Tracking genetically-engineered microorganisms

CONTENTS 1. Problems in Detecting Dormant (VBNC) Cells, and the Role of DNA Elements in This Response ......................................................... 1 James D. Oliver 1.1. The Viable but Nonculturable State ............................................. 1 1.2. Assays Used to Determine Viability .............................................. 2 1.3. Characteristics of Cells in the VBNC State ................................... 3 1.4. Inducing Factors ............................................................................ 4 1.5. Bacteria Known to Enter the VBNC State .................................... 5 1.6. Resuscitation from the VBNC State .............................................. 5 1.7. In situ Evidence of the VBNC Response ....................................... 7 1.8. Use of PCR to Detect Cells in the VBNC State ............................ 9 1.9. Effect of Plasmids on Entry into the VBNC State ........................ 9 1.10. Significance of the VBNC State in the Release of Genetically Modified Bacteria ................................................. 12 1.11. Conclusion ................................................................................... 13 2. The Use of Antibiotic Resistance Gene Markers for Studying Bacterial Populations in Natural Environments ............. 17 Sharon Egan and Elizabeth M.H. Wellington 2.1. Introduction ................................................................................. 17 2.2. Cultivation Based Assays ............................................................. 18 2.3. Direct Molecular Monitoring Methods ...................................... 20 2.4. Choice of Antibiotic Resistance Gene Markers .......................... 20 2.5. Cross-Resistance .......................................................................... 21 2.6. Marking of Bacteria with Antibiotic Resistance Genes .............. 21 2.7. Use of Antibiotic Resistance Genes to Monitor Gene Transfer in Soil ................................................ 22 2.8. Ethical Concerns .......................................................................... 23 2.9. Conclusion ................................................................................... 24 3. Extraction and Analysis of Microbial Community Nucleic Acids from Environmental Matrices ................................................................ 29 Jan Dirk van Elsas, Kornelia Smalla and Christoph C. Tebbe 3.1. Introduction ................................................................................. 29 3.2. Extraction of Microbial Cells from Environmental Matrices .... 33 3.3. Cell Disruption ............................................................................. 35 3.4. Extraction and Purification of DNA ........................................... 36 3.5. Analysis of DNA and Detection of Specific Sequences .............. 38 3.6. Extraction and Purification of RNA ........................................... 39 3.7. Detection of RNA......................................................................... 40 3.8. Quantification of Specific Targets in Environmental Nucleic Acids ................................................. 42 3.9. Conclusion ................................................................................... 44 4. Detection of Bacteria by Their Intrinsic Markers ................................. 53 Éva Tas and Kristina Lindström 4.1. Introduction ................................................................................. 53 4.2. Brief Description of PCR-Based Fingerprinting Methods ......... 54 4.3. Development of Specific Hybridization Probes and PCR Primers.......................................................................... 54 4.4. Case Study: Specific Detection of Rhizobium galegae ................ 56 4.5. Future Prospects .......................................................................... 61 4.6. Conclusion ................................................................................... 63 5. Luminescence-Based Microbial Marker Systems and Their Application in Microbial Ecology ......................................... 69 James I. Prosser, Antonio J. Palomares, Matti T. Karp, Philip J. Hill 5.1. Introduction ................................................................................. 69 5.2. Lux-Based Systems ....................................................................... 69 5.3. Luc-Based Systems ....................................................................... 72 5.4. Methodology ................................................................................ 74 5.5. Applications ................................................................................. 76 5.7. Conclusion ................................................................................... 82 6. The Use of the GUS Reporter System to Study Molecular Aspects of Interactions Between Bacteria and Plants ......................................... 87 M. Lambrecht, A. Vande Broek and Jos Vanderleyden 6.1. Introduction ................................................................................. 87 6.2. The Escherichia coli gusA gene ..................................................... 88 6.3. The GUS Reporter system ........................................................... 89 6.4. Application of the GUS Reporter System in Studies of Plant-Bacterium Interactions ................................ 93 6.5. Conclusion ................................................................................... 97 7. Using Green Fluorescent Protein (GFP) as a Biomarker or Bioreporter for Bacteria ......................................... 101 J. R. Stoltzfus, Janet K. Jansson and Frans J. de Bruijn 7.1. Introduction ............................................................................... 