Skip to main content
SpringerLink
Log in

Molecular methods for environmental monitoring and containment of genetically engineered microorganisms

  • Published:
Biodegradation Aims and scope Submit manuscript

Abstract

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Atlas RM, Sayler G, Burlage RS & Bej AK (1992) Molecular approaches for environmental monitoring of microorganisms. Bio/Techniques: 12: 706–711

    Google Scholar 

  • Bakken LR (1985) Separation and purification of bacteria from soil. Appl. Environ. Microbiol. 49: 1482–1487

    Google Scholar 

  • Balkwill DL, Labeda DP & Casida LEJr. (1975) Simplified procedure for releasing and concentrating microorganisms from soil for transmission electron microscopy viewing as thin-section and frozen-etched preparations. Can. J. Microbiol. 21: 252–262

    Google Scholar 

  • Barkay T (1987) Adaptation of aquatic microbial communities to Hg2+ stress. Appl. Environ. Microbiol. 53: 2725–2732

    Google Scholar 

  • Barkay T & Olson BH (1986) Phenotypic and genotypic adaptation of aerobic heterotrophic sediment bacterial communities to mercury stress. Appl. Environ. Microbiol. 52: 403–406

    Google Scholar 

  • Barkay T, Fouts DL & Olson BH (1985) The preparation of a DNA gene probe for the detection of mercury resistance genes in gram-negative communities. Appl. Environ. Microbiol. 49: 686–692

    Google Scholar 

  • Barkay T, Liebert C & Gillman M (1989) Hybridization of DNA probes with whole community genome for detection of genes that encode microbial responses to pollutants: mer genes and Hg2+ resistance. Appl. Environ. Microbiol. 55: 1574–1577

    Google Scholar 

  • Bej AK & Mahbubani MM (1992) Applications of the polymerase chain reaction in environmental microbiology. PCR Methods and Appl. 1: 151–159

    Google Scholar 

  • Bej AK, Perlin MH & Atlas RM (1988) Model suicide vector for containment of genetically engineered microorganisms. Appl. Environ. Microbiol. 54: 2472–2477

    Google Scholar 

  • Bej AK, Steffan RJ, DiCesare J, Haff L & Atlas RM (1990) Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl. Environ. Microbiol. 56: 307–314

    Google Scholar 

  • Bej AK, Mahbubani MM & Atlas RM (1991) Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. CRC Crit. Rev. Mol. Biol. 26: 301–334

    Google Scholar 

  • Berent SL, Mahmoudi M, Torczynski RM, Bragg PW & Bollon AP (1985) Comparison of oligonucleotide and long DNA fragments as probes in DNA and RNA dot, Southern, northern, colony and plaque hybridizations. Bio/Techniques 3: 208–220

    Google Scholar 

  • Bialkonska-Hobrzanska H (1987) Detection of enterotoxigenic Escherichia coli by dot blot hybridization with biotinylated DNA probes. J. Clinical Microbiol. 25: 338–343

    Google Scholar 

  • Blackburn JW, Jain RL & Sayler GS (1987) Molecular microbial ecology of a naphthalene-degrading genotype in activated sludge. Environ. Sci. Tech. 21: 884–890

    Google Scholar 

  • Blattner FL, Williams BG, Blechl AE, Dennistron-Thompson K, Baber HE, Furlong LA, Grunwald DJ, Kiefer DO, Moore DD, Schumm JW, Sheldon ET & Smithies O (1977) Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science 196: 161–169

    Google Scholar 

  • Boivin R, Chalifour F & Dion P (1988) Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium, and Rhizobium. Mol. Gen. Genet. 213: 50–55

    Google Scholar 

  • Britton RJ & Davidson EH (1985) Hybridization strategy. In: Hames BP & Higgins SJ (Eds) Nucleic Acid Hybridization: A Practical Approach (pp 3–15). IRL Press, Oxford

    Google Scholar 

  • Brown TH, Colwell RK, Lenski RE, Levin BR, Lloyd M, Regal PJ & Simberloff D (1984) Report on workshop on possible ecological and evolutionary impacts of bioengineered organisms released into the environment. Bull. Ecol. Soc. Am. 65: 436

    Google Scholar 

  • Burlage RS, Sayler GS & Larimer FW (1990) Monitoring of naphthalene catabolism by bioluminescence with nah-lux transcriptional fusions. J. Bacteriol. 172: 4749–4757

    Google Scholar 

  • Chaudhry GR, Toranzos GA & Bhatti AR (1989) Novel method for monitoring genetically engineered microorganisms in the environment. Appl. Environ. Microbiol. 55: 1301–1304

