Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.     

Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment.

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.     

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.     

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments.

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.     

U.S. regulatory framework for genetic biocontrol of invasive fish

This paper provides an overview of the U.S. regulatory framework governing genetic biocontrol efforts for invasive fish. Genetic biocontrol refers to the intentional release of genetically modified organisms (GMOs) into the environment to control a target population of a non-native species. The terms “genetically modified” and “genetically engineered” are often used interchangeably, despite the scientific distinctions. A GMO is an organism that has had its genetic material altered or modified by humans through any method, including conventional breeding. Genetic engineering, as defined by the Food and Drug Administration (FDA), is the use of recombinant DNA techniques to introduce new characteristics or traits into an organism. GE organisms are therefore a subset of GMOs. As this paper will discuss, existing laws focus on GE organisms raising significant questions as to whether organisms modified without utilizing rDNA techniques fall within the jurisdiction of any federal agency. Under the 1986 Coordinated Framework for Regulation of Biotechnology, three federal agencies have primary responsibility over biotechnology—the Environmental Protection Agency (EPA), the U.S. Department of Agriculture, and the FDA. Because the EPA has exempted biological control agents from regulation as pesticides and no fish species are currently considered plant pests, the FDA is the agency responsible for approving the use of genetically engineered fish for biocontrol. FDA regulates genetically engineered animals through its New Animal Drug Application (NADA) process. The NADA process presents several challenges to effective and transparent regulation of genetic biocontrol, including the FDA’s focus on drug safety, secrecy provisions potentially limiting disclosure of the results of environmental reviews, and the secondary role of the Fish and Wildlife Service, the federal agency with the most experience with invasive species management. In addition, relying on the NADA process creates a significant regulatory gap as NADA approval is only required for GE organisms. The regulatory framework for GMOs created for genetic biocontrol without rDNA technology is unclear and primary responsibility may fall to the states. Given its extensive experience with hatcheries, invasive fish species control, and environmental reviews, the Fish and Wildlife Service (FWS) is the more appropriate agency to review applications for genetic biocontrol. Efforts should be undertaken now, while genetic biocontrol is still in the theoretical stages, to increase the role of the FWS in the permitting process either through formal regulations or more informal mechanisms such as memorandum of understanding.     

Evolution in Lego ®: A Physical Simulation of Adaptation by Natural Selection

We describe a physical simulation of natural selection in a population of legorgs, six-segment model organisms. Legorg morphology is genetically specified by five alleles on each segment. Legorgs show a simple form of motility that could evolve in originally sessile animals. This motility, the ability to move horizontally on a smooth surface, depends on the morphology and interaction of the six segments that produce different patterns of locomotion. Legorgs are selected for motility and reproduce in proportion to fitness. After just five generations, the average population motility increases 2.5 times. Additionally, we describe a slightly less time-consuming simulation of legorg evolution, where fitness is assigned by comparison with a template. The calculation of gene pools is precisely the same as in the previous simulation and produces very robust increases in fitness during five generations. The simulation is designed as a classroom experiment to explore the mechanism of natural selection. A test of its learning efficiency by evaluating the students’ conception of central aspects of evolutionary theory before and after showed a significant improvement. The surprising power of natural selection in this very simple physical system may also be exploited in more advanced experiments.     

Production of isoprenoid pharmaceuticals by engineered microbes

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.     

Production of microbial chitinases – A revisit

Production of microbial chitinases have received increased attention in recent years due to its potential application in various fields. There has been an all around effort to increase the production of chitinases by using genetically engineered organisms and by incorporating modern fermentation techniques. This review presents a brief outline on the production of microbial chitinases which includes information on random screening and selection of chitinolytic organisms and the rationale behind the use of genetically engineered organisms. The problems and perspectives involved in large scale production of chitinases is discussed with special reference to bioreactor studies and new fermentation methods. The present limitations in the understanding of the chitinase fermentation process is also discussed and the scope for future investigation is outlined in this communication.     

Regulation of genome edited technologies in India

In India, genetically modified organisms and products thereof are regulated under the “Rules for the manufacture, use, import, export and storage of hazardous microorganisms, genetically engineered organisms or cells, 1989” (referred to as Rules, 1989) notified under the Environment (Protection) Act, 1986. These Rules are implemented by the Ministry of Environment, Forest and Climate Change, Department of Biotechnology and State Governments though six competent authorities. The Rules, 1989 are supported by series of guidelines on contained research, biologics, confined field trials, food safety assessment, environmental risk assessment etc. The definition of genetic engineering in the Rules, 1989 implies that new genome engineering technologies including gene editing technologies like CRISPR/Cas9 and gene drives may be covered under the rules. The regulatory authorities if required, may also review the experiences of other countries in dealing with such new and emerging technologies.

     

Model to predict aerial dispersal of bacteria during environmental release

Risk assessment for genetically engineered bacteria sprayed onto crops includes determination of off-site dispersal and deposition. The ability to predict microbial dispersal patterns is essential to characterize the uncertainty (risk) associated with environmental release of recombinant organisms. Toward this end, a particle dispersal model was developed to predict recovery of bacteria on fallout plates at various distances and directions about a test site. The microcomputer simulation incorporates particle size distribution, wind speed and direction, turbulence, evaporation, sedimentation, and mortality, with a time step of 0.5 s. The model was tested against data reported from three field applications of nonrecombinant bacteria and two applications of recombinant bacteria. Simulated dispersal of 10(5) particles was compared with reported deposition measurements. The model may be useful in defining appropriate populations of organisms for release, methods of release or application, characteristics of a release site that influence containment or dispersal, and in developing an appropriate sampling methodology for monitoring the dispersal of organisms such as genetically engineered bacteria.     

