ETDE-DE—1442 Dr. rer. nat. Larissa Hendrickx Natural Genetic Transformation in Acinetobacter sp. BD413 Biofilms: Introducing natural genetic transformation as a tool for bioenhancement of biofilm reactors Berichte aus Wasseigute- und Abfallwirtschaft Technische Universitat Munchen 2002 i k Nr. 171 DISCLAIMER Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. Natural Genetic Transformation in Acinetobacter sp. BD413 Biofilms: Introducing natural genetic transformation as a tool for bioenhancement of biofilm reactors von Dr. rer. nat. Larissa Hendrickx Herausgeber: Prof. Dr.-Ing. Dr. h. c. Peter A. Wilderer Berichte aus Wassergtite- und Abfallwirtschaft Technische Universit&t Mflnchen Berichtsheft Nr. 171 ISSN 0942-914X 2002 Alle Rechtc vorbehalten. Wiedergabe nur mit Genehmigung der Gesellschaft zur FOrderung des Lchrstuhls fttr WassergOte- und Abfallwirtschaft der Technischen Universitat Mtlnchen e.V., Am Coulombwall, 85748 Garehing Dmck: Hieronymus Buchreproduktions GmbH, LerchenstraBe 5,80995 Mtlnchen °D. 'oor mij*i tiade/i 1943-1991 SUMMARY i ZUSAMMENFASSUNG iv ACKNOWLEDGEMENTS ix TABLE OF CONTENTS xii I. INTRODUCTION 2 A. Introduction 4 B. Mission statement 5 H. STATE OF KNOWLEDGE 6 A. Biological enhancement of bioremediation 8 I. Introduction 8 2. Bioenhancement 9 3. Waste water treatment 10 3.1. Types of waste water treatment plants 10 3.2. The advantage of biofilter plants 11 4. Tackling bioenhancement of biofilters 12 Overview 14 Some open questions IS B. Bacterial life in biofilms 16 1. Introduction 16 2. How biofilms develop 17 3. Use of biofilms 18 4. How bacteria communicate 18 5. Signals synthesized by Acinetobacter sp. BD413 20 6. How bacteria defend themselves in biofilms 21 6.1. Diffusion limitation 21 6.2. Physiological limitation 22 6.3. The protected biofilm phenotype 23 6.4. Persisters 23 Overview 24 Some open questions 25 C. Natural genetic transformation 26 1. Introduction 26 2. Mechanisms of natural genetic transformation 27 2.1. Release, dispersal and persistence of the DNA in the environment 28 2.2. The development of competence for DNA uptake by cells in the 29 natural habitat 2.3. The interaction and uptake of DNA with competent cells 31 2.4. The recombination and expression of acquired DNA 33 3. Natural genetic transformation in biofilms 33 4. Regulation of natural genetic transformation 35 4.1. The influence of environmental conditions on natural transformation 36 4.2. Induction of competence for natural genetic transformation 37 4.3. Barriers to natural genetic transformation 38 Overview 40 Some open questions 41 m. MATERIALS AND METHODS 42 A. Used bacterial strains and plasmids 44 B. Media and solutions 44 1. Luria-Bertani medium (LB) 44 2. M9 minimal medium 45 3. Tris minimal medium 45 4. Heat sensitive stock solutions 46 5. Plasmid construction solutions 46 6. Hybridisation solutions (Amann, 1995) 47 6.1. Phosphate buffered saline (PBS) solution 47 6.2. TrisHCl 48 63. EDTA 48 6.4. Hybridization buffer for a certain formaldehyde concentration 48 6.5. Washing buffer for the corresponding formaldehyde concentration 48 in tiie hybridization buffer C. Standard microbiological methods 49 1. Plasmid DNA extraction of £ coli strains based on the method of Bimboim 49 and Doly (Bimboim and Doly, 1979) 2. DNA cloning techniques 49 2.1. DNA restriction analysis 49 2.2. Cloning < 50 3. DNA degradation test 51 4. Triparental conjugation 51 5. Standard natural genetic transformation in pure cultures 51 D. In situ quantification of gene transfer in biofilms 52 1. Tools 52 1.1. The confocal laser scanning microscope 52 1.2. The green fluorescent protein 53 1.2.1. GFP as a tool for in situ detection of gene transfer 53 1.2.2. Monitoring with dual/multiple fluorescence labelling using green 54 fluorescent protein variants and homologues Z Preparation of the samples 55 2.1. In situ natural genetic transformation in biofilms grown on slides 55 2.1.1. Growth of biofilms on slides 55 ; 2.1.2. Transformation on slides 55 2.1.3. Fixation of biofilms on slides (optional) 56 2.1.4. Staining biofilms grown on slides with Syto 17 or Syto 60 nucleic acid 57 stains (Molecular Probes, Eugene, Oregon) 2.1.5. Hybridisation of biofilms on slides with rRNA-directed oligonucleotide 57 probes -v ' ■ 22. In situ natural genetic transformation in biofilms grown in flow cells 57 2.2.1. Growth of biofilms in flow cells 57 2.2.2. Conjugation in flow cells 59 2.2.3. Transformation in flow cells 59 2.2.4. Fixation of biofilms in flowcells 59 2.2.5. Staining biofilms in flow channels with nucleic acid stains 59 2.2.6. Hybridisation of biofilms in flow cells with rRNA-directed 59 oligonucleotide probes 3. Microscopic in situ monitoring of the samples " • ■ 60 3.1. Transformation in a monoculture biofilm 60 3.2. Conjugation betweeaAcinetobacter sp. BD413 and Ralstonia 61 metallidurans CH34 4. Automated image acquisition and semi-automated digital image processing 61 5. Mathematical parameters 62 IV. RESULTS 66 A. Use of gfp and flj>-variants for in situ monitoring of natural genetic 68 transformation in monoculture Acinetobacter sp. BD413 biofilms 1. Introduction 68 2. Plasmid constructions . 