    Google Scholar 

  • Curtiss RIII (1976) Genetic manipulation of microorganisms: potential benefits. Annu. Rev. Microbiol. 30: 507–533

    Google Scholar 

  • Curtiss RIII (1988) Engineering organisms for safety: what is necessary? In: Sussman M, Collins CH, Skinner FA & Stewart-Tull DE (Eds) The Release of Genetically-Engineered Microorganisms. (pp 7–20). Academic Press, New York

    Google Scholar 

  • Curtiss RIII, Szybalski W, Helsinki DR & Falkow S (1976) Workshop on design and testing of safer prokaryotic vehicles and bacterial hosts for research on recombinant DNA molecules. ASM News 42: 134–138

    Google Scholar 

  • Curtiss RIII, Inoue M, Pereira D, Hsu JC, Alexander L & Rock L (1977) Construction and use of safer bacterial host strains for recombinant DNA research. In: Scott WA & Werner R (Eds) Molecular Cloning of Recombinant DNA (pp 99–114). Academic Press, Inc., New York

    Google Scholar 

  • Cuskey S & Bourquin A (1987) Construction of bacteria with conditional lethal genetic determinants for control of released microorganisms in the environment. Abstr. Annu. Meet. Am. Soc. Microbiol. Q127, p 3

  • Datta AR, Wentz BA & Hill WE (1987) Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization. Appl. Environ. Microbiol. 53: 2256–2259

    Google Scholar 

  • Datta AR, Wentz BA, Shook D & Trucksess MW (1988) Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes. Appl. Environ. Microbiol. 54: 2933–2937

    Google Scholar 

  • Drahos DJ, Hemming BC & McPherson S (1986) Tracking recombinant organisms in the environment: β-galactosidase as a selectable nonantibiotic marker for fluorescent Pseudomonads. Bio/Techniques 4: 439–444

    Google Scholar 

  • Echeverria P, Seriwatana J, Chityothin O, Chaicumpa W & Cirapat C (1982) Detection of enterotoxigenic Escherichia coli in water by filter hybridization with three enterotoxin gene probes. J. Clin. Microbiol. 16: 1086–1090

    Google Scholar 

  • Engebrecht J, Simon & M. Silverman M (1985) Measuring gene expression with light. Science 227: 1345–1347

    Google Scholar 

  • Ehrlich HA (Ed) (1989) PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York

    Google Scholar 

  • Ehrlich HA, Gelfand D & Sninsky JJ (1991) Recent advances in the polymerase chain reaction. Science 252: 1643–1651

    Google Scholar 

  • Faegri A, Torsvik VL & Goksoyr J (1977) Bacterial and fungal activities in soil: separation of bacteria by a rapid fractionated centrifugation technique. Soil Biol. Biochem. 9: 105–112

    Google Scholar 

  • Falkenstein H, Bellemann P, Walter S, Zeller W & Geider K (1988) Identification of Erwina amylovora, the fireblight pathogen, by colony hybridization with DNA from plasmid pEPA29. Appl. Environ. Microbiol. 54: 2798–2802

    Google Scholar 

  • Federal Register. June 26, 1986. Office of Science and Technology Policy: Coordinated framework for regulation of biotechnology

  • Festl H, Ludwig W & Schleifer KH (1986) DNA hybridization probe for the Pseudomonas fluorescens group. Appl. Environ. Microbiol. 52: 1190–1194

    Google Scholar 

  • Fitts R, Diamond M, Hamilton C & Neri M (1983) DNA-DNA hybridization assay for the detection of Salmonella spp. in foods. Appl. Environ. Microbiol. 46: 1146–1151

    Google Scholar 

  • Flamm RK, Hinrichs DJ & Thomashow MF (1989) Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe. Appl. Environ. Microbiol. 55: 2251–2256

    Google Scholar 

  • Ford SF & Olson B (1988) Methods for detecting genetically engineered microorganisms in the environment. Adv. Microbial Ecol. 10: 45–79

    Google Scholar 

  • Fuhrman JA, Comeau DE, Hagstrom A & Cham AM (1988) Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies. Appl. Environ. Microbiol. 54: 1426–1429

    Google Scholar 

  • Giovannoni SJ, Britschgi TB, Moyer CL & Field KG (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345: 60–62

    Google Scholar 

  • Grunstein M & Hodgness DS (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 72: 3961–3965

    Google Scholar 

  • Halvorson HO, Pramer D & Rogul M (1985) Engineered Organisms in the Environment: Scientific Issues. American Society for Microbiology, Washington, DC

