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Molecular Characterization of Anaerobic Dehalogenation by Desulfitobacterium Dehalogenans

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Molecular Characterization of Anaerobic Dehalogenation by Desulfitobacterium Dehalogenans MOLECULAR CHARACTERIZATION OF ANAEROBIC DEHALOGENATION BY DESULFITOBACTERIUM DEHALOGENANS HAUKE SMIDT Promotor: prof.dr .W .M .d eVo s hoogleraar inde microbiologic Co-promotoren: dr.A .D .L . Akkermans universitairhoofddocent bij de leerstoelgroep Microbiologic dr.J . van derOos t universitairdocent bij de leerstoelgroep Microbiologic Leden van de promotiecommissie: prof. dr.S .N .Agatho s Universite Catholiquede Louvain, Belgie prof.dr .D .B .Jansse n Rijksuniversiteit Groningen dr.J . R. van derMee r Eidgenossische Anstalt fur Wasserversorgung,Abwasser- reinigungund Gewasserschutz, Zwitserland prof. dr.I . M.C .M . Rietjens Wageningen Universiteit .'•O 29 So MOLECULAR CHARACTERIZATION OF ANAEROBIC DEHALOGENATION BY DESULFITOBACTERIUM DEHALOGENANS HAUKE SMIDT Proefschrift terverkrijgin g vand egraa dva ndocto r opgeza gva nd erecto r magnificus vanWageninge nUniversiteit , prof. dr. ir.L . Speelman, inhe topenbaa rt everdedige n opvrijda g 16maar t200 1 desnamiddag st evie ruu r ind eAula . y?0 \ l£c c* S /(:• The research described in this thesis was financially supported by a grant from the Studienstiftung des Deutschen Volkes and contract BIO4-98-0303 ofth eEuropea n Union. Cover- Graphic: Thomas Klefisch Cover- Design: Dorothee Brinkmann Wolffian Smidt Printing: Ponsen &Looije n B.V., Wageningen Smidt,Hauk e Molecular characterization of anaerobic dehalogenation byDesulfitobacterium dehalogenans. [S.I.:s.n.] Thesis Wageningen University, Wageningen, TheNetherland s- Withref . - With summary in Dutch andGerma n - 200p . ISBN: 90-5808-369-1 Subject headings: anaerobic, reductive dehalogenation, halorespiration, Desulfitobacterium dehalogenans, chlorophenol, genetics,regulatio n fur meineEltern Stellingen 1. Chloorfenol reductief dehalogenase mRNA is een makkelijk aan te tonen marker voor actieve chloorfenol afbraak doorDesulfltobacterium spp . Dit proefschrift 2. De term 'halorespiratie' behoeft geen verdere aanvulling om het proces van ademhaling met gehalogeneerde verbindingen correct weer te geven. Dit proefschrift Holliger, C, Wohlfarth, G., and Diekert, G. (1999) Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiol Rev 22: 383-398. Sanford, R. A., Cole, J. R., Loftier, F. E., and Tiedje, J. M. (1996) Characterization of Desulfltobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro- 4-hydroxybenzoate. Appl Environ Microbiol 62: 3800-3808. 3. Het uitblijven van methylviologeen-afhankelijke enzymactiviteit in hele cellen geeft uitsluitend informatie over de localisatie van het elektronen accepterende of donerende centrum van een oxido-reductase. Jones, R. W., and Garland, P. B. (1977) Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane. Biochem J164: 199-211. 4. Vanwege een opmerkelijke microheterogeniteit tussen kopieen van het 16S ribosomaal RNA gen inhe t genoom is deze als fylogenetische marker voor het defmieren van soorten binnen het genus Desulfltobacterium niet voldoende. Ebersbach, H., Breitenstein, A., and Lechner, U. (2000) All species of Desulfltobacterium contain two types of 16S rRNA genes of different length. Biospektrum, special issue Microbiology 2000 15.P.13.01. 5. Veel xenobiotica zijn deze naamnie t waard. 6. Hygiene is net als een medicijn. Bij goede dosering levensreddend, maar in overmaat gebruikt slecht voor de gezondheid. Diaz-Sanchez, D. (2000) Pollution and the immune-response: atopic diseases - are we too dirty or too clean? Immunology 101: 11-18. 7. Hetwe l ofnie t nasynchroniseren van anderstalige films heeft voornamelijk met geld te maken. 8. Concurrentie is goed, coOperatiei s beter. Stellingen behorende bij het proefschrift 'Molecular characterization of anaerobic dehalogenation by Desulfltobacterium dehalogenans' van Hauke Smidt Wageningen, 16maar t 2001 ABSTRACT Smidt, H. (2001). Molecular characterization of anaerobic dehalogenation by Desulfitobacterium dehalogenans. PhD thesis. Laboratory of Microbiology, Wageningen University, TheNetherlands . Haloorganics such as chlorophenols and chlorinated ethenes are among the most abundant pollutants in soil, sediments and groundwater, mainly caused by past and present industrial and agricultural activities.Du e to bioaccumulation and toxicity, these compounds