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.     

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.     

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.     

Key issues in the deliberate release of genetically-manipulated bacteria

Abstract The deliberate release of a genetically engineered bacterium often requires that a complex pathway be travelled through scientific and regulatory questions. It is important to consider the scientific aim of the release and the nature of the modification (deletion or insertion, site of insertion, level of expression) and its likely effect on survival of the organism and the possibility of gene transfer. In Australia, the Genetic Manipulation Advisory Committee assesses applications and makes recommendations about pre-release testing and procedures for conducting field release. Two examples of field release of genetically manipulated bacteria in Australia are considered. Firstly, the commercial product Agrobacterium strain K1026 (‘NoGall’^TM), a genetically engineered biological control agent for crown gall disease of stone fruits and roses. Secondly, a lacZY -marked derivative of a strain of Pseudomonas corrugata , that can act as a biological control agent against take-all disease of wheat. Prior to release, bacterial survival and competition was tested in soil microcosms. The distribution and survival of the organism were monitored after field release. Since 1992 the marked bacteria have been recovered only after enrichment. Assessment of risk should consider the survival and spread of the genetically manipulated bacterium and its foreign DNA and the impact of the inoculated bacteria on other (‘non-target’) organisms.     

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.     

Dispersal of Engineered Male Aedes aegypti Mosquitoes

Background

Aedes aegypti, the principal vector of dengue fever, have been genetically engineered for use in a sterile insect control programme. To improve our understanding of the dispersal ecology of mosquitoes and to inform appropriate release strategies of ‘genetically sterile’ male Aedes aegypti detailed knowledge of the dispersal ability of the released insects is needed.

Methodology/Principal Findings

The dispersal ability of released ‘genetically sterile’ male Aedes aegypti at a field site in Brazil has been estimated. Dispersal kernels embedded within a generalized linear model framework were used to analyse data collected from three large scale mark release recapture studies. The methodology has been applied to previously published dispersal data to compare the dispersal ability of ‘genetically sterile’ male Aedes aegypti in contrasting environments. We parameterised dispersal kernels and estimated the mean distance travelled for insects in Brazil: 52.8m (95% CI: 49.9m, 56.8m) and Malaysia: 58.0m (95% CI: 51.1m, 71.0m).

Conclusions/Significance

Our results provide specific, detailed estimates of the dispersal characteristics of released ‘genetically sterile’ male Aedes aegypti in the field. The comparative analysis indicates that despite differing environments and recapture rates, key features of the insects’ dispersal kernels are conserved across the two studies. The results can be used to inform both risk assessments and release programmes using ‘genetically sterile’ male Aedes aegypti.     

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.     

The 'biodrug' concept: an innovative approach to therapy

Cell engineering technology using recombinant microorganisms has created new opportunities in the development of innovative drugs. This article presents the use of living genetically engineered microorganisms, such as bacteria or yeasts, as a new delivery vehicle to the gastrointestinal tract. This 'biodrug' concept was demonstrated using recombinant Saccharomyces cerevisiae expressing the plant cytochrome P450 73A1. This enzyme provides a relevant model for potential therapeutic applications, such as 'biodetoxication' in the digestive environment. An artificial gastrointestinal tract simulating human digestion was chosen as a powerful tool to validate the biodrug concept. This approach offers a novel strategy for drug discovery and testing.     

Timing of propagule release significantly alters the deposition area of resulting aerial dispersal

Aim The aim of this study is to determine whether changes to the seasonal and circadian timing of propagule release can a have a significant effect on the area covered by resulting aerial dispersal. Location Western Australia. Methods Using the atmospheric pollution model (TAPM), an existing meso‐scale dispersal model, a range of release patterns was simulated and the resulting deposition compared. Comparisons were based on observations of deposition patterns and the calculated area of deposition. Results Small changes to the timing of propagule release were shown to significantly impact on the area experiencing deposition from the resulting aerial dispersal. Main conclusions Simulations performed in this study show that, for small propagules, changes to the timing of release can lead to alternate, clearly differentiable dispersal events. Small changes in both the seasonal and circadian patterns of release can have significant effects on the area that experiences deposition during the resulting dispersal event. This effect is particularly important at the landscape scale and when there is a need to quantify individual dispersal events. Predictive modelling of aerial dispersal needs to be undertaken with an understanding of the manner in which biological and environmental factors that affect the timing of propagule release can influence results. Results presented highlight the need to characterize the epidemiology of pathogenic organisms of importance to biosecurity as much as possible before they arrive.     