    Google Scholar 

  • Hanahan D & Meselson M (1980) Plasmid screening at high colony density. Gene 10: 63–67

    Google Scholar 

  • Hill WE, Payne WL & Aulisio CCG (1983) Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization. Appl. Environ. Microbiol. 46: 636–641

    Google Scholar 

  • Hodgson ALM & Roberts WP (1983) DNA colony hybridization to identify Rhizobium strains. J. Gen. Microbiol. 129: 207–212

    Google Scholar 

  • Holben WE & Tiedje JM (1988) Applications of nucleic acid hybridization in microbial ecology. Ecology 69: 561–568

    Google Scholar 

  • Holben WE, Jansson JK, Chelm BK & Tiedje JM (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol. 54: 703–711

    Google Scholar 

  • Ingram C, Brawner M, Youngman P & Westpheling J (1989) xylE functions as an efficient reporter gene in Streptomyces spp.: Use for the study of galP1, a catabolite-controlled promoter. J. Bacteriol. 171: 6617–6624

    Google Scholar 

  • Innis M, Gelfand D, Snisky D & White T (Eds) (1990) PCR Protocols: A Guide to Methods and Applications. Academic Press, New York

    Google Scholar 

  • Jain RK, Sayler GS, Wilson JT Houston L & Pacia D (1987) Maintenance and stability of introduced genotypes in ground-water aquifer material. Appl. Environ. Microbiol. 53: 996–1002

    Google Scholar 

  • Jansson JK, Holben WE & Tiedje JM (1989) Detection in soil of a deletion in an engineered DNA sequence by using gene probes. Appl. Environ. Microbiol. 55: 3022–3025

    Google Scholar 

  • King JMH, DiGrazia PM, Applegate B, Burlage R, Sanseverino J, Dunbar P, Larimer F & Sayler GS (1990) Bioluminescent reporter plasmid for naphthalene exposure and biodegradation. Science 249: 778–791

    Google Scholar 

  • King RJ, Short KA & Seidler RJ (1991) Assay for detection and enumeration of genetically engineered microorganisms which is based on the activity of a deregulated 2,4-dichlorophenoxyacetate monooxygenase. Appl. Environ. Microbiol. 57: 1790–1792

    Google Scholar 

  • Knudsen SM & Karlstrom (1991) Development of efficient suicide mechanisms for biological containment of bacteria. Appl. Environ. Microbiol. 57: 85–92

    Google Scholar 

  • Kraus J, Ludwig W & Schliefer KH (1986) A cloned 23S rRNA gene fragment of Bacillus subtilis and its use as a hybridization probe of conserved character. FEMS Microbiol. Lett. 33: 89–93

    Google Scholar 

  • Levine MM, Kaper JB, Lockman H, Black RE, Clements ML & Falkow S (1983) Recombinant DNA risk assessment studies in man: efficacy of poorly mobilizable plasmids in biologic containment. Recomb. DNA Tech. Bull. 6: 89–97

    Google Scholar 

  • Miliotis MD, Galen JE, Kaper JB & Morris JGJr. (1989) Development and testing of a synthetic oligonucleotide probe for the detection of pathogenic Yersinia strains. J. Clin. Microbiol. 27: 1667–1670

    Google Scholar 

  • Molin S, Klemm P, Poulsen LK, Biehl H, Gerdes K & Andersson P (1987) Conditional suicide system for containment of bacteria and plasmids. Bio/Techniques 5: 1315–1318

    Google Scholar 

  • Mullis KB (1990) The unusual origin of the polymerase chain reaction. Sci. Amer. 262(4): 56–65

    Google Scholar 

  • Mullis KB & Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155: 335–351

    Google Scholar 

  • Nortermans S, Chakrobarty T, Leimeister-Wachter M, Dufrenne J, Heuvelman KJ et al. (1989) Specific gene probe for detection of biotyped and serotype Listeria strains. Appl. Environ. Microbiol. 55: 902–906

    Google Scholar 

  • Obukowicz MG, Perlak FJ, Kusano-Dretzmer K, Mayer EJ, Bolten SL & Watrud LS (1986) Tn5-mediatedintegration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads. J. Bacteriol. 168: 982–989

    Google Scholar 

  • Obukowicz MG, Perlak FJ, Bolten SL, Kusano-Kretzmer K, Mayer EJ & Watrud LS (1987) IS50L as a nonself transposable vector used to integrate the Bacillus thuringiensis delta-endotoxin gene into the chromosome of root-colonizing pseudomonads. Gene 51: 91–96

