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.
B. EVALUATION OF THE GENE ENCODING FOR THE ENHANCED YELLOW FLUORESCENT PROTEIN (eyfp) AS A DISADVANTAGEOUS GENE
While gfp and ecfp did not reveal any negative effects on Acinetobacter sp. BD413, the gene eyfp proved to be disadvantageous for BD413. The strain revealed problematic growth when cultivated as a pure culture. Effects on survival during different modes of growth were evaluated. The most severe detrimental effects were observed when pure culture biofilms were attempted to be grown in a flowcell. Massive lysis initiated after only 21 hours. The longest survival period of the BD413 strain carrying eyfp was obtained with growth on minimal medium agar plates without selective antibiotics pressure. The colonies that were found on plates showed viability for up to 20 days. Survival of the stressed strain BD413 carrying eyfp, grown as a biofilm, was supported if other unlabeled cells were present at least in a ratio of 10:1. When compared with the survival of compromised cells grown under pure culture conditions it seemed that uncompromised cells supported the viability of the cells bearing the burden of a negatively influencing gene that reduced the fitness of its host. In the presence of unlabeled cells the survival of the eyfp expressing BD413 strain was ensured for at least 9 days. In conclusion it was shown that both the maintenance of compromised cells and the persistence of detrimental DNA was enhanced when cells containing a disadvantageous gene were allowed to grow in the presence of cells without negatively influencing DNA.
C. In situ QUANTIFICATION OF NATURAL GENETIC TRANSFORMATION IN BIOFILMS
Natural genetic transformation occurred at high frequency in monoculture biofilms. Amounts as little as 1 fg DNA/ml resulted in detectable levels of natural genetic transformation. Overall, biofilms continuously contained a certain fraction of
ii Summary Natural genetic transformation with a disadvantageous gene competent cells. Nevertheless, cells in young and growing biofilms were more readily transformed. Furthermore, cells containing the transferred recombinant DNA did not disappear right away in the biofilm. Transformants resided mostly at the biofilm base, where biofilm density was the largest. Further investigation revealed an influence of porosity on natural genetic transformation. Low porosity positively correlated with the occurrence of transformation events. Another parameter effecting transformation independent of porosity was the metabolic condition of the biofilm cells. Pre-starvation of the biofilm before exposure of the biofilm to DNA-containing nutrient-rich medium induced transformation events to high levels. However, the presence of cells in the bulk fluid and a high porosity of biofilms reduced transformation to a minimum. Overall, the chances for improving bioenhancement by in situ natural genetic transformation in biofilm reactors containing Acinetobacter sp. BD413 by manipulation of the essential parameters appears high.
D. NATURAL GENETIC TRANSFORMATION OF A DISADVANTAGEOUS GENE IN BIOFILMS
Transformation with disadvantageous DNA was also detected in monoculture biofilms. Like experiments using the neutral gene, successful transformation occurred with minuscule amounts of disadvantageous DNA (1 fg DNA/ml). However, in contrast to transformation using a neutral gene, no correlation between the porosity value of the biofilm and the volume of transformants was detected in experiments studying the effect of exposure time to disadvantageous DNA. However, an inverse relationship was found between changes in biofilm porosity and transformation frequency. Increase and decrease of the biofilm porosity coincided with a decrease and increase in transformation frequency, respectively. In conclusion, it was proved that detrimental genes could be transferred to cells growing in biofilms. Transformed cells amplified the DNA and released the disadvantageous gene into the environment, leading to the surprising observation of a net accumulation of transformants in the biofilm although the received genes provided the host with a selective disadvantage.
E. CONCLUSION
The dissertation presented a first look at in situ natural genetic transformation events in monoculture biofilms. It revealed parameters that can influence natural genetic transformation and are useful for biological enhancement of biofilm reactors. Bioenhancement via natural Summary Conclusion genetic transformation of Acinetobacter sp. BD413 can be achieved due to high transfer frequencies and subsequent expression of the transferred gene in the absence of selective advantage. Even if transformants obtained a significant disadvantage after gene uptake and expression, transformation still occurred and transformants were initially stabilized in the biofilm, suggesting applicability of in situ natural genetic transformation in biofilm reactors that need to process wastes with highly variable xenobiotic concentrations.
iv Zusammenfassung Zusammenfassung
ZUSAMMENFASSUNG
Schwerpunkt der vorliegenden Arbeit war die Lokalisierung und die Quantifizierung der natOrlichen genetischen Transformation, wobei der Gentransfer selektiv neutraler und nachteiliger Gene sowie die Etablierung solcher Gene in Monokultur-Biofilmen in Abwesenheit eines Selektionsfaktors untetsucht wurde. Die gewonnenen Daten dienten als erster Schritt fOr die Evaluierung der MSglichkeit, dutch in situ stattfmdende, genetische Transformation cine in einem Reaktor siedelnde Bakteriengemeinschaft mil zusStzlichen katabolischen Eigenschaften auszustatten. Da Stamme von Acinetobacter spp. hSufig in KISranlagen vorkommen, und Acinetobacter sp. BD413 ein hohes MaB an Transformationskompetenz besitzt, wurde die natflrliche genetische Transformation an Biofilmen aus Acinetobacter sp. BD413-Monokulturen untersucht. Die fOr die Transformation benutzten Gene codieren ftlr das „green fluorescent protein" (GFP) und seine Varianten. Das Monitoring von Transformationsereignissen wurde mit Hilfe des confokalen Laser-Scanning-Mikroskops (CLSM) und semi-automatisierter digitaler Bildverarbeitung und -analyse durchgefOhrt Die Arbeit wurde in folgende Teile gegliedert:
A. Einsatz von gfp und g^Varianten Eh das Monitoring nattlrlicher genetischer Transformation in Biofilmen B. Einsctifltzung des Gens, das fOr das ^enhanced yellow fluorescent protein" (eyfp) codiert, als nachteiliges Gen C. Quantifizierung der natOrlichen genetischen transformation in Biofilmen D. Nattiriiche genetische Transformation mit eyfp in Biofilmen gfp und gfp- Varianten fitr das in situ Monitoring
A. EINSATZ VON gfp UND g/^-VARIANTEN FOR DAS MONITORING NATtlRLICHER GENETISCHER TRANFORMATION IN BIOFILMEN
Es warden Plasmide, die entweder aus zwei verschiedenen Vektoren konstruiert warden, die gfp tnigen oder aus ein Vektor der gfp und dessen Varianten eyfp und ecfp (enhanced cyan fluorescent protein) tnigen. Konstmktionen mit dem Plasmid-Vektor pRK415 wiesen die am meisten erwOnschten Eigenschaften auf: die Integration in das Genom erfolgte in Form eines Plasmids; die Wirtszelle exprimierte die erwOnschte Fluoreszenz; die Transferraten lieflen sich einfech messen. Um den gesamten Biofilm sichtbar zu machen, wurden generelle NukleinsSurefSrbungen durchgefQhrt Gleichzeitig konnte die Expression des GFPs und dessen Varianten (Hr das Monitoring von Gentransferereignissen crfasst werden. Das Scannen eines grofien Biofilmvolumens (1.2xl07 pm3) resultierte in verlSsslichen und reproduzierbaren Ergebnissen. Daher konnte ein Monitoring der natOrlichen genetischen Transformation in situ in Monokultur-Biofilmen durchgeftihrt werden.
B. EINSCHATZUNG DES GENS, DAS Ft)R DAS .ENHANCED YELLOW FLUORESCENT PROTEIN" (eyfp) CODIERT, ALS NACHTEILIGES GEN
Wahrend gfp und ecfp keine negativen Auswirkungen auf Acinetobacter sp. BD413 aufwiesen, stellte sich das Gen eyfp als nachteilig fQr BD413 heraus. Der Stamm zeigte Probleme beim Anwachsen, wenn er als Reinkultur angezflchtet wurde. Die Auswirkungen verschiedener Wachstumsbedi ngungen auf das Oberieben der Zellen wurden evaluiert Die schwersten Schfldigungen zeigten sich, wenn man versuchte, cine Reinkultur im Fliefikanal zu zQchten. Bine massive Zelllyse fend schon nach 21 Stunden statL Bei Kultivierung auf einem Minimalmedium ohne den Selektivdruck dutch Andbiodka zeigte der eyfp tragende BD413 Stamm die lflngste Oberiebensdauer. Die Kolonien auf diesen Flatten wiesen eine LebensfShigkeit von bis zu 20 Tagen auf. Die Oberiebensdauer des belasteten, das eyfp tragenden Stamm BD413 in Biofilmen wurde veriSngert, wenn andere, unmarkierte Zellen im Verbaltnis von mindestens 10:1 anwesend waren. Stellte man den Vergleich mit der LebensfShigkeit von Kompromittierten Zellen in enter Reinkultur an, schien es, dass nicht kompromittierte Zellen die LebensfShigkeit von Zellen fSrdem, die ein negativ be-
vi Zusammenfessung Quanltftcierung der naturlichen genetischen Transformation einflussenden Gen besitzen. In Gegenwart der unmarkierten Zellen wurde das Oberleben des ejj$)-exprimierenden Stamms fQr mindestens 9 Tage gesichert. Daher war sowohl die Oberlebensdauer der kompmmittierten Zellen als auch die Gegenwart von nachteiliger DNS lUnger als erwartet, wenn Zellen, die ein nachteiliges Gen besaflen, in der Gegenwart von Zellen ohne negativ beeinflussender DNS wuchsen.
C. In situ QUANTIFIZIERUNG DER NAT0RLICHEN GENETISCHEN TRANSFORMATION IN BIOFILMEN
Es stellte sich heraus, dass die natOrliche genetische Transformation in Monokultur-Biofilmen haufig auftrat Sogar kleine Mengen wie 1 fg DNS/ml resultierte in nachweisbarer, natOrlicher genetischer Transformation. Im Grofien und Ganzen enthielten Biofilme stSndig einen gewissen Anteil kompetenter Zellen. Dennoch warden Zellen in noch j ungen, wachsenden Biofilmen leichter transformiert Zudem konnten Zellen, welche die transferierte, rekombinante DNS enthielten, sich anfenglich im Biofilm etablieren. Transformanten blieben moistens am Boden des Biofilms, wo die Biofilmdichte am hOchsten war. Weitere Untersuchungen deckten den Einfluss der Porositflt auf die natOrliche genetische Transformation auf. Niedrige Porositat korrelierte positiv mit dem Vorkommen von natOrlichen genetischen Transformationsereignissen. Ein weiterer Parameter, der die Transformation unabhSngig von der Porositat beeinflusste, war der metabolische Zustand der Biofilmzellen, Das Aushimgem des Biofilms vor der Exposition des Biofilms zu nahrstoffreichem, DNS-enthaltenden Medium induzierte hSufige Transformationsereignisse. Allerdings reduzierte sowohl die Gegenwart von Zellen in der den Biofilm umgebenden FlOssigkeit als auch cine hohe Porositat des Biofilms die Transformatfonsrate auf ein Minimum. Insgesamt erscheint jedoch die verlockende Aussicht auf cine erfolgreiche Leistungssteigerung durch natOrliche, in situ stattfindende, genetische Transformation in Biofilmreaktoren, die Acinetobacter spp. BD413 enthalten, durch eine ptaktikable, einfach durchzufOhrende Manipulation der wesentlichen Parameter als realistisch.
d . nat Orliche genetische transformation NACHTEILIGER GENE IN BIOFILMEN
Auch die Transformation mit nachteiliger DNS wurde in Monokultur-Biofilmen detektiert. Wie bei den Experimental, bei denen neutrale Gene eingesetzt wurden, fend eine Transformation mit winzigen Mengen nachteiliger DNS (1 fg DNS/ml) statt. Jedoch wurde
vu Schlussfolgenmg
im Gegensatz zur Transformation mit einem neutralen Gen bei Experimenten, bei denen die Auswirkung der Expositionszeit zu der nachteiligen DNS getestet wurde, keine Korrelation zwischen der Porositat des Biofilms und dem Volumen an Transformanten festgestellt Dennoch zeigte sich erne reziproke Beziehung zwischen dem Wechsel der Biofilmporositat und der Transformationsfiequenz. Schliefilich wurde bewiesen, dass nachteilige Gene in Biofilm-Zellen transferiert werden kOnnen. Die transformierten Zellen vervielfiltigten die DNS und gaben das nachteilige Gen in die Umwelt ab. Oberraschenderweise konnte cine netto Anhaufung von Transformanten im Biofilm beobachtet werden, obwohl die aufgenommenen Gene den Wirtszelle keinen selektiven Vorteil brachten.
E. SCHLUSSFOLGERUNG
Im Rahmen dieser Dissertation wurde zum ersten Mai der Blick auf, in situ stattfindende, natOrliche genetische Transformationsereignisse in Monokultur-Biofilmen von Acinetobacter sp. BD413 gewahrt Es wurden Parameter aufgedeckt, welche die natOrliche genetische Transformation entsprechend den BedOrfiiissen der Forscher beeinfiussen und die biologische Leistung von Biofilmreaktoren verbessem kOnnen. Zusammenfassend kann festgestellt werden, dass cine Leistungssteigerung durch natOrliche genetische Transformation von Acinetobacter sp. BD413 aufgrund von hohen Transformationsraten und die Expression des Obertragenen Gens such bei Abwesenheit eines Selektionsfektors erreicht werden kann. Auch wenn die Transformanten einen signifikanten Nachteil durch die Aufiiahme und Expression des Gens erhalten, findet die Transformation dennoch statt und die Transformanten etablieren sich anfinglich im Biofilm. Daher 1st von der Anwendbarkeit von natOrlicher, in situ stattfindender, genetischer Transformation in Biofilmreaktoren, in denen Abwflsser mit start schwankender Belastung von Xenobiotika aufbereitet werden mOssen, auszugehen.
via Acknowledgements Acknowledgements
ACKNOWLEDGEMENTS
This work was performed at the Institute of Water Quality Control and Waste Management in the Technical University of Munich and was supported in part by the Training and Mobility of Researchers (TMR) Research Networks BioToBio (from Biofilms to Bioreactors) (ERB-FMRX-CT97-0114(DG12-MSPS)) for the period of 01.04.98 to 31.03.99. 1 likewise gratefully acknowledge the support by the Research Center for Fundamental Studies of Aerobic Biological Wastewater Treatment, Munich, Germany (SFB411) for the period of01.04.99 to 31.12.01..
I want to express my heartfelt thanks to my supervisor Dr. Stefim Wuertz for getting file idea of the project and for assisting with highly qualified scientific guidance, discussions and advice. I also want to thank him for teaching me the art of writing a scientific paper and for learning never to give up when firings don ’t go as smooth or quick as one had hoped for. Thank you very much for believing in me and for always being optimistic!
I also want to thank Prof. Wilderer, who made it possible to complete my doctoral thesis at the Institute of Water Quality Control and Waste Management in Garching. His open mindedness to allow basic microbiological research in an institute aimed at engineering-based research projects has given me the opportunity to work with a rare blend of scientists with different educational as well as native backgrounds. I will never forget the unique atmosphere nor the joyous times I have experienced during my stay and therefore I would like to thank you with all my heart
’ I would like to address my great thanks to Dr. Martina Hausner for teaching me all about confocal laser scanning microscopy. Thanks for always helping me when I was stuck with my experiments and giving me expert advice to improve my work. Also, I would like to thank you for supporting me as a friend. Even when you were buried in work you always managed to spend some time listening to me when I needed it. Thank you for guiding me through all the highs and lows, dear Martina.
I don ’t want to forget to thank a lot of other people whose contribution to my work was irreplaceable. I would like to thank Dr. Pierre Wattiau and Dr. Leo Eberl for
ix Acknowledgements Acknowledgements the scientific discussions, the critical notes and introducing me with other microbiological techniques, which have helped me tremendously to improve my work. I likewise want to thank Uschi Wallentits and Anja Vieler for their professional assistance in my experimental work. I would like to give special thanks to Dr. Brigitte Helmreich, Annette Schnell, Veronica Niestroy and Suzanne Wiessler for smilingly helping me with tire administrative work during my stay in Germany. Furthermore, I am so happy to have been able to rely on someone like Dieter Brockelt when it came to finding comfort in a hot cup of coffee, to satisfying the culinary needs in our conventions or parties and numerous other things.
However, my stay in Munich would have been a little bit dull if it wasn ’t for so many friends and colleagues with whom I was lucky to work with. Thank you Carola for all the fun discussions and helping me with the procedure of writing my thesis, Martina for sharing my passion for horrifying novels as well as my fear for little spiders, Regina for having found a friend, Sonia for the many laughs and for having had the best PhD. party together, Christina for sharing complaints and joking them away, Christian ‘zfix’ Ekkerlein for teaching me how speak proper ‘Bayerisch’ (especially in nerve-racking situations) and for keeping my computer optimally functional, Yazmina for being a box full of happy surprises and Barbara for all the talks and an Amazing week-end in Vienna. Lisa, Bernard, Ulli, Fall, Bcnze, Michael, Anja, Ushi (1+2), Peter, Roberto, Isabel, Boris, Patrick, Eva, Pierre, Thomas, Anders, Norbert and so many other people, I thank you for all the fun times we spend at the ‘mensa*, at parties and in Munich!
I would also like to thank my friends and family back home. Thank you, Winnie, for all the encouraging e-mails full of advice that helped me through those sometimes rather frustrating situations. I am so happy we’ve stayed in contact and developed a friendship that I wish to keep for a very long time. Dirk, although you made me miss my home country, it was always a pleasure to see a familiar face among the crowd in fully packed conventions and catch up in Flemish. Very special thanks go to my sister Nadine who traveled with me to Munich and made my stay one of the best periods of my life. To my other sister Sonia and my mum, I thank you so much for all the happy times, for always cheering me up, for the broad supportive shoulders and for always believing in me. Last but not least I want to thank my dad. His strong character, his insatiable thirst for knowledge and his dedication to his profession will always be an inspiration to me. Therefore, I would like to dedicate this work to my father. All the sweat, blood and tears that I invested in this project are mirrored at how my father lived his life. Thank you, dad, for the inspiration.
x I. INTRODUCTION
2
I. Introduction Introduction
I. INTRODUCTION
A. INTRODUCTION
Bioaugmentation or bioenhancement can be obtained by ameliorating the efficiency of biotechnological processes with the use of biological tools. One of the problems of bioaugmentation is the poor survival rate of introduced optimally designed microorganisms under the harsh conditions of a bioreactor or polluted soil site where they have to endure biotic as well as abiotic stresses (Roszak and Colwell, 1987). A possible solution to this problem is the introduction of bacterial gene delivery systems that could introduce desired catabolic genes into the indigenous bacterial community which is already adapted to the environmental conditions (Rittmann et a!., 1990). Nevertheless, the persistence of transferred genes, mostly residing on plasmids, seems to be dependent on selective pressure. Once the concentration of a specific compound is reduced to a certain level, the genes may not be maintained, thus creating an unstable waste treatment system or an ineffective attempt to clean polluted soils (Timmis and Pieper, 1999). Immobilization of interesting strains increases the retention time of the strains and enhances the activity of the systems, making bioaugmentation very attractive (Rittmann et al., 1990; Barbeau et al., 1997; Hajji et al., 2000). While biofilms can be compared to immobilized systems with an increased retention time it was hypothesized in this work that transgenic cells grown in biofilms did not need the support of a selective pressure to be established in a biofilm. This hypothesis was set even a step further: was it possible to obtain and establish transgenic cells inside biofilms, even if the introduced DNA offered the transgenic cell a significant disadvantage?
These two hypotheses instigate the following questions: What environmental parameters affect the frequency of exchange of genetic information in biofilms? Where does gene transfer occur? Does gene transfer and subsequent gene maintenance occur in biofilms, even without providing an obvious advantage to the transgenic host? Does gene transfer and subsequent gene maintenance occur in biofilms if the transferred gene offers a significant disadvantage to the transgenic host? In order to find answers to these questions, the in situ quantification of gene transfer events in undisturbed biofilms must be studied. The discovery of the green fluorescent protein (GFP), which provides a label for living bacteria (Chalfie et al., 1994), enabled the in situ microscopic detection of gene transfer events on a single cell level. This approach has until now been limited to the investigation of conjugal gene transfer L Introduction Introduction
in biofilms (Christensen et al., 1996; Dahlberg et al., 1998; Geisenberger et al., 1999; Normander et al., 1998; Hausner and Wuertz, 1999). Although research has been done on infection of biofilms with bacteriophages (Doolittle et al., 1995; Doolittle et al., 1996), no research on gene transfer by phages in biofilms has been done yet And what little is known about natural genetic transformation in biofilms has only been determined on the basis of transformant growth on selective agar media (Wuertz, 2002; Williams et al., 1996).
Acinetobacter strains are ubiquitous, strictly aerobic, non-motile organisms that ran be isolated from samples of soil, water or sewage (Juni, 1978). Acinetobacter was long regarded a key player for enhanced biological phosphate removal (EBPR) (Fuhs and Chen, 1975). With the development of a gene probe targeted against Acinetobacter spp. (Wagner et al., 1994) it was discovered that the classical culture dependent methods strongly selected for Acinetobacter spp., while Acinetobacter spp. were not primarily responsible for the EBPR process (Mino et al., 1998). However, Acinetobacter strains can degrade recalcitrant aromatic and alicyclic compounds, as well as some aromatic amino acids, mineral oils and synthetic polymers (Baumann et al., 1968; Bode et al., 2001; Benndorf et al., 2001; Pleshakova et al., 2001). hr addition, Acinetobacter strains produce biosurfactants, like emulsan (Karanth et al., 1999; Rouse et al., 1994) and alasan (Barkay et al., 1999), that enhance the bioavailability of poorly soluble compounds. Last but not least, Acinetobacter BD413 (Juni and Janik, 1969) is amenable to gene manipulation by conjugation, transformation as well as transduction (Juni, 1978) and this makes tire strain particularly interesting as a tool to biologically enhance catabolic properties of more hazardous waste treatment facilities.
B. MISSION STATEMENT
hr this work horizontal gene transfer by natural genetic transformation was investigated in Acinetobacter BD413 biofilms using neutral and disadvantageous DNA of gjp variants to monitor tire in situ occurrence of transfer events with exogenous free DNA and tire initial establishment of the transformants in biofilms in the absence of selection. Knowledge about the parameters that effect natural genetic transformation in biofilms can be used to improve desired gene transfer inside biofilm reactors for bioenhancement Effective bioenhancement depends on sufficient gene transfer events and subsequent stabilisation of the genes as well as the survival of the transgenic microorganisms, even if the transgenic organisms are discriminated on the basis of selective advantage. Although this initial study involves monoculture investigations, it offers a basis for future research involving defined mixed and natural biofilms.
5 II. STATE OF KNOWLEDGE
6
II. State of Knowledge Biological enhancement of Bioremediation
A. BIOLOGICAL ENHANCEMENT OF BIOREMEDIATION
"The real distinction is between those who adapt their purposes to reality and those who seek to mould reality in the light of their purposes."
Hemy Kissinger (1923- ), American political scientist
1. INTRODUCTION
A great variety of synthetic chemicals have been released into the environment. Hence, serious pollution by toxic and recalcitrant compounds has been created. Biological treatment processes offer a possibility to reduce the amount of xenobiotics in the environment. Terms like bioremediation (waste treatment with use of micro-organisms) (Timmis and Pieper, 1999) and phytoremediation (polluted soil treatment with use of plants) (Gianfreda and Nannipieri, 2001) describe biological treatment processes that are regarded as a cost efficient technology for the clean up of polluted waters or soils. The three most important goals for successful bioremediation are the bioavailability of the pollutant, the subsequent removal of die toxic compound and the stability of the biodegrading organisms in the system.
The accessibility of the pollutant to microbial attack is dependent on the solubility of the xenobiotic. Hydrophobic pollutants, such as PCBs, are poorly water soluble. Enhancing the solubility of the pollutant would make the recalcitrant compound more readily accessible and this can be accomplished by the use of surfactants. Biosurfactants are produced by bacterial strains and have the ability to desorb and disperse poorly soluble compounds in small, high-surface-area micelles within the water phase (Rouse et al., 1994).
Removal of xenobiotics is achievable by mineralization or by bioconcentration (De wolf et al, 1992) of the contaminant followed by extraction. To be able to degrade the xenobiotic compounds, the biocatalysts that are needed for degradation of the specific compound need to be present in the polluted environment (Janssen et al, 1994; Janssen and Schanstra, 1994). This intrinsic biodegradability is dependent on enzyme specificity and
8 n. State of Knowledge Biological enhancement cf Bioremediation activity. Catabolic enzymes and/or regulatory proteins of degradation pathways are specific for certain pollutants or a certain group of pollutants, possessing similar structures. It is unlikely that micro-organisms developed enough enzyme and/or regulatory activity to mineralize all synthetic chemicals. The comparison of catabolic degradation pathways has revealed that micro-organisms are able to develop new catabolic routes in situ by changing the specificity of catabolic functions throughmutagenesis and by recruitment and assembly of novel genetic traits via horizontal gene transfer (Pries et al., 1994; Prijambada et al., 1995; van der Meer et al., 1992).
Maintenance of the microorganisms that possess suitable metabolic capabilities or the persistence of the interesting genes in a system ensures stable processing of bioreactors and effective clean up of polluted sites. Maintenance of the desired traits demands a prolonged retention time in the system, independent of a selectively advantageous character of the trait
2. BIOENHANCEMENT
Bioenhancement can be obtained by the anthropogenic manipulation of the indigenous bacterial community in order to broaden the effective catabolic or anabolic properties and ameliorate degradation, accumulation or detoxification of a certain undesired compound.
Increasing (unavailability is achievable by solubilisation and translocation of the xenobiotic through the addition of (microorganisms (surfactant producing bacteria, arbuscular mycorrhizal fungi, earthworms,...) (Straube et al., 1999; Vosatka, 2001; Singer et al., 2001) or biomolecules (enzymes, biosuriactants, ..) (Zouboulis et al., 2001; Singer et al., 2000; Olivers et al., 2000).
With the addition of exogenous optimally constructed bacteria (Roane et al., 2001) or functionally adapted bacterial consortia (Hajji et al., 2000) and communities (Saravanane et al., 2001; Barbeau et al, 1997), an enrichment in the total gene pool can be obtained. Likewise, the in situ gene pool can be augmented by using in situ genetic manipulation of indigenous bacterial communities (Rittmann et al., 1990). In situ genetic manipulation of the indigenous bacterial community can be obtained by three major horizontal gene transfer mechanisms: conjugation (transfer of DNA based on cell-to-cell contact) (Ehlers and Bouwer, 1999); transduction (transfer of DNA by bacteriophages) and natural genetic transformation (transfer of DNA in which recipient organisms take up free DNA liberated by excretion or by lysis of dying cells) (Levy and Miller, 1989).
Stability can be supported through the enhancement of bacterial diversity, functional redundancy (Yin et al., 2000) and immobilization (Rittmann et al., 1990). While desired
9 n. State of Knowledge Biological enhancement of Bioremediation
genes can be stabilized by inserting the heterologous genes into the chromosome of the host bacteria (de Lorenzo et al., 1998; Kristensen et al., 1995), stabilization of micro-organisms containing the desired genes can be achieved by encapsulation (Weir et a!., 1996; Hajji et at., 2000), if bioaugmentation in activated sludge plants is desired, or by biofilm grown soil particles (activated soil) (Barbeau et al., 1997) for the use of bioremediation of soils.
3. WASTE WATER TREATMENT
It has been known for many years that waste water needs to be purified before disposal into the environment because discharges of organic substances cause extensive growth of micro organisms and oxygen deficiency. Subsequently, nutrients bom from waste water can lead to eutrophication. While first removal of visible contaminants and organic compounds were considered with priority, later the removal of phosphorous and nitrate became evidently more important because the release of the nutrients nitrogen and phosphorous with wastewater discharges were the major cause of the eutrophication problems (Henze et al., 1997). Nowadays waste water treatment can become even more complicated due to the presence of synthetic chemical compounds. Xenobiotic compounds are recalcitrant and potentially toxic. Toxic compounds could inhibit microbiological processes or even cause waste water reactors to break down (Walter et al., 1997; Lens et al., 1998). After a reactor breakdown, months may be needed to revive a stable bacterial community adapted to the initial reactor conditions. Therefore efforts should be directed towards successful biological enhancement which supports the biocenosis of a reactor by effectively degrading toxic pollutants (Rittmann et al., 1990).
3.1. TYPES OF WASTE WATER TREATMENT PLANTS
There are essentially two different types of biological waste water treatment plants: biofilm reactors and activated sludge plants. Many variations exist among these two types (Henze et al., 1997). In biofilm reactors bacteria aggregate on carrier material and the waste water that needs to be treated moves through this material. Microorganisms readily growon substrates like soil or sediment particles as well as on rocks, plastic media and membranes where they can form biofilms. Biofilms are composed of cells, water and extracellular polymeric substances (Costerton et al, 1994). They are ubiquitaiy and present a customized niche for more than 90% of all micro-organisms.
In activated sludge micro-organisms live as suspended cells or as floes. The mass of activated sludge is kept moving in water by stirring or aeration (Henze et al., 1997). In the
10 n. State of Knowledge Biological enhancement of Bioremediation
settling tank the floes are separated from the treated waste water by sedimentation (Henze et al., 1997). Therefore, activated sludge plants do not only need organisms capable of degrading certain compounds, but also a fast and effective floe sedimentation, which is not so important in the functioning of a biofilm reactor because settling of removed biomass from biofilm reactors (abrasion, erosion, sloughing,...) is not returned to the bioreactor.
Selection for micro-organisms that live in activated sludge plants is dependent on the application of electron acceptors and nutrients, settling characteristics of the floes, temperature, growth rate of the organisms and viability of suspended life forms. In the case of biofilm reactors is selection mostly dependent on adhesion characteristics and growth rate at a particular microcondition (Henze et al., 1997). Biofilm reactors carry many unknown parameters. Easy determinable physical reactor conditions like temperature, oxygen concentration or pH are difficult to measure in biofilm reactors. Likewise there is much difficulty in designing mathematical models that can describe the biological processes of a biofilm reactor completely (Falkentoft, 2000). However, biofilm reactors carry a number of interesting advantages in comparison to activated sludge plants and these are described in the next paragraph.
3.2. THE ADVANTAGE OF BIOFELM REACTORS
It can not go unnoticed that many water treatment plants rely on biofilms. One of the advantages of biofilms is compactness. The small footprint area of biofilm reactors is caused by two factors (Falkentoft, 2000): high volumetric biomass concentration (Kwok et cd., 1998) and high biomass retention times (Bishop, 1997). Further, there is no need of a final clarifier for many biofilm processes (Borregaard, 1997). And if clarifiers are needed for the settling of biomass, removed after filter backwashing steps, these settling tanks may be more compact (Falkentoft, 2000). The retention time of the micro-organisms is dependent on the frequency of backwashing. Bacteria that are not readily detached from the carrier material have an extended retention time and this offers the opportunity for slowly growing micro-organisms to establish themselves which would have been difficult in activated sludge plants. Another advantage is the high species variety in biofilm communities. The extended retention time of bacteria growing mar the substratum is a reason why an enhanced variety of micro organisms is allowed to develop in biofilms (Christensen et al., 1998). Another reason is the formation of microniches that posses their individual environmental conditions containing concentration gradients of adsorbed chemical pollutants by diffusion limitation or zonation caused by degradation and usageof certain compounds that are passing through the biofilm by diffusion (Falkentoft, 2000; James et al., 1995). Extended retention time and species richness in biofilm reactors suggest desired conditions for remediation of synthetic pollutants.
11 11. State of Knowledge Biological enhancement of Bioremediation
A high retention time is needed because initial catabolisation of easily degraded carbon sources suppresses utilisation of recalcitrant compounds (Nflsslein et al, 1992; Goldstein et al., 1985). Other factors that inhibit biodegradation of recalcitrant compounds and which could be eliminated by a higher retention time (Hajji et al., 2000) include concentrations which are too low to induce enzymes of catabolic pathways and insufficient densities of microbial degraders (Ramadan etal, 1990; Wiggins etal., 1987).
4. TACKLING BIOENHANCEMENT OF BIOFILM REACTORS
Micro-organisms, living in biofilm reactors are subjected to abiotic (temperature, nutrient availability, etc.) and biotic (predation, competition, etc.) stresses which can limit the survival and growth of introduced bacteria (Roszak and Colwell, 1987; Tiedje et al., 1989; Mehmannavaz et a!., 2001; Bouchez et al., 2000).