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system.

Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.     

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system

Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.     

Risk assessment in environmental biotechnology.

Scientists in academia and industry concur that appropriate oversight and regulation for biotechnology are in the best interests of society. Field trials have not resulted in any uncontrolled hazard. Oversight should continue and useful methods for assessing risk associated with release of genetically engineered organisms to the environment have been proposed.     

Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains

The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis.     

Genetically modified filamentous phage as bactericidal agents: a pilot study

AIMS: To evaluate the ability of a filamentous phage encoding lethal proteins to kill bacteria without host-cell lysis. METHODS AND RESULTS: Bacterial survival was determined after infection of a growing Escherichia coli culture with phage M13 encoding either the restriction endonuclease BglII gene or modified phage lambda S holin genes. The genetically engineered phage exerted a high killing efficiency while leaving the cells structurally intact. When compared with a lytic phage, the release of endotoxin was minimized after infection with the genetically modified phages. CONCLUSIONS: Genetically engineered phage can be used for efficient killing, concomitantly minimizing endotoxin release. SIGNIFICANCE AND IMPACT OF THE STUDY: This feasibility study provides a possible strategy for the use of genetically engineered phage as bactericidal agents by optimizing the advantages and minimizing potential risks such as release of pyrogenic cell wall components.     

Modelling and estimating pollen movement in oilseed rape (Brassica napus) at the landscape scale using genetic markers

Understanding patterns of pollen movement at the landscape scale is important for establishing management rules following the release of genetically modified (GM) crops. We use here a mating model adapted to cultivated species to estimate dispersal kernels from the genotypes of the progenies of male-sterile plants positioned at different sampling sites within a 10 x 10-km oilseed rape production area. Half of the pollen clouds sampled by the male-sterile plants originated from uncharacterized pollen sources that could consist of both large volunteer and feral populations, and fields within and outside the study area. The geometric dispersal kernel was the most appropriate to predict pollen movement in the study area. It predicted a much larger proportion of long-distance pollination than previously fitted dispersal kernels. This best-fitting mating model underestimated the level of differentiation among pollen clouds but could predict its spatial structure. The estimation method was validated on simulated genotypic data, and proved to provide good estimates of both the shape of the dispersal kernel and the rate and composition of pollen issued from uncharacterized pollen sources. The best dispersal kernel fitted here, the geometric kernel, should now be integrated into models that aim at predicting gene flow at the landscape level, in particular between GM and non-GM crops.     

Engineering glycoproteins for use as pharmaceuticals

The fact that glycosylation is not a significant process in prokaryotes means that many of the proteins produced by genetically engineered bacteria are not identical to their eukaryotic counterparts. Although glycosylation affects the physical, chemical and biological nature of proteins, its pharmacological value in potential protein pharmaceuticals is not easy to predict. However, the development of mammalian cell culture methods for expressing recombinant DNA-derived glycoproteins will permit further studies in the field.     

Evaluating the risk of releasing genetically engineered organisms

This article considers the question of a priori assessment of the safety of releasing recombinant DNA engineered organisms. Now and for the foreseeable future, decisions to release such an organism must be based on the results of limited, case-by-case risk assessment studies. The criteria calling for the termination of release programs must be agreed upon in advance of these studies. There is no justification for excluding classes of release organisms from risk assessment. Theory is useful in suggesting a hierarchy of risks, raising the questions that have to be addressed in case-by-case risk assessment and providing protocols for the standardization and execution of these studies. We do not believe that theory can be used to argue categorically for or against the safety of specific releases of recombinant DNA engineered organisms.     

The involvement of European industry in developing regulations

In the belief that the release of genetically engineered organisms will provide significant benefits to our society, European industry is taking an active role in the development of regulations which make both economic and scientific sense. Such regulations will have public support and enable industry to predict the cost of satisfying the requirements for risk assessment and hence be more confident in funding research and development programmes.     

Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.