Model to predict aerial dispersal of bacteria during environmental release.

Risk assessment for genetically engineered bacteria sprayed onto crops includes determination of off-site dispersal and deposition. The ability to predict microbial dispersal patterns is essential to characterize the uncertainty (risk) associated with environmental release of recombinant organisms. Toward this end, a particle dispersal model was developed to predict recovery of bacteria on fallout plates at various distances and directions about a test site. The microcomputer simulation incorporates particle size distribution, wind speed and direction, turbulence, evaporation, sedimentation, and mortality, with a time step of 0.5 s. The model was tested against data reported from three field applications of nonrecombinant bacteria and two applications of recombinant bacteria. Simulated dispersal of 10(5) particles was compared with reported deposition measurements. The model may be useful in defining appropriate populations of organisms for release, methods of release or application, characteristics of a release site that influence containment or dispersal, and in developing an appropriate sampling methodology for monitoring the dispersal of organisms such as genetically engineered bacteria.     

Not all GMOs are crop plants: non-plant GMO applications in agriculture

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.     

A public goods approach to major evolutionary innovations

The history of life is marked by a small number of major transitions, whether viewed from a genetic, ecological, or geological perspective. Specialists from various disciplines have focused on the packaging of information to generate new evolutionary individuals, on the expansion of ecological opportunity, or the abiotic drivers of environmental change to which organisms respond as the major drivers of these episodes. But the critical issue for understanding these major evolutionary transitions (METs) lies in the interactions between environmental, ecologic, and genetic change. Here, I propose that public goods may serve as one currency of such interactions: biological products that are non‐excludable and non‐rivalrous. Such biological public goods may be involved in either the generation of new evolutionary variation, as with genetic sequences that are easily transferred between different microbial lineages, or in the construction of new ecological niches, as with the progressive oxygenation of the oceans and atmosphere. Attention to public goods emphasizes the processes by which organisms actively construct their own evolutionary opportunities. Such public goods may have facilitated some METs.     

Genes in new environments: genetics and evolution in biological control

The availability of new genetic technologies has positioned the field of biological control as a test bed for theories in evolutionary biology and for understanding practical aspects of the release of genetically manipulated material. Purposeful introductions of pathogens, parasites, predators and herbivores, when considered as replicated semi-natural field experiments, show the unpredictable nature of biological colonization. The characteristics of organisms and their environments that determine this variation in the establishment and success of biological control can now be explored using genetic tools. Lessons from studies of classical biological control can help inform researchers and policy makers about the risks that are associated with the release of genetically modified organisms, particularly with respect to long-term evolutionary changes.     

Site-specific selfish genes as tools for the control and genetic engineering of natural populations

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.     

Environments that induce synthetic microbial ecosystems

Interactions between microbial species are sometimes mediated by the exchange of small molecules, secreted by one species and metabolized by another. Both one-way (commensal) and two-way (mutualistic) interactions may contribute to complex networks of interdependencies. Understanding these interactions constitutes an open challenge in microbial ecology, with applications ranging from the human microbiome to environmental sustainability. In parallel to natural communities, it is possible to explore interactions in artificial microbial ecosystems, e.g. pairs of genetically engineered mutualistic strains. Here we computationally generate artificial microbial ecosystems without re-engineering the microbes themselves, but rather by predicting their growth on appropriately designed media. We use genome-scale stoichiometric models of metabolism to identify media that can sustain growth for a pair of species, but fail to do so for one or both individual species, thereby inducing putative symbiotic interactions. We first tested our approach on two previously studied mutualistic pairs, and on a pair of highly curated model organisms, showing that our algorithms successfully recapitulate known interactions, robustly predict new ones, and provide novel insight on exchanged molecules. We then applied our method to all possible pairs of seven microbial species, and found that it is always possible to identify putative media that induce commensalism or mutualism. Our analysis also suggests that symbiotic interactions may arise more readily through environmental fluctuations than genetic modifications. We envision that our approach will help generate microbe-microbe interaction maps useful for understanding microbial consortia dynamics and evolution, and for exploring the full potential of natural metabolic pathways for metabolic engineering applications.     

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。     

Ecological aspects of the evolutionary processes

Darwin in his On the Origin of species made it clear that evolutionary change depends on the combined action of two different causes, the first being the origin of genetically based phenotypic variation in the individual organisms comprising the population and the second being the action of selective agents of the external environment placing demands on the individual organisms. For over a century following Darwin, most evolutionists focused on the origin of inherited variation and its transmission; many workers continue to regard genetics to be the core of evolutionary theory. Far less attention has been given to the exact nature of the selective agents with most evolutionists still treating this cause imprecisely to the detriment of our understanding of both nomological and historical evolutionary theory. Darwin was vague in the meaning of his new concept of "Natural Selection," using it interchangeably as one of the causes for evolutionary change and as the final outcome (= evolutionary change). In 1930, natural selection was defined clearly as "non-random, differential reproduction of genes" by R. Fisher and J.B.S. Haldane which is a statement of the outcome of evolutionary process and which omits mention of the causes bringing about this change. Evolutionists quickly accepted this outcome definition of natural selection, and have used interchangeably selection both as a cause and as the result of evolutionary change, causing great confusion. Herein, the details will be discussed of how the external environment (i.e., the environment-phenotype interaction) serves as selective agents and exerts demands on the phenotypic organisms. Included are the concepts of fitness and of the components of fitness (= adaptations) which are respectively (a) survival, (b) direct reproductive and (c) indirect reproductive features. Finally, it will be argued that historical-narrative analyses of organisms, including classification and phylogenetic history, are possible only with a full understanding of nomological evolutionary theory and with functional/adaptive studies of the employed taxonomic features in addition to the standard comparative investigations.