    Google Scholar 

  • Ogram A, Sayler GS & Barkay T (1987) The extraction and purification of microbial DNA from sediments. J. Microbiol. Methods 7: 57–66

    Google Scholar 

  • Paul JH, Cazares L & Thurmond J (1990) Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments. Appl. Environ. Microbiol. 56: 1963–1966

    Google Scholar 

  • Pettigrew CA & Sayler GS (1986) The use of DNA:DNA colony hybridization in the rapid isolation of 4-chlorobiphenyl degradative bacterial phenotypes. J. Microbiol. Methods 5: 205–213

    Google Scholar 

  • Pillai SD, Josephson KL, Bailey RL, Gerba CP & Pepper IL (1991) Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Appl. Environ. Microbiol. 57: 2283–2286

    Google Scholar 

  • Saiki RK, Glenfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB & Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–494

    Google Scholar 

  • Sayler GS & Layton AC (1990) Environmental application of nucleic acid hybridization. Annual Review of Microbiology 44: 625–648

    Google Scholar 

  • Sayler GS, Shields MS, Tedford ET, Breen A, Hooper SW et al. (1985) Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples. Appl. Environ. Microbiol. 49: 1295–1303

    Google Scholar 

  • Seriwantana J, Echeverria P, Taylor DN, Sakuldaipeara T, Changchawalit C & Chivoratanond O (1987) Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes. J. Clin. Microbiol. 25: 1438–1441

    Google Scholar 

  • Sethabutr O, Hanchalay S, Echeverria P, Taylor DN & Leksomboon U (1985) A nonradioactive DNA probe to identify Shigella and enteroinvasive Escherichia coli in stools of children with diarrhoea. Lancet 2: 1095–1097

    Google Scholar 

  • Sharples FE (1983) Spread of organisms with novel genotypes: Thoughts from an ecological perspective. Recomb. DNA Tech. Bull. 6: 43–56

    Google Scholar 

  • Sommerville CC, Knight IT, Straub WL & Colwell RR (1989) Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl. Environ. Microbiol. 55: 548–554

    Google Scholar 

  • Steffan RJ & Atlas RM (1988) DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl. Environ. Microbiol. 54: 2185–2191

    Google Scholar 

  • Steffan RJ & Atlas RM (1990) Solution hybridization assay for detecting genetically engineered microorganisms in environmental samples. Appl. Environ. Microbiol. 54: 2185–2191

    Google Scholar 

  • Steffan RJ & Atlas RM (1991) Polymerase chain reaction: applications in environmental microbiology. Annu. Rev. Microbiol. 45: 137–161

    Google Scholar 

  • Steffan RJ, Goksoyr J, Bej AK & Atlas RM (1988) Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54: 2908–2915

    Google Scholar 

  • Sussman M, Collins CH, Skinner FA & Stewart-Tull DE (1988) The Release of Genetically-Engineered Micro-Organisms. Academic Press London

    Google Scholar 

  • Torsvik VL (1980) Isolation of bacterial DNA from soil. Soil Biol. Biochem. 12: 15–21

    Google Scholar 

  • Torsvik VL & Goksoyr J (1978) Determination of bacterial DNA in soil. Soil Biol. Biochem. 10: 7–12

    Google Scholar 

  • Torsvik VL, Goksoyr J & Daae FL (1990) High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56: 782–787

    Google Scholar 

  • Tsai YL & Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57: 1070–1074

    Google Scholar 

  • Tsai YL & Olson BH (1992) Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Appl. Environ. Microbiol. 58: 754–757

    Google Scholar 

  • Ward DW, Weller R & Bateson MM (1990) 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345: 63–65

    Google Scholar 

  • Weller R & Ward DM (1989) Selective recovery of 16S rRNA sequences from natural microbial communities in the form of cDNA. Appl. Environ. Microbiol. 55: 1818–1822

    Google Scholar 

  • Zehr JP & McReynolds LA (1989) Use of degenerate oligonucleotides for the amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl. Environ. Microbiol. 55: 2522–2525

    Google Scholar 

  • Zukowski MM, Gaffney DF, Speck D, Kauffmann M, Findeli A, Wisecup A & Lecocq J-P (1983) Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc. Natl. Acad. Sci. USA 80: 1101–1105

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, University of Louisville, 40292, Louisville, KY, USA

    Ronald M. Atlas

Authors
  1. Ronald M. Atlas
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Atlas, R.M. Molecular methods for environmental monitoring and containment of genetically engineered microorganisms. Biodegradation 3, 137–146 (1992). https://doi.org/10.1007/BF00129079

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00129079

Key words

Search

Navigation