69 3. Integration of the fluorescence reporter genes 71 4. Application form of transforming plasmid 71 5. Colocalization 72 6. Spectral overlap 73 7. Reproducibility of in situ transformation using CLSM 74 8. Conclusions . 75 B. Evaluation of eyfp as a disadvantageous gene in Acinetobacter sp. BD413 76 1. Introduction 78 2. Viability and fluorescence of transformants and transconjugants 82 3. Survival of the compromised transformant in a mixed biofilm 82 4. Stability of the recombinant plasmids 84 5. Dissemination of plasmids by transformation in suspended cultures 85 6. Dissemination of plasmids by transformation in biofilms 85 7. Dissemination of plasmids by conjugation in a predefined biofilm 86 8. Conclusions 87 C. In situ quantification of natural genetic transformation in monoculture 88 Acinetobacter sp. BD413 biofilms 1. Introduction 88 2. Effect of biofilm age on natural genetic transformation 89 3. Effect of the concentration of added free DNA 92 4. Effect of biofilm development on natural genetic transformation 94 5. Effect of biofilm ontogenesis on natural genetic transformation 96 6. Conclusions 98 D. Natural genetic transformation of a disadvantageous gene in monoculture 100 Acinetobacter sp. BD413 biofilms 1. Introduction 100 2. Effect of DNA concentration on transformation with a disadvantageous gene 101 2.1. Presence office transformable DNA? 104 2.2. Inhibition of competence development of host cells and/or DNA 105 uptake from the environment? 2.3. Problematic incorporation of the gene, expression of the 107 incorporated genes and/or maturation of the gene product? 2.4. Limiting transformant survival? 109 3. Prolonged challenge with detrimental DNA 110 4. Conclusion 113 V. DISCUSSION 114 A. Use of gfp and ^-variants for the use of monitoring natural genetic 116 transformation in biofilms B. Evaluation of eyfp as a disadvantageous gene in Acinetobacter sp. BD413 118 C. In situ quantification of natural genetic transformation in monoculture 120 Acinetobacter sp. BD413 biofilms D. Natural genetic transformation of a disadvantageous gene in monoculture 124 Acinetobacter sp. BD413 biofilms E. Outlook 128 VI. CONCLUSION 130 A. Main points 132 B. Answers to some of die open questions in: 133 1. Chapter C: Horizontal gene transfer by natural genetic transformation 133 2. Chapter B: Natural genetic transformation and transformant cells in a biofilm 134 3. Chapter A: Natural genetic transformation as a tool for bioenhancement of 134 biofilm reactors VD. REFERENCES 136 Summary Use of gfp and gfp-variants for in situ monitoring SUMMARY This study focussed on the localization and quantification of natural genetic transformation using neutral and disadvantageous genes in monoculture biofilms to investigate gene transfer and expression of the transferred genes in the absence of a selective advantage. Data obtained by this investigation were regarded as initial steps for evaluating the applicability of adding catabolic traits into the indigenous bacterial community of biofilm reactors by in situ natural genetic transformation. Because Acinetobacter spp. strains are readily found in waste water treatment plants and because Acinetobacter sp. BD413 possesses a high effective level of competence, natural genetic transformation was investigated in monoculture Acinetobacter sp. BD413 biofilms. The genes used for transformation encoded for the green fluorescent protein (GFP) and its variants. Monitoring of transformation events were performed with the use of automated confocal laser scanning microscopy (CLSM) and semi automated digital image processing and analysis. The study was divided into four parts: A. Use of gfp and g^-variants for in situ monitoring of natural genetic transformation in monoculture Acinetobacter sp. BD413 biofilms B. Evaluation of the gene encoding for the enhanced yellow fluorescent protein (eyfp) as a disadvantageous gene C. In situ quantification of natural genetic transformation in biofilms D. Natural genetic transformation of a disadvantageous gene in biofilms A. USE OF gfp AND ^-VARIANTS FOR MONITORING NATURAL GENETIC TRANSFORMATION IN BIOFILMS Plasmids were constructed with two different vectors, carrying gfp, or with one vector carrying either gfp or the gfp variants eyfp and ecfp (the gene encoding for the enhanced cyan fluorescent protein). Constructs built on plasmid vector pRK415 gave the most desirable features: integration in the genome was established as a plasmid; the host expressed i Summary Evaluation of eyfp as a disadvantageous gene desired fluorescence; and transfer rates were in a measurable range. General nucleic acid stains were employed for the visualization of the total biofilm and could be combined with green fluorescent protein expression and variants for monitoring gene transfer events. Scanning of large volumes of the biofilm (1.2x10? pm3) resulted in reliable and repeatable results. Hence, natural genetic transformation could be monitored in situ in monoculture biofilms.