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Engineered Microorganism:
fter all the preliminary experiments, the etbical debates, the government red tape, and the inevitable legal battles, field tests of genetically engineered microorganisms (GEMS) are f d y under way. As biotechnology steps out of the controlled environment of the research laboratory and into the real world, analyticalteehkniaues for detecting and monitoringreed GEMs musckeep pace. Theirsnal analytical methoda of microbisuch as counting bacterial coioniw a[n a petri dish, will have to be techniques for monitorcoupled 5 qt DNA. ing recorn.. y field experiments Many of enwonmental imare looking at I the release 01 pact, if any, fl examining hoa specific GEM) their presence anems other microbial anmal corn. and, ultimately, plan munities. ' . Analytica. yythods musi provide answers to ques how successfully GEMs c(
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Plant Physiology at the University of California, Berkeley, directed release experiments with Ice- Pseudomonas syringae, a genetically engineered bacterium designed to reduce frcat dammts. Two strains of this GEM ed on potato plant seedlings an experimental agricultnrfilelake, CA. br .,acteria differ from wild-type r. syringae by the partial deletion of the gene encoding an ice-catalyzing protein. Pure water can supere001 to -40 OC unless a template of appropriate molecules or particles promotea ice formation. In most plants, freezing at "Cis catalyzed hy number of common the expression of the ice protein in wild-type P.syringae (labeled Ipe'). Introducing engineered Ice- bacteria before Ice+ colonizes the seedlings uld minimize wild-type popnlat' d c e + though - p e t i t i o 4 v y e d plants ta sho1
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SEPTEMBER 1. 1989
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experience one-third leas frost damage
metal resistance, the expression of precursors to a chemical pigment, or an The California researchers followed enzyme for bioluminescence. The use the fate of Ice- P. syringae by collect- of unique reporter genes could provide ing analysis samples weekly or biweekresearchers with rapid and accurate ly at 32 locations scattered in and methods for identifying GEMS. Howaroundtheirteat .te.Aplant-freemne ever, these procedures can fail if the extending 2@3$m around the test organism becomes dormant, though plot, dispossble clothing, spraying in still viable in the field, or if it cannot be calm air, and other protwols reduced grown in d t u r e . the spread of Ice- bacteria beyond the Immunology offers a solution to test bed. these problems and yet another strateCollected samples were sonicated to gy for identifying engineered microordislodge bacteria, which were then ganisms. Antibodies coupledwith a fluplated for analysis on appropriate meorescent dye, such as fluorescein isodia containing 100g / dof the antibi- thiocyanate, or with an enzyme for an otic rifampicin. The drug generally ELISA procedure can bind and mark eliminated all other bacteria except the particular organisms. Monoclonalantireleased strains of Ice- P. syringae, bodies, which are homogeneous and which are resistant to the drug (2). therefore generate fewer false-positive Another method for tracking GEMs testa, provide the best analyses. relies on reporter genes that uniquely Monoclonals are formed hy injecting announce the presence of a microormice with purified antigen from a GEM ganism. For instance, researehers from and then harvesting the spleens of the Clemson University and Monsanto animals. Antibody-producing B lymhave begun field-testing lac73 Pseuphocyte cells, a type of white blood cell, domonas aureofaciens, a modified are collected from the organ and fused form of a root-colonizing bacterium with mouse tumor cells. The hyhrid that unlike other fluorescent soil pseucells produced by this procedure manudomonads can utilize l a c h e as its sole facture the monoclonal antihodies in carbon source. The GEM contains the large amounts. IacZ and lacy genes from Escherichia The fluorescent antibody teats can coli that express, respectively, ,%galacbe coupled with direct microscopy, and tosidase and lactose permease. These specific GEMS can be detected (6). genes enableP. aureofacienato metabFurthermore, the immunological oliie lache. Furthermore, the engiprobes could be targeted to an antigen neered organism will metabolize the expressed by an inserted or modified chromogenic substrate X-Gal (5gene. c~oro-4-bromo-3-indolyl-~-~-gala~-However, this procedure also bas pyranoside), which stains the lacZY limitations. Antibodies do not always colonies blue @, 4). distinguish between living and dead The IacZY genes can be inserted escells,and they suffer from environmensentially permanently into the chromo- tal interferencessuch as naturally fluosomes of many different bacteria using rescing materials or organic slimes that a special DNA sequence called a traneprevent antibody binding. Sensitivity poson (5).Once inserted, these markers is also a concern (7),as is the potential can be combined with other factors, for cross-reaction with other organsuch as antibiotic resistance, to identiisms. fy a parti& GEM. Immunology and cnltwe methodsInserted lache metabolism genes the means of identifying organ’L8m& offer a more sensitive analytical deter- can be combined with analytical techminant than antibiotic resistance. The niques that directly target the recomhibackground levels of resistance among nant DNA. Beginning in 1986,British soil bacteria to the antibiotic rifampi- field teats conducted by the Natural cin,for example, can run as high as 105 Environment Research Council’s InstiCFU (colony-forming units)/g soil. tute of Virology in Oxford tracked a (Total bacterial levels u s d y exceed virus that had been marked by a syn108 CFWg soil.) This natural back- thetic oligonucleotideinserted into the ground of antibiotic resistance could organism’s genome. The virus, Autointerfere with the detedion of low lev- g r a p h californica, only infectscertain els of GEMS.On the other hand, pseu- insect species, including unwanted catdomonads marked with the lacZY erpillars. Genetic engineers hope evengenes can be detected at < 10 CFU/g tually to accelerate the deadly effeds soil (3).Furthermore, because E. coli of this virus and thereby provide an can be found in the gastrointestinal “environmentally d e r ” alternative to tract of most animals, the IacZY trait chemical pesticides (8). may not pose any health hazards. The inserted oligonucleotide, 80 Other genetic markers are also being base-pairs in length, neither added nor studied or proposed, such as heavy subtracted from the genetic activity of
than untreated seedlings (I).