In tiie literature, bioaugmentation of biofilm reactors is scarce. Nevertheless some of these sporadically reported investigations are optimistic. A bioaugmented fixed bed bioreactor was effectively used to remove toxic pollutants (Duba et al., 1996; Knapp and Faison, 1996) by the addition of degrading bacteria. Also stimulation of translocation of bacterial cells in soil minicolumns with the use of biosurfactants sufficed to obtain small bioactive zones over a distance of hi order to obtain effective bioaugmentation by in situ genetic manipulation three important conditions need to be met (Nflsslein et al., 1992): first, transfer of the recombinant gene needs to occur readily to introduce the gene in the collective gene pool (Fulthorpe and Wyndam, 1991; Christensen et al, 1998); secondly, the transgenic genes enable their hosts to degrade the target chemicals made available for microbial attack via the introduced modified catabolic pathways (Janssen et al., 1994; Janssen and Schanstra, 1994; Rouse et al, 1994); 12 n. State of Knowledge Biological enhancement of Bioremediation and thirdly, the transgenic indigenous strains need to survive and grow under the environmental and processing conditions (Winkler el al., 1995; NOsslein et al, 1992; Bouchez et al., 2000). It seems that biofilms present good conditions for bioaugmentation by in situ genetic manipulation. Survival of strains that do not have a direct competitive advantage need an environment that offers a high microbial diversity and time to grow and to mineralize recalcitrant compounds. Both conditions are offered by biofilms. The biodiversity of biofilms also offer an ideal niche to obtain complete mineralization of recalcitrant compounds, which is in many cases accomplished by the help of bacterial consortia (Watnick and Roller, 2001) certainly when metabolization of certain compounds leads to the production of even more toxic intermediate compounds. Furthermore, tire transfer of plasmids or transposons by conjugation from a donor to a recipient cell is likely to be prevalent in biofilms due to the relative spatial stability of bacterial cells within the extracellular polymeric substance (EPS) matrix (Hausner, pets. com.). Similarly, natural genetic transformation, in which competent recipient organisms take up free DNA liberated by excretion or by lysis of dying cells, may readily occur in biofilms, since the accessibility of free DNA may be increased as a result of its sorption to and accumulation in tire EPS matrix, where it may be bound and protected (Palmgren and Nielsen, 1996). The potential for transduction to infect cells in biofilms has been investigated only sporadically (Doolittle et al., 1995; Doolittle et al., 1996; Hughes et al., 1998). It has been mentioned that EPS functions as a barrier, preventing adsorption of the phage to the host (Costerton et al., 1999). Further investigation on transduction as a means of gene transfer is certainly needed because as of yet only the influence of lytic phages on biofilm cells has been studied. Therefore the importance of gene transfer by transduction could be underestimated (McLean et al., 2001). Biofilms possess ideal circumstances wherein a stable genetic enrichment in tire indigenous gene pool of a biofilm based reactor could be obtained. 13 II. State of Knowledge Biological enhancement of Bioremediation OVERVIEW Effective bioenhancement is obtained bv addition of biological tools that help to: - stimulate the bioavailability of recalcitrant compounds to bacterial attack - improve the biodegradability of these compounds by accruement of the needed biocatalysts (via addition of genes, strains, consortia or communities) - extend effective bioconcentration and extraction of toxic compounds for subsequent easy removal - stabilize the bioaugmented process. Problems while obtaining bioaugmented biological processes include: - inhibitory degradation by easily metabolizable carbon sources - degradation resulting in even more toxic intermediates - instability of introduced biocatalysts - low densities of microbial degraders - low solubilization of the pollutant - low adsorption of the pollutant - insufficient induction of, catabolic pathways by low xenobiotic concentration Bioenhancement of bioremediation bv biofilm reactors looks promising because of the: ...... - prolonged retention time of biocatalysts and xenobiotics - increased gene transfer - support of slowly growing organisms - stabilization and symbiosis of consortia - increased resistance against toxic compounds - enhanced bacterial diversity - enhanced functional redundancy 14 IL State of Knowledge Biological enhancement of Bioremediation SOME OPEN QUESTIONS Can the treatment processes of biofilm reactors be augmented by amelioration of the solubility of a xenobiotic by the addition of enzymes, biosurfactants or higher organisms? Will it be possible to completely mineralize low concentrations of xenobiotics in biofilm reactors? Can effective degradation of recalcitrant compounds occur in the presence of inhibitory concentrations of easily metabolizable carbon sources? Can stored toxic compounds easily be extracted from biofilm reactors? Can biofilm reactors undergo in situ genetic manipulation using natural genetic transformation or transduction? Is natural genetic transformation using Acinetobacter sp. BD413 in natural biofilms effective enough to be used for bioaugmentation? Can biocatalysts be adequately stabilized in biofilm reactors? And is this stabilization possible even if the desired degraders possess a less fit phenotype and seem to have a negative competitive advantage compared with other members of the biofilm community? What level of bacterial diversity and functional redundancy is needed to obtain stable processing? 15 II. State of Knowledge Bacterial life in biofilms B. BACTERIAL LIFE IN BIOFILMS "A little and a little, collected together, become a great deal, the heap in a bam consists of single grains, and drop and drop makes an inundation." Moslih Eddin Saadi (1184-1291), Persian poet 1. INTRODUCTION A natural biofilm is a multi-species microbial community which consists of single bacterial cells as well as fungi, algae, protozoa, debris and corrosion products, embedded in an extracellular polymeric matrix (Costerton et al., 1995; Watnick and Kolter, 2000). Biofilms grow at interfaces and are adapted to the needs of the multi-species community (Marshall, 1994). Metabolical tasks are divided among the members of the biofilm and genetic material is exchanged at high rates (Hausner and Wuertz, 1999; Watnick and Kolter, 2000). Biofilms protect microorganisms against washout of slow growing bacteria under low hydraulic residence times (Bishop, 1997), offer higher nutrient concentrations (Flemming, 1995) and high retention times of recalcitrant compounds due to adsorption in tire extracellular polymeric matrix (Spath et al., 1998; Wuertz, 2000). Furthermore, biofilms allow interspecies interaction by split-second signaling and nutrient cycling (James et al., 1995), offer resistance to antimicrobial compounds (Lewis, 2001), to physical stress like extremes of pH or temperature (Flemming, 1994) and to desiccation (Roberson and Firestone, 1992; Wuertz, 2000). Watnick and Kolter (2000) referred to a biofilm as ‘a city of microbes’, with the organized chaos of a human city. A city with its own hierarchical structures, where its inhabitants and communities interact with each other, with a prepared defense system and which can expand in times of wealth or die out during deflation. This chapter will focus on biofilm development, the use of biofilms, the possible ways of communication between the microorganisms in biofilms and their defense mechanisms against bactericidal challenges. 16 n. State of Knowledge Bacterial life in biqfllms 2. HOW BIOFILMS DEVELOP The early imageof biofilms, long before the confocal laser microscope was developed, was that of a plain and dull amorphous layer of bacteria that were packed and squeezed on top of each other. This view was mostly supported by the investigation of biofilms after harsh deforming and dehydrating preparations that were needed to investigate biofilms with the scanning electron microscope. Ever since biofilms were investigated with the confocal layer scanning microscope (CLSM) a multifaceted biofilm architecture was discovered (Lawrence et al., 1991; Costerton et al., 1995). Costerton (1995) defined biofilms thereafter as ‘the highest phenotypic expression of the bacterial genome* and suggested that the organized structure of this bacterial community could not be mere accidental. Biofilms are an ubiquitous life form and their organized structure allows penetration of nutrients and oxygen deep inside. It did not last very long before numerous workgroups concentrated on the development and tiie characterization of biofilms (Flemming, 1994; Costerton et al., 1995; van Loosdrecht et al., 1997; Lawrence and Neu, 1999; O’Toole et al., 2000; Watnick and Kolter, 2000; Wuertz, 2000 ). Biofilms grow, after initial aggregation at an inviting colonizing substratum, into a complex three-dimensional architecture (Costerton et al., 1995). Like inoculated bacterial suspensions, a biofilm develops in three phases: the induction phase, the logarithmic accumulation phase and a stabilization phase (Flemming, 1994). The irreversible adsorption of macromolecules to substrata diminishes the critical surface tension and this can lead to a net slightly negative charge (Flemming, 1994). This adsorption of macromolecular particles creates a conditioning film that influences the initial attachement of the bacteria. After an initial reversible attachment phase, an irreversible attachment phase is entered, whereafter growth exponentially increases (O’Toole et al, 2000). A biofilm is formed with an architecture dependent on the applicated shear forces, growth rate of bacterial mixtures, nutritional loading rate (Watnick and Kolter, 2000), signals produced by the growing microorganisms (Davies et al., 1998), amount and composition of produced extracellular polymeric substances (EPS) (Flemming, 1994) and induced biotic/abiotic stresses (Davey and O’Toole, 2000). The biofilm may contain channels and is built with optimally positioned microbial consortia (Watnick and Kolter, 2000). During growth of the attached bacteria, other microorganisms like fungi and protozoa start to aggregate on the surface to form a wild jungle of microbes in a microbial Amazon forest Just like the nature of the Amazon forest is molded by the influence of physical (storms, soil type, wind, temperature, rain), chemical (human intervention) and biological influences (competing photosynthesizing organisms, prey- predator competing organisms); a mature biofilm is shaped by comparable physical (shear, the nature of the substratum (easy/difficult detachment), aspects of gas formation in the anaerobic 17 n. State of Knowledge Bacterial life in biqfttms parts of the biofilm, temperature conditions, pH), chemical (presence of EPS complexing agents, presence of bactericidal agents) and biological influences (nutritional status, concentration gradients of electron acceptors or nutrients, lysis of cells, presence of predators) (Flemming, 1994). 3. USE OF BIOFILMS Although biofilms are responsible for many problems (biofouling, clogging, chronic infections, etc.) and many efforts are undertaken to eradicate biofilms (Flemming, 1994; Lewis, 2000), biofilms have many beneficial uses. The attraction in using biofilms for biotechnological applications resides in the extended retention time, prolonged metabolical activity, easy and Stable process handling and a reduced biomass (Flemming, 1994). As mentioned in the previous chapter biofilms are utilized under various forms for waste water treatment (Flemming, 1994). Flemming (1994) regarded also bacterial aggregates in activated sludge systems as a special case of ‘suspended ’ biofilms. Next to treatment of waste waters, biofilms are used for the cleanup of to be exhausted contaminated air (Ergas et al„ 1999), contaminated soil sites (Semple el al., 2001), remediation of solid wastes (Mailer and Bardtke, 1988) and preparation of potable water (bioregeneration) (Flemming, 1994). Furthermore, biofilms are used in the production of enzymes, fuels (Kunduru and Pometto, 1996; Obgonna et al., 2001) and other commercial products (Flemming, 1994; Kargi et al, 1990; Ho et al, 1997). Also in the extraction of heavy metals (Hutchins et al., 1986) or coal fine recovery (Sharma et al., 1999), biofilms have been successfully employed. Future applications could even be the cleanup and regeneration of water and air in enclosed systems for creating viable settings in extra orbital space (Ichikawa et al., 1999). 4. HOW BACTERIA COMMUNICATE Cell-to-cell communication can be defined as a phenotypic response to signals produced by micro-organisms induced by a certain condition. Cell-to-cell communication signals do not enclose the signals restricted to intracellular signal-response cascades that respond to extracellular environmental signals with no cellular origin like chemotaxis (Bren and Eisenbach, 2000), nitrogen starvation (Stock et al., 1989) or entry into the stationary phase in E. coll Salmonella or Vibrio (Kolter et al., 1993). Extracellular cell-to-cell signaling molecules are used by both Gram negative and Gram positive bacteria to regulate a variety of physiological functions. Particularly quorum sensing, 18 n. State of Knowledge Bacterial life in biofilms the cell density dependent signaling mechanism, has been under extensive investigation. Quorum sensing is a signaling mechanism that is dependent on population density based on the accumulation of small extracellular signaling molecules (Swift et al, 1996). Quorum dependency for the expression of certain traits is an elegant way of bacteria to save energy (bioluminescence), outsmart the host or competing organisms (virulence factor production, antibiotic production) or have access to desired components after safety clearance (competence development, conjugal transfer) and prepare for or escape suboptimal environmental conditions (starvation response, survival, swarming motility, biofilm formation) (Eberl, 1999; Msadek, 1999; Rowbury, 2001). The nature of cell-to-cell communication signals as well as its inductive conditions are diverse. While some W-Acyl-homoserine lactones (AHL) and peptides are induced by a threshold quorum (Kaplan and Greenberg, 1985; Diep et al., 1996), others are induced by environmental factors (Flavier et al., 1998; Klecrebezem et al., 1997). The autoinducer AI-2 has until now only been connected with quorum sensing (Bassler, 1999; Burette et al., 1999). Alarmones are constitutively expressed signals that are only activated under certain environmental conditions (Rowbury, 2001) and the A-signal is a hybrid signal which is dependent on a threshold quorum and starvation conditions (Kaplan and Plamann, 1996). Among other signals Streptomyces strains synthesize y-butyrolactones which structurally resemble AHLs and are involved in the control of streptomycin production (Horinouchi and Beppu, 1992). Another signal is a diffusable extracellular fatty acid derivative in the plant pathogen Xanthomonas campestris that regulates exoenzyme production and virulence (Barber et al., 1997) or the volatile extracellular factor (3- hydroxypalmitic acid methyl ester) was found to regulate virulence gene expression in wild type Ralstonia solanacearum (Flavier et al., 1997). Cell-density-dependent gene expression was also observed to be regulated through a glycoprotein (conditioned medium factor) that is secreted in response to starvation (Clarke and Corner, 1995). Diketopiperazines and quinolones, isolated from Pseudomonas and other gram-negative bacteria were found capable of activating or antagonizing AHL biosensors at high concentrations (Holden et al., 1999; Pesci et al., 1999). While universal signaling themes exist, variations in the design of the extracellular signal, the signal detection apparatus and the biochemical mechanisms of signal relay have allowed cell-cell signaling systems to be exquisitely adapted for their varied uses (Bassler, 1999). Over the past years it has become apparent that several bacteria employ more than one quorum sensing system. Thoroughly studied examples are the hierarchical autoinduction cascade regulating multiple phenotypes in P. aeruginosa (Eberl, 1999) or the convergent 19 II. State of Knowledge Bacterial life in biofilm sensing pathways that mediate response to competence factors and inducing expression of several other phenotypes in B. subtilis (Solomon et al., 1995; Msadek, 1999). Whereas it is generally assumed in the literature that gram negative bacteria communicate using AHL signaling molecules and gram positive bacteria communicate with peptide signals, it should be noted that gram negatives could likewise be able to use peptide signals (Michiels et al., 2001). That gram positives could use AHL molecules, has not been observed yet although production of y-butyrolactone molecules, which are AHL structural homologues, by Streptomyces species have been reported (Horinouchi and Beppu, 1992), but it was reported to be unlikely that this regulatory mode involved activity of LuxR or LuxI homologues (Kleerebezem et al, 1997). 5. SIGNALS SYNTHESIZED BY Acinetobacter SP. BD413 Although Acinetobacter strains can cause serious infections as nosocomial pathogens in hospitalized patients (Dijkshoom et al., 1990) and are readily found in soil (Juni, 1978), most investigations on cell-to-cell communication have been limited to human pathogens like Pseudomonas (Wang et cd„ 1996; Glessner et al., 1999; Pesci and Iglewski, 1999; Chancey et al., 1999), Yersinia (Throup et al., 1995), Aeromonas (Swift et al., 1997), or Burkholderia strains (McKenney et al., 1995); plant pathogens like Erwinia (Bainton et al., 1992), Xantkomonas (Barber et al., 1997); plant associated bacteria like Rhizobitim strains (Gray et al., 1996) and soil bacteria like Bacillus subtillis (Msadek, 1999), Pseudomonas strains (Pierson et al, 1994; Dunny and Winans, 1999), Ralstonia solanacearum (Flavier et al, 1998), Agrobacterium (Fuqua et al, 1995) and Pantoea stewartii (Von Bodmann and Farrand, 1995). The properties that are regulated by autoinduction in soil bacteria vary between carbapenem production (Bainton et al., 1992), antibiotic production for plant protection against pathogens (Pierson et al., 1994), aggregation into a symbiotic state with Leguminosae (Gray et al., 1996), conjugation (Piper et al., 1993) and competence for natural transformation (Msadek, 1999). Only a single study reports AHL production in Acinetobacter strains (Gonz&les et al., 2001). The signals were detectable using an Agrobacterium tumefaciens reporter strain. Acinetobacter sp. BD413 was able to produce 4 signals when grown in minimal medium. Phenotypes induced by these signal molecules as well as the characterization of the signals are still unknown. 20 H. State of Knowledge Bacterial life in biafibns 6. HOW BACTERIA DEFEND THEMSELVES IN BIOFILMS Biofilm cells me less affected by bacteriophage attack and physical stress like heat or extremes of pH. They may also survive periods of starvation better than planktonic cells. Adaptive responses against nutrient limitation supported by the biofilm mode of growth simulate in a way the fight or flight response encountered in higher organisms by scaffolding on the EPS that surround the biofilm cells and detachment of cells or aggregates in order to find more suitable niches (Allison et al., 1998). Starvation could also signal cells in biofilms to adopt a more starvation resistant state. When grown at high cell density, Rhizobium leguminosarum was better adapted for survival in a nutrient deficient environment compared to cells starved at low cell density (Thome and Williams, 1999). Biofilms can also be resistant to chemical stress, like hydrogen peroxide (Liu et al., 1998), monochloramine (Huang et al., 1995) or biological stress in the form of antibiotics or immunogenic responses (Costerton et al., 1999). Resistance to these more acute challenges have been explained by a number of theoretical models, each of which explain the ability to conquer bactericidal assault in different ways. Resistance in these models has been conferred to biofilm-dependent barriers against harmful components at die multicellular level (penetration limitation through the biofilm) (Costerton et al., 1999), cellular level (physiological limitation in metabolically downregulated cells) (Brown and Gilbert, 1993), genetical level (the protected biofilm phenotype expressed by cells grown in biofilms) (Costerton et al., 1999) or an extracellular barrier (persisters embedded in the glycocalyx) (Lewis, 2001). 6.1. DIFFUSION LIMITATION Failed penetration into the biofilm may lead to survival of biofilm cells residing at deeper layers of tire biofilm, which are protected against tire challenge of an antimicrobial agent Diffusion limitation can be accomplished in several ways. Extracellular polymeric substances for example can shield bacterial cells against opsonisation or phagocytosis (Costerton et al., 1999). The action of bacteriophages can be limited in the same way and phages that yield polysaccharide depolymerases during bacterial lysis may penetrate more readily into a biofilm (Sutherland and Wilkinson, 1965). Penetration limitation can also be due to a reaction-diffusion mechanism. Synthesis of an antibiotic-degrading enzyme such as a p-lactamase enzyme (Nichols et al., 1989), sorption of an antibiotic to biofilm components (Stewart, 1996), consumption of active chlorine by surface layers of the biofilm before full penetration can occur (DeBeer et al., 1994) can all lead to concentration gradients of the antibacterial agent in tire biofilm. 21 n. State of Knowledge Bacterial life in biofilm 6.2. PHYSIOLOGICAL LIMITATION This resistance mechanism is built on the hypothesis that physiological gradients within the biofilm enable the presence of cells in different physiological states, both resistant and vulnerable to antimicrobial challenge (Brown and Gilbert, 1993). Possible factors responsible for this resistant state are growth rate (Evans et a!., 1991), biofilm age (Anwar el al., 1992) and starvation (Jenkins et al., 1988). Gradients of the physiological status within a biofilm have been demonstrated (Huang et al., 1995; Sternberg et al, 1999). The hypothesis of physiological limitation goes as follows: The more metabolically active and more susceptible cells reside at the biofilm-medium interface, while cells that are metabolically downregulated by nutrient limitation or product inhibition hide in the deeper layers of a biofilm, near the substratum (Sternberg et al., 1999). When challenged with a growth rate-dependent antimicrobial agent, only metabolically active cells will be killed. Cells in deeper layers of a biofilm would not be affected by the bactericidal challenge by virtue of their existence in a non-growing state. Hence, at least one part of the biofilm would escape killing and the biofilm would have survived the treatment This hypothesis, however cannot explain biofilm resistance. If antimicrobial agents kill biologically active cells residing at the biofilm-medium interface, then those cells are expected to lyse and, as a result parts of the biofilm detach. This would nourish the metabolically downregulated cells now located much nearer to the bulk fluid. Hence the next layer of foe biofilm would be more susceptible to foe antimicrobial agent and this would continue until all layers in the biofilm have been killed (Xu et al., 2000). Xu etal. (2000) give two possible alternatives to this hypothesis. Slowly growing or non-growing cells may not be stimulated by replenishment of nutrients alone. The authors suggested the action of cell-to-cell communication for entry into the dormant or protected phenotypic state. This, however, would lead to the same problem as before. Killing and removal of active cells in the upper layers of a biofilm would lead to contact between layers residing more deeply within foe biofilm. Cell-to-cell signals inducing a certain protective phenotype would eventually diffuse out, leaving cells to exit the dormant state and hence be susceptible to killing by foe antimicrobial agent P. putida R1 cells residing in foe centers of microcolonies, which were metabolically down-regulated by a general exhaustion of foe nutrient supply responded as quickly as cells located at foe surface of microcolonies to foe addition of an easily metabolizable carbon source (Sternberg et a!., 1999). The other alternative could be more correct It was speculated that some treated cells would continue to use nutrients for a prolonged time although they were irreparably compromised in their ability to reproduce. These damaged cells, residing at foe outer layers of foe biofilm, would not detach, still use nutrients but not divide. There, they would shield underlying cells from 22 n. State of Knowledge Bacterial life in biofilms nutrient exposure that would stir them from their non-growing state and render them susceptible to killing. 6.3. THE PROTECTED BIOFILM PHENOTYPE This hypothesis is based on the assumption that certain traits are overexpressed when cells are grown in a biofilm mode (Costerton et at., 1999). Although several studies involve identification of genes induced solely under biofilm conditions (Ding et al., 2001; Parkins et al„ 2001), this protection mechanism can not yet be accepted or dismissed. This model, however, presents cells, expressing this mystical protected biofilm phenotype, as a sort of little Rambos, genetically prepared to conquer any possible challenge put upon them. And this makes the suggested hypothesis very hard to prove. 6.4. PERSISTERS In 1944, Joseph W. Bigger published an important discovery in the journal ‘The Lancet* (Bigger, 1944; Lewis, 2001). The author reported that treatment of a staphylococcal culture with penicillin still left a small number of “persisters** which survived the treatment Furthermore he speculated that these persisters had either a higher heritable resistance to growth or were variants that had die same susceptibility to growthinhibition by penicillin as the bulk of the cells but are insensitive to killing by penicillin. In a further experiment he observed that the persisters, which survived the first treatment with penicillin, were similarly sensitive to growth inhibition and produced new persisters. Hence persisters were not mutants. Persisters also do not represent a special stage in the cell cycle, nor are they in a special dormant state of no growth(Lewis, 2001). Lewis (2001) proposes herewith a model, where biofilm cells do not have more resistance than planktonic cells. Paradoxically, persisters are inhibited in growth due to the presence of the antibiotic itself, thereby inhibiting the bactericidal effect of the antibiotic. The inaccessibility, however, of immunity factors towards these persisters, not necessarily increased in number inside the biofilm, hidden in tire glycocalix, is responsible for the subsequent reformation of the biofilm (Lewis, 2001). 23 II. State of Knowledge Bacterial life in biofilms OVERVIEW Biofilms offer: - high nutrient concentration - conditions for high gene exchange - high retention times of nutrients and micro-organisms - ideal juxtaposition of microbial consortia - resistance against physical, chemical and biological stress Biofilm development: - the induction phase on a conditioned surface - the logarithmic accumulation phase into a multi species organized microbial community - the stabilization phase with attachment, detachment, growth and decay of the microbes Beneficial uses of biofilms: - treatment of waste water, contaminated soil and other solid wastes - prepare potable water - produce commercially interesting products - bioleaching Bacteria talk with: - signals induced by a threshold quorum - signals induced by environmental conditions - constitutively expressed signals - signals induced by a threshold quorum under certain environmental conditions Bacteria signal to: - save energy - outsmart the host or competing organisms - check for safety - prepare for harsh conditions - escape harsh conditions Hypotheses why bacteria can resist challenges in biofilms are: - failed penetration of the bactericidal agent - slowly growing or non-growing state of embedded organisms - induction of resistance genes in the attached state of cells - persistent ghost cells interrupted in metabolism before cell death 24 H. State of Knowledge Bacteria1 life in bicfihns 25 n. State of Knowledge Natural Genetic Transformation C. NATURAL GENETIC TRANSFORMATION "The essence of success is that it is never necessary to think of a new idea. It is far better to wait until somebody else does it, and then to copy him in every detail except his mistakes." Aubrey Menen (1757-1827), English poet L INTRODUCTION In 1944, Avery et al. (1944) discovered that DNA acted as the carrier of heritable traits after reexamining an experiment done by Griffith (1928) involving a peculiar phenomenon: the expression of a new phenotype in one microbial strain after being in proximity of another microbial strain possessing this specific phenotype. It was the first report of horizontal gene transfer by natural genetic transformation. Natural genetic transformation is defined as the active uptake of free DNA, released in flie environment through lysis or excretion, by bacterial strains that are naturally competent for transformation. Natural genetic transformation has been observed in a wide range of organisms (Lorenz and Wackemagel, 1994). Next to natural genetic transformation, bacteria can also extend their genetic variability by two other major horizontal gene transfer mechanisms: bacterial conjugation and transduction via bacteriophages. A few reports on alternative ways of acquiring new genes have been published. Among these are retrotransfer (Mergeay et al, 1987; Blanco et al., 1991), vesicle or bleb mediated horizontal gene transfer (Yaron et al., 2000; Dorward et al., 1989) or horizontal gene transfer with gene transfer agents (Bertani, 1999; Lang and Beatty, 2001). They can be regarded as slightly different variants of the three major horizontal gene transfer mechanisms. Retrotransfer is a special plasmid-specific form of conjugation, involving reciprocal genetic exchange that has been observed in a number of bacteria (Mazodier and Davies, 1991). Vesicle mediated transfer is an adapted form of natural genetic transformation. Haemophilus strains, for example produce vesicles with DNA-binding activity, that are shed into the medium (Goodgal, 1982) or located on the cell surface (Kahn et al., 1983). After competence development they are able to take up 26 H. State of Knowledge Natural Genetic Transformation DNA from these vesicles, having first rescued free DNA from the DNAse sensitive state (Kahn et al., 1983). Gene transfer agents (GTA) are bacteriophage-like particles with a head and a tail, but they lack plaque-forming activity (Mans, 1974) and comprise only bacterial DNA (Yen et al., 1979). Genes encoding for the formation of GTA particles reside on the genome of GTA producing strains and they are not included in the genome of the particle itself (Lang and Beatty, 2001). Off all force horizontal gene transfer processes, natural genetic transformation has foe least requirements (Lorenz and Wackemagel, 1994). To allow conjugation and transduction a lot more conditions need to be met. Conjugation is a horizontal gene transfer mechanism that requires two cells to be in contact with each other when genes are passed from one to the other. The mechanism needs a living and active donor and recipient that are spatially and temporally located very near to each other (Ippen-Ihler, 1989). Hence, extrachromosomal units or plasmids can be transferred from the donor to foe recipient Transduction is mediated by foe transfer of DNA between cells via bacteriophage particles through injection of packaged prokaryotic genomic DNA into foe recipient cell. Transduction does not need an active recipient to infect but donor cells infected by phages need to be active to build the progeny phage. The phage acts as a DNA vehicle between the related donor and recipient that do not need to be near each other nor present in the same location at a certain time (Stotzky, 1989). For transformation to occur there is no need for the donor to be alive. Spatial and temporal separation will only be a problem when DNA is released in an environment where it cannot be adsorbed on minerals, (Galori et al., 1994), humic acids (Tsai and Olson, 1992) or other components (Lorenz and Wackemagel, 1994), where it is shielded against DNase attack. As long as DNA is not degraded it can potentially be used for natural genetic transformation of recipient cells on the condition that the cells possess tire ability to change into a state conferring competence for transformation (Lorenz and Wackemagel, 1994). Natural genetic transformation can be regarded as the genuine bacterial gene transfer process, because genes necessary for transformation are located on the chromosome whereas genes necessary for conjugation are carried by plasmids, and genes instigating transduction reside on phages (Lorenz and Wackemagel, 1994). 2. MECHANISMS OF NATURAL GENETIC TRANSFORMATION The process of transformation has been divided in the literature into the following steps (Wackemagel et al., 1992; Lorenz and Wackemagel, 1994; Yin and Stotzky, 1997; Wuertz, in press) : (i) release of DNA from cells; (ii) dispersal and (iii) persistence of the DNA in foe environment; (iv) the development of competence for DNA uptake by cells in the natural 27 n. State of Knowledge Natural Genetic Transformation habitat; (v) the interaction of cells with DNA and the uptake of DNA; and (vi) the expression of an acquired trait following DNA uptake. 2.1. RELEASE. DISPERSAL AND PERSISTENCE OF THE DNA IN THE ENVIRONMENT DNA can be excreted abundantly by essentially all bacteria examined so far (Lorenz and Wackemagel, 1994). It was shown in some cases, for example in Bacillus subtilis, that DNA excretion is genetically determined (Sinha and Iyer, 1971) and thereby an energy requiring process. On the other hand large amounts of DNA are released during death phase in pure cultures by loss of cell wall integrity (Lorenz and Wackemagel, 1994). This DNA may not be attacked immediately, but could persist longer than was presumed (Thomanetz, 1982) before. DNA released by induced lysis (Kloos et al, 1994) as well as DNA released by death in the late stationary phase (Lorenz et al., 1992) persisted for hours under laboratory conditions. DNA has been detected practically everywhere in the environment: in freshwater (DeFlaun et al., 1986), marine waters (DeFlaun et al., 1987), waste water (Palmgren and Nielsen, 1996) or in soils and sediments (Steffan et al, 1988). The largestamounts of DNA (6.0 to 44.0 mg/liter) extracted from environmental samples are found in marine aquatic environments (DeFlaun et al., 1987; Paul et al., 1987). Also in activated sludge large amounts of DNA (>20 mg/liter) are detectable and DNA is suggested to be accumulated in and protected by the extracellular polymeric substances (Palmgren and Nielsen, 1996). In a much earlier work (Catlin, 1956) DNA was already found in culture slime in amounts exceeding 40% of the dry material. Moreover, the slime-associated DNA could still transform Neisseria meningitidis (Catlin, 1960). The half-life of free DNA ranges from 0.017 to 235 hours in aquatic environments and between 9.1 to 28.2 hours in terrestrial environments (Lorenz and Wackemagel, 1994). It has been found that nucleases are inhibited by the presence of humic compounds (Tebbe and Vahjen, 1993). Persistence of DNA could also be attributed to the adsorbance to minerals, clay particles or humic acids (Lorenz and Wackemagel, 1994). Other studies attribute the DNase-resistant state of adsorbed DNA to conformational inaccessibility of DNases to attack adsorbed DNA (Khanna and Stotzky, 1992; Lorenz and Wackemagel, 1992, Paget et al, 1992). However the spatial separation of immobilized DNases and DNA, adsorbed on particles also prevented degradative action on DNA by the enzymes (Demarche et al, 2001). Likewise, it could be speculated that the reduction of enzymatic activity would originate in the masking of the active site upon binding of the enzyme to the surface of the 28 n. Slate of Knowledge Natural Genetic Transformation particle or to conformational changes, negatively affecting kinetic properties of the enzyme (Sarkar eta!., 1989). The amount of extractable DNA, however does not even insinuate the actual amount of DNA available for transformation. Reliable extracellular DNA extraction methods that detect adsorbed DNA and avoid disruption of cells in samples still need to be refined. In addition to that, adsorbance of DNA might, depending on the substratum, DNA concentration or surrounding medium, inhibit or stimulate DNA uptake. Studies indicate that transformation in soil occurs at lower (Lorenz et al„ 1992; Chamier et al., 1993; Paul et al, 1991), comparable (Lorenz and Wackemagel, 1990) as well as higher frequencies (Lorenz and Wackemagel, 1987) compared to liquid suspensions. Aquatic solution flow with a high salt concentration in die environment could temporarily desorb DNA from its matrix (Demandche et al., 2001). This would allow DNA fluxes in soil and thereby create the possibility for other bacterial communities to encounter a great variety of genes. However, transformation itself might be stimulated in environments with low salt concentrations. Transformation of Acinetobacter sp. BD413 residing in silt loam soil, for example, was higher under more stringent salt concentrations (Nielsen et al., 2000). 