101 7.2. Properties of Wild-type GFP ..................................................... 101 7.3. Improving GFP Fluorescence .................................................... 102 7.4. Detection of GFP ....................................................................... 104 7.5. GFP as a Biomarker in Bacteria ................................................. 105 7.6. GFP as a Bioreporter in Bacteria ............................................... 111 7.7. Conclusion ................................................................................. 112 8. Monitoring Persistence and Risk Assessment Following the Field Release of Pseudomonas fluorescens SBW25EeZY6KX........ 117 Mark J. Bailey, Tracey M. Timms-Wilson, Richard J. Ellis, Ian P. Thompson and Andrew K. Lilley 8.1. Introduction ............................................................................... 117 8.2. Construction of P. fluorescens SBW25EeZY6KX and Detection and Monitoring Methods ................................. 118 8.3. Case Study of the Persistence of P. fluorescens SBW25EeZY6KX in Sugar Beet Crops ........... 118 8.4. Survival of Recombinant in Bulk Soil under Glasshouse Conditions ................................................... 119 8.5. Field Sites, Treatment and Sampling of Plants and Soil .......... 120 8.6. Survival of GMM on Over-Wintering Sugar Beet Secondary Growth, Resown Crop Plants and Volunteer Weeds ............... 120 8.7. Would Persistence of a Modified Indigenous Strain in the Soil Environment Pose a Threat? .................................... 122 8.8. Conclusion ................................................................................. 123 9. Use of luc-Tagged Genetically Modified Microorganisms (GMMs) to Study Rhizobial Ecology in Soil Columns, Field Lysimeters and Field Plots ........................................................... 127 Christoph C. Tebbe 9.1. Introduction ............................................................................... 127 9.2. Construction and Properties of GMM Strains L1 and L33 ..... 128 9.3. Experimental Design: What Can Be Achieved with Each System? ...................................................................... 129 9.4. Techniques and Consequences of GMM Soil Inoculations..... 132 9.5. Survival and Spread of GMMs in Soil Columns, Field Lysimeters and Field Plots ................................................ 133 9.6. Conclusion ................................................................................. 135 10. The Field Release and Monitoring of Rhizobial Strains Marked with lacZ and Mercury Resistance Genes ............................................ 139 Viviana Corich, Alessio Giacomini, Elena Vendramin, Patrizia Vian, Milena Carlot, Andrea Squartini and Marco P. Nuti 10.1. Introduction ............................................................................... 139 10.2. Designing the Markers ............................................................... 139 10.3. Construction of GMM Strains 1110, 1111 and 1112 ............... 140 10.4. Microcosm Studies of GMMs ................................................... 140 10.5. Field Trial I; Fate and Impact of an Allochtonous GMM ........ 141 10.6. Field Trial II; Fate and Impact of a GMM Native to the Site .................................................................................... 143 10.7. Conclusion ................................................................................. 144 11. The Field Release and Monitoring of GUS-Marked Rhizobial Strain CT0370 ........................................... 145 Penny R. Hirsch, Tom A. Mendum, Alfred Pühler and Werner Selbitschka 11.1. Introduction .............................................................................. 145 11.2. Construction of GMM Strain CT0370 .................................... 145 11.3. Detection of Strains CT0370 and RSM2004 ........................... 146 11.4. Inoculant Preparation .............................................................. 147 11.5. Field Release .............................................................................. 147 11.6. Screening for pSym Acquisition by CT0370 in the Field ....... 148 11.7. Plasmid Transfer from RSM2004 to CT0370 .......................... 148 11.8. Conclusion ................................................................................ 149 12. Regulatory Aspects ................................................................................ 153 Kersti Gustafsson 12.1. Introduction .............................................................................. 153 12.2. Historical Aspects of the Regulation of Deliberate Releases of GMOs ............................................... 153 12.3. The European Union and the European Free Trade Association .............................. 155 12.4. Canada ....................................................................................... 156 12.5. Switzerland ................................................................................ 157 12.6. United States ............................................................................. 157 12.7. Risk Assessment ........................................................................ 158 12.8. Concern about the Insertion of Genes Coding for Antibiotic Resistance .......................................................... 159 12.9. Concern about the Insertion of Genes Coding for Mercury Resistance ............................................................. 159 12.10. Concern on the Use of Hazardous Chemicals in Connection with the Deliberate Release of a GMM ........... 159 12.11. Ethical Aspects .......................................................................... 160 12.12. Information on Field Releases and Products .......................... 160 Index....................................................................................................... 163