因工程构建方法的研究进展及其在生物降解中存在的问题。     

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。     

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。     

Intact Soil-Core Microcosms for Evaluating the Fate and Ecological Impact of the Release of Genetically Engineered Microorganisms

Intact soil-core microcosms were studied to determine their applicability for evaluating the transport, survival, and potential ecosystem effects of genetically engineered microorganisms before they are released into the environment. Soil-core microcosms were planted with wheat and maize seeds and inoculated with Azospirillum lipoferum SpBr17 and SpRG20a Tn5 mutants, respectively. Microcosm leachate, rhizosphere soil, plant endorhizosphere, insects, and xylem exudate were sampled for A. lipoferum Tn5 mutant populations. A. lipoferum Tn5 populations, determined by most-probable-number technique-DNA hybridization, varied from below detection to 10⁶ g of dry root⁻¹ in the rhizosphere, with smaller populations detected in the endorhizosphere. Intact soil-core microcosms were found to maintain some of the complexities of the natural ecosystem and should be particularly useful for initial evaluations of the fate of plant-associated genetically engineered bacteria.     

Coexistence of Genetically Engineered Escherichia coliStrains and Natural Microorganisms in Experimental Aquatic Microcosms

In experimental aquatic microcosms (AMCs), the population of the Escherichia colistrain Z905 harboring the recombinant plasmid pPHL7 (Ap^rLux⁺) was found to gradually accumulate AMC-adapted cells, which retained the plasmid but differed from the original cells in some biochemical and physiological characteristics. Both the original and the AMC-adaptedE. colicells could coexist with the native AMC microflora for one year or longer. When introduced into AMCs together with native pseudomonads, the AMC-adapted E. coliZ905-33 (pPHL7) cells were more competitive than the nonadapted cells.     

Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.     

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.     

Photoquenching of the Bioluminescence of the Genetically Engineered Escherichia coli TG1 (pXen7) Strain in the Presence of Photodithazine

The photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain was studied in the presence of the photosensitizer photodithazine, a glucosamine salt of chlorin e ₆. The photosensitized quenching of the bioluminescence was found to correlate with the colony-forming ability of the strain. The data obtained are discussed from the standpoint of using biosensor luminescent bacterial systems for the assessment of the efficiency of photosensitizers in antimicrobial photochemotherapy.     

Bioluminescence Activity and Degradation Kinetics of Genetically Engineered Strain P. putida mt-2 RB 1401 in Soil System with m-Xylene

Strain RB1401 contains a plasmid with a fusion between the upper pathway promoter of the TOL plasmid xyl genes and a lux gene cassette. Bioluminescence activity appeared in the early period of biodegradation of m-xylene, and was observed at all tested conditions of temperature (15, 27, 35°C) and pH (5, 7, 9), showing in the range of 300 ～ 6100 RLU. Biokinetics of strain RB1401 in the presence of m-xylene were as follows: Y 0.56 mg/mg, μ_max 2.67 day^-1, and K_s 19.25 mg/L. A ratio of C:N(KNO₃):P(KH₂PO₄) = 100:8:1.5 and 0.01% (w/v) of H₂O₂ were chosen for optimum biostimulation conditions of RB1401 with m-xylene in soil system. These results demonstrate the capability of such a genetically engineered bacteria for the development of biomonitoring and bioremediation in soil environments contaminated with specific volatile organic compounds.     

基因工程抗体融合蛋白的构建

抗体融合蛋白可以具有抗体的特性和所融合的功能蛋白的活性,可广泛用于免疫治疗、免疫诊断、抗体纯化及抗体和抗原的分析定量等,特别可用于免疫导向药物的制备。基因工程抗体融合蛋白比传统的化学交联的抗体融合蛋白具有更多的优越性。本文就基因工程抗体融合蛋白的构建和性质做一综述 。     

制备遗传工程小鼠的技术改进

遗传工程小鼠是当今生命科学领域集成度最高的研究体系之一。特别在“人类基因组计划和小鼠基因组计划”完成后,遗传工程小鼠在制备人类疾病模型、药物开发和评价、基因功能分析以及比较基因组学中发挥着越来越重要的作用。由此,也推动了遗传工程小鼠相关技术的快速发展。就遗传工程小鼠制备的现况、存在的问题以及新策略等相关问题进行了总结。     

Genetically Engineered Animal Models of Alzheimer's Disease

The application of transgenic research has proven to be a powerful and popular tool for investigating the contribution of specific genes known or suspected to be involved in the pathology of Alzheimer's disease. Many different experimental approaches have been pursued in an effort to mimic one or more of the numerous and diverse features characterizing Alzheimer's disease. Results have been variable but not without successes. Some of the cardinal hallmarks of this disorder have been recapitulated through the manipulation of a single gene, providing information on the interrelationship between several pathological events. Also, through the generation of such transgenic animals, potential models are being established for this disease that will be valuable for development of intervention strategies.     

转基因植物的生态风险

转基因植物已在很多国家大规模商业化种植,并且取得了显著的经济效益。同时有关转基因植物潜在的生态风险已引起广泛的关注。本文从转基因植物人侵危害、对非靶标有益生物直接和间接的影响、害虫对抗虫转基因植物产生抗性、抗病毒转基因植物带来的潜在风险等方面论述了转基因植物可能潜在的生态安全性问题。