22. THE DEVELOPMENT OF COMPETENCE FOR DNA UPTAKE BY CELLS IN THE NATURAL HABITAT During competence development, cells express specialized proteins that bind and take up DNA. The mechanism of competence development is in most microbial strains still unknown. Some authors divide the mechanism of competence development into two major subgroups: competence development in gram negative and in gram positive bacteria. It should be noted, though, that competence induction of strains belonging to one group share common features with competence induction of the other group. Furthermore it could also be that competence induction of strains sharing the same group is completely different As examples of the various ways competence is induced, four strains will be discussed. Bacillus subtillis has been studied very thoroughly concerning competence development and natural genetic transformation. It is a gram positive soil bacterium. As gram positive pathogenic bacterium, Streptococcus pneumoniae was chosen. It was the first strain in which transformation was discovered and its transformation system has already been thoroughly investigated (Sicard et al., 2000). The description of the natural transformation system in gram negative bacteria is still sketchy. As a pathogenic gram negative bacterium Haemophilus influenzae is introduced, while Acinetobacter sp. will be discussed as an example of a gram negative soil bacterium. In Bacillus subtillis competence development is regulated by a competence pheromone ComX. ComX pheromone is synthesized as a 55-residue protein that is processed and 29 n. State of Knowledge Natural Genetic Transformation released in the extracellular medium as a 10 amino-acid peptide (Tortosa and Dubnau, 1999). The competence switch is turned off by the competence and sporulation factor (CSF) after internalization and inhibition of phosphorylated ComA phosphatase (Lazazzera and Grossman, 1998). Extensive variability was observed between four Bacillus isolates in ComQ, ComX and in the region of Corn? encoding the segment positioned at the surface of the outer membrane. This ensured sexual isolation of the strain because every Com? responded only to its cognate ComX signal peptide. ComX does not only regulate competence. It has also been observed to control a number of different growth processes, such as the production of surfactin (srfA) or degradative enzyme synthesis (degQ) (Msadek, 1999). In Streptococcus pneumoniae competence is also regulated by a competence stimulating peptide (CSP), that is synthesized as part of a precursor protein, ComC (Tortosa and Dubnau, 1999). Again among 60 S pneumoniae isolates different and specific CSP’s were isolated, that were associated with their own cognate ComD receptor, ensuring isolation (Whatmore et al., 1999). Specificity would tend to ensure development only in the presence of related organisms. The risk of gene disruption or the synthesis of foreign toxic proteins would be diminished (Tortosa and Dubnau, 1999). Quotum sensing acted in this case as well as in the case of B. subtilis as a safety mechanism to avoid the uptake of detrimental DNA. Although it might be advantageous to adopt new traits by integrating novel genes, this sexual isolation should have been important enough for evolution to have selected for (Tortosa and Dubnau, 1999). Cells of Haemophilus influenzae are able to develop into the competent state without any regulatory input from other cells (Solomon and Grossman, 1996). Competence is induced under conditions that inhibit growth and practically all cells can become competent When nutrients become less available, the synthesis of cAMP increases. High intracellular cyclic AMP levels signal competence development (Solomon and Grossman, 1996). There is another signal, related to nutrient depletion, which is involved in competence development The protein encoded by say could respond to the signal. The signal as well as the gene product are still unknown (Williams et al., 1994). Competence does not always have to be regulated. For example, Neisseria gonorrhoeae, expresses the state of competence constitutively (Lorenz and Wackemagel, 1994). Likewise, tiie present state of knowledge about competence development in Acinetobacter sp. BD413 does not support the idea of regulated expression of competence genes. Attempts to extract a competence inducing peptide had been failed (Juni, 1978). BD413, however, does not express competence constitutively. High competency of Acinetobacter sp. BD413 is induced immediately after the transition from the lag phase to the exponential phase and gradually declines thereafter (Lorenz et al., 1991). Information about the molecular basis of the competence development in BD413 is scarce. Only a handful of competence genes have been 30 n. State of Knowledge Natural Genetic Transformation discovered. Among these are die comP (PorstendOrfer et al., 1997), comC (Link et al., 1998), ComE (Busch et al., 1999), ComF (Busch et al., 1999), ComB (Herzberg et al., 2000) and ComA (Friedrich et al., 2001). ComE, ComF, ComB and ComP are similar to prepilins of type IV pili and to pilin-like components of the protein translocation machinery (Busch et al., 1999; Herzberg et al., 2000; PorstendOrfer et al, 1997), whereas ComC is similar to various type IV pilus biogenesis factors (Link et al., 1998). ComA displays significant similarities with ComEC in Bacillus, suggesting that ComA might function in DNA transport through the cytoplasmic membrane (Friedrich et al., 2001). Surprisingly, a growth-phase dependency of the expression of ComP, Com B and ComA was found in a strain regarded to become competent without regulative mechanism. Even more surprising was that expression started at mid-exponential phase and continued long after stationary phase was reached, Le. expression of ComP, ComB and ComA was induced when cells did not seem to be competent for natural genetic transformation (PorstendOrfer et al., 2000; Herzberg et al., 2000; Friedrich et al., 2001). Thermus thermophilus, an aerobic, rod-shaped, gram-negative bacterium that grows at temperatures between SO and 82°C also possesses the ability to take up free DNA from the environment (Hidaka et al., 1994). The fact that gram positive, gram negative, pathogenic, soilbome, extremophile bacteria and archaeae (Worrell et al., 1988) share all mechanisms that can be used to expand their gene diversity indicates that the trait is very ancient and seems to be advantageous for the strains capable of it 2.3. THE INTERACTION AND UPTAKE OF DNA WITH COMPETENT CELLS A simplified representation of the interaction of DNA and the competent cell can be summarized as the binding of double stranded DNA, of a certain minimal sequence length, to die surface of a competent cell, still sensitive to assault with nucleases. Thereafter one strand is transported across the membrane while the other strand is degraded and released into the medium. This description is in many cases still uncertain or incorrect (Dubnau and Prowedi, 2000). For example, it is true that double stranded DNA is preferably needed for uptake, but for some organisms, like Haemophilusinfluenzae, single stranded DNA can also be taken up without a problem (Postel and Goodgal, 1966). Furthermore, it is still not known whether the single strands are produced externally to the membrane, within a water-filled pore or in the cytoplasm (Dubnau and Prowedi, 2000). Concerning the internalization of the DNA strand, bacteria use competence proteins, related to the main terminal branch of the general secretory pathway required in gram-negative bacteria (Pugsley, 1993). The secretory pathway requires secretin, which is also required for transformation of gram negative bacteria (Russel, 1998; Marciano et al., 1999), but only restricted to the steps that take place outside the cell membrane (Dubnau and Prowedi, 2000). Dubnau and Prowedi (2000) speculate further that 31 II. Slate of Knowledge Natural Genetic Transformation in the secretion systems the proteins needed for pilus formation, secretion, twitching motility and competence proteins are required in an analogous way for macromolecular passage across tile membrane. In that sense it could be assumed that transformation can be compared to secretion in reverse (Dubnau and Prowedi, 2000). Some organisms, like Haemophilus influenzae, restrict their DNA uptake to homologous DNA, whereas other organisms (Acinetobacter sp., S. pneumoniae, B. subtillis) have no preference (Lorenz and Wackemagel, 1994). There is a trend that bacteria which are capable of taking up DNA without sequence preference regulate competence development by cell-to-cell communication. Strains that do not regulate competence by signal molecules have sequence specificity in DNA uptake to undergo sexually isolated horizontal gene transfer. Perhaps organisms that are promiscuous in DNA uptake all utilize cell-to-cell communication or other forms of regulation to ensure species specific transformation (Solomon and Grossman, 1996), thereby offering them uptake of suitable DNA for the use of DNA repair while avoiding uptake of useless or even detrimental DNA. Naturally, DNA uptake requires energy. In B. subtil is, S. pneumoniae and H influenzae, uptake of DNA only occurs in competent cells, possessing a high energy state (Palmen and Hellingwerf, 1997). Furthermore, it was demonstrated that competence induction is dependent on a high intracellular ATP concentration (Clavd and Trombe, 1989). Palmen and Hellingwerf (1997) have put forward a model that explains energisation of DNA uptake by ATP (Fig. 1). dsDNA. nucleotides ATP ss DNA Fig. I. DNA uptake according to a model by Palmen and Hellingwerf (1997). After hydrolysation of ds DNA by an endonuclease and liberation of nucleotides, is DNA internalised as ss DNA via an ATP-dependent uptake machanism. (After Palmen and Hellingwerf 1997). 32 IL State of Knowledge Natural Genetic Transformation High ATP levels of transformable competent cells, inhibited transformation in ATP- poor competent cells, and consensus sequences of competence genes for ATP binding all point to flic functionality of this model (Palmen and Hellingwerf, 1997). Still, further investigation is needed to support the ATP-mediated DNA translocation model. 2.4. THE RECOMBINATION AND EXPRESSION OF ACQUIRED DNA The process of natural genetic transformation in bacteria can only be completed after successful incorporation of the internalized DNA. DNA strands are integrated via homologous recombination. Also plasmids that do not contain homologous DNA can be integrated by recombining two strands of the plasmids that have been cut at different sites (Palmen et a!., 1993). It seems that in some cases the recipient ’s genome prepares itself for homologous recombination. DNA internalization is escorted by an enhanced level of single strand gaps in the genome of competent cells of H. influenzae (McCarthy and Kupfer, 1987) and B. subtillis (Harris and Barr, 1971). This enhances the chance of successful homologous recombination. Once a gene is incorporated into tire genome on the chromosome or as an extrachromosomal unit, tire gene can be expressed and the recipient has acquired a new trait (Lorenz and Wackemagel, 1994). 3. NATURAL GENETIC TRANSFORMATION IN BIOFILMS It is difficult to extrapolate observed phenomena from in vitro studies to real life. In the natural habitat of a biofilm bacteria encounter rough and variable growth conditions. The temperature, humidity, availability of oxygen and nutrient sources are most probably not optimal. The studied properties of bacteria under predefined optimized laboratory conditions may obscure hidden responses that are only detectable in the in situ environment Nevertheless, one has to start with investigating a simplified model, a known culture medium, defined strains and preset temperature and pH to answer the following question: What environmental parameters in biofilms favor transformation (or each step towards the integration of free DNA into the genome of recipient cells) and what conditions prevent transformation? DNA entrapment and protection inside extracellular polymeric substances (Palmgren and Nielsen, 1996), together with the spatial stability of the bacterial cells can lead to the assumption that natural genetic transformation is most likely to happen inside a biofilm. 33 n. State of Knowledge Natural Genetic Transformation However, natural genetic transformation in biofilms has been reported only on very few occasions. Although cell aggregates growing on soil and sediment particles can be regarded as thin biofilms, the various published studies on transformation at the soil/sediment bulk interface do not really involve transformation in biofilms. In most cases recipients harvested after an overnight incubation in liquid culture were added to sterile or non-sterile soil particles in combination with DNA. Natural genetic transformation was only on very few occasions assessed in a biofilm grown on soil particles in a batch run microcosm (Nielsen et al., 1997). The authors first incubated the recipient strain for certain periods of time ranging between 1 and 24 hours before adding DNA. The study revealed an increase in transformation frequency after a 6 hour incubation whereafter the soil microcosm gave rise to lower transformation frequencies (Nielsen et al., 1997). The authors suggested that the transformation frequency increase depended on a nutrient upshift. Regrettably the study did not involve a comparison with a liquid suspension incubated under identical conditions to check if the increase in transformation frequency solely depended on nutrient addition. Further no attempt was made to wash away the cells that failed to aggregate or grow on tire soil particles. True natural genetic transformation of Acinetobacter sp. BD413 in biofilms was investigated in situ in river epilithon. In that report, a mutant BD413 derivative strain, that was unable to grow in the absence of histidine, was able to be transformed to the wild type genotype when grown into river epilithon in the presence of donor BD413 cells at rates comparable to sterile microcosms under laboratory conditions on filters (Williams et al., 1996). Natural genetic transformation of Streptococcus rnutans was investigated in artificial biofilms grown in microtiter plates or in a chemostat-based fcrmentor (Li et al., 2001). & rnutans was shown to control genetic competence with the use of a peptide pheromone system (Li et al., 2001). Because peptide pheromone systems control genetic competence in a cell density dependent way, it was no surprise that the authors could detect transformation at up to 600-fold higher rates than in planktonic S. rnutans cell suspensions. To summarize, there is still not much known about natural genetic transformation in naturally grown biofilm. It would be wrong to already speculate about the importance and prevalence of this type of horizontal gene transfer in biofilm niches such as biofilms grown in soil, the phylloplane or the rhizosphere, in waste water reactors (Wuertz, in press), the mucus layer of the gut, tubing, medical implants, etc. It is possible that natural genetic transformation can play an important role in natural biofilms, but the truth of foe matter remains that we have absolutely no clue. 34 n. State of Knowledge Natural Genetic Transformation 4. REGULATION OF NATURAL GENETIC TRANSFORMATION Evolution is guided by the survival and procreation of the fittest as a result of random mutation and natural selection. Hence, a variety of species came into existence which were fully adapted to live in perfect interspecies balance (Darwin, 1859). Yet a closer look at bacterial genetic variability reveals that all species may simply be used as hosts for parasitic and selfish genes (Orgel and Crick, 1980). While the survival of the individual gene host is less important, die persistence of genes and their evolution takes all nature ’s attention. Young (1992) interpreted bacterial evolution as a result of failure on the part of the bacterial cell: the failure to prevent replication errors (mutation); failed attempts to correct these errors by recombination (transformation, genome rearrangement); and the failure to prevent the activities of selfish elements (conjugation and transduction). These failings increased the genetic variability for the otherwise poorly adaptable clonal organisms. Without criticizing the evolutionary consequences, one could say that the enhancement of bacterial genetical variability is hence a passive and random mode of acquisition of new traits. This view of bacterial evolution changes rapidly when considering complex genetically encoded systems that can control bacterial genome plasticity. The still controversial theory of adaptive mutagenesis (Foster, 1993) presents a less random and more directed version of tire origin of mutations in certain bacterial strains, called mutators. Adaptive mutations are spontaneous mutations that occur in microorganisms, during periods of prolonged stress in non-dividing or very slowly dividing populations and that are specific to the environmental challenge that causes that stress. This theory, however, is beyond the scope of this thesis and therefore tire interested reader is referred to excellent reviews concerning adaptive mutagenesis (Foster, 1993; Rosenberg et al„ 1995; Hall, 1998; Janion, 2000). This section will address the regulation of horizontal gene transfer by natural genetic transformation. It has been found that bacteria do not always act as passive actors that are subjected to involuntary gene transfer events. Some (if not all) bacteria tend to limit horizontal gene transfer on those occasions where gene exchange would be advantageous (or at least less hazardous) by having evolutionary developed defence mechanisms against invading heterologous DNA (f.e. DNA restriction enzymes, insertion elements, requirement of an origin of replication) or control mechanisms that are regulated by environmental stimuli to signal cells that circumstances are optimal for exchanging genes (f.e. nutrient condition, light intensity, octopines). In other cases bacteria have developed complicated mechanisms based on cell-to-cell communication to regulate their genome variability (f.e. peptides, AHLs). These regulatory systems are found in all three horizontal gene transfer mechanisms: conjugation, transformation and transduction. In this thesis, however, we will only concentrate on natural genetic transformation. 35 II. State of Knowledge Natural Genetic Transformation 4.1. THE INFLUENCE OF ENVIRONMENTAL CONDITIONS ON NATURAL TRANSFORMATION In Bacillus subtilis competence development is under temporal, genetic and environmental regulation (Dubnau, 1991; Tortosa and Dubnau, 1999). Competence induction starts after the point of transition from exponential growth to stationary phase, controlled by a complex set of genetic switches in the presence of certain nutritional requirements (Dubnau, 1991). Nutritional requirements play an important role. Growth in minimal medium leads to the highest transformation frequency in B. subtilis (Dubnau, 1991). The need for minimal medium to transform is also shared by Azotobacter vinelandii (Page and Sadoff, 1976) and Haemophilus influenzae (Harriott et al., 1970). However, minimal medium is not a common requirement for transformation to occur. In other strains transformation occurs solely (S. pneumoniae) (Morrison et al., 1983) or preferably in rich media (Acinetobacter sp. BD413) (Lorenz et al., 1991; Nielsen et al., 1997). Hence, a nutrient-limiting condition most likely to take place in natural environments, will probably affect transformation either positively or negatively. Acinetobacter sp. BD413 needs energy to become competent, to be able to take up and integrate new DNA strands and express the newly acquired genes (Stotsky, 1989). Azotobacter, which is a nitrogen-fixing strain, can only become competent under iron limitation and when nitrogenase is protected from inactivation by oxygen (Page, 1982). Starvation at Acinetobacter sp. BD413 for a single nutrient using a chemostat decreased transformation frequency (Palmen et al., 1994). On the other hand, cells of Azotobacter vinelandii, starved for iron and molybdenum, reached an even higher competence level than did cells in medium limited for iron only (Page, 1985). It was noted (Lorenz and Wackemagel, 1994) that the rhizosphere of plants is a nutrient enriched but iron-limited habitat (O’Sullivan and O’Gara, 1992). This would rather indicate that transformation in Azotobacter is supported in an nutrient rich environment with a dense bacterial population, instead in a condition of stress. P. stutzeri and H. influenzae, however, become highly competent when growth ceases (Lorenz and Wackemagel, 1994). It is speculated that this reaction results in an attempt to . use nucleotides as a energy source (Macfayden et al., 2001; Kroer et al., 1994) although it is not practiced by all bacteria (toner etal., 1994). The influence of other kinds of stress (extremes of pH and temperature, oxidizing agents, osmotic pressure, high pressure, irradiation, toxic metal ions, antibiotics, electrophiles, alkylating agents, detergents, bacteriophages), present in the environment have only scarcely been investigated. The effect of temperature, pH or salinity at suboptimal values always reveal a decrease in transformation frequencies (Lorenz and Wackemagel, 1994). 36 n. State of Knowledge Natural Genetic Transformation It was popular to speculate that transformation is increased due to the stressful character of nutrient limitation in an attempt to obtain new metabolic traits via gene transfer and escape the stressful conditions (Dubnau, 1999). But one has to keep in mind that the development of competence, the ability to take up, internalize, integrate and express DNA is an energy consuming process. In the cyanobacterium Agmenellum quadruplicatum, energy limitation by exclusion of light resulted in loss of competence (Essich et al, 1990). Besides the stress of nutrient limitation, stress did not enhance transformation frequency. These observations suggest that a high energy load of the competent cell is strictly needed. It could be possible that tiie non-growing competence cells (P. stutzeri) exhibit a high individual energy load for cell maintenance but restricted for cell growth, because growth cessation by nutrient limitation that resulted in high transformation frequencies was only limited to those conditions where cells were starved for one nutrient only (Lorenz and Wackemagel, 1994). 4.2. INDUCTION OF COMPETENCE FOR NATURAL GENETIC TRANSFORMATION Until now regulation of competence development was only associated with the use of signaling peptides. In Bacillus subtillis competence development and sporulation is regulated by two interconnected signaling peptides: a competence pheromone ComX and the competence and sporulation factor CSF. The competence pheromone is synthesized and released in the extracellular medium as a peptide which can activate Com? that in his turn can activate ComA (Tortosa and Dubnau, 1999). When active and phosphorylated, ComA-p induces the expression of the competence genes. The competence switch is turned off by CSF after internalization and increased intracellular accumulation (Lazazzera, 2000). At low intracellular concentrations of CSF, dephosphorylating enzyme RapC is inhibited in dephosphorylating the active and phosphorylated ComA-p. In addition Com? can phosphorylate ComA without interruption and this leads to induction of the competence genes. However, when CSF accumulates inside the cell, Com? is inhibited in phosphorylating ComA and the inhibition of RapC is suspended, allowing dephosphorylation of activated ComA-p (Lazazzera and Grossman, 1998) and the induction of sporulation genes (Lazazzera, 2000 ). In Streptococcus pneumoniae the competence stimulating peptide (CSF) is synthesized and secreted in the environment (Tortosa and Dubnau, 1999). ComD responds to CSF in the environment and phosphorylates ComE (Tortosa and Dubnau, 1999). Next, ComE-p drives the transcription of the cornCDE, the cornAB operons as well as that of ComX, which gene product activates the transcription of the late competence proteins (Tortosa and Dubnau, 1999). 37 U. State of Knowledge Natural Genetic Transformation The induction of competence for uptake of free DNA is an example of how bacteria can actively sense the right conditions to transform by communicating with each other. The receptors reacted specifically to their cognate signaling peptides. The feet that peptides from other strains could interfere with the recognition of the specific peptides by their cognate receptor proteins (Muscholl-Silberhom et al., 1997), means that bacteria could sense the presence of other bacteria (Otto et al., 1999), thereby shutting of competence development also in de presence of the threshold quorum wherein competence was favored. Sensing the presence of others and blocking gene transfer would only increase useful natural genetic transformation. 4.3. BARRIERS TO NATURAL GENETIC TRANSFORMATION With the use of PCR, cloning and large-scale genome sequencing it became clear that random mutation did not play the most important role in bacterial evolution. Horizontal gene transfer contributed far more to the evolutionary change of bacteria. Mosaic gene structures and rapid spread of antibiotic resistance genes resulted from horizontal gene transfer events, not mutagenesis (Smith et al, 1992; Dowson et al., 1997). Hence bacterial evolution is regarded to be dependent on both horizontal gene transfer and random mutations. If, however, horizontal gene transfer occurred frequently between species, recombination would lead to a sequence homogenizing effect on bacterial genomes (Nielsen, 1998). Barriers to recombination by sexual isolation are thus necessary. They would lead to sequence divergence and lower recombination rates (Nielsen, 1998). Barriers against natural genetic transformation can be active at different phases of the transformation process: the exposure of free DNA in the environment, the uptake of genes, the stabilization of genes inside the host, the expression of the integrated gene and the selection of the fittest (Nielsen, 1998). In a first phase DNA needs to persist long enough to encounter naturally competent cells. Adsorbance of DNA into the DNAse resistant state to particles, humic acids and extracellular polymeric substances can protect DNA long enough to overcome spatial and temporal barriers (see chapter II.C.2.1.). Unprotected DNA can be degraded very quickly by nucleases and DNA-degrading microorganisms, which can be found at high concentrations in seawater and sediments (Maeda and Taga, 1974). Uptake of DNA can be limited in various ways. At the cellular level DNA restriction, a defense mechanism used by bacteria to avoid invasion of foreign genes by digesting it, can prevent subsequent recombination. For example, Neisseria gonorrhoeae, does not accept DNA synthesized by K coli (Stein et al, 1988). Acinetobacter sp. BD413, does not have this defense mechanism, it takes up DNA indiscriminately (Palmen and Hellingwerf, 1997). Haemophilusinfluenzae uses another mechanism to ensure itself of the uptake of homologous 38 n. Stite of Knowledge Natural Genetic Transformation DNA. It only takes up DNA that contains a certain recognition sequence (Sisco and Smith, 1979). And as stated previously, B. subtilis and S. pneumoniae defend themselves against heterologous DNA by signaling the induction of competence only at a certain cell density of their own kind. But not all bacteria build high barriers against uptake of free DNA. Certain bacteria have been shown to transform even at overshoot frequencies (Lorenz and Wackemagel, 1994). Among these are the mutant strain B. subtilis 168 Marburg (Mulder and Venema, 1982), Acinetobacter sp. BD4 (and derivatives among which BD413) (Juni and Janik, 1969), and Vibrio strain WJT-1C (Frischer et al„ 1990; Lorenz and Wackemagel, 1994). Once DNA is taken up it needs to be retained in the host's genome. The new sequence can only be stabilized if the host is able to replicate the introduced DNA, i.e., it needs to be linked to an origin of replication either on the chromosome or on a plasmid (Nielsen, 1998). If the sequence does not contain an origin of replication, sequence homologyis a prerequisite to obtain recombination in the chromosome (Lorenz and Wackemagel, 1994). If a gene that has been taken up fails to integrate via homologous recombination, it will loose its stability and eventually be lost In laboratory experiments, the appearance of an altered phenotype serves as evidence of a transformation event Expression of an acquired trait does not always lead to a more fit state (Lorenz and Wackemagel, 1994). Natural transformation of genes affecting the host negatively has been investigated, leading to the killing of 55% of the population exposed to detrimental DNA (Albritton et a!., 1984) or significant growth limitation after integration of the new trait (Click et al., 1985) and poor survival of these transformants. It can be said that as soon as new transformants have established themselves in their bacterial community, they have overcome the last barrier that prevented genetic diversification via natural genetic transformation. 39 II. State of Knowledge Natural Genetic Transformation OVERVIEW Successful natural genetic transformation is defined as: ‘Uptake of free DNA, that resisted degradation after release in the environment via excretion or lysis, whereafter, the accepted gene is integrated into the genome by cells that can develop natural competence for transformation, whereafter the transformant can express the newly acquired trait and pass it on to its siblings.’ Conditions that influence natural genetic transformation - an environment favorable/unfavorable for the persistence of DNA - nature and origin of free DNA - nutritional requirements that influence competence development - signal-response mechanisms that influence competence development - discriminating DNA uptake mechanisms - intra- and extracellular DNA restriction - linkage of recombinant sequence to an origin of replication - sequence homology - resulting effect of the altered phenotypeon the cells metabolism Natural genetic transformation in Acinetohacter so. BD413: - BD413 takes up every DNA strand indiscriminately - it possesses a high effective level of competence which starts at early log and decreases during the exponential phase - BD413 can be transformed in minimal medium, but it is more readily transformed in rich medium - addition of nutrients after a starvation period can induce transformation frequency to even higher levels - in natural biofilms, BD413 was shown to obtain comparable transfer frequencies as under sterile laboratory conditions 40 II. State of Knowledge Natural Genetic Transformation SOME OPEN QUESTIONS Is it possible that natural genetic transformation can play a role in natural biofilms? Can natural genetic transformation be controlled in biofilms? What are the parameters that influence natural genetic transformation in monoculture Acinetobacter sp. BD413? What are the parameters that influence natural genetic transformation in mixed species biofilms containing Acinetobacter sp. BD413? How does natural genetic transformation with advantageous/neutral /disadvantageous DNA occur inside a biofilm? Do transformants persist in biofilms? What are the ecological effects of natural genetic transformation? Does DNA persist in biofilms? How long does it persist intra- and/or extracellulaHy? Does natural genetic transformation play a role in the persistence of advantageous/neutral/disadvantageous genes? 41 III. MATERIALS AND METHODS 42 m. Materials and methods Used bacterial strains and plasmids A. USED BACTERIAL STRAINS AND PLASMIDS Acinetobacter sp. BD413 (Juni and Janik, 1969) was used for growing monoculture biofilms. Escherichia coli strains GM16, GM25 and EC63 were used for extracting pGARl (Hausner and Wuertz, 1999), pGAR2 (Hendrickx et al., 2000) and pGAR39 respectively by an alkaline extraction method (Sambrook et al., 1989). pGARl is pRK415 (Keen et al., 1988), the mob* tra" Inc PI plasmid, carrying the wild type gfp (Clontech, Palo Alto, CA) under regulation of a Plac promotor. pGAR2 and pGAR39 are identical to pGARl, but carry respectively eyfp (Clontech, Palo Alto, CA) or ecfp (enhanced cyan fluorescent protein) (Clontech, Palo Alto, CA), instead of gfjp. The E. coli strain EC1 was used as a cloning vehicle, E. coli strain EC7 contained pRK2013 (Figurski and Helinski, 1979) encoding the cognate conjugation system and was therefore used as helper strain in standard triparental mating procedures. B. MEDIA AND SOLUTIONS All media were autoclaved during 20 min at 121eC in a Vapoklav 400 autoclave (Vapoklav, Oberschleissheim, Germany). Heat sensitive components (antibiotics, X-gal) were separately prepared in concentrated stock solutions and sterilized by filtration with a 0.2 pm non-pyrogenic, cellulose acetate filter (Schleicher & Schuell, Dassel, Germany). Filtered solutions were added to the medium after heat sterilization and cooling down to at least 60°C. For the preparation of agar plates, 16 g agar-agar (Gibco BRL, Eggenstein, Germany) and for tire preparation of soft agar 10 g agar-agar were added per liter medium before autoclaving. 1. LURIA-BERTANI MEDIUM (LB) (SAMBROOK ETAL, 1989) Tryptone 10 g Yeast extract 5g Nad 5g HgOjoubdist ad 1000 ml PH 7.2 44 QI. Materials and methods Media and solutions 2. M9, MINIMAL MEDIUM (SAMBROOK ETAL., 1989) Stock solution A NazHPO* x 2 H20 42.48 g KH2P04 15 g NaCl 2.5 g NH4CI 5.0 g H^doubdia ad 1000 ml Stock solution B lMMgSO« (12.4 g MgSOVlOO ml or 24.65 g MgSO« x 7 H2O/100 ml) Stock solution C 1M CaCh (11.1 g CaCiyiOO ml or 14.7 g CaCl2 x 2 H2O/IOO ml) M9 medium HhOdouMia (sterile) 798 ml Stock solution A 200 ml Stock solution B 2 ml Stock solution C 0.1 ml Add the stock solutions to the sterile water at 50-60°C under sterile conditions. Afterwards a carbon source from a concentrated sterile stock solution can be added. As carbon source, usually 10 ml 20% gluconate solution was added per 1000 ml medium to obtain a 0.2 % gluconate enriched minimal medium, unless otherwise stated. 3. TR1S MINIMAL MEDIUM S17 solution 25%HC1 1.3 ml ZnCl 2 0.07 g MnCl 2x2H20 0.1 g H3BO3 0.062 g C0CI2 x 6 H20 0.19 g Cu C12x 2H20 0.017 g NiCl2x 6HzO 0.024 g Na2Mo04x2H20 0.036 g ^Odoubdist ad 1000 ml Tris medium was set to pH 7. Addition of a carbon source or antibiotics occurred after sterilization of the medium. 45 III. Materials and methods Media and solutions As carbon source, usually 10 ml 20% gluconate solution was added per 1000 ml medium to obtain a 0.2% gluconate enriched minimal medium, unless otherwise stated. Tris medium: TrisHCL 6.06 g NaCl 4.68 g KC1 1.49 g NHaCl 1.07 g Na2S04 0.43 g MgCl2x6H20 0.20 g CaCl2 x 2 H20 0.03 g NaH2P04x2H20 0.04 g Fe(III)NH4-citrate 0.048 g S17 solution 10 ml HtOdoubdist ad 1000 ml 4. HEAT SENSITIVE STOCK SOLUTIONS To select the strains, antibiotics at certain given concentrations were used. To detect LacZ activity, 5-bromo-4-chloro-3-indolyl-p-D-gaIactopyranoside (X-gal) was added to the medium. Breakdown of X-gal leaves blue colored breakdown components, visible by the naked eye. Stock solution Concentration (mg/ml) Solvent Tetracycline Tet 5 Methanol Ampiciline Amp 100 H20 X-gal 20 5. PLASMID CONSTRUCTION SOLUTIONS Chloroform-isoamylalkohol solution (24:1 v/v; solution should be stored in a light-protected vessel at 4°C) Ethidium bromide solution (1% ethidium bromide in H2Odoubdist(v/v); solution can be stored in light-protected vessels at room temperature) 46 ffl. Materials and methods Media and solutions Electrophoresis buffer (EY) (20x) Tris 48.5 g EDTA 7.4 g HzOdoobdia ad 1000 ml pH 7.9 SOLI solution glucose 0.9 g EDTA 0.37 g Tris 0.3 g HzOdodbdia ad 100 ml pH (with HC1)8 SOL2 solution lOMNaOH 0.2 ml 20%SDS 0.5 ml HiOdoMist (sterile) ad 10 ml This solution should be prepared fleshly, every time. SOL3 solution 5 M Na-aeetate 60 ml glacial acetic acid 11.5 ml HzOiodMia ad 100 ml pH 4.8 The solution should be stored at 4°C. Phenol-Chloroform solution (1:1 v/v; phenol pH 7.1 fiom Sigma-Aldrich (STAD, Germany) 6. HYBRIDISATION SOLUTIONS (AMANN, 1995) 6.1. PHOSPHATE BUFFERED SALINE fPBSl SOLUTION lx PBS 3 x PBS NaCl 8g 24 g KC1 0.2 g 0.6 g Na2HP04.2H20 1.78 g 5.34 g NaH2P04.H20 0.23 g 0.69 g pH 7.0 7.0 47 III. Materials and methods Media and solutions 62. TRIS HCL (PH 7.4) (0.01 M) Add 1 ml of TrisHCl stock solution (1 M) to 99 ml FfoOdoubdia (sterile). 6.3. EDTA. DISODIUM SALT (PH 81 fO.OOl Ml Add 400 ;il of stock solution EDTA (0.25 M) to 100 ml HzOdoubdist (sterile). 6.4. HYBRIDIZATION BUFFER FOR A CERTAIN FORMALDEHYDE CONCENTRATION Formaldehyde SMNaCl TrisHCl H2O hides! Formaldehyde 10% SDS 0% 360 nl 40 |il 1600 |il 0|il 2 |il 10% 360 nl 40 |il 1400 |il 200 |il 2 |il 20% 360 ill 40 |il 1200 |il 400 |il 2 111 25% 360 fil 40 |il 1100 |il 500 |il 2 |il 30% 360 |il 40 |il 1000 |il 600 |il 2 111 35% 360 |tl ' 40 |il 900 |il 700 |il 2 |il 40% 360 |il 40 |il 800 |il 800 |il 2 |*1 70% 360 |il 40 |il 1400 |il 1400 |il 2 Ml 6.5. WASHING BUFFER FOR THE CORRESPONDING FORMALDEHYDE CONCENTRATION IN THE HYBRIDIZATION BUFFER Formaldehyde TrisHCl NaCl EDTA HiO bides! 10 % SDS 0% 1 ml pml 0|il ad 50 ml 50 |il 10% 1 ml 4500 |il 0 111 ad 50 ml 50 |il 20% 1 ml 2150 |il 500 |il ad 50 ml 50 |il 25% 1 ml 1490 |il 500 |il ad 50 ml 50 |il 30% 1 ml 1020 |il 500 |il ad 50 ml 50 |il 35% 1 ml 700 |il 500 |il ad 50 ml 50 |il 40% 1 ml 460 |il 500 |il ad 50 ml 50 |il 70% 1 ml 0 |il 350 |il ad 50 ml 50 |il 48 HI. Materials and methods Standard microbiological methods C. STANDARD MICROBIOLOGICAL METHODS 1. PLASMID DNA EXTRACTION OF E COU STRAINS BASED ON THE METHOD OF BIRNBOIM EN DOLY (BIRNBOIM AND DOLY, 1979) 1. Grow a culture of 5 or 50 ml in LB medium overnight at 37°C, 100 rpm. 2. Centrifuge the 5 ml culture during 5 min at 11000 rpm at room temperature. A 50-ml culture is centrifuged at 5000 rpm during 5 min at 4°C, whereafter the pellet is resuspended in 10 ml 0.01 M MgSOs and centrifuged again, for another 5 min, at 4°C and 5000 rpm. 3. Add 100 pi or 2 ml SOLI to the pellet and resuspend by vortexing. Incubate during 5 min at room temperature. 4. Add 200 pi or 4 ml of freshly prepared SOL2 and mix by converting the tube a few times. Incubate for 5 min on ice. 5. Add 150pl or 3 ml SOLS and mix by converting the tube a few times. Incubate for 15 min on ice. 6. Centrifuge the mixture for 10 min at 12000 rpm in an 1.5 ml tube or 30 ml glass corex tube that can resist centrifugation at high speed. Save the supernatant 7. Extract the supernatant with 1 ml (9 ml) phenol-chloroform solution and separate the phases by centrifugation (20 min, 10000 rpm, 4°C). 8. Extract the water phase with 1 ml (9 ml) chloroform-isoamylalkohol solution and separate the phases by centrifugation (20 min, 10000 rpm, 4°C). 9. Rescue the water phase again and add 2.5 volumes of 100% EtOH (-20°C). Incubate the mixture for 30 min at -20°C. 10. Centrifuge the mixture during 10 min, at 10000 rpm and 4°C. Wash the pellet with 70% EtOH, and centrifuge again 10 min, at 10000 rpm and 4°C. 11. Dry the pellet completely. Then add respectively 50 pi or 200 pi sterile HiObMca- DNA extracts can be stored at -20°C. 2. DNA CLONING TECHNIQUES 2.1. DNA RESTRICTION ANALYSIS DNA restriction occurred with restriction endonucleases and buffers of Hybaid-AGS (Heidelberg, Germany) which were used according to the manufacturer ’s instructions. After agarose gel electrophoresis on a 0.8% (w/v) agarose gel, amounts and length of the fragments 49 in. Materials and methods Standard microbiological methods could be estimated by comparison with a standard DNA ladder (Hybaid-AGS , Heidelberg, Germany). 2.2. CLONING (SAMBROOK et aL 19891 1. Vector DNA and insert DNA was cut with appropriate restriction enzymes, whereafter the vector DNA was dephosphorylised with 1 pi calf intestinal phosphatase (Hybaid-AGS , Heidelberg, Germany) during 30 min at 37°C as recommended by the producer. 