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ANALYTKXL CWEMISTRY, VOL. 81, NO. 17, SEFTEMBW 1. 1989
the viruses. The modified DNA of A. californica was detectable by a procedure laheled dot-blot analysis (8). The DNA was denatured, split into single strands, and identified with a 32P-laheled oligonucleotide probe matched to the unique gene sequence. DNA that binds to the probe sticks to a fdter, such as nitrocellulose, whereas unreacted prohes are washed off. Autoradiography identifies the labeled DNA (2). A useful variation of dot-blot analysis, known as colony hybridization, lyses cells growing directly on a nitrocellulose filter embedded in an appropriate medium on a petri dish. The exp o d DNA is picked up by the fdter and identified with radioactive probes. This p d u r e can identify a single GEM colony in 106 background colonies (7). DNA hybridization was used m a sionally to test for Ice- P. syringae. The procedure probed a unique DNA sequence in the GEM created by the deletion of the ice gene and the reconnection of the cut DNA ends (2). Probably one of the most powerful of the direct DNA techniques is the polymerase chain reaction (PCR). In a few hours PCR can reproduce more than 10 million DNA copies from an initial DNA sequence. Microorganisms with concentrationsas low as 1cell/g soil can be detected following amplification by this method. Other methods exist for probing DNA, such as restriction enzyme digests, Southern blots, and nucleic acid sequencing. Many of these sophisticated analysis procedures are, at present, too expensive and time-consuming for routine analysis. The interest in monitoring GEMS is driving research toward new methods that could accelerate and simplify the proceas. For instance, University of Maryland researchers have developed a protocol for collecting DNA from aquatic microorganisms that can easily be performed in the field. A large volume of water is r m through a fdter membrane that traps and concentrates the microorganisms. The cells are then lysed; the crude material, removed from the filter, can either be purified and concentrated immediately or stored and prow e d later. Yields of 1 ng DNA/lOG cells were calculated (9).Other protocols for the collection and concentration of microorganisms from soil have also appeared in the scientific literature. New approaches for analyzing microbes are also being developed. David Stahl and his co-workers at the University of Illinois at Urbana-Champaign have been using ribosomal RNAs
(rRNAs), which are found in all living organisms, for sorting out microbial populations. At present, rRNAs offer the most comprehensive database of sequenced nucleic acids, particularly for the 5s and 16s rRNAs (Srefers to the sedimentation value following centrifugation). The nucleic acid analysis finds that rRNAs are highly conserved in both sequence and structure; however, variable sections offer a means to differentiate microorganisms by species and genus. Once again, identification depends on radioactively labeled oligonucleotides (IO). The future progress of GEM field studies is clearly tied to the continued development of these analytical procedures. Early experiments are providing the data necessary to understand the ecological impact of geneticallyaltered microorganisms. However, scientific (and, ultimately, public) approval for any commercial GEM products will depend on how well the analytical tools enable researchers to unravel the complex environmental effects. Alan R . Newman
References (1) Baum, R. Chem. Eng. News 1989,April 24,3632. (2)Lindow, S. E.; Panopoulos, N. J. In The Release of Genetically-Engineered Micro-Organisms; Sussman, M.; Collins, G. H.; Skinner, F. A.; Stewart-Tull, D. E., Eds.; Academic Press: London, 1988; pp. 121-38. (3) Drahos, D. J.; Hemming, B. C.; McPherson, S. BiolTechnology 1986, 4, 43944. ( 4 ) Drahos, D. J.; Barry, G. F.; Hemming, B. C.; Brandt, E. J.; Skipper, H. D.; Kline, E. L.; KlueDfel, D. A.; Hughes, T. A.; Gooden, D. T. In The Release of Genetically-Engineered Micro-Organisms; Sussman, M.; Collins, G. H.; Skinner, F. A.; Stewart-Tull, D. E., Eds.; Academic Press: London, 1988,pp. 181-91. (5) Barry, G.F. BiolTechnology 1986, 4, 446-49. (6) Colwell, R. R.; Somerville, C.; Knight, I.; Straube, W. In The Release of Genetically-Engineered Micro-Organisms; Sussman, M.; Collins, G. H.; Skinner, F. A.; Stewart-Tull, D. E., Eds.; Academic Press: London, 1988; pp. 47-60. (7) Glaser, D.; Keith, T.; Riley, P.; Chambers, G.; Manning, J.; Hattingh, s.;Evans, R. In Biotechnology in the Enuironment; Omenn, G. S.; Teich, A. H., Eds.; Noyes Data Corp.: Park Ridge, NJ, 1986; pp. 114-64. (8) Bishop, D.H.L.; Entwistle, P. F.; Cameron, I. R.; Allen, C. J.; Possee, R. D. In The Release of Genetically-Engineered Micro-Organisms; Sussman, M.; Collins, G. H.; Skinner, F. A.; Stewart-Tull, D. E., Eds.; Academic Press: London, 1988;pp. 143-79. (9) Sommerville, C. C.; Knight, I. T.; Straube, W. L.; Colwell,R. R. Appl. Enuiron. Microbiol. 1989,55,548-54. (10) Stahl, D. A.; Flesher, B.; Mansfield, H. R.; Montgomery, L. Appl. Enuzron. Microbiol. 1988.54, 1079-84.
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 17, SEPTEMBER 1, 1989
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