UseofBioluminescence forDetection ofGenetically Engineered Microorganisms Released intotheEnvironment (original) (raw)
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Applied and Environmental Microbiology, 1992
The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mistand wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.
Journal of Applied Microbiology, 1996
Dispersal and persistence of a pathogenic strain of Xanthomonas campestris pv. campestris, genetically engineered to bioluminesce, was followed in and on host and non-host plants in the field environment. Black rot susceptible cabbage plants were mist inoculated with the bioluminescent strain only, or were mist inoculated with X. campestris pv. vesicatoria or a weakly pathogenic strain of X. c. campestris 1 week before challenge inoculation with the bioluminescent strain. Growth of the bioluminescent strain was detected with a low-light, charge-coupled device camera or through bioluminescence measurements of broth-enrichment cultures of leaf disk samples. Bioluminescent X. c. campestris could often be observed as populations on symptomless leaves or in lesions, and persisted as a vascular endophyte for more than 6 months throughout the winter growing season. Dispersal to cruciferous and non-cruciferous weeds was frequently detected. Pre-inoculation with X. c. vesicatoria or the weakly pathogenic X. c. campestris did not significantly affect the movement and persistence of the bioluminescent strain nor reduce the incidence of black rot disease.
Frontiers in Plant Science, 2021
Imaging technology can provide insight into biological processes governing plant-pathogen interactions. We created and used a bioluminescent strain ofXanthomonas hortorumpv.gardneri(Xgb) to quantify infection processes in plants using tomato as a model. AnX. hortorumpv.gardneriis one of the fourXanthomonasspecies that causes bacterial spots in tomatoes. We used Xgbto quantify bacterial growthin planta, to assess disease severity in resistant and susceptible tomato lines, and to observe infection routes in leaves. A positive and significant linear correlation r (67) = 0.57,p≤ 0.0001 was observed between bioluminescence signals emitted by Xgbin plantaand bacterial populations determined through dilution plating. Based on bioluminescence imaging, resistant and susceptible tomato lines had significantly different average radiances. In addition, there was a positive and significant correlation r = 0.45, p = 0.024 betweenX. hortorumpv.gardneri-inoculated tomato lines evaluated by biolumin...
Novel method for monitoring genetically engineered microorganisms in the environment
Applied and environmental microbiology, 1989
A method has been devised for directly detecting and monitoring genetically engineered microorganisms (GEMs) by using in vitro amplification of the target DNAs by a polymerase chain reaction and then hybridizing the DNAs with a specific oligonucleotide or DNA probe. A cloned 0.3-kilobase napier grass (Pennisetum purpureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments was inserted into a derivative of a 2,4-dichlorophenoxyacetic acid-degradative plasmid, pRC10, and transferred into Escherichia coli. This genetically altered microorganism, seeded into filter-sterilized lake and sewage water samples (10(4)/ml), was detected by a plate count method in decreasing numbers for 6 and 10 days of sample incubation, respectively. The new method detected the amplified unique marker (0.3-kilobase DNA) of the GEM even after 10 to 14 days of incubation. This method is highly sensitive (it requires only picogram amounts of...
Containment of a genetically engineered microorganism during a field bioremediation application
Applied Microbiology and Biotechnology, 1999
A ®eld release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were ®lled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas uorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio ®scheri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10 14 bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low.
Lux gene technology - a strategy to optimize biological control of soil-borne diseases
New Phytologist, 1996
Lux gene technology is based on the transformation of environmental bacteria with genes of the lux operon from the marine hacteria Vibrio fischeri and V. harveyi. Transformed strains are bioluminescent and thus provide a rapid and very accurate tool for the study of population dynamics, metabolic activity and spatial distribution of specific bacteria in environmental samples. This review summarizes the recent progress io using iux-marked rhizobacteria to optimize biological control methods for soil-borne diseases. Special emphasis has been placed on the advantages and limitations of the new technology.
Archives of microbiology, 2016
Fluorescent proteins have been used to track plant pathogens to understand their host interactions. To be useful, the transgenic pathogens must present similar behaviour than the wild-type isolates. Herein, a GFP marker was used to transform two plant pathogenic bacteria, Agrobacterium and Xanthomonas, to localize and track the bacteria during infection. The transgenic bacteria were evaluated to determine whether they showed the same fitness than the wild-type strains or whether the expression of the GFP protein interfered in the bacterial activity. In Agrobacterium, the plasmid used for transformation was stable in the bacteria and the strain kept the virulence, while Xanthomonas was not able to conserve the plasmid and transformed strains showed virulence variations compared to wild-type strains. Although marking bacteria with GFP to track infection in plants is a common issue, works to validate the transgenic strains and corroborate their fitness are not usual. Results, presented...
BMC Microbiology, 2008
Background Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight disease, is a serious pathogen of rice. Here we describe a fluorescent marker system to study virulence and pathogenicity of X. oryzae pv. oryzae. Results A fluorescent X. oryzae pv. oryzae Philippine race 6 strain expressing green fluorescent protein (GFP) (PXO99GFP) was generated using the gfp gene under the control of the neomycin promoter in the vector, pPneo-gfp. The PXO99GFPstrain displayed identical virulence and avirulence properties as the wild type control strain, PXO99. Using fluorescent microscopy, bacterial multiplication and colonization were directly observed in rice xylem vessels. Accurate and rapid determination of bacterial growth was assessed using fluoremetry and an Enzyme-Linked ImmunoSorbant Assay (ELISA). Conclusion Our results indicate that the fluorescent marker system is useful for assessing bacterial infection and monitoring bacterial multiplication in planta.
Applied and Environmental Microbiology, 2012
ABSTRACTBacterial blight, caused by the phytopathogenPseudomonas cannabinapv.alisalensis, is an emerging disease afflicting important members of theBrassicaceaefamily. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogenPseudomonas syringaepv.maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant “light”-tagged reporter phage was generated by integrating bacterialluxABgenes encoding luciferase into the genome ofP. cannabinapv.alisalensisphage PBSPCA1. The PBSPCA1::luxABreporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxABrapidly and sensitively detectsP. cannabinapv.alisalensisby conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens ofBrassicaspecies such asP. syringaepv...