2. The DNA fragments needed to be purified. After inactivation of the restriction enzymes and phosphatases in a 10 min incubation at 65°C, sterile water was added to obtain 50 pi. DNA was extracted with 50 pi phenol-chloroform solution. After rescue of the DNA via centrifugation during 10 min at 13000 rpm, the supernatant was recuperated and DNA was precipitated with 1.5 volumes of 100% EtOH (-20°C). After precipitation and a washing step with 70% EtOH, DNA was dried in a vacuum centrifuge for 3 min and resuspended in 10 pi. 3. After estimation of the fragment length and concentration via agarose gel electrophoresis, 100 ng insert DNA was mixed with 50 ng vector DNA in a fresh 1.5 ml tube. Involuntary ligation of the fragments was diminished after a short (5 min) incubation of the fragments at 45°C. Immediately thereafter, the tube was transferred to ice. T4 DNA ligation buffer, T4 DNA ligase, DTT and ATP solutions (Hybaid-AGS , Heidelberg, Germany) where added following the product instructions. The ligation was performed overnight at 16°C. 4. The ligation mixture was purified by precipitation with 2 volumes of 100% EtOH. After centrifugation and drying, the ligated DNA sample was resuspended in 10 pi sterile aqua bidest 5. The ligated DNA sample was introduced into £ coli EC1 by electroporation. A100 ml LBEC1 culture was harvested at mid log phase, centrifuged, washed 3 times with cold (4°C) sterile aqua bidest and incubated in ice to obtain fresh competent EC1 cells. One microliter of the DNA sample was added to 50 pi of competent cells and subjected to a pulse of2000 volt with an BTX ECM399 electroporation system ( Genetronics Inc., stad, land). 6. After electroporation, the cells were left to recuperate in 1 ml LB at 37°C for lh. Thereafter the suspension was diluted by a factor of 10 and 100. Hundred microliters of the diluted and undiluted suspensions were plated on selective agar plates and incubated overnight at 37°C. 7. Randomly, 10 colonies were picked from the selective plates and checked for the desired plasmid composition by restriction analysis. 50 III. Materials and methods Standard microbiological methods 3. DNA DEGRADATION TEST Cultures of BD413, BD413(pGAR2) and BD413(pGAR39) were grown overnight in 40 ml LB. Twenty milliliter of every culture underwent harsh ultratorax sonication (Bandelin, Sonopuls HD70, Berlin, Germany) at full power during 16 min, on ice, to obtain lysates that were not hampered in enzyme activity by heat, biological or chemical treatment Twenty milliliter of every culture remained untreated. Both sonicated and untreated cultures were centrifuged at 4000xg during 15 min whereafter the supernatant was collected. One milliliter of every supernatant was filtered with a 0.2 pm non-pyrogenic, cellulose acetate filter (Schleicher & Schuell, Dassel, Germany). Zero point five microgram, plasmid DNA was incubated in 40 pi of the filtered and non-filtered, ultra torax-sonicated and untreated supernatants, fresh LB and H%0 bidest for 3 and 7 h at room temperature. Degradation was estimated via comparison with freshly thawed control DNA by gel electrophoresis. 4. TRIPARENTAL CONJUGATION Hundred ml of an overnight culture of the donor strain, the helper strain and the recipient strain grown under selective conditions was dropped on an LB agar plate and incubated overnight Next the mating patch was removed from the agar plate, resuspended in a 0.01M MgSO* solution and diluted up to 10"*. Hundred milliliters of the dilutions were plated on selective agar plates to select for the donor, the recipient or the transconjugants. 5. STANDARD NATURAL GENETIC TRANSFORMATION IN PURE CULTURES An overnight culture of Acinetobacter sp. BD413 was diluted 1/25 in desired medium containing a certain amount of free DNA. After 1-3 hours transformation was stopped by adding 1 unit RQ1 Rnase-fiee Dnase per lpg added DNA (Promega, Madison, USA) and incubating die solution for 10 minutes at 37°C. Afterwards different dilutions have to be plated out on plates that select for recipients and transformants. 51 m. Materials and Methods In situ quantification of gene transfer D. In situ QUANTIFICATION OF GENE TRANSFER IN BIOFILMS "REALISM,The art of depicting nature as it is seen by toads. The charm suffusing a landscape painted by amok, or a story written by a measuring worm." Ambrose Bierce (1842-1914), American journalist In ‘The Devils Dictionary', 1911 1. TOOLS 1.1 . THE CONFOCAL LASER SCANNING MICROSCOPE In this study, biofilms were monitored with the confocal laser scanning microscope LSM410 (Zeiss, Jena, Germany). Confocal laser scanning microscopy (CLSM) allows direct investigation of microbial biofilms without outside disturbance (Caldwell el al., 1992). The possibilty to observe 3D biofilm morphogenesis in situ was accomplished with the development of CLSM by which it was possible to eliminate interference from out-of-focus objects and to create digital reconstructions of the investigated biofilm (Hartmann et al., 1998). Elimination of out-of-focus objects is accomplished by restricting light to a certain confocal plane when the image is formed. This results in obtaining an optodigital thin Section parallel to the microscopic cover glass (x-y plane) with a thickness approaching the theoretical resolution of the light microscope (Caldwell et al., 1992). Consequently a series of 2D (x-y) images perpendicular to the attachement surface (z-direction) can be formed from the scanned object This series of 2D images can be used to create a 3-D reconstruction of the investigated specimen (Caldwell et al., 1992). Afterwards, gray scale images can be enhanced by transformation of the gray level distribution, filtration operations, image calculation operation, object erosion and object dilatation. In a next step the image is converted into a digitized image and ready to be automatically processed (Caldwell et al., 1992; Kuehn et al., 1998; Wuertz, 2000). 52 in. Materials and Methods After automated image acquisition, image processing and digital image conversion, investigations based on confocal laser scanning microscopy allow automated quantification of areas occupied by biofilm cells as a stack of 2D images in biofilms divided into z-sections to obtain the biovolume of the investigated sample. The calculation of the accumulating biovolumes is obtained with a numerical integration algorithm (Kuehn et al., 1999), which is integrated in the macro routine of the image analysis software system Quantimet 500IW (Leica Imaging Systems Ltd., Cambridge, England). Further mathematical calculation of the quantified data is thereafter done in EXCEL worksheets (Microsoft) (Kuehn et al., 1999). The combination of in situ microscopic measurement of the biofilm with the programmed quantification of the obtained data hence produce an objective picture of the investigated biovolume, the biofilm architecture and the microbiological events occurring in the biofilm. 1.2. THE GREEN FLUORESCENT PROTEIN The green fluorescent protein is a biologically formed fiuorophore responsible for the green emitted light of the bioluminescent system of the jellyfish Aequorea victoria (Chalfie et al., 1994). Since its discovery, GFP proved to have many advantages as a detection marker for living eukaryotic as well as prokaryotic cells. Unlike other commonly used reporter genes, no additional substrate is needed to allow direct in vivo monitoring of GFP labeled cells. Labeled cells can simply be detected by visual inspection upon irradiation (Prasher, 1995). With a size of only 238 amino acids, GFP is easily amenable to mutation resulting in the creation of GFP variants with different excitation or emission spectra, improved brightness (Heim and Tsien, 1996, Yang et al., 1998), destabilized fluorescence expression (Andersen et al., 1994) or temperature desensitized fluorescence expression (Kimata et al., 1997). 1.2.1. GFP as a tool for in situ detection of gene transfer The use of GFP as a biological marker displays many advantages. Most importantly, there is no selective pressure needed to observe gfp expressing bacteria. Bacterial communities can be investigated without any outside mechanical, chemical or biological disturbance. This allows simultaneous localization and detection of gene expression. It even allows detection of non-culturable cells (Cho and Kim, 1999; Hausner and Wuertz, 1999). Although gfp and its derivatives allow monitoring of living cells, the effect on the metabolism of the labeled cells due to the expression of this artificial protein remains unclear. The choice of GFP to investigate in situ horizontal gene transfer is an obvious one, considering the simple detection of GFP expression after genes encoding GFP have been received by bacterial cells via horizontal gene transfer. 53 in. Materials and Methods In situ quantification of gene transfer 1.2.2. Monitoring with dual/multiple fluorescence labeling using green fluorescent protein variants and homoloeues The creation of different GFP variants has opened up the possibility of using fluorescent proteins for dual/multiple labeling. With dual fluorescence labeling using GFP variants, gene transfer can be monitored over time without interruption of the process. Until now dual labeling in prokaryotic cells was established with GFP mutants excitable in the UV range (Lewis and Marston, 1999; Yang et a!., 1998). Considering the mutagenic character of UV radiation, it is more desirable to use GFP variants excitable at a longer wavelength. Promising variants with separable fluorescence spectra and excitable in the non UV range have already been engineered. EYFP (Clontech, Palo Alto, Cal., USA) has an excitation peak at 513 nm and an emission spectrum in the yellow-green region (527 nm). ECFP (Palo Alto, Cal., USA) has a fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501) spectrum compatible with EYFP for the purpose of dual fluorescence labeling. Recent discoveries of biological tags with features similar to GFP are reported to be useful for multilabeling in combination with GFP variants. The recently discovered GFP homologue drFP583 (RFP) (Matz et al., 1999) from Discosoma sp. with an absorption maximum at 558 and emission maximum at 583 can be used as a reporter protein in combination with the red shifted GFP variant PI 1 (Heim et al., 1994) which has an excitation peak at 489 nm and an emission peak at 511 nm. Kjaergaard eta!. (2000) studied the effect of E. coli antigen 43 (a surface displayed autotransporter protein) expression with a GFP tagged E. coli strain on the 3D-structuring of biofilm development when grown in combination with RFP (Clontech, Palo Alto, Cal., USA) labeled P. fluorescens. Bloemberg et al. (2000) used triple labeling by combining ECFP, EYFP and RFP for real time monitoring of population dynamics of differently labeled rhizosphere-colonizing P. fluorescens WCS365. It has to be mentioned however, that RFP possesses a long maturation time that exceeds 48h at room temperature, when expressed in E coli (Mizuno et al., 2001). Therefore, the wild type RFP can only be used as a non dynamic fluorescence tag i.e. for the labeling of a certain established bacterial population. This at first glance disadvantageous characteristic of RFP inspired Terskikh et al. (2000) to create a drFP853 mutant that changes its emission spectrum over time, where the change in color was independent of protein concentration, so as to be able to trace time-dependent protein expression. Another disadvantage of RFP is its complex fluorescence spectrum. The absorption spectrum is broad with several shoulders and peaks other than the main peak at 558 nm (Mate et al., 1999). Currently new GFP variants and homologues are being developed for a whole range of different applications. And although their uses are numerous, one has to bare in mind the 54 m. Materials and Methods limits of using GFP. The inability to detect GFP (Tsien, 1998) because of failed gfp expression, foiled GFP maturation, disturbing background signals, gfp overexpression associated toxicity effects in the host (Endow and Piston, 1998), inter- and intraspecies variations in gfp expression (Wuertz et at., 2001) or the difficulty in staining certain gfp expressing cells with oligonucleotide probes or nucleic acid dyes (Geisenberger et at., 1999; Wuertz et at., 2001) are just a handful of foe practical problems that should be overcome in each new experimental design separately. Nonetheless, GFP and other fluorescent proteins provide important information at foe cellular level which could not be otherwise detected. 2. PREPARATION OF THE SAMPLES To investigate horizontal gene transfer, GFP can be combined with fluorochrome tags such as general nucleic acid stains (Hendrickx et at., 2000) or oligonucleotide probes (Geisenberger et al., 1999; Hausner and Wuertz 1999). General nucleic acid dyes are available with differing cell permeability, fluorescence enhancement upon binding nucleic acids, excitation and emission spectra, DNA/RNA selectivity and binding activity. While some nucleic acid stains differentiate between live and dead cells, others do not Fluorescently labeled oligonucleotide probes can be obtained with varying degrees of selectivity and can be conjugated to any desired fluorochrome. When biofilm samples are to be stored for future investigation or hybridized with gene probes, the biofilm needs to undergo paraformaldehyde fixation. If tire biofilm will be investigated immediately without hybridization, no fixation step is required. The following instructions were followed (Wuertz et al., 2001): 2.1. In situ NATURAL GENETIC TRANSFORMATION IN BIOFILMS GROWN ON SLIDES 2.1.1. Growth of hinfilms on slides Grow a biofilm on a glass microscopic slide in a Petri dish filled with the appropriate medium and inoculate with one colony of a bacterial strain on a slowly tilting table until foe desired state of foe biofilm is reached (Fig. 2A). 2.1.2. Transformation on slides 1. Remove the medium in tire Petri dish and rinse carefully with 0.01 M MgS04 (Fig. 2B). 2. Add M9 medium containing a certain concentration of DNA and incubate at room temperature on a slowly tilting table during a certain period of time (Fig. 2C). 55 in. Materials and Methods In situ quantification of gene transfer 3. Remove the DNA containing medium and rinse carefully with 0.01 M MgS04 (Fig. 2D). 4. Add M9 medium without DNA and allow expression of the transferred genes while incubating overnight on a slowly tilting table, at room temperature (Fig. 2E). Fig. 2. Work scheme for the in situ quantification of natural genetic transformation on slides. 2.1.3. Fixation of biofilms on slides (optional! 1. Gently rinse the biofilm-covered slide with PBS. 2. The biofilm is submerged in a 4% paraformaldehyde (PFA) solution in a fresh Petri plate for Ih at RT. The PFA solution is prepared as described by Amann (1995). 3. Wash the slide with PBS. 4. Drying the slide for 10 min at 65°C will additionally fix the biofilm. 5. When necessary, dehydration can be carried out by incubating the slides for 3 min in ethanol solutions of increasing purity (50%, 75%, 100%). After air drying of the microscopic slides, the samples can be stored in an air-tight vessel at -20°C or stained immediately. 56 m. Materials and Methods In situ quantification ofgene transfer 2.1.4. Staining biofilms grown on slides with Svto 17 or Svto 60 nucleic acid stains (Molecular Probes. Eugene. Oregon! 1. Gently rinse the slides with 0.01 M MgSO, (Fig. 2F). At the manufacturers' recommendation, buffers containing phosphate need to be avoided as they may increase unspecific background signals. 2. Incubate 1 cm2 of the microscopic slide for 1-30 min at RT with 100 pi of a SO nM-20 pM Syto solution diluted with 0.01 M MgSO« (Fig. 2G). 3. Rinse the biofilm with 0.01 M MgSO« (Fig. 2H). 4. Non-fixed biofilms can be directly monitored after covering them with a microscopic cover slip (Fig. 21). A non-fixed biofilm cannot be stored after microscopic monitoring. Fixed biofilms need to be air dried. Dried, fixed and Syto-stained biofilms can be stored during several months in a sealed 50-ml disposable plastic tube at -20°C. For microscopic investigations, apply a drop of antifading agent (e g. Citifluor Ltd., London, U.K.) to the slide. Note that antifading agents may contain toxic or carcinogenic components and should be handled and applied in a fume hood while wearing gloves. After microscopic monitoring, the antifading agent can be gently rinsed off the biofilm-covered slide with distilled water. The waste is collected according to laboratory safety rules for hazardous compounds. After drying, the biofilm can be stored in a disposable plastic SO ml tube at -20°C. 2.1.5. Hybridisation of hinfilms on slides with rRNA-directed oligonucleotide probes Fixed biofilms on slides are dotted with 16 pi of hybridization buffer (Manz et al, 1992) mixed with 2 pi (100 ng) of oligonucleotide probe. Biofilms are hybridized in a moisture chamber at 46°C for 1.5 h, washed and prepared for microscopy as described previously (Manz ef of, 1992). 2.2. In situ GENE TRANSFER IN BIOFILMS GROWN IN FLOWCELLS 2.2.1. Growth of biofilms in flow cells 1. Construct a stainless steel (Kuehn et at., 1998) or plexiglass (Wolfaardt et al, 1994) continuous flow cell system consisting of four separate flow-through channels (40 mm long, 4 mm wide, 8 mm in height), sealed with 0.2 mm thick cover slips (24 x 50 mm) (Fig. 3) and supplied with medium via inlets and outlets connected with Tygon or Teflon tubes. The entire flow cell including tubing can be sterilized by autoclaving or by rinsing with 0.5% sodium hypochlorite. 2. Under sterile conditions, introduce medium into the flow-through channels using a peristaltic pump (Ismatec, Glattburg-ZOrich, Switzerland). 57 III. Materials and Methods In situ quantification of gene transfer Cover dips Fig. 3. Technical draft of the flow cell used in the experiments. Measurements are depicted in mm. The flow cell design and die technical draft was made by Martin Kahn. 3. Disconnect the medium flow and clamp the tubing connecting the inlets of the flow cell with the medium reservoir. 4. Sterilize the surface of the tubing near the outlet of the flow cell with ethanol, insert a sterile syringe tip connected to a sterile syringe containing an overnight culture of the recipient strain through the sterilized tubing into the flow channel and inoculate the channels with 200 pi of the culture. 5. Cell attachment is dlowed to take place during the following 2-3 hours. 6. Reconnect medium flow and allow the biofilm to develop on the cover slips for a desired time period, depending on the strain and medium used. Fig. 4. Picture of the experimental setup. The inlet medium was connected to the flowcell. The peristaltic pump pulled the medium through die flowcell and pumped it thereafter into the outlet flask. A 2 ml syringe was used for inoculation of the flow channels. 58 IIL Materials and Methods In situ quantification of gene transfer 2.2.2. Conjugation in flow cells 1. Introduce donor and if needed helper cells to the recipient biofilm by pumping the cell solution into the channels. 2. Disengage pump, allow 2 h for plasmid transfer between donor and recipient cells. 3. Wash for an additional 12 hours with phosphate buffered saline (PBS) to allow GFP expression in recipient cells. 2.2.3. Transformation in flow cells 1. Pump medium containing a desired amount of specific DNA prepared with a standard alkaline DNA extraction method (Sambrook et al., 1989) into the flow channel. 2. Wash the channels with mineral medium (or 0.01 M MgSO*) without DNA for an additional 12 hours to allow GFP expression in transformant cells. 2.2.4. Fixation of biofilms in flowcells 1. Rinse the biofilm by pumping PBS for 30 min through the flow channels 2. Replace PBS in the flow channels with 4% PFA using the pump. Fix biofilms for 1 h atRT. 3. Wash biofilms by pumping PBS through the channels for 1 h. 2.2.5. Staining biofilms in flow channels with nucleic acid stains 1. Pump 10 ml of an 50 nM-20 pM Syto solution diluted with 0.01 MgSO* into the flow channels. 2. Rinse overnight with 0.01 MgSO* by pumping at the same rate. 2.2.6. Hybridisation of biofilms in flow cells with rRNA-directed oligonucleotide probes 1. To precondition the fixed biofilms, hybridization buffer (Manz et al., 1992) containing the appropriate formamide concentration is pumped into the flow channels. 2. Desired oligonucleotide gene probes are added to hybridization buffer in a separate vial to obtain a final concentration of 6.25 ng gene probe/pl hybridization buffer. The preconditioning hybridization buffer is pumped out of the flow channels. 3. The buffer/probe mix is pumped into the flow channels so that the biofilms on the lower cover slip are covered. 4. The inlet and outlet tubes are clamped with metal clamps. The flow cell including tubing is transferred to a sealable plastic dish. Hybridization is carried out for 1.5 hat 46°C. 59 in. Materials and Methods In situ quantification of gene transfer 5. Following hybridization, the flow cell is reconnected to the pump. Appropriate washing buffer (Manz et al., 1992), prewanned to 48°C, is pumped through the channels to displace the hybridization buffer/probe solution. 6. The flow cell is again transferred to a sealable plastic dish and incubated for 30 min at 46°C. 7. The washing buffer is displaced with PBS and the hybridized biofilm can be observed with the microscope. 3. MICROSCOPIC in situ MONITORING AND QUANTIFICATION OF THE SAMPLES 3.1 TRANSFORMATION IN A MONOCULTURE BIOFILM To investigate natural transformation in a monoculture biofilm discrimination between two cell types is needed: recipients and transformants. While recipients can be detected with a general nucleic acid dye that helps visualize the total biofilm, transformants will only be detected after receiving and expressing genes encoding a GFP variant In this work plasmids pGARl (Hausner and Wuertz, 1999), pGAR2 (Hendrickx et al., 2000) and pGAR39 (Hendrickx et al., 2001) were used containing the gfp (green fluorescent protein) insert from pGFP (Clonthech, Palo Alto, Cal., USA), the eyfp (enhanced yellow fluorescent protein) insert from pEYFP (Clontech, Palo Alto, Cal., USA) and the ecjp (enhanced cyan fluorescent protein) insert from pECFP (Clontech, Palo Alto, Cal., USA), respectively, for transformation of the highly naturally competent A. calcoaceticus BD413. Biofilm cells were visualized with the general nucleic acid stain Syto 60 (Molecular Probes, Eugene, Or., USA) and were detected with an LSM410 CLSM (Zeiss, Jena, Germany). The 633 nm laser line and a 665 run longpass emission filter were employed to detect cells stained with Syto 60. The 488 nm laser line and a 515 nm longpass emission filter were used for the detection of cells expressing eyfp, ecjp and gfp (Clontech, Palo Alto, Cal., USA). With the same laser line, cells expressing ecjp could also be detected using a 515-540 bandpass emission filter with the appropriate threshold settings to avoid detection of EYFP signals. Epifluorescent microscopy studies were carried out with a Leitz Aristoplan microscope (Leica, Wetzlar, Germany) equipped with a 100x/1.32 oil immersion lens. 60 TO. Materials and Methods In situ quantification of gene transfer 3.2. CONJUGATION BETWEEN Acinetobacter SP. BD413 AND Ralstonia metallidurcms £H34 Investigation of conjugation in a biofilm implies discrimination between at least three cell types: donors, recipients and transconjugants. While donors carry and express the transferable genes encoding a GFP variant, recipients can be labeled with an oligonucleotide probe specific for the recipient strain. Recipients that have accepted and expressed the GFP variant, will be detected as labeled cells expressing GFP. Note that triparental conjugation involves a fourth player, the helper strain. In the conjugation process it acts as a silent partner providing the donor with tire necessary genes encoding the cognate conjugation system to transfer the plasmid carrying a GFP variant to the recipient strain. When the GFP variant carrying plasmid is transferred to the helper strain, cells will express fluorescence and be rightfully counted as donor cells. To visualize the recipient cells tire biofilm was hybridized with the rRNA-directed oligonucleotide probe BET42a (Manz et al„ 1992), labeled with the fluorescent dye Cy 5, which is specific for the p subgroup of Proteobacteria. The hybridized biofilms were investigated with tire CLSM using the 488 and 633 nm laser lines, in combination with the 515 to 540 nm band-pass and 665 nm long-pass emission filters to detect GFP expressing donors and Cy5-labeled recipients, respectively. Transconjugants were recognized as Cy5- labeled cells expressing GFP fluorescence. 4. AUTOMATED IMAGE ACQUISITION AND SEMI-AUTOMATED DIGITAL IMAGE PROCESSINGfWUERTZ eta!.. 2001) Intact biofilms are three-dimensional (3D) structures. Thus, gene transfer must be quantified in a biofilm volume unit Such an approach necessitates the use of CLSM (Caldwell et a/., 1992) which allows for the collection of confocal xy-optical sections obtained in stacks along tire z-axis. To minimize the subjective choice of a microscopic field to be scanned (i.e., repeatedly finding a position in the biofilm where a high number of transconjugants or transformants are apparent), an automated scanning procedure based on macro routines designed to perform automated investigations of biofilms using image acquisition and image analysis techniques (Kuehn et a!., 1998) should be employed. The key components of this method are tire on-line collection of confocal two-dimensional (2D) optical images in the xy-plane from a 3D domain of interest followed by the off-line analysis of these 2D images. 61 in. Materials and Methods In situ quantification cf gene transfer 1. Mount the flow cells with the stained or hybridized biofilm to the specimen support flame incorporated into the motorized stage. 2. Check the biofilm parameters (the staining or hybridization quality, transconjugant or transformant frequency, gfp expression, thickness and density of the biofilm) with the microsope in the fluorescence mode and find a suitable starting position. 3. Perform a pre-scan of the selected area to locate the biofilm-glass interface, the “z = 0” starting plane, and to optimize the scan parameters. These include contrast, brightness, pinhole size, laser intensity and possibly the use of the average filter during image collection. 4. The image collection macro is activated. Deliver information on the image edge length, the number of stacks in the domain of interest, the vertical step interval (Az) between consecutive vertical xy-sections and the file name for image storage. 5. Process the collected gray image with a Quantimet 570 image processing software (Leica, Cambridge, U K.) and use the macro routine that allows for semi-automated processing of image data (Kuehn et a!., 1998). The automatic macro routines involve image acquisition, image transformation with the user-specified filters, imagebinarization using the user-selected thresholds, co-localization (i.e. detection of common pixels in two corresponding images), quantification of areal fraction (the ratio of detected pixels to the total pixel number in the measured flame) in binarized images, and data output into a user- specified table, which can be read into a Microsoft-Excel file. Thereafter data can be analysed using mathematical formulae. 5. MATHEMATICAL PARAMETERS The mathematical formulae describing volumes, transformation frequency and normalised mean location are listed below: The calculation of the volume of transformants and the volume of recipients was based on the following equation: (a) XfV = total volume [pm3] X=area covered by cells of interest at position i [pm*] Zi = distance from the substratum at position i [pm] i = scanning position in the z direction starting at the biofilm substratum 62 in. Materials and Methods In situ quantification of gene transfer e = last scanned position in a biofilm in the z direction . The equation was adapted with the trapezoidal rule used to obtain a more correct approximation of the numerical integral after Kuehn et e/.(1998): £ = £~1/2x(Z| ~r»)x^+ -z,)x-y+l/2x(z,-r,^1))xX (b) Transformation frequency was the fraction of transformant volume/total cell volume. TF=V/V i>/ a, TF - transformation frequency V — volume of transformants obtained by formula (b) [nm 1] V = volume of recipients obtained by formula (b) [pm3] To obtain a reproducible estimate of biofilm porosity, total cell volume and biofilm thickness in the investigated biofilm area, we limited the total cell volume and biofilm thickness to contain 98% of tire scanned biomass (V) starting from the substratum towards the biofilm/bulk fluid interface within the investigated biofilm volume. '-fe-hVi V a Kx0.98 ** ** P — biofilm porosity [pm3/pm3] V — scanned volume limited to position k [pm5] jK = volume of recipients calculated with formula (b) limited to 98% of the total scanned biomass V [pm3] Kjl A = total scanned substratum surface area [pm3] k = position in a biofilm when V reached 98% of V KJt ks 63 m. Materials and Methods In situ quantification of gene transfer The normalized distance from the substratum was calculated as the distance from the biofilm attachement surface divided by biofilm thickness dj = Zj/zk for V S Vx0.98 R* V* = volume of recipients calculated with formula (b) limited to 98% of the total scanned biomass V [pm3] dj = normalized distance fiom the substratum at position i [pm] Zj = distance fiom the substratum at position i [pm] Zfc = biofilm thickness or distance fiom the substratum at position k [pm] k = position in a biofilm when V reached 98% of V R* Rf The normalized mean location (NML) was a parameter that described the normalized distance fiom the substratum where most transformants were localized inside the biofilm. The NML ranged between 0 (biofilm substratum) and 1 (biofilm top) and was based on the following equation describing the normalized position of the main weightpoint of residing transformants: T = area covered by transformants at position i [pm2] V = volume of transformants calculated by formula (a) limited to position k [pm3] 64 IV. RESULTS 66 IV. Results In Situ Monitoring of Natural Genetic Transformation A. USE OF gfp AND ^-VARIANTS FOR in situ MONITORING OF NATURAL GENETIC TRANSFORMATION IN MONOCULTURE Acinetobacter SP. BD413 BIOFILMS “Nothing tends so much to the advancement of knowledge as the application of a new instrument. The native intellectual powers of men in different times are not so much the causes of the different success of their labours, as the peculiar nature of the means and artificial resources in their possession." Sir Humphrey Davy (1778-1829), English Chemist 1. INTRODUCTION The use of in situ detection methods to monitor natural genetic transformation with gfp and variants as transforming DNA in combination with nucleic acid staining to visualize biofilm recipients using CLSM requires some preparative work and control experiments. First, the choice of chromosomal or extrachromosomal DNA as transforming DNA needs to be made. Because Acinetobacter sp. BD413 does not discriminate between homologous or heterologous DNA, the need for specially designed gfp and g^t-derivative tagged sequences of chromosomal DNA is not necessary. This thesis will concentrate on the in situ investigation of natural genetic transformation with plasmid DNA, which required less complicated cloning procedures. The recircularisation of the reporter plasmid after DNA uptake will be verified in this chapter. The possibility exists namely, that the reporter gene could be inserted unforeseeably into the chromosome by recombination. Further control experiments include the testing of the applicability of the nucleic acid stain in combination with the used fluorescent protein reporter genes. Also combinations with the different gfp- variants need to be tested if they are intended to be used simultaneously in an experiment And last but not least, it is important to test the reproducibility of results when working in a heterogeneous system such as biofilms. Disruption and homogenization of the investigated biofilms is not an option to obtain reliable reproducible results for in situ investigations. The use of an optimally sized investigated sample together with the investigation of a number of biofilms only slightly differently treated will allow for the retrieval of reliable data. 68 IV. Results In Situ Monitoring of Natural Genetic Transformation 2. PLASMID CONSTRUCTIONS Tagging bacteria with genes encoding fluorophores is advantageous in three ways compared to classical plating methods. Firstly, it allows detection of cells without selection pressure. If the expression of gfp and gjj-variants does not inhibit the cell’s metabolism, cell communities can be observed directly without any outside disturbance. Secondly it is possible to detect the location of the labeled cells inside bacterial communities. And thirdly, the use of fluorescent reporters could even detect viable but non culturable cells (Cho and Kim, 1999). The eyfp and ecfp genes of pEYFP and pECFP (Clontech, Palo Alto, Cal., USA) were cut out with Hindi II and EcoRI restriction enzymes, and ligated into the mob" tra+ IncPl broad host range vector pRK415 (Keen et al, 1988) likewise digested with Hindlll and £coRI.To select for the rightconstructs, ligated samples were introduced into E coli EC1 by elecroporation. Colonies were selected for tetracycline resistance, brightfluorescence upon UV radiation and the desired restriction pattern detected. The resulting plasmids pGAR2 (pRK415::%$?) and pGAR39 (pRK415::eg£>) could be used for the detection of in situ transformation (Fig. 4). Fig. 4. Physical and genetic map of pGAR2 and pGAR39. Plasmid pGAR2/pGAR39 consists of the broad-host-range tra" mob* teracyclinc resistant vector pRK4I$ (Keen et al., 1988) and the 0.79 kb EcoRI-ffWHI fragment carrying eyfp and ecfp, respectively. The introduced genes are under control of the Lac promoter. The constructed plasmid offers the host tetracycline resistance and fluorescence expression. 69 IV. Results In Situ Monitoring of Natural Genetic Transformation To construct pGAR38, primers were designed to add restriction sequences (HindlU, EcaiRI) to the gfp expression cassette of Tombolini et al. (1997) which consisted of a ribosomal binding site, the PpsbA promotor (Elhai, 1993) and the red-shifted gfp variant PI 1 (Heim et al., 1994). The forward primer containing a HintBR restriction sequence (S’-aaa gag ctt gta aaa cga egg cca gt-3’) and the reverse primer containing an £coRI restriction sequence (5’-aaa caattc aac age tat gac cat g-3’) were used to amplify the gfp expression cassette and after digestion with HindlU and EcoRI the fragment was inserted into the pMLIO vector (Labes et al., 1990) likewise digested with the same restriction enzymes. After ligation and electroporation into E. coli strain EC1, the desired construct was selected based on the tetracycline resistance marker, the expression of fluorescence and the correct restriction pattern (Fig. 5). San BamH Hndm BamH PpsbA —Aval BstEH Aval Fig. 5. Physical and genetic map of pGAR38. Plasmid pGAR38 consists of die broad-host-range tra" mob* gentamycin and tetracyline resistant vector pMLIO (Labes et o/„ 1990) and the 0.85 kb gfp expression cassette (Tombolini et al., 1997) under control of the PpsbA promoter (Elhai, 1993). The constructed plasmid offers the host gentamycin and tetracycline resistance as well as g^>-fluorescence expression. 70 IV. Results In Situ Monitoring of Natural Genetic Transformation 3. INTEGRATION OF THE FLUORESCENCE REPORTER GENES In a first step, fire desired expression of the reporter gene, residing on the recombinant plasmids, after DNA uptake by transformation was tested. Of each plasmid (pGARl, pGAR2, pGAR39 and pGAR38) 1 pg DNA/ml was used to transform Acinetobacter sp. BD413 by inoculation of 1/25 volumes of an overnight BD413 culture into fresh medium containing 1 pg DNA/ml as described by Palmen et aL (1993). Transformants were selected on selective minimal medium agar plates containing tetracycline. Thereafter, transformed colonies where checked for fluorescence. While all tetracycline resistant colonies grown on selective plates after transformation with pGARl, pGAR2 or pGAR39, fluoresced upon UV illumination, only maximally 40% of the antibiotic resistant colonies obtained by transformation with pGAR38 were fluorescent It is possible that a part of the plasmid pGAR38, containing the resistance gene, was inserted into the chromosome instead of recombining into a circular plasmid, thereby losing the fluorescence reporter gene. Hence, in these experiments, pGAR38 was not very useful although foe colonies that did express gfp, were brightly fluorescent 4. APPLICATION FORM OF THE TRANSFORMING PLASMID To test whether the desired phenotypic trait by addition of the pRK415 derivative plasmids was obtained due to integration into foe Acinetobacter sp. BD413 genome by recombination of foe fluorescent reporter gene into foe chromosome or remained an extrachromosomal unit by recombination of two linearized plasmid copies, a grown biofilm was exposed to plasmid DNA in different forms. Biofilms were transformed with 1 pg plasmid pGAR2 DNA/ml that was either digested with EcoRI or digested with HindtH. Furthermore foe addition of a mixture of 0.5 pg pGAR2/ml linearized by EcbRl and 0.5 pg pGAR2 DNA/ml linearized by HindHl was tested. Also transformation with 1 pg/ml crude undigested pGAR2 was checked in a separately grown biofilm. There was no observable difference in transformation frequency when undigested (5.92x10*), mixed digested (8.86x1 0"04) and HindlVL digested (8.08x1 O'04) pGAR2 DNA was used as transforming DNA. Digestion of foe plasmid with EcoRI decreased the transformation frequency almost 100-fold (9.10x10*). Gel electrophoresis of foe digested samples revealed partial digestion of the HindSi digested sample, leaving enough ‘background ’ uncut plasmid DNA. Even just a tenfold uncut 71 IV. Results In Situ Monitoring of Natural Genetic Transformation DNA (100 ng/ml) could still leave enough DNA for standard transformation. £coRI digestion, on the other hand, was complete although the DNA sample probably did contain some uncut contaminant plasmids. The lower frequency indicates, however, that cells were unable to recircularize the singular cut plasmid DNA due to the impossibility to piece overlapping single strands together, leading to lower transformation frequencies. The results indicated that Acinetobacter sp. BD413 was able to take up circular and linearized plasmids as was also shown by Palmen et al„ (1993). Furthermore, pGAR2 recircularized in BD413 as a plasmid and was not integrated into the chromosome, because two differently cut linearized plasmids are required to obtain the EYFP fluorescence phenotype (Saunders and Guild, 1981). Because BD413 was able to take up circular plasmids by cutting them randomly to obtain the linearized form, necessary for DNA uptake (Lorenz and Wackemagel, 1994), crude extracts of undigested plasmids were used in future experiments. 5. COLOCALIZATION Control experiments were designed to test detection of false positive fluorescent protein signals. The effect!vity of colocalization of fluorescent protein expressing nucleic acid stain labeled cells was tested. When no DNA was added to the medium, no signals were detectable using CLSM settings for detection of EYFP/ECFP/GFP signals in two separate tests. In an additional test, signals were obtained using EYFP settings, but no overlap was detectable with the Syto 60 settings. These false EYFP signals might be due to autofluorescent impurities in the inlet medium. Pure culture BD413(pGARl), BD413(pGAR2) and BD413(pGAR39) biofilms grown in selective minimal medium (M9, 0.2% gluconate, 20 pg/ml tetracycline) were stained with Syto 60 to test colocalization of both signals. Approximately 18.5% of the signals in the BD413(pGARl) biofilm were colocalised GFP and Syto 60 signals, 22.6% were single Syto 60 signals and 58.9% were single GFP signals (Fig. 6 A). Likewise even fewer signals (7.8%) were colocalised in a pure culture BD413(pGAR39) biofilm as ECFP expressing Syto 60 stained cells; 47.5% were only detected with the ECFP settings and 44.7% only with the Syto 60 stain settings. Therefore, in transformation experiments total cell volume was obtained by adding Syto 60 signals and GFP/ECFP signals and subtracting the overlapping signals. GFP/ECFP signals were regarded as transformants. To avoid overestimation of GFP/ECFP due to autofluorescent impurities, images needed to be checked manually to discard possible false positive signals on the basis of form and size. The potential underestimation of 22.6% GFP signals and 44.7% ECFP signals is not considered in the calculation of the results because it was not possible to check whether cells still contained the introduced plasmid. 72 IV. Results In Situ Monitoring of Natural Genetic Transformation Fig. 6. Picture depicting pure culture biofilms BD413(pGARl) (A) and BD413(pGAR2) (B) grown in selective medium (M9, 03% gluconate, tetracycline) during 46 hours, whereafter the biofilm was stained with Syto 60. Images A and B depict superimposed single optical sections of Syto 60 stained cells (red darnel) or fluorescent protein expressing cells (green channel) in a microscopic field of approximately 288pmx288pm and 1 lSpmxl 15 pm, respectively. In the pure culture BD413(pGAR2) biofilm a 55.1% Syto 60 and EYFP overlap was obtained (Fig. 6 B). 39.6% of the signals were from the Syto 60 stain alone and only 5.3% represented single EYFP signals. Because the biofilm was very fragile and lysis was observable after only 21 h of growth, it is possible that cells lost pGAR2 when dividing. Transformants were regarded as Syto 60 stained, (^-expressing cells. Total cell volume was regarded as Syto 60 stained biomass. This way any possible false positive EYFP signal could be avoided. 6. SPECTRAL OVERLAP Detection of possible misinterpreted EYFP or ECFP signals by overlapping emission tails in experiments using both signals, was tested in a control experiment In a control biofilm containing a mixture of BD413 and BD413(pGAR39), only 14% of the ECFP signals could be misinterpreted as EYFP signals. Hence, there was a possible underestimation of ECFP signals by 14%. Using contrast threshold and filter settings for ECFP detection in biofilm containing eyfp expressing cells, no signals were detected. It can be concluded that signals detected using the ECFP microscopic detection settings could only be produced by ecjp expressing cells. 73 IV. Results In Situ Monitoring of Natural Genetic Transformation 7. REPRODUCIBILITY OF in situ TRANSFORMATION USING CLSM In situ transformation experiments in monospecies Acinetobacter sp. BD413 biofilms were performed as previously described (see Materials and Methods). First BD413 biofilms were grown for 3 days in rich medium in a continuous flow-through cell. Natural transformation was allowed to occur in situ in mineral medium containing gluconate during lh by adding 1 pg plasmid pGAR2 DNA/ml to the influent medium. After transformation, biofilms were incubated in mineral medium without DNA to allow expression of the obtained plasmid DNA overnight The flow rate in the system remained unchanged at 2.4 ml/h during growth, transformation, incubation, staining and washing to minimize shear forces that could obscure the effects of biofilm behavior after introducing the detrimental gene. Following staining, the biofilms were subjected to microscopic monitoring with CLSM, automated image acquisition and semi-automated digital image processing and analysis. Transformation frequencies were obtained by calculating volumes (Section mD.4.). Hence, transformation frequencies present a value in pm1 transformants/pm 1 recipients. For reasons of readability these units will not be stated in the following sections unless the transformation frequency was obtained by other means than as a resulting volume fraction. Standard transformation frequencies from 7 separate experiments ranged between 1.64x1 O'04 and 8.86x1 0"04, or between the logarithmic values -4.16 and -3.06, with a mean value of 2.46xlO'04 or logarithmic value -3.61 and a standard deviation of 0.29. In contrast to the standard method using planktonic cells (Palmen et al., 1993), the in situ method using CLSM offered reliable and repeatable results. In all 7 experiments a minimum volume of 1.2x10? pm1 was scanned. Hence, as a rule of thumb 1.2x10? pm* was used as the minimal size needed to be scanned in future transformation experiments in biofilms. 8. CONCLUSIONS The plasmids pGAR2, pGAR39 and pGAR38 were successfully constructed. Together with pGARl (Hausner and Wuertz, 1999) they offered the possibility to work with different ^-variants that could be excited and emitted at different wavelengths. The constructs were under control of different promotors and carried by different vectors. All plasmids were tested on Acinetobacter sp. BD413 as a host Plasmids derived from pRK415, (pGARl, pGAR2 and pGAR39) could be recircularized inside the host On the other hand could chromosomal integration of the tetracycline resistance marker of pGAR38 have 74 IV. Results In Situ Monitoring of Natural Genetic Transformation interfered with plasmid integration by recircularization. Nevertheless, further genetic investigation is necessary before this assumption can be accepted. . If biofilms were scanned for monitoring transformation events the following rules were obeyed: - gfptecfp expressing cells are detected as GFP/ECFP signals - eyfp expressing cells are detected as EYFP signals overlapping Syto 60 signals - recipients in biofilm experiments using pGARl or pGAR39 are the detected Syto 60 stained plus gfp/ecfa expressing cells minus overlapping signals - recipients in biofilm experiments using pGAR2 are the detected Syto 60 stained cells - g$r-variants eyfp and ecfp which possess different excitation and emission wavelengths can be combined in the same biofilm experiment - a minimum volume of 1.2x107pms should be scanned in every investigated biofilm 75 IV. Results Evaluation of eyfp as a disadvantageous gene B. EVALUATION OF eyfp AS A DISADVANTAGEOUS GENE IN Actnetobacter SP. BD413 "There must be some kind of unsatisfactory scientific explanation." Leif Panduro (1923-1977), Danish author 1. INTRODUCTION Lethal genes have been largely investigated for use in biological containment systems (Molin et al., 1987; Cuskey, 1992; Knudsen and Karlstrdm, 1991; Molin et al., 1993; Ronchel et al., 1995). These ‘containment ’ genes were developed to avoid potential risks associated with unintentional releases of genetically engineered micro-organisms as well as with the unpredictability of their behaviour in natural ecosystems (Ronchel et al., 1995; Molin et al., 1993). To obtain a containment system, a suicide mechanism is used based on the expression of a lethal gene, which is induced either randomly, resulting in killing a fraction of the recombinant cells per unit time (Molin et al., 1987), or which is triggered by certain conditions like the transfer of recombinant genes to wild-type strains (Molin et al., 1987, Munthal et al., 1996), growth temperature (Ahrenholtz et al., 1994) or absence of a chemical compound (Jensen et al., 1993; Kaplan et al., 1999; Ronchel et al., 1995). Next to lethal genes like hot and gef (Poulsen et al., 1989), which encode a cell-killing function, products of foreign genes can likewise posses toxicity when expressed inside host cells (Nieboer et al., 1993; O’Connor and Timmis, 1987). Also certain native genes can be detrimental to the host when these are overexpressed or when mutant derivatives are used (Beck and Bremer, 1980; Joyce and Grindley, 1983; O’Connor and Timmis, 1987). In the past studies involving survival of containment strains and persistence of genes that should not be disseminated in the environment were performed with the use of selective plating techniques (Cuskey, 1992; Knudsen and KarlstrSm, 1991; Molin et a!., 1993; Molin et al., 1987; Ronchel et al., 1995). As of yet, no report exists on in situ unselective monitoring of cells/strains carrying to-be-contained genes in a multi-species defined or undefined bacterial community. 76 IV. Results Evaluation of eyfp as a disadvantageous gene In this chapter, the gene for the enhanced yellow fluorescent protein, eyfp was evaluated as a detrimental gene in the host strain Acinetobacter sp. BD413 using both in situ quantitative microscopy and ex situ selective plating. The objectives were to study the survival of «%gr-expressing BD413 cells and the dissemination of a plasmid carrying eyfp in suspended cell cultures and biofilms. When comparing the use of different gfp variants for labeling BD413, eyfp had a peculiar, almost toxic effect on Acinetobacter sp. BD413. When BD413 cells accepted the eyfp carrying plasmid pGAR2 in mineral medium containing gluconate by horizontal gene transfer, certain death (Fig. 7) occurred in a time span of a few hours to several days, depending on the mode in which transformants were grown. Fig. 7. Picture depicting the lysis of a transformed cell. Matured EYFP is set free and clouds the surrounding non-transformant cells. The detection of EYFP (red) reveals the coccoid forms of the cells neighboring the lysing cell. The size bar measures approximately 1.5 pm. It has been suggested that the lethal effect of GFP and its derivatives expressed by some bacterial strains could be due to toxicity associated with overexpression of the protein (Endow and Piston, 1998). In this chapter, eyfp was evaluated as a detrimental gene in the host strain Acinetobacter sp. BD413. Furthermore, the survival of «%$?-expressing BD413 cells and the persistence and dissemination of pGAR2 was followed in predefined mixed strain biofilms. Maintenance of the compromised strain was evaluated by testing viability and survival of the strain grown in various modes. Persistence of the detrimental gene was evaluated by investigating plasmid stability in the host and the spread of plasmids towards 77 IV. Results Evaluation of eyfp as a disadvantageous gene ejg$>-expression sensitive strains on petri plates or in biofilms. Furthermore, dissemination of the disadvantageous gene was investigated from compromised ey^t-expression sensitive donors towards potential recipients that were more tolerant to the gene product. 2. VIABILITY AND FLUORESCENCE OF TRANSFORMANTS AND TRANSCONJUGANTS In the strain BD413(pRK415) no fluorescence was observed when compared with strain BD413. Upon epifluorescent microscopy again no fluorescence was observed. Weak fluorescence was observed by transconjugant and transformant strain BD413(pGAR2) on the transilluminator as well as with undiluted plated suspensions by transconjugant and transformant strains BD413(pGARl) and BD413(pGAR39) (Table 1). This weak fluorescence could indicate compromised metabolism of the labeled bacteria by expression of the recombinant marker. Transconjugant strains Transilluminator Epifluorescent microscopy Weak fluorescent colonies Either strong or no fluorescence expression of single cells Bright fluorescent colonies Strong homogeneous fluorescence expression of single cells BD413(pG AR39) Bright fluorescent colonies Strong homogeneous fluorescence expression of single cells BD413(pRK415) No fluorescent colonies when No fluorescent single cells compared with autofluorescence ofBD413 Table. 1. Evaluation of fluorescence produced by fluorescent protein expressing BD413(pGAR2), BD413(pGARl), BD413(pGAR39) and BD413(pRK415); observed on the transilluminator and with the use of epifluorescent microscopy. BD413(pGAR2) colonies grown on plates with tetracycline displayed even weaker fluorescence compared to colonies produced on plates with no tetracycline, from the same pure culture suspension. Although selective plates would counterselect against recipients, donors or helper cells, microscopical investigation often reveals a different story (Hausner, pers. com.). A colony 78 IV. Results Evaluation of eyfp as a disadvantageous gene picked from a plate that would select for transconjugants after plating out the suspended mating patch does not solely contain transconjugants. Only after repeated subculturing on selective plates, it is possible to obtain pure culture transconjugant colonies. Hence it was no surprise to detect a mixed population of E. coli (donor and helper strain) and Acinetobacter sp. cells when transconjugant colonies were picked from the selective plates for microscopic investigation. Purification of the transconjugants resulted in pure culture transconjugants, except for BD413(pGAR2). BD413(pGAR2) did not survive the third subculturing step on fresh tetracycline containing mineral medium plates (Table 2). Mode of growth Medium Observation Flowcell M9 ,02 % gluconate Gas bubbles after 21h Biofilm sloughs off within 46h No fluorescence after 46h M9, 0.2 % gluconate, Gas bubbles after 21h tetracycline Biofilm sloughs off within 46h Fluorescence after 46h Liquid culture Tris, 0.2 % gluconate Tetracycline resistent cfiis detectable when BD413 (pGAR2) was subcultured several times in fresh medium incubated overnight and streaked on Tris glue tetr agar plates +/-fluorescence Tris, 02 */o gluconate No tetracycline resistant cfiis detectable when tetracycline BD413 was inoculated twice in fresh medium with tetracycline, incubated overnight and streaked on (Tris glue tetr agar) plates +/-fluorescence Agar plates Tris, 0.2 % gluconate Viable during +/- 20 days Fluorescence Tris, 02 % gluconate Viable during +/-14 days or 2-3 purification tetracycline steps Fluorescence Table. 2. Observation on survival and fluorescence by eyfp-exprcssmg Acinetobacter sp. BD413 cells grown in different modes and in the presence or absence of selective pressure. Viability was checked by picking up a colony, streaking it on a flesh selective plate and checking for growth after a 2-day incubation at 30°C. cfiis - colony forming units 79 IV. Results Evaluation of eyfp as a disadvantageous gene In transformation experiments, donor or helper cells would not longer be needed and hence transgenic colonies could not be contaminated with the presence of these strains. Investigation of purified transformant colonies showed that BD413(pGARl) and BD413(pGAR39) transformants had strong homogeneous fluorescence expression upon investigation with the epifluorescent microscope, whereas transformant strain BD413(pGAR2) displayed a decidedly heterogeneous fluorescence. In the latter case only some cells fluoresced brightly while others did not fluoresce at all, because the colony consisted of a mixture of transformed BD413(pGAR2) cells and nontransformed BD413 cells. It can be suggested that an ey^labeled cell could only grow when these uncompromised unlabeled cells were present because purification by repeated streaking on flesh tetracycline containing plates led again to the loss of the viability of the strain containing the compromised plasmid pGAR2. When transformant strain BD413(pGAR2) was grown as a pure culture using medium with or without tetracycline in a flowcell, lysis was observable in the biofilm after only 21h. Gas bubbles started to appear in the flowcell. After 46h the whole flowcell was filled with gas bubbles. The biofilm itself was hardly detectable with the naked eye. It consisted of slimy viscous debris, which could easily be detached from the surface. The cells of a monospecies 46 h pure culture BD413(pGAR2) biofilm could only fluoresce clearly when grown under tetracycline pressure upon investigation with the CLSM (Table 2) (Fig. 8). The settled cells of a pure culture BD413(pGAR2) biofilm, grown without tetracycline pressure, might have lost the plasmid or blocked the expression of eyfp. Growing BD413(pGAR2) in a flowcell lead to extreme overall cell lysis within 2 days. Growth was also very unstable in liquid medium containing tetracycline. After subculturing twice in fresh medium, it was observed that no BD413(pGAR2) colony was able to grow on selective agar plates, although the added antibiotics in the liquid medium selected for plasmid maintenance. Likewise it was impossible to grow and purify BD413(pGAR2) long enough on agar plates to be able to process a culture for preservation in liquid nitrogen. Hence it was always necessary to transform BD413 with pGAR2 DNA using standard methods (Palmen et a!., 1993) every time a ‘pure culture’ BD413(pGAR2) was needed. When ‘pure’ culture strain BD413(pGAR39) was grown as a biofilm under selective (M9, 0.2% gluconate, tetracycline) as well as under non-selective (LB) conditions during 46h, it was found that the strain was very stable. Under non-selective conditions cells still maintained pGAR39, shown by an approximately equal volume of eq$Mransformants compared with the total volume of cells in biofilm (Table 3). Under selective conditions it was observed that the strain could likewise grow a biofilm without a problem. The total 80 IV. Results Evaluation of eyfp as a disadvantageous gene volume of cells growing under selective conditions was comparable to the total volume of cells under non-selective conditions (Table 3). The same, however, could not be observed for the BD413(pGAR2) biofilm. Under non- selective conditions cells rapidly lost their plasmid pGAR2 or blocked the expression of eyfp, shown by a dramatical reduction in the number of ey£>-expressing cells compared with the total number of cells (Table 3). And under selective conditions almost all cells lysed, leaving only a very few ejj^exprcssing cells (Table 3). Fig. 8. Confocal laser scanning microscope images of Syto 60 stained pure culture Acinetobacter sp. strains BD413(pGARl), labeled with g/p (B) or BD413(pGAR2), labeled with 81 IV. Results Evaluation of eyfp as a disadvantageous gene BD413(pGAR2) BD413(pGAR39) e%$»-expressing total volume of ecj$7-expressing total volume of cells (pm3) cells (pm3) cells (pm3) cells (pm3) LB 3.6 2.1x10" 5.1x10" 5.3x10" M9, glue., tetr. 2.5x10* 5.7x10* 2.6x10" 4.7x10" Table 3. Total volume of cells as well as the volume of ey^j-expressing cells and egfi-expressing cells in a pure culture BD413(pGAR2) and BD413(pGAR39) biofilm respectively, grown under selective (M9, 0.2% gluconate, 20pg/ml tetracycline) and non-selective (LB) conditions during 46 hours. The volume of die eyfp- expressing cells, eq^>-expressing cells and the total volume of cells were extracted from a total scanned volume of 1.8X10 ’pm3. 3. SURVIVAL OF THE COMPROMISED TRANSFORMANT IN A MIXED BIOFILM The fate of transformants, expressing a detrimental gene, was investigated on line and in situ in a preset volume of interest in a three species mixed biofilm grown in a flowcell fed with rich LB medium. Therefore an almost pure culture suspension of BD413(pGAR2) (only subcultured once) that would only contain labeled and a little fraction of unlabeled BD413 was diluted,into an unlabeled BD413 culture and used as an inoculum for biofilm development in flowcells. Also BD413 labeled with ecfp were added to the mixture to be able to check biofilm growth and normal strain development inside a biofilm community. Therefore, different dilutions of Actnetobacter sp. BD413 labeled with either eyfp or ecfp were mixed with unlabelled BD413. BD413 labeled with ecfp was diluted three times more than BD413 labeled with eyfp, which was diluted either 10-fold, 100-fold, 1000-fold or 10,000-fold into the undiluted unlabelled culture. Thereafter, the mixtures were used as an inoculum to grow a biofilm, by injection of the mixture into the flowcell. After a settling time of 3 hours the peristaltic pump was engaged and the mixed biofilm was allowed to develop. Actnetobacter sp. BD413 cells labeled with either fluorescence marker steadily established themselves in the biofilm (Fig. 9). It seemed that Actnetobacter sp. BD413(pGAR2) needed the presence of other, not compromised cells to survive inside the biofilm. This was already observable with the microscopic investigation of BD413(pGAR2) colonies, grown on selective agar plates. 82 IV. Results Evaluation of eyfp as a disadvantageous gene Fig. 9. On line measurement of volume accumulation of ecfp- (•) and qg^t-expressing (o) cells inside a mixed biofilm consisting of three strains (BD413; BD413(pGAR2); BD413(pGAR39)) during growth for 9 days. Fkweells were inoculated with culture mixtures containing strains BD413(pGAR2) and BD413(pGAR39) diluted in an unlabeled BD413 suspension. Graphs A, B, C, and D depict respectively an inoculated mixture with 10-fold diluted BD413(pGAR2) and 30 fold diluted BD413(pGAR39) (A); 100-fold diluted BD413(pGAR2) and 300-fold diluted BD413(pGAR39) (B); 1000-fold diluted BD413(pGAR2) and 3000-fold diluted BD413(pGAR39) (C); and 10,000-fold diluted BD413(pGAR2) and 30,000-fold diluted BD413(pGAR39) (D). As long as the colonies were not purified, BD413(pGAR2) cells could live in the presence of uncompromised neighboring cells (donor cells, helper cells, non-transformed cells). After purification BD413(pGAR2) colonies were cured from cells that did not contain pGAR2. These colonies, however, did not contain viable BD413(pGAR2) cells for a long time. Also, in biofilms Acinetobacter sp. BD413(pGAR2) did not seem to suffer dramatically from negative selection when it was allowed to grow at diluted numbers in a mixed strain biofilm. This was surprising considering the fact that it was not possible to grow a pure culture BD413(pGAR2) biofilm. The harm caused by EYFP, in biofilms grown in rich medium in the presence of ecfp labeled and unlabeled Acinetobacter sp. BD413, did not cause extinction of compromised transformants. It has been noted that an enhanced diversity in a bacterial community favors bioaugmentation by added exogenous strains (Dejonghe et al„ 2001). These results indicate that the biofilm mode of growth and the increased diversity of a three-species biofilm, compared to a monospecies biofilm, plays a 83 IV. Results Evaluation of eyfp as a disadvantageous gene possible role in stabilizing the survival of strains in an established bacterial community that have a selective disadvantage over other members of the biofilm. It was even more surprising to detect practically no effect of dilution in the starting volumes of eyfp expressing cells, which were monitored after only 6 hours of growth. The first scanning of the biofilm grown with 10-fold, 100-fold, 1000-fold or 10,000-fold dilutions, measured respectively 2256 pm1,712 pm1, 605 pm1 and 591 pm1 volumes of eyfp expressing cells (Fig. 9). Within 6 hours the volume of eyfp expressing cells must have rose dramatically in flow cells containing a 1000-fold or 10,000-fold dilution of eyfp expressing cells in the inoculum. Subsequently the volume of eyfp expressing cells stayed approximately status quo, with regular fluctuations in volume measurements due to cell growth and cell death. The respective final volumes of eyfp expressing cells were 6747 pm1, 2024 pm1,3400 pm1, and 14872 pm1. The volumes of ecfp expressing cells behaved as expected (Fig. 9). The starting volumes of ecfp expressing cells were 986 pm1,224 pm1,8 pm1 and 0 pm1 with the 30-fold, 300-fold, 3000-fold and 30,000-fold dilutions respectively. They grew steadily towards an endvolume of respectively 20823 pm1, 17719 pm1, 30584 pm1 and 28912 pm1. One can speculate that the development of volumes of eyfp expressing cells was essentially the same, except that the exponential growth phase was already reached within the first six hours. Once a certain optimal volume of the introduced bacterial strains was reached, the number of cells fluctuated due to temporal growth spurts and subsequent death or detachment but remained essentially the same. 4. STABILITY OF THE RECOMBINANT PLASMIDS Singer et al. (1986) observed high instability of pRK290 (from which pRK415 is derived) in BD413 and speculated that the deletion of the RK2 DNA to form pRK290 specified DNA primase and was strictly required for stable plasmid maintenance. Low level selection with tetracycline (1 to 5 pg/ml) stabilized pRK290 in BD413 cultures, but resulted in host cells that were osmotically fragile and exhibited lysis upon mixing or dilution (Singer et al., 1986). Therefore, the stability of the plasmids in the transconjugant strains BD413(pGARl), BD413(pGAR2) and BD413(pGAR39) was tested. The plasmid stability dropped for every transconjugant after 30 generations. All tested plasmids were equally stable. Even the detrimental plasmid pGAR2 was not less stable than the other transconjugants. 84 IV. Results Evaluation of eyfp as a disadvantageous gene 5. DISSEMINATION OF PLASMIDS BY TRANSFORMATION IN SUSPENDED CULTURES To test variations in DNA uptake, dependent on the gfp variant, transformation was tested. In transformation experiments 1 pg DNA/ml of untagged pRK415 or recombinant plasmids pGARl, pGAR2 and pGAR39 was used. It was a surprise that a high transfer frequency was observed when the adverse plasmid pGAR2 was used for testing transformation (Table 4). It is important to note that variation between two separate tests using the same plasmids exceeded variation between two separate tests using different plasmids (Table 4). However, in both tests initiated with different starting cultures, pGAR2 exhibited high transformation frequency in comparison with the unlabelled plasmids. Plasmid Transformation Table 4. Transformation frequencies of Acinetobacter sp. (T/R) BD413 using lfig Plasmid DNA (pGARl, pGAR2, pGAR39 and control plasmid pRK415) /ml. pRK415 2.40x10"; 2.73x10"*" a and b are frequencies resulting from different BD413 culture experiments. pGARl 3.60x10"; 3.10x10" T - transformant cfus; R ™ recipient cfus pGAR2 7.20x10"; 4.30x10" pGAR39 6.00x10^6.88x10'"" 6. DISSEMINATION OF PLASMIDS BY TRANSFORMATION IN BIOFILMS To test the effect of gfp variant on natural genetic transformation in biofilms grown in flowcells, biofilms were subjected to transformation with 0.2 pg pGARl, pGAR2 or pGAR39 DNA/ml during 15 minutes, and transformation frequencies were obtained with quantitative microscopy. Transformation using pGAR39 resulted in the highest transformation frequency. While transformation using pGARl gave rise to an intermediate transformation frequency, transformation using pGAR2 delivered the lowest transformation frequency (Table 5). The same trend was seen in a repeated experiment It was surprising to observe that while transformation frequencies obtained with the addition of pGAR39 were the lowest in transformation experiments using suspended cultures, 85 IV. Results Evaluation of eyfp as a disadvantageous gene transformation experiments in biofilms lead to the highest frequencies with this ecfp carrying plasmid. The reverse could be observed when transformation with pGAR2 in suspended cultures was compared with biofilm cultures. While transformation with pGAR2 was relatively high in suspended cultures, transformation with pGAR2 in biofilms resulted in the lowest detected frequency compared with transformation using other plasmids. Hence, successful transformation in biofilms will be dependent on other parameters than those that influence transformation in suspended cultures. Table 5. Transformation fre- Plasmid Transformation frequency quentcies of Acinetobacter sp. BD413 in biofilms using pGARl 7.67x1 O'4,6.84x1 04; 3.47xl04 plasmids pGARl, pGAR2 and pGAR39, obtained by CLSM. pGAR2 7.76x10"5; 8.88xl0" 5 pGAR39 8.74x1 0"3; 6.46x10* 3 The few transformants that were formed with pGAR2 transformation (compared with transformation with pGARl and pGAR39) were very sensitive. Every attempt to obtain eyfp- transformant colonies on plates after mechanical removal of the biofilm failed and hence the measured «%j$Mransfbrmation frequencies could only be detected with CLSM. Lowder et al. (2000) studied the use of OFF as a tool to monitor bacteria in the viable-but-nonculturable state, the authors observed that the majority of dead cells did not display GFP fluorescence, due to the loss of membrane integrity. This suggests that in the present study, at least a fraction of eyjiMransformants could be regarded as viable but nonculturable on selective agar plates. In situ detection of cells expressing detrimental DNA could henceforth reveal possible persistence of cells previously thought to be disappearing very quickly. 7. DISSEMINATION OF PLASMIDS BY CONJUGATION IN A PREDEFINED BIOFILM To evaluate the persistence of a detrimental gene compared with the dissemination of a neutral gene in a biofilm environment, the possibility of triparental conjugation was investigated between the compromised transformant BD413(pGAR2) or the noncompromised transformant BD413(pGARl) as the donor and Ralstonia metattidurans CH34 (Houba, 1976) as the recipient strain, which was able to express eyfp or gfp without any observable negative effects (Hendrickx, unpublished data). Unfortunately, there was no triparental conjugation observed when Ralstonia eutropha CH34 strain was mated with 86 IV. Results Evaluation of eyfp as a disadvantageous gene Acinetobacter sp. BD413(pGARl) or Acinetobacter sp. BD413(pGAR2) as donors and in the presence of helper strain EC7. This might be due to the reported low conjugation frequencies, with.dc/Reto6acfer sp. BD413 as a donor (Towner and Vivian, 1976). 8. CONCLUSIONS The expression of eyfp by Acinetobacter sp. BD413 imposed a burden on the strain and caused death after a certain period of time. The negative influence of (^-expression together with its fluorescence properties offered the opportunity to investigate maintenance and dissemination of a disadvantageous gene in situ. Maintenance of compromised BD413(pGAR2) cells was enhanced in the presence of uncompromised cells. Cells retained viability much longer when they were allowed to grow colonies on agar plates in the presence of unlabeled BD413 cells or E coli strains that were present due to the mating procedure or the standard transformation experiment In monoculture BD413(pGAR2) biofilms, cells started to lyse within one day. Under non- selective conditions they seemed to lose the plasmid very rapidly, which was not the case for monoculture BD413(PGAR39) biofilms. Selective pressure, on the other hand, instigated almost complete destruction of the biofilm. However, when diluted in a culture of isogenic strains that did not carry the eyfp gene, BD413(pGAR2) cells grew well and were maintained during the entire observation period (9 days). They demonstrated a rapid initial increase in biomass volume during the first six hours. This could be an indication that expression of EYFP harmed cells by inducing elevated and insufferable cell metabolism. Although cells expressing eyfp suffered mote and lysed more readily it was observed that the detrimental plasmid could be regarded as stable as plasmids containing neutrally affecting plasmid variants. Transformation of the adverse gene occurred at increased frequencies in suspended cultures, while the opposite was true for transformation of the adverse gene in biofilms. In biofilms, transformation with the adverse gene resulted in the lowest transfer frequencies. The plasmid containing disadvantageous DNA could readily be transferred to BD413 but thereafter the host did not seem to be able to transfer the complete plasmid carrying the detrimental gene to a tolerant recipient However, the latter problem did not originate in the challenged metabolic character of the compromised cell. Conjugation with the use of CH34 as a recipient strain and the pRK415 derivative plasmid was difficult regardless of the (health) condition of the donor. 87 IV. Results In situ quantification of natural genetic transformation C. In situ QUANTIFICATION OF NATURAL GENETIC TRANSFORMATION IN MONOCULTURE Acinetobacter SP. BD413 BIOFILMS "Seek simplicity and distrust it" Alfred North Whitehead (1861-1947), English mathematician and philosopher 1. INTRODUCTION Until now transformation has rarely been investigated in biofilms. The factors that influence natural genetic transformation in biofilms are therefore yet unknown. In this chapter Acinetobacter sp. BD413 was chosen as a model bacterial strain to take a first glance at natural genetic transformation in situ in biofilms. Biofilms are formed by micro-organisms conditioned to adhere to solid substrata. After initial attachment, bacteria, fungi, algae and protozoa start to form micro-colonies and consortia to develop a mature and optimally adapted biofilm (Davey and O’Toole, 2000). Changing conditions will reshape and restructurize the biofilm and biofilm cells (Watnick and Kolter, 2000). The formation of biofilm architecture in space and time is described by the morphogenesis of a biofilm. Hence, it does not only describe the three-dimensional spatial structure consisting of channels, pores and the juxtaposition of microcolonies and bacterial consortia. It also describes the development of a biofilm: the initial attachment and tire lag phase of a biofilm, growth and development and the subsequent stable phase of a biofilm where detachment, growth and attachment are balanced (Davey and O’Toole, 2000). Microbial community shifts due to external or internal changing parameters likewise take part in biofilm development and morphogenesis. Even the morphological changes on a microscale, the morphotype of a bacterial cell, shape biofilms in space, time and maybe even in physiological properties. It is possible that natural genetic transformation will be affected by the spatial and temporal changes and conditions in biofilms. In this chapter in situ natural genetic transformation was monitored using the gene gfp, residing on the autonomously replicating plasmid pGARl, that did not provide the recipient with advantageous or disadvantageous traits, in monoculture Acinetobacter sp. BD413 biofilms. The following conditions were 88 IV. Results In situ quantification of natural genetic transformation investigated to elucidate the effect on natural genetic transformation: biofilm age, free DNA concentration, biofilms grown in the absence or presence of suspended cells. 2. EFFECT OF BIOFILM AGE ON NATURAL GENETIC TRANSFORMATION Standard transformation experiments, using Acinetobacter sp. BD413, require cells to be transformed in a state of competence (Palmen et al., 1993). Acinetobacter sp. BD413 reaches competence at early log phase. The cells remain competent during the log phase until competence drops to almost zero, when the stationary phase is reached (Lorenz et al., 1991). Biofilms, however, cannot be compared to batch cultures. Because of the continuous feed the biofilm mode of growth shows some similarity with the constantly exponentially growing steady state batch or turbidostat cultures of Palmen et al. (1994) who could still detect a decreased transformation frequency after 3 days compared to initial transformation frequencies. In standard batch culture experiments (Lorenz et al., 1991) or soil microcosms (Nielsen et al., 1997) additional transformation events were not detectable after a prolonged period that exceeded 12 hours. To test transformability of biofilm cells at different growth stages, experiments were conducted using 0.2 pg pGARl DNA/ml as a function of bipfilm age (Table 6). The response of cells to DNA exposure was measured with two different exposure times, wherein cells were allowed to come in contact with DNA. Flow cell experiments were performed with 1- day-old and 3-day-old biofilms. Biofilms grown as described in ‘Materials and Methods ’ reached maturation after 3 days. A 1-day-old biofilm could therefore be regarded as an actively growing biofilm and a 3-day-old biofilm could be considered a matured biofilm. Although cells in young and growing biofilms are more readily transformed, the biofilm heterogeneity in 3-day-old biofilms still ensured the presence of a significant fraction of competent cells that are able to take up DNA (Table 6; Fig. 10 A, B). Exposure time to DNA Biofilm age 1 day 3 days 15 min 1.9x10"* 7.7x10"* 45 min 1.3x10"* 7.4x10"4 Table 6. Transformation frequencies obtained in differently aged biofilms, and exposed to 02 pg plasmid pGARl DNA/ml for different periods of time. 89 IV. Results In situ quantification of natural genetic transformation Furthermore, cells responded as quickly as within 15 min to the addition of exogenous DNA. Palmen and Hellingwerf (1997) discovered that cells were able to take up DNA within 1 minute. Detectable expression, however, could be delayed for an extended period (Lorenz, 1992). It was more difficult to explain why there was no evidence of an increased transformation frequency with the 3 times longer exposure period of 45 min compared to the 15 min exposure to DNA. All explanations involving the DNA’s gradual inaccessibility towards cells inside a biofilm (change from reversible to irreversible adsorption on EPS or on (non) competent cells, easy dilution, degradation, etc.) will not answer this question. In all cases it would suggest that for biofilms exposed to 45 min, 3 times more DNA was available. The answer will involve a certain characteristic of the competent cells or the environment surrounding the cells that will result in an amount of transformants that is limited to the fraction of cells that are transformable at the offered conditions. And this limit will already have been reached within an exposure time of only 15 min. Fig. 10. Pictures depicting pGARl transformants in 3-day-old (A) and 1-day-old (B) Acinetobacter sp. BD413 biofilms. Gray images show GFP signals (I) or Syto 60 stained cells (II). Superimposed single optical images (HI) show g£rtransformants (yellow, green) in a background of recipient Acinetobacter sp. BD413 biofilm cells (red). 90 IV. Results In situ quantification of natural genetic transformation This way only a fraction of the cells within biofilms take up available DNA until they have reached a saturation level, regardless of whether this would be dependent on the time of incubation in DNA containing medium or the resulting amount of DNA after the incubation period. The transformation frequency would only be dependent on the initial fraction of cells inside the biofilm that can take up and integrate DNA at the offered conditions. In matured biofilms it seemed that the fraction of competent cells was evenly spread throughout the entire biofilm because the profile of the volume of transformants paralleled the profile of the volume of recipients (Fig.ll). The extension of the incubation time resulted in a slight increase in the volume of transformants in the middle or upper part of the biofilm (Fig.11). Hence, early transformation occurred at the biofilm base. ounce ton wtxtreun dm) Otetance eom eubsMun (pm) Fig. 11. Distribution profiles of volumes of recipients (closed symbols) and volume of transformants (open symbols) when either a growing (A) or a mature (B) monoculture Acinetobacter sp. BD413 biofilm was exposed to 02 pg pGARl DNA/ml during 15 min (O) or 45 min (□). It seems, however, odd that the first transformation events take place at the biofilm base. The observation insinuates that free DNA first has to diffuse through the biofilm before competent cells catch them and subsequently integrate the DNA strand in their genome. It is possible that the immobilisation of the cells were to a certain point responsible for the occurrence of the first transformation events at the bottom of the biofilm. The location of transformation seemed to be simply a matter of chance that rises with increasing cell density at a certain distance from the substratum. And in case of monoculture Acinetobacter sp. BD413 biofilms the cell density was the largest near the biofilm medium interface. 91 IV. Results In situ quantification of natural genetic transformation 3. EFFECT OF THE CONCENTRATION OF ADDED FREE DNA To investigate the influence of DNA concentration on transformation frequency and transformant location in a mature biofilm of the highly competent Acinetobacter sp. BD413 with a neutrally effecting gene, natural genetic transformation was investigated in biofilms that were exposed during 1 h to a specific pGARl DNA load. The DNA concentration in the inlet medium ranged from lxlO"9 pg pGARl/ml to 1.5 pg pGARl/ml. Transformation frequency increased as a function of DNA concentration within the investigated range of DNA concentration (Fig. 12). Fig. 12. Transformation in mono-culture BD413 biofilms with pGARl DNA at varying DNA concentrations in the inlet medium. The insert shows the same data points on a log-log scale. pGAR1 cone (pg/ml) The observed relationship between DNA concentration and transformation frequency corresponds to previous studies involving batch transformation experiments (Palmen et a!., 1993). Transformation frequency rose likewise with DNA concentration. However, a saturation point was not reached yet in these experiments. Further examinations using DNA concentrations in the range of 100 pg/ml and above would be needed to see if a saturation point is reached or if transformation keeps rising until 100% of the cells are transformed. This however was not done because amounts of DNA as large as 100 pg DNA/ml are not very likely to happen in nature. With low concentrations of pGARl in the feed, transformants were only formed at the biofilm attachment surface. Note that exposure to increasing amounts of pGARl was displayed as gradual accumulation of transformants at the bottom of the biofilm, where biofilm density was the largest (Fig.13) and not in the middle or upper part of the biofilm. If it was true that the fraction of competent cells was homogeneously distributed inside the biofilm, most transformants would be found in layers with the highest biofilm density and the transformation frequency would be equally high throughout the biofilm. However, this was 92 IV. Results In situ quantification of natural genetic transformation only occasionally observed (5 out 10 times). Hence other factors may have contributed to the distribution of the detected frequencies. Fig. 13. Distribution profile of die volume of gjjMiansformants as a function of the normalized distance from die substratum, when exposed to 1 x 10"9 pg DNA/ml (O); lx 109 fig DNA/ml (□); 1X10"* fig DNA/ml (O); 1 x 10"' fig DNA/ml (A) and 1.5 fig DNA/ml (*) 0 0.2 0.4 0.6 0.8 1 NoimaBzad distance from the substratum The influence of porosity on transformation frequency and henceforth its indirect effect on the establishment of transformants at a certain location inside the biofilm was investigated. Therefore, the porosity of a volumetric slice at a certain distance from the substratum in the biofilm was evaluated with respect to transformation frequency in that particular biofilm slice. It was important to know if a certain optimal porosity was needed for transformation to occur. Figure 14 shows that low porosity creates an environment where transformation frequency reaches a maximum at almost any DNA concentration used, with the exception of IX10"9 pg pGARl DNA/ml or lx 10'7 pg pGARl DNA/ml. Minimum frequency is obtained in areas that possess high porosity (Fig.14). Fig. 14. Transformation frequency as a function of volumetric porosity in biofilm slices (Az = 1.5 pm), when exposed to lxlO"9 pg DNA/ml (O); 1X10"7 pg DNA/ml (□); lx iff4 pg DNA/ml (O); 1X Iff' pg DNA/ml (A) and 1.5 pg DNA/ml (*) 93 IV. Results In situ quantification of natural genetic transformation Hence, particularly in dense areas of the biofilm, transformation is most likely to occur. These dense areas are, in the case of the homogeneously structured monoculture Acinetobacter sp. BD413 biofilms, near the biofilm substratum. And it was observed that most transformants were primarily situated at the biofilm base. This suggests that in a tightly packed biofilm, a biofilm with a low porosity value, the chances are the highest for transformation to occur. This, however, does not mean that porous biofilms are ill fitted to allow transformation. Young biofilms, for example, are very porous (compare Fig. 10 A with Fig. 10 B), and they allowed transformation at increased frequencies due to enhanced competent levels during exponential growth. Porosity only plays a major role in mature biofilms. 4. EFFECT OF BIOFILM DEVELOPMENT ON NATURAL GENETIC TRANSFORMATION Biofilms are complex structures that are constantly shedding cells, growing new colonies and sculpturing channels. Due to the dynamics of biofilm development it could be speculated that at a certain stage a fraction of cells would develop competence for transformation in an extended time period. It was shown that 3-day-old biofilms could give rise to transformants, while no difference was observed between an exposure time of 15 min and 45 min. Would extending the exposure time to DNA allow new transformation events to occur while the biofilm was developing? To observe natural genetic transformation during biofilm development in extended stationary phase, 3-day-old biofilms were exposed during increasing periods of time to pGARl DNA varying from 15 minutes to 41 hours. Porosity was calculated as the normalised volume that was not occupied by biomass in the specific scanned volume, limited to contain 98% of the volume of the scanned biofilm (see Materials and Methods). After calculation of transformant frequency a periodical rise and fall of the fraction of the volume of transformants was observed (Fig. 15) and these fluctuations correlated negatively with the porosity. Almost always when transformation frequency increased, the porosity in the biofilm decreased. This indicates that the reduction in biomass effected an enhanced decrease in the volume of transformants while biofilm growth enhanced the increase in volume of transformants. The increased fluctuations of tire volume of transformants was indeed observable by plotting the absolute volumes of transformants detected during biofilm development (data not shown). The enhanced increase in volume of transformants during biofilm growth can be explained by the same reason why young and growing biofilms revealed greater transformation frequencies compared to older and established biofilms. 94 IV. Results In situ quantification of natural genetic transformation Fig. 15. Transformation frequency and biofilm porosity in bio films as a function of exposure time to 0.1 |ig pGARI DNA/inl. Here, growth induction stimulated competence development and therefore could more transformation events occur inside the biofilm (Palmen et a!., 1993). The reason for the dramatical reduction of the volume of transformants upon porosity increase, could be greater sensitivity of the gfp expressing cells. The unproportional change of the volume of transformants in comparison to the change in total biofilm volume could also be reflected in correlation tests between volume of recipients and transformation frequency. The volume of transformants was independent of the total number of recipients (data not shown) (R* = 0.05). On the other hand the volume of transformants revealed a logarithmic correlation with biofilm porosity (RMI.55) (Fig. 16). Fig. 16. Porosity is a function of volume of transformants in biofilms tint underwent transformation with lpg pGARI DNA/ml during different times of exposure. 5 4 4.6 5 5 Log Vokxne of Transformants (pnf) 95 IV. Results In situ quantification of natural genetic transformation The compactness of a biofilm with decreased porosity during biofilm growth could also have influenced transformation frequency and hence the volume of transformants. Therefore both growth induction and biofilm compactness positively influenced transformation inside mature biofilms. The lower transformation frequencies obtained in biofilms with high porosity values can be explained by absence of stimulation of transformation because recipients neither accumulated (no growth impulse) nor was the biofilm optimally compact (high porosity). Together with a reduction in g/p-transformants due to lysis and/or detachment this would lead to the observed low transformation frequencies. 5. EFFECT OF BIOFILM ONTOGENESIS ON NATURAL GENETIC TRANSFORMATION The initiation of a biofilm starts with attachment of free floating bacteria. How would a biofilm that had developed in the presence of bacterial cells in the bulk phase, respond to tire addition of free DNA? To answer this question, flow cells were continuously fed with medium inoculated with Acinetobacter sp. BD413. Thereafter, biofilm inlet tubes were rinsed to prevent transformation in the bulk phase before cell-free minimal medium containing 0.1 pg pGARl DNA/ml was fed into the flowcell. Incubation was allowed to occur overnight, whereafter biofilms were stained, washed, microscopically investigated and quantified. Acinetobacter sp. displays two different morphotypes: the bacillar morphotype and the coccoid morphotype (James et a!., 1995b). In biofilms fed with cell-free medium, both the bacillar and the coccoid form were detected, while biofilms having emerged in the presence of cells in the bulk fluid consisted predominantly of long bacillar formed cells. The cells that possess the coccoid morphotype remain firmly attached while bacillus-shaped cells are more readily detached and are preferably found in the drifting fraction (James et al., 1995b). James and coworkers (1995b) discovered that upon starvation, bacillus shaped cells revert to tire coccoid form by reduction-division resulting in conservation of biomass but increased cell number. When biofilms were grown in the presence of cells in the bulk phase and were thereafter subjected to starvation conditions by feeding the flow cell with a continuous supply of minimal medium without a carbon source or a salt solution (MgSO 96 IV. Results In situ quantification of natural genetic transformation cells grown into the stationary phase (data not shown). And stationary phase cells are not competent for transformation. It is as if all signs suggest that the coccoid cell is the competent one and the bacillar cell is not competent for DNA uptake. Fig. 17. Pictures de picting die shape of Acinetobacter sp. BD413 cells in a monoculture biofilm at various distances from the biofilm sub stratum: 2pm (A); 16 pm (B); 35 pm (C) and 50 pm (D). All pictures depict a microscopic field of 128pmxl28pm. Still, when cell shape was checked as a function of distance from the substratum, it was observed that cells at the biofilm substratum possessed the bacillar shape while the prominent morphotype of a cell at the biofilm/medium interface was coccoid (Fig. 17). In the previous paragraphs, however, most transformants were formed at the biofilm substratum, hence where most bacillus shaped cells resided. Further experiments are therefore needed to establish if the fraction of bacillus shaped cells/cocci shaped cells or the absolute amount of coccoid cells can be correlated with transformation frequency. 97 IV. Results In situ quantification of natural genetic transformation 6. CONCLUSIONS Young and growing biofilms allowed more transformation events compared with older and established biofilms. Palmen et al. (1993) showed that exponentially grown cultures revealed increased transformation frequencies and continuously grown steady state batch or chemostat cultures exhibited, similar to continuously grown flowcell cultures, decreased but measurable transformation frequencies. While early transformants are found at the biofilm substratum, subsequent transformation events occur a little bit further away from the biofilm substratum. Transformation was observed at minute DNA loads (2.4 fg DNA/h) and a saturation point was not yet reached when using 3.6 pg DNA/h. While additional DNA added to the biofilm due to extended exposure time to DNA did not increase transformation frequency, additional DNA added to the biofilm by an increased DNA concentration in the inlet was positively correlated with increasing transformation frequency. It should be stressed that additional DNA due to increased DNA concentration in the inlet resulted in accumulation of transformants at the biofilm substratum. Furthermore transformation frequency was dependent on DNA concentration and not on the amount of DNA that is added to the biofilm. This suggests that the competence level of cells may play a role in DNA take up. There was a correlation between the volume of transformants and biofilm porosity. The more compact the biofilm, independent of the volume of recipients, the higher were the detected volumes of transformants in older and established biofilms. Transformation frequency was independent of exposure time to DNA. However, biofilm development had a direct influence on the dynamics of the volume of transformants. Although the volume of transformants was independent of tire volume of recipients, changes in volume of transformants coincided with changes in recipient volume. These fluctuations, however, did not lead to a net accumulation or a net decrease of transformants in the biofilm, once a peak transformation frequency was recorded.. Biofilms that were grown in the presence of cells growing in the bulk fluid, did not give rise to a single transformation event If these biofilms, on the other hand, were starved before being exposed to DNA, transformation events were observed. Microscopic examination revealed a change in morphotype of the biofilm cells from the bacillar to the coccoid form upon starvation. 98 IV. Results In situ quantification of natural genetic transformation In conclusion the following set of parameters were extracted that can be used for controlling natural genetic transformation: DNA concentration, porosity, nutrient load. Exposure time to DNA does not need to be extended for a prolonged period, a short period of 15 to 60 min will transform most of the transformable cells in a biofilm. Much more important is the DNA concentration. High DNA concentrations are needed to obtain high frequencies of transformation. Low porosity was correlated with high volumes of transformants. Hence, all parameters that lead to a biofilm with low porosity will increase the chance for transformation. An induction of transformation can be achieved by pre-starvation of the biofilm. Thereafter, the nutrient addition should be resumed in combination with DNA in tire inlet to obtain increased transformation frequencies. 99 IV. Results Natural genetic transformation of a disadvantageous gene D. NATURAL GENETIC TRANSFORMATION OF A DISADVANTAGEOUS GENE IN MONOCULTURE Acinetobacter SP. BD413 BIOFILMS "But the truth is... as may well be expressed in the tale ... of the philosopher, that while he gazed upwards to the stars Jell into the water; for if he had looked down he might have seen the stars in the water, but looking aloft he could not see the water in the stars." Francis Bacon (1561-1626), English politician 1. INTRODUCTION In this chapter natural genetic transformation of an adverse gene was investigated in monoculture Acinetobacter sp. BD413. The gene for the enhanced yellow fluorescent protein (EYFP) was used as the negatively effecting gene, which resided on a heterologous, self- replicating plasmid pGAR2 (Section IV.B.). Acinetobacter sp. BD413 was observed to be very sensitive to the expression of the enhanced yellow fluorescent protein (EYFP) (Section IV.B). Traditional natural genetic transformation experiments (Palmen et at, 1993) using planktonic batch cultures of Acinetobacter sp. BD413 (Juni and Janik, 1969) and the enhanced yellow fluorescent protein (EYFP) encoding plasmid pGAR2 (Hendrickx et al., 2000) gave rise to unstable and fragile transformants (Section IV.B.). Expression of EYFP had a lethal effect on BD413 transformant cells. The unfavorable effects of EYFP, together with its fluorescence properties, provided a means for the in situ investigation of natural genetic transformation with an adverse gene in a model bacterial biofilm with quantitative confocal laser scanning microscopy (CLSM). In this way the response of the biofilm faced with a disadvantageous gene could easily be recorded by CLSM in terms of variations in biovolume, transformation frequency and transformant location, which would not have been noted by selective plating techniques. 100 IV. Results Natural genetic transformation of a disadvantageous gene 2. EFFECT OF DNA CONCENTRATION ON TRANSFORMATION WITH A DISADVANTAGEOUS GENE To investigate the biofilm response to a disadvantageous gene, equally conducted experiments as with the neutrally affecting plasmid pGARl (Section IV.C.) were performed in which biofilms were exposed to a DNA concentration in the influent ranging from lxlO"9 to 5 pg pGAR2Zml. In contrast to an expected overall low transformation frequency due to a small fraction of surviving transformants, a log-log plot of transformation frequency as a function of pGAR2 DNA concentration displayed a complete scattering of data with no deductible information (Fig. ISA). However, when pGAR2 DNA concentration was set on a normal scale it emerged that, within the investigated range of DNA concentration, higher amounts of DNA added to the medium resulted in lower maximally obtainable transformation frequencies (Fig. 18B). It was a surprise that increased DNA concentration would lead to a decrease in the maximally detectable transformation frequency (Fig. 19). To explain the scatter of high and low transformation frequencies, it is possible that new transformants first amplified the received DNA before releasing it into the environment via cell lysis. The notion that cell lysis by eyfp- expressing cells could occur inside a biofilm was supported by the observation of massive lysis of qgjr-expressing cells in an attempt to grow a pure culture BD413(pGAR2) as a biofilm (Section IV J$.). -to -a Log pGAR2 cone (pgftnl) PGAR2 cone (pg/ml) Fig. 18. Transformation frequency in monoculture Adnetobacter sp. BD413 biofilms using pGAR2 DNA as ■function of varying DNA concentrations in the inlet medium, logarithmically plotted with respect to transformation frequency and DNA concentration (A); or where transformation frequency is plotted on a logarithmical and DNA concentration on a normal scale (B). 101 IV. Results Natural genetic transformation of a disadvantageous gene Kloos et at (1994) showed that the release of plasmids by cell lysis produced DNA that can be used for transformation as efficiently as added alkaline extracts of the plasmid. In the present section the possible release of DNA could have resulted in a transformation frequency as high as 6xl0"3 pm* of transformants/pm 3 of recipients (Fig. 19, Ifi.C.), obtained using minute amounts of only 10 fg pGAR2/mI in the feed. This transformation frequency exceeded the estimated observable transformation frequency, based on pGARl transformation, 200-fold (Fig. 19). Therefore, it is safe to presume that lysis of transformants resulted in more transferably active DNA present in the biofilm than was actually added to the inlet medium in all experiments involving transformation with pGAR2. I H HI IV Fig. 19. Acinetobacter sp. BD413 biofilms transformed with pGARl (1,11) or pGAR2 (HI,IV) at minute concentrations of 0.1 pg DNA/ml (I); 10 fg DNA/ml (IH) or saturating concentrations of 1.2 pg DNA/ml (II) and 1 |tg DNA/ml (TV). Gray images depicted GFP or YFP signals (A) or the total biofilm stained with syto60 stained biofilm (B). Superimposed single optical images (C) show gfp-transformants (green-yellow) or eyfp- transfonnants (yellow) in a background of recipient Acinetobacter sp. BD413 biofilm cells (red). Images were obtained with the 40* oil immersion objective representing biofilm areas of 65 pm%58 pm. 102 IV. Results The decreasing maximal transformation frequency during increasing DNA concentration was not easily explainable. Furthermore, spatial investigation revealed a unique distribution of transformants inside the biofilm. The mean location of transformants was dependent on the DNA concentration of the inlet medium (Fig. 20). With increasing pGAR2 DNA concentration the distribution profile of ey$>-transformants inside the biofilm shifted from the biofilm base to the top of the biofilm (Fig. 20). To paint an objective picture on where the shifting relative locations of qj$>-transformants are inside the biofilm, the investigated biofilms were normalized and a mathematically quantifiable parameter, describing the location where most transformants were detected, was put forward. This parameter was called the normalized mean location. It presented the mean location of transformants residing in a biofilm as a number ranging from 0 to 1 (Fig. 21). 300 -10 «8 «e -4 -2 o 2 NoonaBzed distance from the substratum Log DNA cone (pgAnl) Fig. 20. Distribution profile of the volume of €yjp~ Fig. 21. Normalised mean location (NML) of transformants as a function of the normalized ey^Hransformants (O) and g$Mransformants distance from the substratum, when exposed to (•) inside a biofilm as a function of the DNA 1 x 10* |!g DNA/ml (O); 1 x lO"’ |ig DNA/ml (□); concentration used in die inlet medium during 1 h IX Id 4 jig DNA/ml (O); IX10 ' |ig DNA/ml (Ah 1 DNA exposure. fig DNA/ml (*) and 4 fig DNA/ml (-); In contrast to the mean location of g^vtransformants inside the biofilm, which seemed to oscillate around 0.21 (Fig. 21), there was a trend of a shifting optimal ejg^r-transformant location depending on added DNA concentration. At low DNA concentrations in the inlet medium, most transformants were found near the substratum. With increasing DNA concentration, the transformant distribution peak profile shifted further towards the 103 IV. Results Natural genetic transformation of a disadvantageous gene biofilm/medium interface so that no more transformants were detected in an expanding area at the biofilm attachment surface. It is clear that successful and stable transformation using a detrimental gene was hampered in the lower parts of a biofilm. In addition it was shown that absence of effective transformation increased with increasing pGAR2 concentration. Inefficient transformation with the use of adverse genes can be due to the presence of barriers against gene transfer at any stage of natural genetic transformation. The different stages of natural genetic transformation can be divided as follows: (i) presence of free transformable DNA; (ii) development of competence of the recipient cells; (iii) uptake of transformable DNA; (iv) incorporation in the genome of the host; (v) expression of the incorporated genes; (vi) maturation of the synthesized protein; (vii) survival of transformants. The cause of inhibition resulting in ineffective transformation can reside in all these stages. Furthermore, unsuccessful transformation was only obtained when negatively influencing DNA was used. A barrier at stage (i) is based on external limiting factors: penetration limitation or physiological limitation. A barrier at stage (iv), (v) and (vi) is dependent on intracellular factors: the hosts internal gene and protein processing functions. The incorporation will follow the same pattern in a recipient cell that has taken up either neutral or detrimental DNA. The expression and the maturation of the negatively affecting protein compared with the neutral protein could be different under the same conditions. While the expression of a gene can be dependent on the gene sequence or be influenced by the presence of the synthesized protein, maturation of recombinant proteins can vary if the protein needs certain limiting compounds to fold correctly into a functional protein. A barrier at stage (vii) will propose the most probable explanation. It implies that natural genetic transformation with a detrimental gene will occur in essentially the same way as natural genetic transformation would occur using a neutral gene. The only difference would be the resulting limited survival of transformants that have taken up detrimental genes. A barrier at stage (ii) or (iii) is difficult to detect but can still not so easily be dismissed either if the local conditions in a biofilm at a microscale are regarded. In the paragraphs searching for barriers at other stages then in stages (i), (iv), (v) and (vi), the hypothesis to explain the absence of transformants based on transformant survival (vii) will compete with an introduced daring but nevertheless possible hypothesis: the hypothesis of inhibited competence development (ii) or hindered DNA uptake (iii) indirectly caused by the presence of adverse DNA in the environment 2.1. PRESENCE OF FREE TRANSFORMABLE DNA? It is not possible that the decreased detected transformation frequency is caused by the absence of transformable DNA at the bottom levels of the biofilm due to diffusion limitation. 104 IV. Results Natural genetic transformation of a disadvantageous gene Transformation with neutral DNA (pGARl) occurred readily and even preferably at the biofilm substratum (Section IV.C). It could have been possible that DNA was made unusable for transformation by in situ degradation occurring more often when biofilm cells took up detrimental DNA and lysed after expression of the incorporated lethal sequences. DNA released by £ coll S17-1 and P. stuzeri JM300 containing the broad-host-range lysis system plasmid pDKLOl with the lysis gene E from bacteriophage $X174, had a half-life of less then 1 h because of the nuclease activity induced with the cellular autolysis system (Kloos et at., 1994). On the other hand, it has been observed that DNA can persist for an extended period in pure cultures. Lorenz et d. (1991) still detected high amounts of biologically active DNA 40 h after the laboratory culture reached stationary phase and natural lysis was progressing. To test the hypothesis whether exogenous detrimental DNA could be degraded by lysis of (^-transformants inside the biofilm, the degradation capacity of BD413 and derivative lysates was investigated on pRK415 DNA. There was no degradation detectable in the sonicated lysates of BD413, BD413(pGAR2) or BD413(pGAR39) cultures, even after an 7 h incubation period. 12. INHIBITION OF COMPETENCE DEVELOPMENT OF THE HOST CELLS AND/OR DNA UPTAKE FROM THE ENVIRONMENT? A certain fraction of cells in a biofilm were shown to be competent for natural genetic transformation and plasmid DNA was integrated resulting in the expression of the accepted genes (Section IV.C.). Hence, cells in a biofilm were generally not hindered to perform successful transformation. Is it possible that the presence of transformants carrying a bad gene influence transformation in other potential recipient cells? The hypothesis of an active and inducible protection of biofilm cells against unwanted gene transfer was tested using the combination of a neutral and a detrimental gene in tire same transformation experiment. The model BD413 biofilm was treated with Ipg pGAR2 DNA/ml for 15 minutes. Thereafter, the pretreated biofilm was subjected to Ipg pGAR39 DNA/ml for 15 or 30 min intervals. A 133-fold reduction of the ecj^-transformation frequency was observed after a 15 min exposure to pGAR39 with pGAR2 pretreatment (7x10"*) compared to 15 min ecjp- transfonnation with pGAR39 without pGAR2 pretreatment (9x1 0"z) (Fig. 23). After 30 minutes, the egjMransformafion frequency already rose by a factor of 8 (5xl0* 3). When the eyfp treated biofilm was exposed to a mixture of lpg/ml pGAR2 and lpg/ml pGAR39 during 15 minutes, egfr-transformation dropped to 4x1 O'7. 105 IV. Results Fig. 23. Volumes of ec/^-transformants obtained when eyfp and ecfp were added consecutively. Volume of transformants in a biofilm that underwent transformation using an inlet concentration of lug pGAR39/ml during 15 min without pre-exposure of die biofilm to pGAR2 (O); volume of ecfp- transfonnants that underwent transformation by addition of 1 |ig pGAR39/ml in die inlet medium during 15-min after a 15-min pre-exposure of the biofilm to 1 pg pGAR2Anl (D); volume of ecfp- transformants in a biofilm that underwent transformation using 1 pg pGAR39/ml in the inlet during 30 min with 15 min pre-exposure of the biofilm to 1 pg pGAR2/ml (A). Distance from substratum (pm) Additional exposure to pGAR39 DNA lead to an increase in the number of ecfp- transformants in the outer half of the biofilm (Fig. 22) as it was likewise observed when biofilms were subjected to 45 min of exposure to pGARl compared with 15 min of exposure to pGARl (Section IV.C.). , In light of the hypothesis suggesting an active and inducible protection mechanism the above results can be explained as follows: first, competent cells took up the added detrimental DNA, thereby triggeringthe induction of the protection phenomenon in a biofilm challenged with detrimental DNA. This protection phenomenon could be based on cell-to-cell communication between compromised cells having taken up the detrimental gene and recipient cells that have not taken up DNA yet According to the hypothesis based on a certain kind of protection against unwanted gene transfer, the produced signal would inhibit competence development and limit successful natural genetic transformation. Highreduction in the transformation frequency is observed when biofilms are pre-treated with adverse DNA. Even if transformation already reached saturating levels after 15 min of pre-exposure to detrimental DNA, it was possible to observe a massive decrease in the .transformation frequency recorded in the pre-treated biofilm exposed to a mixture of pGAR39 and pGAR2 in comparison to the biofilm exposed to pGAR39 alone. While the 15 min treatment of the pre exposed biofilm with pGAR39 resulted in a transformation frequency of 7x10"4, the treatment of the pGAR3 9/pGAR2 mixture resulted in a transformation frequency as low as 4xl0"7. If the chance to obtain an ecj^-transformant is equal to the chance to obtain an eyfp- transfonnant, this high drop would indicate the presence of an active and inducible protection mechanism in biofilms. Although this statement is correct, it is important to consider the possibility to obtain double transformants (i e. transformants carrying both the eyfp and the 106 IV. Results Natural genetic transformation of a disadvantageous gene ecfp gene), which can reach levels of 100% doubly transformed cells (Lorenz and Wackemagel, 1994). Let us make a few simplifications. The chance to recirculate two strands in either pGAR2 or pGAR39 is the same. Two pGAR2 strands will be recirculated as a pGAR2 plasmid and two pGAR39 strands can reconstruct a pGAR39 plasmid. If a pGAR2 strand recombinates with a pGAR39 strand the chance will be the same to obtain a pGAR2 plasmid or a pGAR39 plasmid. Furthermore, the presence of one single replicable pGAR2 plasmid in die transformant, containing a number of tecirculised plasmids, will result in transformant death. If all reconstructed plasmids in one transformant cell are pGAR39 plasmids, the cell will survive and be detected as an eg^-plasmid. The fiaction of the transformation frequency of the pre-treated biofilm exposed to the mixture pGAR2/pGAR39 (4x1 O'7) and the pre treated biofilm exposed to pGAR39 alone (7x10"*) indicates hence the chance that all reconstructed plasmids inside the transformant are pGAR39 plasmids. What is the minimum number of plasmids in the transformant to obtain a survival rate of (4x 10"7/7x 10‘4 = 6x1c4)? And is that number reasonable? The chance to obtain only pGAR39 plasmids in a collection of independently emerged plasmids that could either be pGAR2 or pGAR39 is 1/2°, where n equals the number of the reconstructed plasmids. Solving n out of the equation 2"” = bxlO"4, reveals a value of 15.9. Hence a mean number of 16 plasmids should be reconstructed and replicated in the transformant cell. And although this number might look very high, it is not so unlikely as it seems. Acinetobacter sp. BD413 cells can take up to 750 DNA strands (Palmen and Hellingwerf, 1997). So in essence about 375 plasmids could be reconstructed, which of course is not very likely to happen. Furthermore, cells can carry up to 250 copies of a plasmid in the case of multi copy plasmids and up to 60 copies of medium copy plasmids (like pRK415 and derivatives). An estimated mean value of 16 reconstructed plasmids is therefore not necessarily immediately dismissive. Hence it is possible that the observed low transformation frequency detected in a biofilm exposed to both pGAR2 DNA as well as pGAR39 was simply because generally both types of plasmids were integrated in the transformed cells. 2.3. PROBLEMATIC INCORPORATION OF THE GENE. EXPRESSION OF THE INCORPORATED GENES AND/OR MATURATION OF THE GENE PRODUCT? Plasmid pGAR2 differs only in the inserted sequence coding for the GFP-variant EYFP. Henceforth the integration of the disadvantageous plasmid will occur in exactly the same way as with the neutral plasmid. The integration of the latter did not seem to be problematic. Furthermore it was shown that the plasmid was integrated in the genome as an 107 IV. Results Natural genetic transformation of a disadvantageous gene extrachromosomal unit (Section IV.B.). Could the failure to detect ey^-transformants, residing deep within a biofilm subjected to high DNA concentrations, originate in the absence of expression of the integrated eyfp gene by downregulated metabolism or incomplete maturation of the expressed protein by low oxygen concentration? This was not very likely because eyfp expression was detected at the substratum when low amounts (1 fg DNA/ml) were added to a biofilm that was of comparable thickness to the biofilm that was subjected to 4 pg DNA/ml, that is 27 pm and 26 pm, respectively. Was it possible that abundant presence of eyfp expressing cells had altered the conditions in a biofilm in such a way that expression or maturation of EYFP was inhibited in cells at the substratum? Experiments with eyfp expressing cells diluted in unlabeled BD413 cultures revealed that the ejj^-expressing cells were detectable during the entire observation period (see section IV.B.3.). In a repeated experiment an overnight BD413(pGAR2) culture was diluted 100-fold into an unlabeled overnight BD413 culture and used as inoculum for biofilm cultivation. 100000 Fig. 23. Growth of eyfp (□) and ecfp (o) expressing 10000 cells, monitored as biovolumes as a function of time (days), in a cultivated biofilm, that was initially inoculated as a mixture of unlabeled, 100-fold diluted eyfp-\tSx\ed and 1000-fold diluted ecfp- labeled Acinetobacter sp. BD413 cultures. , The inoculum was spiked with a 1000-fold dilution of an overnight BD413(pGAR39) culture to check biofilm growth and fluorescence detection of the expression of a harmless fluorescence protein during cultivation (Fig. 23). . the biofilm was followed during 7 days by measuring a predefined volume of interest (Fig. 23, 24). eyfp expressing cells were detectable during the seven days, throughout the entire biofilm depth (Fig. 24). Also the cells at the bottom of the biofilm were able to synthesize EYFP or ECFP. During the seven days the volume of BD413(pGAR39) cells increased steadily from about 10 pm* to 3400 pm1, while the volume of BD413(pGAR2) cells remained approximately stable ranging from 593 pm3 to 988 pm3 (Fig. 23). It can be ruled out that the same inoculated BD413(pGAR2) cells remained present in the biofilm during the entire observation period because the volume of ey£>-expressing cells changed within the same volume of interest at different time points (see section IV.B.3.). The shift of the 108 IV. Results distribution profile ofEYFP containing cells on day 5 and day 6 (Fig. 24), also observable for the ECFP containing cells on day 6 (data not shown), is probably due to growth of unlabeled cells underneath the labeled cells. Even if it would indicate lack of expression of integrated genes or failed maturation of the protein, this problem could not have occurred within one overnight incubation period during which biofilms were incubated to allow gene expression Fig. 24. Time course spatial distribution profiles m 1000 of volumes of eyfp expressing cells in a biofilm, cultivated for seven days. The day on which each profile was obtained is indicated on the respective curve. Distance tom substratun &jn) When EYFP signals disappeared, probably due to lysis and loss of integrity of the cell membrane, most surviving transformants remained detectable at the substratum (Fig. 24). The appearance of new eyfp and ecfp expressing cells could be the result of cell division or transformation, but not of conjugation. Plasmids pGAR2 and pGAR39 are tra" plasmids. Without genes encoding tire cognate conjugation system it is impossible that pRK415 derivatives could be transferred via conjugation. No matter how new eyfp and ecfp expressing cells were formed, the genes encoding the fluorescence proteins needed to be expressed and die proteins needed to mature in situ inside the biofilm. From the extracted data, there is no evidence that expression of eyfp and maturation ofEYFP was more problematic compared to ecfp expression or ECFP maturation. It should be noted, however, that these data do not prove that eyfp expression was allowed under the physiological conditions of a biofilm challenged with detrimental DNA. They merely indicate the absence of problematic eyfp expression or EYFP maturation in cells containing pGAR2 and residing at the substratum of a biofilm that was not subjected to the addition of free DNA. 2.4. LIMITING TRANSFORMANT SURVIVAL? It seems confident to assume that transformation was taking place the same way as it did in experiments using pGARl. Also the cells near the substratum underwent transformation. However, these transformants should have lysed prior to being detected by CLSM. This 109 IV. Results Natural genetic transformation of a disadvantageous gene would be in contrast to the assumption that cells further away from the biofilm/medium interface are less metabolically active in comparison to cells near the biofilm/medium interface. In comparison with transformants at the biofilm top, transformants residing at the biofilm attachment surface would produce less of the toxic EYFP. According to the above assumption, one would expect more surviving transformants near the biofilm substratum. This, however, was not observed. Instead of overall limiting transformation frequency, an increased area inside the biofilm was detected where no transformant was detected when increased pGAR2 concentrations were added to the medium. The shifting distribution profile of ey£»-transfonnants suggested again the triggering of an inducible protection phenomenon that was as strongly stimulated as the challenge of the adverse DNA load put upon the biofilm. It led to an increased area inside the biofilm, devoid of unwanted gene transfer. Still it may be possible that early transformation occurs at the biofilm substratum. Additional transformation occurs further away from the substratum. In the meantime the first transformants could lyse while new transformants are formed further away from the biofilm substratum. Furthermore, it was observed that a large number of ey^-expressing cells are not tolerated in a biofilm. It was possible to maintain cells expressing eyfp in a biofilm if diluted with unlabeled cells (Section IV.B.). Only when the number of eyfp labeled cells grew too high, was a decrease in number of transformants observed. For example the drop in eyfp- expressing cells from day 2 to day 3 not only documents the intolerance of a volume of 4.5x10* pm3 cells/107pm3 scanned volume in a biofilm but also a disappearance of eyfp expressing cells at the bottom of the biofilm (Fig. 24). The volume of git-transformants produced in a biofilm treated with 0.1 pg pGARl DNA/ml nears the limit value of tolerance for eyi'-expressing cells in a biofilm (l^xlO3 pm3 *cells/107pm3 scanned volume). Up to lpg pGAR2 DNA/ml gave rise to comparable transformation frequencies (next to lower and higher transformation frequencies) as with biofilm cells transformed with pGARl (Fig. 18) (Section IV.C.). That transformation frequency decreased below normal levels, estimable by pGARl transformation, can therefore be explained due the high number of transformants emerging with high DNA concentration. Hence both the decreasing maximal level of transformation frequency and the reduction in the local number of transformants at a certain distance from the substratum can be explained by limited survival of emerging transformants. 3. PROLONGED CHALLENGE WITH DETRIMENTAL DNA When Acinetobacter sp. BD413 biofilms were exposed to increasing amounts of adverse DNA an increasing area was observed wherein successful natural genetic transformation was restricted. Thereby total maximal transformation frequency decreased in the challenged biofilm. As biofilms are compared with multicellular organisms, being able to handle tasks 110 IV. Results Natural genetic transformation of a disadvantageous gene like degradation of certain recalcitrant compounds by bacterial consortia or resistance against environmental influences more efficiently (Davey and O’Toole, 2000; Watnick and Kolter, 2000), it can be Suggested that biofilms would also be able to endure prolonged stress. To investigate the efficiency of a biofilm to handle prolonged exposure to detrimental DNA, time-resolved experiments were set up to investigate the response to a pGAR2 DNA load of 0.24 pg DNA/h in the range of 15 min to 48 h. Continuous exposure to transforming DNA revealed a stable increase in transformation frequency after an initial phase of alternating high and low transformation frequencies (Fig. 25). During exposure tire biofilms architectural changes correlated with the varying transformation frequencies. Biofilm cells, transformed with pGARl, presented also an oscillating pattern of increased and decreased transformation frequency concurrent with a respective decrease and increase in biofilm porosity (see section IV.C.3.). However, in contrast to pGARl transformation, no correlation could be observed between biofilm porosity and transformation frequency or the volume of transformants when pGAR2 was added to the biofilm. Only the rise and fell of biofilm porosity, irrespective of its value, influenced the oscillating data points of tire transformation frequency. Biofilm porosity decreased as transformation frequency increased. It could be possible that after a porosity peak, tire biofilm was temporarily out of balance and this may have induced cell growth, restoring cell density. 1.00E-01 0.8 1.00E-03 0.6 8. S 1.00E-04 -♦-transformation frequency 1.00E-05 -e- biomass porosity Ome(h) Fig. 25. Effect of transformation frequency (volume of transformants (|un*yvolume of recipients (pm*)) and biomass porosity (non bacterial volume (pm*)/scanned volume (pm*)) on exposure time to 024 pg qy^containing plasmid DNA/h. Ill IV. Results Natural genetic transformation of a disadvantageous gene After a high transformation frequency interval, an increase in biofilm porosity coincided with a decrease in transformation frequency. When transformation frequency decreased, transformant cell volumes could have been reduced due to cell lysis or cell detachment Again porosity decreased due to biofilm cell growth (Fig. 25). The brief high levels of transformation frequency may have been caused by secondary transformation with Acinetobacter amplified plasmid DNA (i.e. as a result of lysis). In combination with the in situ amplification of the adverse gene also the induced cell growth might have triggered competence development and enhanced DNA uptake (Palmen et al., 1994). The recurring pattern of high and low transformation frequencies finally ended with an extended period of high transformation frequency, until the biofilm fragmentized and became viscous. The volume of recipients in the scanned volume dropped by two orders of magnitude during the 48 hours of exposure to pGAR2 (from 2.9x1 06 pm3 recipient volume to 4.0x104 pm3 recipient volume). In the case of pGARl transformation, however, there was no significant drop in recipient volume after 41 hours of exposure to pGARl (from 6.2x10* pm3 recipient volume to 4.0x10* pm3 recipient volume). 0.1 - Time(h) Fig. 26. Normalized mean location of ygjMransformants in biofilms exposed toO.lpg pGAR2 DNA/ml during varying time periods. ' A unique transformant distribution pattern was observed within the biofilm exposed to pGAR2 (Fig. 26). While transformants were already found at the bottom of the biofilm after less than 15 min of exposure to pGARl (Section IV.C.5.), not a single transformant could be detected at the biofilm attachment surface at early exposure times to the unfavorable gene eyfp (Fig. 26). During a transition phase most ej^r-transformants were found in the center of 112 IV. Results Natural genetic transformation of a disadvantageous gene the biofilm. Only after a prolonged incubation time of about 12 hours were «%$Mransfbrmants accumulating near the substratum. It can be speculated that the prolonged challenge with detrimental DNA and the continuous in situ concentration increase of the adverse gene (due to repeated lysis) led to a net accumulation of transformants, which was detectable even after most transformants had lysed. 4. CONCLUSIONS Like transformation using neutral genes, natural genetic transformation of detrimental genes took place at highfrequencies in biofilms. Minute amounts as little as 1 fg pGARl/ml sufficed to obtain detectable transformation frequencies. Again, no optimal growth phase was needed for natural transformation to be detectable throughout the observation period. The stage at which successful transformation was hampered using detrimental DNA inside biofilms, could not be conclusively established. It was shown that the added DNA was not degraded by lysis of compromised BD413(pGAR2) cells. DNA remained very stable in the tested lysates of BD413(pGARl), BD413(pGAR2) and BD413(pGAR39) suspensions. The lack of eyfp transformants was not caused by failed maturation of EYFP or inhibited expression of eyfp, due to metabolic conditions of cells residing at the biofilm substratum. Whether unsuccessful transformation was due to poor survival of the transformant or by induced protection could not be determined. In contrast with transformation using pGARl, there was no positive correlation between transformation frequency and DNA concentration when detrimental genes were introduced in the biofilm. A decreasing maximal transformation frequency was observed together with an enlarging area inside the biofilm where no successful transformation seemed to occur. Furthermore no correlation between biofilm porosity and volume of transformants was detected. Rather the dynamic character of biofilm development influenced transformation frequency through changing biofilm porosity. In contrast to transformation using a neutral gene, the fluctuations in transformation frequencies, dependent on an induced transformation frequency by growth upshift of biofilm cells together with the release of amplified pGAR2 plasmid after lysis of the transformants, resulted in a net accumulation of transformants. During continuous exposure to detrimental DNA, the volume of recipients dropped two orders of magnitude, whereas this was not observed with transformation using the neutral gene. 113 V. DISCUSSION 114 V. Discussion Use of gfp and gfp-varlants for in situ monitoring A. USE OF gfp AND ^VARIANTS FOR MONITORING NATURAL GENETIC TRANSFORMATION IN BIOFILMS Investigating biofilms is not as easy as studying suspended cultures. The investigated event should preferably be observed without disturbing the biofilm and without losing the certainty of obtaining reliable results that are more easily collected when homogenisation of the investigated sample is allowed. Direct in situ detection of large areas in a number of equally treated parallel biofilms are therefore desired for biofilm studies. A study for determining statistically representative areas of Pseudomonas fluorescens biofilms showed that a minimal area of 1x10s pm2 should be scanned to obtain reproducible results in biofilm investigations (Korber et al., 1992). hi the present study a minimal area of 2.4x10s pm2 was scanned to monitor a biofilm volume of at least 1.2xl07 pm3. Comparing 7 parallel biofilms, inoculated with cells harvested from suspended cell cultures of BD413, treated following exactly the same protocol, resulted in repeatable results if a volume of at least 1.2xl07 pm3 was scanned. The homogeneous character of the tightly packed monoculture Acinetobacter sp. BD413 biofilm offered an easy reproducible medium for the investigation of in situ natural genetic transformation. Reproducibility of biofilm experiments is not always that evident Comparison of biofilm architecture at varying conditions will need both the scanning of a large investigated area and several parallel experiments that are treated equally in every possible little detail (Heydom et al., 2000). Therefore it is essential to test reproducibility in all experiments involving biofilms before the investigation of the desired phenomenon is started. Reproducibility tests of the to situ CLSM detection method take much effort and are much more work-intensive than ex situ detection methods where homogenisation of samples is allowed. However, an important vantage of CLSM investigation is the direct manner of monitoring the investigated event Indirect detection discriminates on culturability of cells when selection is based on culturing with selective agar plates, and this could result in false interpretations of the investigated event The misinterpretation of the role played by Acinetobacter spp. in phosphor elimination is a good example for these kind of incorrect conclusions after indirect experimental observation (Wagner et al., 1994). In situ detection methods have their limitations. Immunological detection methods need a lot of preparative work (Wipat et al., 1992) and can lead to the occurrence of false signals 116 V. Discussion Use cf gjp and gfp-variants far in situ monitoring due to unspecific or hindered binding (Moter and GObel, 2000). PCR and PCR based methods can discriminate on the basis of DNA accessibility. Therefore DGGE band intensity may not always give reliable estimates for the amount of a certain species in the environment or its importance. Also the in situ hybridisation technique using rRNA targeted fluorescent probes can result in false positive signals (hybridisation with non target cells), false negative signals (inaccessibility of the rRNA target site, loss of target cells by repeated spins and washing steps, low rRNA levels due to low cell metabolism) and disturbance of the architecture due to preparation and hybridisation treatments of the sample (Moter and GObel, 2000). PNA probes promise to lessen the drawbacks implicated using die FISH technique, because of the higher specificity, easy target accessibility, the broad range of conditions wherein PNA probes hybridise and hence the subsequent lowering of die impact on integrity and characteristics of cells and cell communities (Ray and Norddn, 2000). Nevertheless, direct in situ methods are certainly needed when gene transfer is investigated inside biofilms. By using die green fluorescent protein and/or its mutants in combination with nucleic acid dyes, single transfer events could be recorded inside the biofilm, even if the expression of the received trait is transient Direct monitoring is not restricted to monitoring those gene transfer frequencies that result in transgenic cells that are able to produce daughter cells and/or are culturable by classic plating methods which have been used in previous studies (Cresswell and Wellington, 1992; Pickup, 1992). A discrepancy between direct CLSM methods and selective plating techniques has been observed (Hausner and Wuertz, 1999). This already suggested that gene transfer using detrimental genes Would not be easily monitored using indirect methods, while direct visualisation of transfer events would allow the detection of gene transfer events before tire cells could suffer from the disadvantages after gene expression. Another advantage of using gfp in combination with CLSM is the ability to detect the location of the gene transfer event, which is important to understand the influence of architecture or the effect of biofilm development Location of gene transfer events will give insight into morphogenic parameters that affect and are affected by gene transfer. Next to investigating the influence of biofilm architecture (How does EPS affect gene transfer in biofilms? How does biofilm architecture effect gene transfer (Hausner et al„ in prep)? Does gene transfer affect biofilm architecture (Ghigo, 2001)?) it would also reveal favourable and unfavourable biological and physical micro-conditions both in space and time (Does cell activity of the donor/recipient play a role in gene transfer? Where does early/late gene transfer occur? Can gene transfer be regulated by cell-to-cell communication?). 117 V. Discussion Evaluation of eyfp as a disadvantageous gene B. EVALUATION OF eyfp AS A DISADVANTAGEOUS . . GENE „ . While the expression of the wild type green fluorescent protein (GFP) did not seem to influence growth or metabolic activity of BD413, the expression of a GFP derivative, the enhanced yellow fluorescent protein (EYFP), decreased cell viability. It was impossible to obtain a stable pure eyfp labeled BD413 culture, eyfp could therefore be used as a disadvantageous gene. The use of in situ quantitative microscopy seemed to be an ideal alternative method for detecting natural genetic transformation of a disadvantageous gene in monoculture biofilms because it was impossible to obtain ey^-transformants from biofilms via selective plating. The use of the gfp marker for investigating viable but non culturable cells was also used by Cho and Kim (1999). It could be argued that ey£>-transformants were not selectable using plating methods due to lack of viability. This turned out to be not the case. Viability of eyfp- expressing cells was observed during a 9 day observation period in a 3 strain biofilm. Because attempts to produce a monoculture eyfp expressing Acinetobacter sp. BD413 biofilm failed, with or without selective pressure, it was speculated whether the presence of uncompromised cells supported viability and survival of compromised ey^-expressing cells. As of yet no report exists on the follow up of competitively disadvantageous strains in biofilms. Although {^-expressing cells were only monitored for a total of 9 days in the present study, it still showed that mixed species biofilms could extend the survival period of compromised cells by at least a factor of 5 times. It is necessary to investigate stability and viability of the compromised strains as well as the persistence of the disadvantageous gene over a longer period and in biofilms possessing a bountiful microbial diversity to conclude if support of healthy and happily growing cells attribute to survival of less fit cells on the long run. In various studies survival of added recombinant strains in natural niches was studied with detection methods like plating (Barcina et al, 1997; Dejonghe et al., 2000), PCR based techniques (BjdrklSf et al., 2001; De Clercq et a!., 2001), bioluminescence detection (Tebbe, 2000), gfp fluorescence detection (Eberl et al., 1997) or with fluorescent in situ hybridization (FISH) (Bouchez et al., 2000). But these reports investigated actively growing strains. Follow up of unfit cells could not be accomplished by all these methods. Monitoring survival of less fit strains should preferably be performed with direct in situ methods using gfp and/or gfp- variants or FISH in combination with CLSM. 118 V. Discussion Evaluation of eyfp as a disadvantageous gene In the present study the follow up of the volume of cells possessing either a negative or a neutral competitive advantage rose toward and subsequently fluctuated around a certain volume of cells inside the biofilm. Similar findings were observed by German et at. (1987). The authors studied residual numbers of added bacteria in a soil ecosystem based on a model introduced by Gompertz (1825). The investigation illustrated that the introduced bacteria reached a level of about 10* colony forming units/g soil, regardless of the amount of bacteria added to the soil ecosystem (Gorman et al., 1987). One can speculate that the development of the number of introduced strains in a biofilm would lead to the same stabilisation. Still, stabilisation of introduced bacteria are regarded to be one of the biggest problems when it comes to obtaining successful bioaugmentation of polluted soils or activated sludge systems (Goldstein et aL, 1985; Van Limbergen et al., 1998), as well as in stabilising an optimally constructed biocontrol agent in soil ecosystems. Nevertheless it has been observed that active potentially pollutant removing organisms can be stabilised in the rhizosphere or phytosphere (Ramos et al., 2000) without having an impact on the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable community. Furthermore were introduced strains active enough to improve phytoremediation in polluted soil sites (Kamnev and van dcr Leiie, 2000). It was also observed that to-be-contained Pseudomonas strains designed to disappear in the absence of 3-methylbenzoate survived for much longer in soil microcosms or in the rhizosphere when compared with the observed high killing rate in liquid cultures (Ronchel and Ramos, 2001). This suggests a role of stabilisation of possibly competitively weak organisms by biofilm communities. As of yet it is not known if increased bacterial diversity supports survival of unfit and selectably disadvantageous bacterial strains. In some papers the positive effect on survival of introduced bacteria has been suggested to be attributed by a higher bacterial diversity (NOfilein et al., 1992, Dejonghe et a!., 2001). Sporadic reports, using mathematical models, seem to support that suggestion. For example, it was observed that an increase in the number of species in a microbial habitat lead to increased unpredictability of the outcome of the repartitioning of the introduced strains in a neutral environment (Huisman and Weissing, 2001). It was even more interesting, that the mathematical models used to describe competitive behaviour between planktonic species always predicted that it was impossible to completely eradicate any species, even if it had all odds against his existence in that particular environment (Huisman and Weissing, 1999). 119 V. Discussion In situ quantification of natural genetic transformation C.In situ QUANTIFICATION OF NATURAL GENETIC TRANSFORMATION IN MONOCULTURE Acinetobacter SP.BD413 BIOFILMS When natural genetic transformation using pGARl in biofilms is compared with natural transformation in suspended cultures, a number of interesting differences is observed. Natural genetic transformation occurred readily at high frequencies in monoculture Acinetobacter sp. BD413 biofilms. Using 12 pg pGARl/ml for transforming cells grown in a biofilm, a transformation frequency as high as 2.4x10"3 was observed. With standard transformation methods, lpg pGARl/ml could only result in a maximal transformation frequency of 3.1xl0'5 transformants/recipients. Furthermore, it was possible to obtain detectable transformation frequencies with minute amounts as little as 1 fg pGARl/ml in biofilms. The lowest tested DNA concentration used for transformation of Acinetobacter sp. BD413 in the literature was not lower than 1 ng DNA/ml (Palmen et al., 1993). In young and actively growing biofilms the transfer frequency was very high. In older and established biofilms the transfer frequency was less pronounced, but still a certain fraction of cells was competent for transformation. Hence, no optimal growth phase was needed for natural transformation to be detectable throughout the observation period as it was required for the transformation of cells grown as a suspended culture. Transformation of cells using planktonic cultures only occurred during the exponential growth phase (Lorenz et al., 1991). The biofilm mode of growth could therefore be compared to the constantly exponentially growing steady state batch or turbidostat cultures of Palmen et. al. (1994) who could still detect a decreased transformation frequency after 3 days compared to initial transformation frequencies, which was impossible in standard batch culture experiments (Lorenz et al., 1991) or soil microcosms (Nielsen et al., 1997). While the culture age of a bacterial community still possessing a significant fraction of competent cells was extended for at least three days when grown as a biofilm, the time during which DNA was taken up was largely reduced. Within 15 minutes of incubation with free DNA the competent fraction of the biofilm cells took up DNA whereafter no immediate DNA take up was observed. In suspended cultures DNA take up already occurred after 1 minute. And in contrast with biofilms all competent cells in a cell suspension continued to take up DNA for an extended time period of 3 hours (Palmen et al., 1993). 120 V. Discussion In situ quantification of natural genetic transformation Increased DNA concentration lead to increasing transformation frequencies in biofilms, as it was shown in transformation experiments using planktonic Acinetobacter sp. BD413 cultures (Palmen et al., 1993). Maximal transfer frequencies (3.26x1 O'3) were obtained with 1.5 pg pGARl DNA within the concentration range used in this work, but no saturation level was yet reached. It could therefore be possible to obtain even higher transfer frequencies if a feed with increased DNA concentrations were added to the biofilm. The question remains if it is necessary to have maximal transformation frequencies when bioaugmentation is planned using in situ genetic manipulations. If it is possible that transformants can establish themselves in the reactor, low but significant transfer frequencies will suffice to obtain bioaugmentation. For effective bioaugmentation it is not very dramatic if the added strains disappear when they are able to transfer genes to more competitive recipient organisms. Dejonghe et al. (2000) used the ability to transfer genes by added exogenous genetically manipulated microorganisms to directly enhance biodegradability of the autochtone bacterial community in a 2,4-D polluted soil microcosm. Biofilm cells grown in a continuous mode have a longer life span than batch grown cultures (Christensen et al., 1998). The stable phase of a biofilm cannot be compared with the stationary phase of a planktonic culture. While biofilms still develop during the stable phase, the stationary phase of a batch culture will represent a stage were culture growth and cell metabolism has ceased. During biofilm development, new transformants are formed and old transformants disappear due to lysis or detachment The volume of transformants followed the oscillating pattern of accumulation and decrease of the volume of recipients. Although the volume of transformants increased and decreased much more than the volume of recipients, a certain fraction of transformants did essentially maintain itself in the biofilm. A short term follow up of the survival of recombinant cells proved their ability to be initially established in a monoculture biofilm. This of course does not yet assume survival of recombinant cells in natural biofilms or that maintenance of recombinant cells is assured for a prolonged period. Nevertheless is the maintenance of a certain optimally constructed organism is less important if the genes, encoding a desired catabolic trait, persist by horizontal gene transfer into microorganisms that are more adapted in a certain environment Therefore, emphasis should be put on increasing the possibility of gene transfer to recipient organisms that are stably maintained in bacterial communities when attempting effective bioaugmentation and hence an understanding is necessary of the parameters that influence gene transfer to bacteria living in biofilms. In the case of natural genetic transformation in biofilms of Acinetobacter sp. BD413 a strong correlation was observed between biofilm porosity and transformation frequency. It could be concluded that most transformants were formed at locations in the biofilm where the biofilm was the densest 121 V. Discussion In situ quantification of natural genetic transformation So, biofilm density positively influenced natural genetic transformation events without even selecting for the resulting transformants. Hence manipulating the factors that decrease biofilm porosity can lead to enhanced occurrence of transformation events. Van Loosdrecht (1997) presents flow rate, nutrient loading rate and growth rate of the biofilm cells as parameters that influence biofilm thickness and porosity. Next to these parameters also cell- to-cell signalling molecules will shape biofilm structures (Davies et a!., 1998). Preliminary experiments revealed a restructuring of the monospecies BD413 biofilms in globular subclusters with high local cell density when the biofilm was grown in the presence of signals extracted from BD413 cultures grown in rich medium. The location of the areas in the biofilm, possessing a high local compactness of cells overlapped with those locations were most transformants were found (data not shown). Hence it could be said that low porosity effectively stimulated the local occurrence of natural genetic transformation events inside Acinetobacter sp. BD413 biofilms. Also the conditions in which a biofilm emerged influenced transformation frequency. The initiation of a biofilm starts with attachment of free floating bacteria. Thereafter biofilms develop by subsequent growth or recurring attachment of other bacteria. In certain cases biofilms may encounter a lot of passing free floating bacteria. The presence of high numbers of BD413 cells in the surrounding medium inhibited transformation in biofilms (Hendrickx, unpublished results). Likewise, if biofilms developed in the presence of high numbers of cells in the suspension but were rinsed to discard cells present in the surrounding medium prior to be exposed to naked DNA, transformation was strongly inhibited. This might be due to an increased attachment of bacterial cells that have entered the stationary phase (and hence became non-competent) in the surrounding medium. This makes natural genetic transformation not very easy for in situ genetic manipulation of Acinetobacter sp. BD413 residing in certain bioreactors. Bioreactors like sequencing batch biofilm reactors, rotation disc reactors or expanded bed reactors not only contain bacterial cells in the biofilm fraction but also in the suspended fraction. On the other hand tubular reactors, trickling filters or polluted soil sites could offer the right conditions for genetic manipulation of Acinetobacter sp. BD413. Even if bioaugmentation by transformation of Acinetobacter sp. BD413 would be used for the latter waste treatment facilities it should be noted that the activity of suspended cells is not a superfluous luxury. Yu et al. (2000) observed that while the suspended bacteria were less than 1% of the total amount of biomass in an aerobic circulating-bed biofilm reactor, they were nevertheless responsible for the turnover of up to 30% of the recalcitrant product into its intermediates. Hence the negative influence of the presence of suspended cells is may pose a problem. 122 V. Discussion In situ quantification of natural genetic transformation However, the problem of inhibited gene transfer due to the presence of suspended cells during biofilm ontogenesis can be solved very easily. It has been shown that starvation periods induced transformation (Nielsen et al., 1997; Section IV.C.2.). This implies that a simple trick would easily instigate transformation very efficiently: first the biofilm needs to be washed to clean it from most cells present in the drifting fraction, thereafter the biofilm needs to be starved by feeding the reactor with water or a salt solution without nutrients, and in a last step in situ transformation can be allowed through the addition of nutrient containing substrate with a certain concentration of desired DNA. A last but very important parameter still needs to be verified: the efficiency of transformation of Acinetobacter sp. BD413 in the presence of other waste water microorganisms. The presence of the natural community has been shown to have mixed effects on transformation. The endogenous community either reduced the frequency of transformation of a marine Vibrio strain or had no effect in water column microcosms (Paul et al., 1991). It reduced the transformation frequency of P. stutzeri (Stewart and Sinigalliano, 1990) and prevented transformation of Vibrio strains (Paul et al., 1991) in sediment microcosms. On the other hand, transformation with Acinetobacter sp. BD413 lead to equally high frequencies in non-sterile groundwater and wet soil microcosms as it was found to be under sterile conditions (Lorenz et aL, 1992). Likewise, the transformation of Acinetobacter sp. BD413 embedded in river epilithon was not inhibited by indigenous organisms (Williams et al., 1996). So it could be speculated that the presence of an ambient community in waste water treatment systems will have little or no negative effects on transformation of Acinetobacter sp. BD413 cells. The presented results indicated that transformation has a potential to be used as a tool for bioaugmentation of biofilm reactors. Up till now the use of transformation as a way of introducing a new genetic trait in a reactor has had very little if not none attention at all. Future research is needed to elucidate the efficiency of transforming Acinetobacter sp. BD413 cells residing in a natural biofilm inhabited by waste water treatment micro-organisms under reactor conditions, before the effective applicability of transformation for bioaugmentation can be proved or rejected. 123 V. Discussion Natural genetic transformation with a disadvantageous gene D. NATURAL GENETIC TRANSFORMATION OF A DISADVANTAGEOUS GENE IN MONOCULTURE Acinetobacter SP. BD413 BIOFILMS It is perhaps misleading to apply the term ‘transformation frequency’ in the experimental observations using a detrimental gene. Transformation events could only be detected after eyfp expression, EYFP maturation and before the host would lyse. Hence, the absolute transformation frequency could not have been detected. Only the resulting efficient transformation frequency as the volume of established ey$7-expressing transformants/volume of recipients was measurable. At what stage efficient transformation frequency was inhibited could not be elucidated yet, although it seems that lysis after uptake and expression of the detrimental gene is the most logical explanation. A satisfying answer will only be obtained by on line in situ observation of transformation in the biofilm. The amount of the occurrence and subsequent disappearance of EYFP signals will shed a light on what was causing unsuccessful transformation at the biofilm substratum. If the volume of appearing EYFP signals at high DNA concentrations is large enough to reach those levels that are obtainable with gfp transformation and if they are found at the biofilm substratum, it could be concluded that unsuccessful transformation was due to limited survival of the transformants. If the volume of appearing and disappearing signals is too low to account for DNA uptake levels as detected with pGARl transformation, than an inducible protective phenomenon against the uptake of disadvantageous genes can be considered. Still, if biofilms offer the cells a certain advantage by offering conditions wherein undesirable gene transfer can be avoided, the reasons why transformation of biofilm cells occurs differently with neutral compared to disadvantageous DNA remain speculative. The protective character in the presence of negatively influencing DNA could be explained by a certain way of cell-to-cell communication that directly or indirectly influenced transformation of cells residing in a biofilm. Acinetobacter sp. BD413 has been shown to produce as many as 5 signal molecules when grown in minimal medium (Gonz61es et al., 2001). Acinetobacter sp. BD413 can therefore sense and produce signals. Stressed BD413 biofilm cells may produce less of a certain signal molecule by hampered metabolic activity, thereby inhibiting competence development. Whether or not the production of certain activating or repressing signals in Acinetobacter sp. BD413 can be induced or inhibited under stress conditions, and whether these signals directly or indirectly regulate natural genetic transformation remains to be discovered. Detection of transformation frequencies, however, demand the visualization of both transformants and recipients. General nucleic acid stains can not be used for on line 124 V. Discussion Natural genetic transformation with a disadvantageous gene measurements of living biofilms. Double fluorescent protein labeling techniques that require one cell to be doubly labeled (instead of two different strains of a mixture labeled each with a different fluorescent protein) require careful selection of the fluorescent proteins and intensive testing of the spectral interactions of the fiubrophores (Hausner, pers comm). Vital fluorescent staining methods (Fuller et al., 2000) depend on interaction of the fluorescent stain with the bacterial membrane and were shown to not adversely affect viability or adhesion of the stained cells. These stains can be an interesting alternative to detect transformation frequency in situ, on the condition that the stain will not disturb normal competence development and DNA uptake. Still, if only low amounts of transformation events are observed at the biofilm substratum in an on line experiment one should be careful in concluding that cells residing in a biofilm can be switched to a non-competent state due to alarming signals from neighboring sjgjMransformants. Death of transformants could be anticipating EYFP fluorescence. This does not mean that the maturation of the EYFP protein is compromised in BD413 cells, under tire local conditions inside a biofilm. It means that immature EYFP could cause the negative effects on an «gj§>-transfonnant before it can be detected accordingly. When an inlet medium with pGAR2 DNA was added to the biofilm measurable transformation frequencies were detected. Like transformation using neutral DNA, transformation with detrimental DNA was observed using very little amounts of disadvantageous DNA (1 fg pGAR2/ml). Furthermore it was shown that diluted numbers of «3g$r-expressing cells did not inhibit subsequent initial establishment of the compromised cells. Hence transformation with a gene that offers biofilm host cells a less fit phenotype resulted in the production of transformants that could be potentially maintained inside the biofilm. This indicated that it was possible to obtain and initially establish transgenic cells that had a competitive disadvantage compared with other cells in the biofilm. But while increasing DNA concentrations led to increasing transformation frequencies when neutral DNA was used, it was shown that the maximal obtainable transformation frequency diminished with increasing DNA concentration Lower transfer frequencies of lethal or detrimental genes have been observed in studies where limiting spread of genes was intended (Molin et al., 1987; Diaz et al., 1994; Knudsen et al., 1995; Torres et al., 2000). Conjugation or transformation (via electroporation) frequencies were quantified using selective plating techniques. Hence it would only detect those transconjugants and transformants that were able to produce daughter cells. When the number of «%$»-transfbrmants in biofilms was obtained using these classical plating methods, the transformation frequency fell below the detection limit It is therefore important to note that the above mentioned containment studies overlooked an important detail: the existence of transgenic cells that are unable to produce colonies. 125 V. Discussion Natural genetic transformation with a disadvantageous gene While in situ genetic manipulation of cells in waste water treatment reactors necessitates the spread of degradation genes in the indigenous bacterial community, one should not forget that dissemination of recombinant genes is not always desired. Especially in those systems where introduced transgenic organisms (biocontrol agents, herbicide resistant agricultural plants, etc.) are used to help reducing the amounts of unwanted organisms (pathogenic strains, weeds, etc.) there is a potential hidden risk to obtain undesirable consequences (antimicrobial agent resistant pathogens, herbicide resistant weeds, etc.), which are not always predictable. It should be kept in mind that next to the unpredictable character of unintentional spread of ‘harmless’ genes (Malone and Pham-Delegue, 2001), containment is also important for process protection/process optimisation reasons (Torres et al., 2000) or to protect patented constructs for unintentional misuse (Monsanto, 1998). With ‘containment ’ two types of blockage against the spread of recombinant genes are considered: biological containment which involves the reduction of dissemination of the transgenic strain; and gene containment which aims at the elimination of possible transfer of the gene itself. Both types of containment cannot be accomplished 100%. A severe reduction of the persistence of the transgenic strain or the recombinant gene in comparison with not to be contained strains or genes will tried to be achieved instead. Common knowledge on evolution predicts that genes that can only be sporadically transferred at negligible rates due to their detrimental effects on the host will eventually disappear in a bacterial community. The hosts which harbour such a gene will not be fit enough to compete with other members in the community and furthermore, the expression of detrimental genes will prevent the production of offspring. Hence hosts accepting detrimental genes would be nonviable and disappear after a certain time unless the detrimental gene had been mutated to a harmless derivative of the gene. The existence of a horizontal gene transfer mechanism like transformation, however, can steer the fate of detrimental DNA into a totally different and unexpected outcome. When the fraction of neutral transformants was followed in a biofilm that was exposed to neutral plasmid DNA for extended periods, it was observed that the net transformant frequency remained approximately the same after 4 or 41 hours, although interrupted with several high transformant frequency peaks due to biofilm growth (Section IV.C.). If however, biofilms were exposed to disadvantageous plasmid DNA for extended time periods, an increase in the volume of transformants was observed after initial raises and drops in transformant frequency (Section IV.D ). It seems even that this accumulation of transformants containing a disadvantageous gene could only have been achieved due to the detrimental character of its gene product, because cells first amplified the disadvantageous gene before releasing it into the environment via cell lysis. Amplification of the detrimental gene could also be observed when only minute amounts of negatively influencing DNA was used in transformation experiments. When using low amounts of DNA regularly higher transformation frequencies were observed than could be estimated using neutral DNA in parallel transformation experiments. Hence, death of a host containing disadvantageous DNA does not always imply 126 V. Discussion Natural genetic transformation with a disadvantageous gene disappearance of its genetic traits. It seems that, in biofilm systems, transformation monopolises an important way of safekeeping those genes that otherwise would be condemned to disappear as soon as they have become detrimental. Just like viruses spread by a mechanism based on parasiting on cellular metabolism in order to amplify and be protected from degrading conditions whereafter release of the amplified factor necessitates death of the host, detrimental genes can spread as long as potentially competent cells are present Torres et al. (2000) recognized the problem of the persistence of the recombinant gene itself. By designing a containment system based on the EcoRI type H restriction-modification system, they have engineered a barrier against unwanted gene transfer at the genetic level. It should be stressed however that such a system may only be safe if the recombinant genes will fail to be replicated and hence the restriction modification-system should be adapted to fulfill separation from an origin of replication. Preservation of selectively disadvantageous genes during evolution has untill now never been considered. Clearly this anti-Darwinistic consideration does not seem logical. Next to the dissemination of detrimental genes via transformation it was observed in a previous chapter that the presence of uncompromised cells supported die viability of cells expressing disadvantageous genes in biofilms. The expression of eyfp, however, was not immediately lethal. Viability of eyfp expressing colonies (a mixture of eyfct-expressing and unlabeled cells) could be observed for up to 20 days when grown on non-selective plates (Section IV.B.). It would be interesting to investigate how long cells, containing disadvantageous and even lethal genes can persist in biofilms and if those genes can be horizontally transferred to other members of a natural biofilm, that are more tolerant towards the gene product If biofilms were able to keep tire number of cells containing detrimental and even lethal genes at significant levels and if biofilms could extend the persistence of the disadvantageous gene itself (e.g. by several transformation and lysis steps), undesired gene sequences could be preserved much longer than expected. While the existence of a gene offering its host a certain advantage in the battle of evolution might be secured by disseminating the gene as much as possible in the collective bacterial gene pool, the persistence of an undesired gene can be maintained by another strategy. Low copy numbers, limited spread, gene silencing, transformation with genes originating from dead donor cells and slow growth of hosts containing negatively influencing genes may safeguard these temporarily disadvantageous genes from vanishing too quickly. The unfavorable effects of a gene sequence in one bacterial strain and/or in one environmental niche might be a valuable genetic asset to another strain and/or in other circumstances. Seen in this light it is advantageous for bacterial evolution to keep cells carrying undesirable genes in a bacterial community and hence protect the existence of undesirable genes in tire microbial gene pool rather than to let those genes disappear as soon as they have lost their immediate benefit or have even become detrimental, only to repeat the whole cycle of mutation and selection to regain the lost characteristics. 127 V. Discussion Outlook E. Outlook Bioaugmentation of biofiim reactors by in situ natural genetic transformation requires in depth knowledge of both microbiology and engineering. In a first step, research of natural genetic transformation should be shifted from defined monoculture Acinetobacter sp. BD413 biofilms to defined mixed species biofilms and even undefined natural biofilms. Also other environmental strains like Pseudomonas species should be tested for the ability to take up free DNA in natural biofilms. It would be interesting to see if transformation using certain strains can be controlled by cell-to-cell communication either directly (triggering competence development) or indirectly (influencing biofilm architecture). Furthermore the role of EPS on DNA protection, DNA storage or DNA availability for transformation will have to be investigated Another important aspect is the modeling of the frequency and location of transformation events in natural biofilms. The influencing parameters (biofilm thickness, presence of certain organisms, biofilm porosity, ...) should be extracted from quantitative microscopical investigations. These data will help the experimenter to develop protocols that stimulate in situ gene transfer by simple external manipulation (backwashing, nutrient addition or interruption, temperature, addition of signal producing organisms, ...) of the influencing parameters. In a last stadium a lab scale biofilm reactor of choice will have to be run in order to determine effective bioaugmention. Here not only data of natural genetic transformation events should be recorded. Next to optimization of the physical processing parameters, the degradation efficiency and the stability of the transgenic strain will play the leading role. Furthermore it is necessary to see if these transgenic cells are active enough to fulfill their purpose in biofilm reactors. Effective bioaugmentation necessitates stable processing during fluctuating concentrations of the recalcitrant product in the feed. Optimizing process conditions will also include the degradation of very low amounts of the xenobiotic compound or degradation of the contaminant in the presence of easy metabolizable carbon sources. Only if in situ genetic transformation is able to enhance effective biodegradation in the reactor, the final goal will be reached. 128 VL CONCLUSION 130 VI. Conclusion Main points A. MAIN POINTS Control experiments revealed that it was possible to distinguish between gj$>-expressing Acinetobacter sp. BD413 cells and Syto 60 stained recipients cells as well as between ecjp- and ex6>expressing Acinetobacter sp. BD413 cells. Hence, it was possible to investigate natural genetic transformation m situ in monoculture Acinetobacter sp. BD413 biofilms with the use of gfp and its variants in combination with the confocal laser scanning microscope. Due to the monoculture ’s homogeneous structure, reproducible results were obtained. Expression of gfp and ectfp did not hamper Acinetobacter sp. BD413’s viability while the expression of eyfp did. The negative effects of expressing eyfp were most evident when monoculture Natural genetic transformation occurs in monoculture Acinetobacter sp. BD413 biofilms. Minute amounts of DNA concentrations in the fg range could give rise to detectable natural genetic transformation. On the other hand a maximal transfer frequency was not yet reached using 1.5 pg DNA/ml. Within 15 min all competent cells had taken up DNA and this indicated that prolonged incubation periods with DNA containing medium was unnecessary to obtain higher transfer frequencies. Parameters that could influence natural genetic transformation in the investigated model biofilm were DNA load, porosity and nutrient load. Exposure to high DNA concentrations, foe use of dense biofilms and biofilm pre-starvation enhanced the observed transfer rates. If biofilm reactors are intended to be bioaugmented by in situ genetic manipulation, only the porosity parameter and foe nutrient load can be used to control in situ natural genetic transformation because a practicable DNA load is limited by the reactor dimensions. Hence 132 VI. Conclusion Answers to some open questions subsequent establishment of transformed cells is important to obtain stable in situ gene pool enrichment Natural genetic transformation with selectively disadvantageous DNA also occurred inside the model biofilm. The negative influence of the expression of the detrimental gene even allowed accumulation of compromised transgenic cells due to repeated amplification of the detrimental gene and lysis of the transgenic cells after gene expression. It can be concluded that in the absence of selection and even when a detrimental gene is introduced, natural genetic transformation occurs and transfer frequencies can be influenced inside a model monoculture biofilm. Initial establishment of both transgenic cells having no selective advantage and transgenic cells with a selective disadvantage were also observed in the model biofilms. B. ANSWERS TO SOME OF THE OPEN QUESTIONS IN: 1. CHAPTER C : Horizontal gene transfer bv natural genetic transformation (base 401 The role of natural genetic transformation as a mechanism for horizontal gene transfer has not been regarded as important as conjugation. In Actnetobacter sp. BD413 cells, however, natural genetic transformation proceeds very efficiently. In biofilms a fraction of cells is continuously competent for transformation and this results in significant transfer frequencies. The controlling parameters of natural genetic transformation in monoculture Actnetobacter sp. biofilms include biofilm porosity, DNA load and nutrient load. On the other hand may natural genetic transformation of Acinetobacter sp. BD413 residing in natural biofilms reveal different or additional controlling parameters. Surprisingly, transformants containing a detrimental gene accumulated during biofilm development under continuous exposure to detrimental DNA while no accumulation of transformants was observed when biofilms were exposed to neutral DNA during an extended period. This was probably due to amplification of the detrimental gene in the biofilm after repeated uptake, amplification of the plasmid, expression of the detrimental gene and subsequent release after lysis of the compromised transformant It is difficult to predict the implication of the accumulation or at least the persistence of a detrimental gene in nature yet It can be presumed, however, that genes will not immediately disappear or that less fit organisms are not readily outcompeted in a natural biofilm. Hence, care should be taken when a to-be-contained recombinant organism (with a genetically based suicide mechanism) is introduced in the environment In any case, horizontal gene transfer by natural genetic 133 VI. Conclusion Answers to some open questions transformation supports persistence and spread of negatively/neutrally/positively influencing DNA whereas conjugation is responsible for the spread of neutral and certainly advantageous genes. Up till now too little is known about gene transfer by bacteriophages to speculate about its role in the spread of disadvantageous/neutral/advantageous genes. 2. CHAPTER B: Natural genetic transformation and transformant cells in a biofilm (page23) After uptake of neutral or detrimental DNA initial establishment of the transgenic cells was observed. So, regardless of the fitness of the transgenic cells, at least a certain fraction of transgenic cells initially survived in the model biofilm. It is still unknown how long disadvantageous/neutral/ advantageous genes can persist in a biofilm, either entrapped in the EPS or stored inside dead/living cells. This implies the persistence of negatively influencing DNA in a biofilm environment and affirms the unpredictable character of the fate of DNA. Biofilms have until now been observed to offer ideal circumstances for horizontal gene transfer to occur by transformation as well as by conjugation. 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Chemosphere 44:1103-1108. 163 -i Schriftenverzeichnis In der Schriftenreihe des Lehrstuhls for WassergOte- und Abfallwirtschaft der Technischen Universitat MOnchen sind bisher folgende Berichtshefle Im Eigertverlag erechienen: (Vergriffene Hefte sind Ober die Bibllotheken-Femleihe zu erhalten) Nr. 1 Pecher, R. Hilfstafeln zur hydraulischen Berechnung von offenen kOnstlichen Gerinnen (1969) 17 Seiten, 8 Beispiele, 6 Anlagen Nr. 2 Pecher, R. Der AbfluBbeiwert und seine AbhSngigkeit von der Regendauer (1969), 140 Seiten, 18 Abb., 46 Anlagen Nr. 3 Pecher, R. Die Bemessung von Regenbecken In der Stadt- vergriffen entwSssenjng (1970), 101 Seiten, 26 Abb.,19 Anlagen Nr. 4 1. Abwassertechnisches Berechnungsmethoden for AbwasserkanSle, vergriffen Seminar RegenObertaufe und Regenbecken (1970) 200 Seiten, 65 Abb., 4 Anlagen Nr. 5 2. Abwassertechnisches Ausgewahlte Kapitel zur Technik der Abwasser- vergriffen Seminar reinigung (1971), 217 Seiten, 80 Abb. Nr. 6 3. Abwassertechnisches Akute Probleme der Abwassertechnik und Abfall- Seminar beseitigung (1973), 144 Seiten, 38 Abb. Nr. 7 4. Abwassertechnisches Neue Aspekte zur Kanalbemessung und Leistungs- Seminar verbesserung biologischer KBranlagen (1974) 180 Seiten. 56 Abb. Nr. 8 6. Abwassertechnisches Entwicklung bel kleinen KISranlagen und bei der Seminar Automation In der Abwassertechnik (1975) 193 Seiten, 83 Abb. Nr. 9 Brunner, P. G. Die Verschmutzung des Regenwasserabflusses im Trennverfahren. Untersuchungen unter besonderer BerOcksichtigung der Niederschlagsverhaitnisse im voralpinen Raum (1975) 200 Seiten, 18 Abb., 41 Anlagen Nr. 10 Bischofsberger, W. Entwicklung und Tatigkeit des Lehrstuhls und PrOfamtes Im Zeitraum 1966 -1975, (1976) 52 Seiten, 7 Abb., 2 Tabelien Nr. 11 Neumann, W. Der NiederschlagsabfluB In stadtischen Einzugs- Mam, G. gebleten (1976), 268 Seiten, 52 Abb., 9 Anlagen Nr. 12 6. Abwassertechnisches Verbesserter Gewasserschutz durch Leistungs- Seminar steigerung In der Kldrtechnik (1976) 195 Seiten, 54 Abb. Nr. 13 Bischofsberger, W. Anwendungen von Failungsverfahren zur Verbesse- vergriffen Hegemann, W. tung der Lelstungsfahigkeit biologischer Anlagen Ruf, M. (1976), 560 Seiten, 80 Abb., 56 Anlagen, Anhang Overath, H. Nr. 14 1. Wassertechnisches Seminar Femwasserversorgung (1977) 226 Seiten, 90 Abb., 8 Tafeln -l- Nr. 15 Ottmann, E. Untersuchungen Ober den Einsatz, die Bemessung und Lelstung von Erdbecken und Oxldattonstelchen (1977), 290 Seiten, 56 Abb. Nr. 16 Hmschka, H. Untersuchungen Ober den Einsatz von ProzeB- Meyer, T. rechnem auf KBranlagen (1979) 218 Seiten, 24 Abb., 10 Tabellen Nr. 17 7. Abwassertechnisches Planungsgrundlagen und Planungskonzepte In der Seminar Kanalisations- und KBrtechnik (1977) 190 Seiten, 62 Abb. Nr. 18 Veits, G. ElnfluB der Vorkldrung auf die biologische Stufe und auf die Wiitschaftlichkeit von Belebungsanlagen (1977), 160 Seiten, 38 Abb. 36 Anlagen Nr. 19 1. MOIItechnlsches Seminar Problems# der Ablagerung fester Abfallstoffe (1977) 131 Seiten, 12 Abb., 24 Tabellen Nr. 20 2. Wassertechnisches Seminar Wasserspeicherung (1977) 195 Seiten, 120 Abb., 3 Tabellen Nr. 21 Billmeier, E. Verbesserte Bemessungsvorschiage tor horizontal durchstrOmte NachklSrbecken von Belebungsanlagen (1978) 180 Seiten, 36 Abb., 15 Tabellen, 25 Seiten Anhang Nr. 22 Bischofsberger, W. Anwendung von Failungsverfahren zur Verbessemng Ruf, M. der Leistungsfahigkeit blologlscher AnlagenTeil II. Hmschka, H. Elsen(ll)-Salz und Kalk (1978) Hegemann, W. 220 Seiten, 38 Abb., 43 Tabellen Nr. 23 GOttle, A. Ursachen der Regenwasserverschmutzung und Ein- fluBgrtSBen auf die AbfluBbeschaffenheit im Trenn- verfahren (1978) 405 Seiten, 42 Abb., 34 Tabellen, 41 Anlagen Nr. 24 Seminar a us WassergOte-und 3. Wasser-, 8 . Abwasser- und 2. MOIItechnlsches Abfallwirtschaft 1978 Seminar - Institutseinweihung 1978 (1978) 477 Seiten, 149 Abb., 37 Tabellen Nr. 25 9. Abwassertechnisches Erfahrungen mit der weitergehenden Abwasser- Seminar behandlung durch Failungsreinigung (1979) 395 Seiten, 139 Abb., 36 Tabellen Nr. 26 3. MOIItechnlsches Seminar Abgasreinigung und Gewasserschutz bel der thermlschen Abfallbehandlung (1980) 256 Seiten, 60 Abb., 29 Tabellen Nr. 27 4. Wassertechnisches Seminar WasserfOnderung - Planung, Bau und Betrieb von Pumpwerken (1980) 190 Seiten, 107 Abb., 1 Tabelle Nr. 28 10. Abwassertechnisches BelOftungssysteme und Energlehaushalt bel der Seminar Abwasserreinigung (1980) 353 Seiten, 145 Abb., 35 Tabellen Nr. 29 Reach, H. Untersuchungen an vertikal durchstrtSmten Nach- kiarbecken von Belebungsanlagen - Neue Gesichts- punkte for Bemessung und Betrieb (1981) 250 Seiten, 105 Abb., 17 Tabellen Nr. 30 Dauschek, H. BeeintrSchtigung von OberflSchen und Grundwasser Blschofsberger, W. durch Auftausalze in Schutzzonen (1986) 150 Seiten, 36 Abb., 15 Tabellen, 264 Anlagen Nr. 31 5. Wassertechnlsches Seminar Wasserverteilung - Planung, Bau und Betrieb von vergriffen Rohmetzen (1981) 251 Seiten, 49 Abb., 7 Tabellen Nr. 32 4. Molltechnisches Seminar Behandlung und Verwertung von metallhaltigen ROckstanden (1981) 208 Seiten, 38 Abb., 27 Tabellen Nr. 33 11. Abwassertechnisches Biologische Stablllsierung von Schiammen und Seminar hochkonzentrierten Abwassem (1981) 254 Seiten, 90 Abb., 40 Tabellen Nr. 34 Blschofsberger, W. Herkunft und Verblelb von Schwermetallen Im vergriffen Ruf, M. Abwasser und Kiarschlamm (1981) Winkler, R. 252 Seiten, 57 Abb., 108 Tabellen Nr. 35 Hruschka, H. Optimlerung derchemischen Failung mit Metallsalzen Marr, G. durch Steuerung der Failmittelzugabe (1982) ; Overath, H 173 Seiten, 58 Abb., 27 Tabellen Trommsdorff, K. U. Nr. 36 6. Wassertechnlsches Seminar Wasseraufbereitung - Planung, AusrOstung und vergriffen . ■ , Betrieb von Wasseraufbereitgngsanlagen (1982) 166 Seiten, 63 Abb., 3 Tabellen Nr. 37 5. MOIItechnlsches Seminar Gemelnsame Behandlung von M0II und Kiarschlamm (1982), 202 Seiten, 36 Abb., 16 Tabellen Nr. 38 12. Abwassertechnisches Schlammbehandlung unter besonderer BerOcksIchtl- Seminar gung von Schadstoffen Im Kiarschlamm (1982) 284 Seiten, 68 Abb., 34 Tabellen Nr. 39 Hruschka, H. ProzeBfOhrung auf KBranlagen durch Elnsatz elektro- nlscher Rechner (1983) 178 Seiten, 30 Abb., 21 Tabellen Nr. 40 Neumann, W. Kompostierung von Abfailen In elnem Bio-Tunnel- Tran tier, J. Reaktor (1983), 166 Seiten, 43 Abb., 48 Tabellen Nr. 41 Sampson, G. Auswirkungen der Failung und Flockung auf den Schlammanfall und die Kosten der Schlamm behandlung (1983) 211 Seiten, 48 Abb., 51 Tabellen, 84 Anlagen Nr. 42 7. Wassertechnlsches Seminar Automatisierung in Wasserwerken (1983) vergriffen 204 Seiten, 51 Abb., 12 Tabellen Nr. 43 6. Molltechnisches Seminar Behandlung und Beseitigung von Sonderabfailen (1983), 234 Seiten, 51 Abb., 36 Tabellen -Iv- Nr. 44 13. Abwassertechnisches Das Niederschlags-AbfluBverhalten stadtischer Ge- vergritfen Seminar blete (1983), 404 Seiten, 146 Abb., 30 Tabellen Nr. 45 Hajek, P.-M. Untersuchungen zum Sauerstoffhaushalt in FlieB- Neumann, W. gewSssem (1983) Bischofsberger, W. 286 Seiten, 112 Abb., 64 Tabellen Nr. 46 8 . W assertechnisches Seminar Grundwassergewinnung - Planung, Bau und Betrieb (1983), 198 Seiten, 72 Abb., 7 Tabellen Nr. 47 7. MOIItechnisches Seminar Beseitigung von Reststoffen aus der MQII- und Kiarschlammbehandlung (1984) 235 Seiten, 43 Abb., 35 Tabellen Nr. 48 Bischofsberger, W. Schiamme aus Hauskiaranlagen (1987) Resch, H. 163 Seiten, 28 Abb., 32 Tabellen, 25 Anlagen Baumgart, P. Nr. 49 GOnthert, F. W. Ein Beitrag zur Bemessung von Schlammraumung und Elndickzone in horizontal durchstrOmten ninden Nachkiarbecken von Belebungsanlagen (1984) 203 Seiten, 65 Abb., 20 Tabellen, 141 Anlagen Nr. 50 Geiger, W. F. MIschwasserabfluB und dessen Beschaffenheit. Ein Beitrag zur Kanalnetzplanung (1984) 253 Seiten, 57 Abb., 41 Tabellen, 154 Anlagen Nr. 51 14. Abwassertechnisches Lelstungssteigerung und Lelstungsgrenzen biolo- Seminar gischer Kiaranlagen (1984) 303 Seiten, 165 Abb., 19 Tabellen Nr. 52 Hajek, P.-M. Stickstoffoxklation in FlieBgewassem - Ein Beitrag zur Bedeutung, den Abhangigkeiten und der mathe- matischen Modellierung der Nitrifikation (1984) 230 Seiten, 55 Abb., 30 Tabellen, 78 Anlagen Nr. 53 Wechs, F. Ein Beitrag zur zweistufigen anaeroben Kiarschlamm vergriffen behandlung (1985) 203 Seiten, 72 Abb., 68 Tabellen, 52 Anlagen Nr. 54 Lessel.T. Optimlerung des Verfahrens zur Gammabestrahlung von Kiarschlamm (1985) 239 Seiten, 64 Abb., 17 Tabellen, 50 Anlagen Nr. 55 Zflschke, W. ErmitUung optimaler Tragfahigkeitsreihen vorge- vergriffen fertigter Rohre fOr Abwasserkandle (1985) 180 Seiten, 43 Abb., 35 Tabellen, 26 Anlagen Nr. 56 Bischofsberger, W. Warmeentnahme aus Abwasser (1984) Seyfried, C. F. 331 Seiten, 59 Abb., 15 Tabellen, 12 Anlagen Damman, P. Nr. 57 9 . Wassertechnisches Seminar Rohmetz und Rohrwerkstoffe (1985) 225 Seiten, 77 Abb., 23 Tabellen Nr. 58 8 . MOIItechnisches Seminar UmweltelnflOsse von Abfalldeponien und Sonder- mOllbeseitigung (1985) 412 Seiten, 95 Abb., 49 Tabellen Nr. 59 15. Abwassertechnisches Klelne KlSranlagen - Planung, Bau und Betrieb (1985) vergriffen Seminar 380 Seiten, 131 Abb., 33 Tabellen Nr. 60 Verschiedene Autoren Berichte aus dem Forschungsbereich des Lehrstuhls vergriffen fOr WassergOtewirtschaft (Festschrift fOr Prof. Dr.-lng. W. Bischofsberger)(1985) 488 Seiten, 115 Abb., 23 Tabellen Nr. 61 2. Kalkseminar * KostengOnstige Verfahren In der Abwassertechnik Ried, M. unter Einsatz von Kalk - EinfluB des Energieelntrages Hegemann, W. auf FSIIung und Flockung von kommunalen AbwSssem (1985) 349 Seiten, 131 Abb., 33 Tabellen, Anhang Nr. 62 Leonhard, K. Die Wirkung von Schwermetallen im Kiarschlamm - Pfeiffer. W. Kupfer, Zink und Silber (1985) Hegemann, W. 160 Seiten, 41 Abb., 28 Tabellen Nr. 63 Bischofsberger, W. Ein Beitrag zur Entwicklung und den Ursachen des Weigelt, R. Chloridanstiegs Im Grundwasser (1985) Klebe, S. 194 Seiten, 93 Abb., 8 Tabellen Nr. 64 Lindner, P. Physlkalischer Sauerstoffeintrag in gestaute FlieB- Riederer, E. gewOsser (1987) Bischofsberger, W. 148 Seiten, 31 Abb., 27 Tabellen, 21 Anlagen Nr. 65 10. Wassertechnisches Projektlerung von Wasserwerken (1985) vergriffen Seminar 229 Seiten, 83 Abb., 9 Tabellen Nr. 66 9 . MOIItechnisches Seminar Konzepte fOr Gewinnung von Wertstoffen aus Haus- mOII (1985), 346 Seiten, 77 Abb., 40 Tabellen Nr. 67 Temper, U. Stand und Entwlcklungspotentiale der anaeroben Pfeiffer, W. Abwasserrelnigung (1986) Bischofsberger, W. 737 Seiten, 188 Abb., 180 Tabellen Nr. 68 Merkl, G. Tauwasserbildung in T rlnkwasserbehaitem - LOftungs- vergriffen Huyeng, P. und wamietechnlsche MaBnahmen (1986) 207 Seiten, 56 Abb., 9 Tabellen, 30 Anlagen Nr. 69 16. Abwassertechnisches Abwasserbehandlung in mehrstufigen blologischen Seminar KHranlagen (1986) 373 Seiten, 134 Abb., 52 Tabellen, 2 Anlagen Nr. 70 Bischofsberger, W. Entwicklung und Tatigkeit des Lehrstuhles und PrOf- amtes fOr WassergOtewirtschaft Im Zeitraum 1976 - 1985 (1986) 176 Seiten, 19 Abb., 5 Tabellen Nr. 71 Neumann, W. ErmltUung von NlederschlagskenngrOBen zur Be- Brummer, J. schreibung von Modellregen fOr die Bemessung von Kanalnetzen (1986) 236 Seiten, 75 Abb., 29 Tabellen Nr, 72 Hofmann, H. Konzeption und Bemessung der vorgeschalteten Denitrifikation beim Belebungsverfahren (1986) 285 Seiten, 64 Abb., 47 Tabellen, 44 Anlagen Nr, 73 11. Wassertechnisches Trinkwasserbereitstellung - Speicherung und FOrde- vergriffen Seminar rung (1987), 287 Seiten, 92 Abb., 14 Tabellen - vl - Nr. 74 10. MQIItechnisches Seminar Energetische Nutzung von Abfallstoffen und 2. Fach- gesprflchKonzepte und Verfahren in der SondermOII- beseitigung (1987) 333 Seiten, 53 Abb., 27 Tabellen Nr. 75 17. Abwassertechnisches Planung, Bau und Betrieb von Regenentiastungen und Seminar FachgesprSch: Kanalnetzsteuerung und Regen entiastungen (1987) 548 Seiten, 183 Abb., 19 Tabellen Nr. 76 Fachseminare Chemische Failung und Flockung mlt Metallsalzen - Konditionierung und Entwasserung kommunaler Ab- wasserschiamme (1987) 399 Seiten, 156 Abb., 26 Tabellen Nr. 77 Becker, M. Auswirkungen verschiedener MaBnahmen auf den Brummer, J. AbfluB In Kanalnetzen (1987) Geiger, W. F. 193 Seiten, 61 Abb., 28 Tabellen, 9 Anlagen Nr. 78 Geiger, W. F. Bewirtschaftung eines stadtischen EntwSsserungs- PflOgler, H. systems durch AbfluBsteuerung an einem Regen- Schindler, H. rOckhaltebecken (1987) 260 Seiten, 128 Abb., 19 Tabellen, 22 Anlagen Nr. 79 12. W assertechnisches Schadstoffe Im Grundwasser - Auswirkungen und Seminar MaBnahmen zur Entfemung (1988) 496 Seiten, 140 Abb., 55 Tabellen Nr. 80 Bischofsberger, W. Siedlungswasserwirtschaft Im Wandel der Zeiten (1987), 160 Seiten, 60 Abb., 2 Tabellen Nr. 81 11. MQIItechnisches Seminar Integrierte Konzepte der Abfallentsorgung (1988) 168 Seiten, 22 Abb., 15 Tabellen Nr. 82 18 . Abwassertechnisches Wasserrechtllcher Vollzug - Mindestanforderungen, Seminar Skherer Klaranlagenbetrieb (1988) 344 Seiten, 84 Abb., 12 Tabellen Nr. 83 Rothmeier, F. ErmitUung von Belastungen for die Kanalnetzberech- nung - stochastische Modelle und abfluBorlentierte Optimlerung (1988) 230 Seiten, 29 Abb., 12 Tabellen, 26 Anlagen Nr. 84 Orth, P. Auswirkungen von Abwasser und Niederschlags- vergriffen Ebers, T. versickerung auf Boden und Grundwasser (1988) 304 Seiten, 73 Abb., 38 Tabellen Nr. 85 13. Wassertechnisches QualitatsQberwachung von Roh- und Trinkwasser - Seminar Messung, Analyse und Bewertung (1989) 268 Seiten, 66 Abb., 25 Tabellen Nr. 86 12. MQIItechnisches Seminar Behandlung und Beseitigung organischer Abfaile und 3. Fachgesprach SondermOII - Chemisch- physlkalische Behandlung von Sickerwasser aus Sonderabfalldeponien (1989) 352 Seiten, 75 Abb., 75 Tabellen Nr. 87 Pfeiffer, W. Verfahrensvariante der Faulung und Entseuchung von Kiarschiamm - Leistungsvergfelch (1990) 350 Seiten, 49 Abb., 38 Tabelian, 28 Anlagen - vii - Nr. 88 Steinmann, G. Sedimentations- und Koagulationsvorgange In Nach- klflrbecken von Tropfkflrpem mit Vorschlagen for die Bemessung (1989) 278 Seiten, 113 Abb., 47 Tabellen, 148 Anlagen Nr. 89 Blschofsberger, W. Verfahrens- und umwelttechnische Analyse neuer vergriffen Bom, R. thermischer Prozesse In der Abfallwirtschaft - Phase I: Pyrolyse (1989) 290 Seiten, 27 Abb., 60 Tabellen, 44 Anlagen Nr. 90 Blschofsberger, W. Verfahrens- und umwelttechnische Analyse neuer vergriffen Bom, R. thermischer Prozesse In der Abfallwirtschaft - Phase II: Wirbelschichtfeuerung (1989) 310 Seiten, 60 Abb., 47 Tabellen Nr. 91 19 . Abwassertechnisches Weitergehende Abwasserreinigung - Stickstoff- und vergriffen Seminar Phosphorelimlnation (1989) 275 Seiten, 75 Abb., 39 Tabellen Nr. 92 Ried, M. Schwermetallelimination aus KlSrschlamm - Kritische Beurteilung der MSglichkelten eines Saureverfahrens (1990), 208 Seiten, 95 Abb., 59 Tabellen, 107 Anlagen Nr. 93 SchOnberger, R. OpUmlerung der biologlschen Phosphorelimlnation bei vergriffen der kommunalen Abwasserreinigung (1990) 255 Seiten, 65 Abb., 28 Tabellen Nr. 94 Blschofsberger, W. EinfluB des Mischwasserzufiusses aufdasT ropf- Steinmann, G. kOrperverfahren (1990) 116 Seiten, 48 Abb., 16 Tabellen. 18 Anlagen Nr. 95 14. Wassertechnisches Neuere Technologlen In der Trinkwasseraufbereitung Seminar (1990), 332 Seiten, 85 Abb., 29 Tabellen Nr. 96 13. MOIItechnlsches Seminar Thermische Nutzung der Energlelnhalte von Abfall- stoffen (1990), 293 Seiten, 78 Abb., 22 Tabellen Nr. 97 Rettinger, S. Wasser- und Stoffdynamik bei der Abwasserperkola- tlon (1992) 289 Seiten, 47 Abb., 15 Tabellen, 6 Anlagen Nr. 98 Ebers, T. Lelstungssteigerung von Meinkiaranlagen (1992) Blschofsberger, W. 532 Seiten, 172 Abb., 35 Tabellen, 44 Anlagen Nr. 99 20. Abwassertechnisches Abwasserbehandlung In den letzten 20 Jahren - Ent- Seminar wicklung und Zukunftsperspektiven (1990) 246 Seiten, 48 Abb., 14 Tabellen Nr. 100 Verschiedene Autoren Beltrage aus den Forschungsbereich des Lehrstuhls for WassergOtewirtschaft und Gesundheitsingenleur- wesen, (Festschrift Prof. Dr.-lng. W. Blschofsberger) (1990), 517 Seiten, 113 Abb., 29 Tabellen Nr. 101 15. Wassertechnisches Sicherung der Trinkwasserversorgung - Aktuelle vergriffen Seminar und Fragen von der Gewinnung bis zur Verwendung. Das 2. Wasserrechtiiches Seminar Recht des Gmndwasserschutzes, seine Konkretisie- rung durch technische Standards und seine Durch- setzung (1991), 375 Seiten, 63 Abb., 8 Tabellen -vill- Nr. 102 14. MOIItechnisches Seminar Strategien und MOglichkeiten der Abfallvermeidung und -verwertung. 4. FachgesprSch SondermOII "Rest- stoffe aus SondemiOllbehandlungsanlagen - Anfall und Entsorgung" (1991) 402 Seiten, 54 Abb., 43 Tabellen Nr. 103 Drummer, J. Kurzfristige Niederschlagsvorhersagen mil Zellen- schatzungen und ihr Einsatz bei der AbluBsteuerung (1991), 176 Seiten, 29 Abb., 7 Tabellen, 7 Anlagen Nr. 104 Bischofsberger, W. Weitergehende Abwasserreinlgung Z Seminarvortrage vergriffen Deininger, A. zum Fortbildungsstudium (1991) 618 Seiten, 177 Abb., 62 Tabellen Nr. 105 21. Abwassertechnisches Planung von Kiaranlagen zur Nahrstoffelimination. Seminar Verfahrenstechnische Umsetzung der Bemessungs- richtiinien (1991) 214 Seiten, 77 Abb., 17 Tabellen Nr. 106 Huyeng, P. Entfemung organischer Schadstoffe aus Abwasser mil Weigelt, R. pulverfOrmiger Aktivkohle und nachfolgender Merkl, G. Flockungsfiltration (1991) Bischofsberger, W. 293 Seiten, 48 Abb., 12 Tabellen, 49 Anlagen Nr. 107 16. Wassertechnisches Wasseraufbereitung bei kleinen Wasserwerken Seminar (1991), 167 Seiten, 67 Abb., 10 Tabellen Nr. 108 15. MOIItechnisches Seminar Sanierung kontaminierter BOden (1991) 230 Seiten, 69 Abb., 29 Tabellen Nr. 109 MitsdCrffer, R. Charakteristika der zweistufigen thermophilen / meso- philen Schlammfaulung unter BerOcksichtigung kine- tischer Ansatze (1991) 241 Seiten, 68 Abb., 13 Tabellen, 14 Anlagen Nr. 110 22. Abwassertechnisches Konzepte und Methoden der Klarschlammverwertung vergriffen Seminar (1992), 181 Seiten, 31 Abb., 53 Tabellen Nr. 111 Cichon, W. Entwicklungspotential der Wirbelschichtfeuerung tor die Emlsslonsminderung bei derthermlschen Abfall- behandlung (1992), 221 Seiten, 32 Abb., Anlagen Nr. 112 17. Wassertechnisches Wasserbehaiten Instandhaltung - Fertigteilbauweise vergriffen Seminar (1992), 242 Seiten, 88 Abb., 8 Tabellen Nr. 113 16. MOIItechnisches Seminar Die Deponie des 21. Jahrhunderts (1992) 186 Seiten, 30 Abb., 13 Tabellen Nr. 114 23. Abwassertechnisches Abwassertechnik In Europa (1993) Seminar 150 Seiten, 23 Abb., 5 Tabellen Nr. 115 18 . Wassertechnisches Vorbeugende Instandhaltung in der Wasserverteilung Seminar unter BerOcksichtigung modemer Rohrieitungstechnik (1993), 214 Seiten, 50 Abb., 22 Tabellen Nr. 116 Tiefel, H. Entsorgung von Reststoffen am Beispie! von Ein- dampfkrlstallisat elner Slckerwasser-Relnlgungs- anlage (1994) 160 Seiten, 27 Abb., 7 Tabellen, 96 Anlagen - bt • Nr. 117 24. Abwassertechnlsches KostendampfUng in der Abwasserrelnigung - M6g- Seminar lichkeiten und Grenzen (1994) 204 Seiten, 69 Abb., 22 Tabellen Nr. 118 17. MOIItechnlsches Seminar Inertisienjng durch thermische Abfallbehandlung (1994), 169 Seiten, 41 Abb., 31 Tabellen Nr. 119 19 . Wassertechnisches Ausglelch und Verbund in der Wasserversorgung Seminar (1994), 191 Seiten, 71 Abb., 10 Tabellen Nr. 120 Steger, M. Th. Untersuchungen zum Abbau halogenorganischer Ver- bindungen be! der Niedertemperaturkonvertiemng von Kiarschlamm (1994), 116 Seiten, 25 Abb., 30 Tabellen Nr. 121 18 . MOIItechnlsches Seminar Praxis der biologischen Abfallbehandlung (1995) 159 Seiten, 43 Abb., 23 Tabellen Nr. 122 25. Abwassertechnlsches Additive in der Abwasserreinigung (1995) Seminar 158 Seiten, 63 Abb., 29 Tabellen Nr. 123 Netter, R. StrOmung In horizontal durchflossenen, bewachsenen Bodenfiltem und deren EinfiuB auf die Abwasser reinigung (1995), 190 Seiten, 49 Abb., 16 Tabellen Nr. 124 20. Wassertechnisches Praxlsbezogene Forschung for die Wasserversorgung vergriffen Seminar (1995), 248 Seiten, 93 Abb., 52 Tabellen Nr. 125 Festschrift zum 70. Geburtstag WassergOte- und Abfallforschung - Innovation zu Jeder von em.Prof.Dr.-Ing. W. Zeit (1996), 145 Seiten, 57 Abb., 10 Tabellen Bischofsberger Nr. 126 Fachtagung Perepektlven der Kiarschlammentsorgung (1996) 147 Seiten, 60 Abb., 10 Tabellen Nr. 127 Borho.M. Arsenentfemung In Grundwasserwerken durch opti- mlerte Kopplung von Oxidations- und Failungs-/ Flockungsverfahren (1996) 119 Seiten, 42 Seiten Anhang, 41 Abb., 10 Tabellen Nr. 128 Kolb, F. R. Blologische Relnlgung von Xenobiotica-haKfger Abwasser In elnem Aktivkohle-Festbett-Schlaufen- reaktor mit Membran-StoffObertrager (1997) 131 Seiten, 16 Seiten Anhang, 57 Abb., 9 Tabellen Nr. 129 Fachtagung Altlastensanierung in Bayern (1997) 181 Seiten, 51 Abb., 13 Tabellen Nr. 130 26. Abwassertechnlsches Blofilm-Technologle zur Relnlgung kommunaler Seminar Abwasser Mode Oder Modeme? (1997) 230 Seiten, 88 Abb., 38 Tabellen Nr. 131 Deinlnger, A. Geschwlndlgkeits- und Feststoffverteilung in radial durchstrOmten Nachkiarbecken (1997) 156 Seiten, 96 Seiten Anhang, 50 Abb., 23 Tabellen Nr. 132 21. Wassertechnisches Kostensparende MaRnahmen in der Wasserver Seminar sorgung (1997), 244 Seiten, 651 Abb., 15 Tabellen X Nr. 133 Wasserrechtliches Seminar Neue Welchenstellung bei der Abwasseneinigung - Rechtllche und technische Fragen fOr heute und morgen - (1997), 145 Seiten, 10 Abb., 4 Tabellen Nr. 134 Adamietz, E. EinfluB der ProzeRfOhrung bei blologlscher Behandlung von Papierfabrikabwasser auf die Zusammensetzung organischer Stoffe Im Abflauf und der Biomasse (1997) 113 Seiten, 31 Abb., 46 Tabellen Nr. 135 Hellge, S. Mlkrobleller Abbau schwerabbaubarer Verblndungen In niedrigen Konzentrationen am Beisplel von DibenzofUran und Dibenzo-p-dioxln (1997) 125 Seiten, davon 30 Seiten Anhang, 33 Abb., 17 Tabellen Nr. 136 Eisner, P. Steigenjng des Warmedurchgangs und der Destillat- qualitat bei der Elndampfung von hochbelasteten Abwassem (1997) 116 Seiten, davon 3 Seiten Anhang, 56 Abb., 15 Tabellen Nr. 137 3. Fachtagung Thermische Abfallbehandlung (1998) 554 Seiten, 163 Abb., 92 Tabellen Nr. 138 27. Abwassertechnisches Dezentrale Abwasserbehandlung fOr landliche und vergriffen Seminar urbane Geblete (1998) 206 Seiten, 74 Abb., 28 Tabellen Nr. 139 Ebert, H. Nichtlineare hydrologische Konzeptmodelle for den KanalabfluB und tire Kalibrierung (1998) 171 Seiten, 27 Abb., 35 Tabellen Nr. 140 Schindler, U. Dioden-Faser-Laser-Doppler-Anemometrie zur Untersuchung der StrBmung in Biofilmreaktoren (1998), 207 Seiten, 100 Abb., 17 Tabellen Nr. 141 Kabalk), H.-P. Das Sequencing Batch Biofilm Reactor (SBBR) Ver- fahren zur Reinigung von chlororganisch belasteten Abwassem im Leistungsvergleich mil einem bauglei- chen kontinuieriichen Biofilmverfahren (1998) 157 Seiten, 58 Abb., 21 Tabellen Nr. 142 DOBerer, J., Biologlsche Behandlung von Slckerwassem aus Sonderabfalldeponien mittels schubweise beschickter, Oberstauter Festbettreaktoren (1998) 121 Seiten +10 Seiten Anhang, 28 Abb., 29 Tabellen Nr 143 Borho, Merid Wiederverwendung von FilterrOckspOlwassem aus der Grundwasseraufbereitung (1999) 120 Seiten, 58 Abb., 11 Tabellen Nr. 144 22. Wassertechnisches Planung und Bau vonTrinkwasserbehaitem im H in- Seminar Mick auf die europaische Normung (1998) 280 Seiten, 88 Abb., 16 Tabellen Nr. 145 Morgenroth, E. Enhanced Biological Phosphorus Removal In Biofilm Reactors (1998) 148 Seiten, 59 Abb., 18 Tabellen -jd- Nr. 146 Dreher, P. 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EinfluB der ProzeBfOhrung auf Menge und Zusam- mensetzung von Proteinen und Polysacchariden im Ablauf von Sequencing - Batch - Reaktoren (1999) 128 Seiten, davon 11 Seiten Anhang, 42 Abb., 9 Tab. Nr. 153 Verschledene Autoren WassergOte- und Abfallwirtschaft, gestem - haute - morgen (Festschrift fOr Prof. Dr.-lng. Dr. h. c. Peter Wilderer) (1999), 124 Seiten, 31 Abb., 7 Tabellen Nr. 154 28 . AbwasserV 19 . MOIItechni- Prozessabwasser aus der Bloabfallvergarung sches Seminar . (1999), 132 Seiten, 33 Abb., 30 Tabellen Nr. 155 Schreff, D. Nutzung Intemer Kohlenstoffquellen bei der Stickstoff- ellmination In mehrstufigen Kiaranlagen (2000) 124 Seiten ♦ 16 Seiten Anhang, 64 Abb., 26 Tabellen Nr. 156 24. Wassertechnisches BetriebsfOhrung unter Einsatz von GIS-Geographi- Seminar schen Informatlonssystemen (2000) 185 Seiten, 47 Abb., 18 Tabellen Nr. 157 Status-Seminar Angewandte Membrantechnologle In Wasserwerken (2000), 124 Seiten, 46 Abb., 20 Tabellen Nr. 158 29 . Abwassertechnlsches TropfkOrper, TauchkOrper, Biofilter: Stand derTechnik Seminar und neue Entwicklungen (2000) 209 Seiten, 63. Abb., 36 Tabellen Nr. 159 Gebert, W. EinfluBfaktoren auf die Leistungsfahigkeit kunststoff- gefOllter TropfkOrper (2001), 65. Abb., 7 Tabellen Nr. 160 Merkl, G. Trinkwasser-Notversorgung unter besonderer BerOcksichtigung militarischer und ziviler Aspekte (2000), 102 Seiten, 16. Abb., 13 Tafeln, 1 Tab., Anhang 1-13 - xfl - Nr. 161 30. Abwassertechnisches DEBAR Seminar /13. DECHEMA- Klelne Ktiranlagen und Wasserwiederverwendung FachgesprSch Umweltschutz (2001), 379 Seiten, 96 . Abb., 54 Tabellen Nr. 162 6. Fachtagung Thennlsche Abfallbehandlung (2001), 502 Seiten, 135 Abb., 63 Tabellen Nr. 163 25. Wassertechnisches Wasserversorgung In der Zukunft unter besonderer Seminar Berilckslchtigung der Wasserspeicherung (2001) 236 Seiten + 21 Seiten Anhang, 76 Abb., 13 Tabellen Nr. 164 Amz, P. Biological Nutrient Removal from Municipal Waste water In Sequencing Batch Biofilm Reactors (2001), 107 Seiten, 44 Abb., 14 Tabellen Nr. 165 JOigens, L. Lbsungs- und Optimierungsansatze bei der AlUasten- bearbeitung in Mittel- und Osteuropa (2001) 226 Seiten, 86 Abb., 56 Tabellen Nr. 166 Kappen, J. Kennwerte als Werkzeuge zur Minlmlerung des Wasserbedarfs bei der Papiererzeugung - ein Beitrag zum prozessintegrierten Umweltschutz (2001) 146 Seiten, 60 Abb., 45 Tabellen Nr. 167 Janknecht, P. Characterization of Ozone Transfer into Water through Porous Membranes (2001) 97 Seiten, 42 Abb., 5 Tabellen Nr. 168 Burtscher, C. Einsatz der Pdymerasekettenreaktion (PCR) tor den Nachweis pathogener Bakterien In Bioabfallproben (2002), 150 Seiten, 27 Abb., 31 Tabellen Nr. 169 B6hm, ,B. Nitritblldung bei der Denitrifikation in Biofiltem mit extemen Kohlenstoffquellen (2002) 102 Seiten + 10 Seiten Anhang, 45 Abb., 10 Tabellen Gesellschaft zur FOrderung des Lehrstuhls for WassergOte- und Abfallwirtschaft derTechnischen Universitat MOnchen e.V., Am Coulombwall, D-85748 Garching