threaten the integrity of the environment, and human and animal health. A recently discovered, phylogenetically diverse, group of anaerobic so-called halorespiring bacteria is able to couple the reductive dehalogenation of various haloorganic compounds to energy conservation and hence to growth, significantly contributing to in situ dehalogenation processes in anoxic environments. The observed persistence of halogenated pollutants in untreated ecosystems andth e accumulation of degradation intermediates during bioremediation, however, indicated the need for engineering of process conditions and/or augmentation with efficient degraders. This thesis describes genetic approaches towards a thorough understanding of the molecular basis of anaerobic reductive dehalogenation in order to enable the further optimization of cleanu pprocedure s for contaminated anoxic environments. The Gram-positive Desulfitobacterium dehalogenans, capable of degrading o- chlorophenols, PCE and hydroxylated PCB's, was used as model organism throughout a major part of this study. The key enzyme o-chlorophenol reductive dehalogenase (CPR) was isolated and characterized at the biochemical and molecular levels, and comparison with known chlorophenol- and chloroalkene-reductive dehalogenases indicated that these enzymes constitute a yet unknown, but evolutionary ancient family of corrinoid-containing iron-sulfur proteins, which in addition share a twin-arginine signal sequence. Transcriptional analysis of the CPR-encoding gene cluster revealed that dehalogenation activity is strongly regulated at the transcriptional level. Efficient gene cloning, and random and specific gene inactivation systemswer e developed to enable (i)th e elucidation of additional components involved in the anaerobic dehalogenation process and (ii) the study of their structure and function within the respiratory network. Halorespiration-deficient mutants were isolated following random chromosomal integration, and their characterization lead to the identification of genes encoding proteins possibly involved in structure, maturation and regulation of respiratory complexes. Reductive dehalogenase-encoding gene targeted (RT-)PCR-based molecular approaches were developed that were useful for the detection of reductive dehalogenation potential and activity inpur e cultures ofpotentiall y halorespiring microorganisms. The results obtained in this study provide valuable knowledge on the molecular basis of anaerobic reductive dehalogenation and might serve as a sound basis for the further exploitation of halorespiring bacteria as dedicated degraders in biological remediation processes. CONTENTS Preface 1 CHAPTER 1 Halorespiring bacteria - Molecular characterization of 3 key enzymes and detection CHAPTER 2 Purification and molecular characterization of ortho- 33 chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans CHAPTER 3 Transcriptional regulation of the cpr gene cluster in 53 OAT/70-chlorophenol respiring Desulfitobacterium dehalogenans CHAPTER 4 Random transposition by TnS/6 in Desulfitobacterium 75 dehalogenans allows for isolation and characterization of halorespiration-deficient mutants CHAPTER 5 Molecular characterization of redox complex-encoding 95 genes in halorespiring Desulfitobacterium dehalogenans CHAPTER 6 Development of a gene cloning and inactivation 105 system for the halorespiring Desulfitobacterium dehalogenans CHAPTER 7 Diversity and expression of reductive dehalogenase- 125 encoding genes from halorespiring bacteria CHAPTER 8 General discussion 143 Summary 159 Samenvatting 163 Zusammenfassung 173 Nawoord 183 About the author 185 List of publications 187 PREFACE Solid evidence has emerged almost a decade ago that strictly anaerobic bacteria are able to conserve energy that is derived from the reductive dehalogenation of haloorganic compounds in a respiration-type of metabolism. Invie w of their favorable degrading capacities, i. e. high dehalogenation rate and low residual concentration ofth e contaminant, itha s been anticipated that these halorespiring microorganisms should be of utmost significance for highly efficient biological remediation of halogenated hydrocarbons in anoxic environments. Previous studies mainly focused on the isolation of halorespiring microbes from various environments and their characterization at the (eco-)physiological level. Nevertheless, only little information was available onth e structure, function and regulation ofth enove l respiratory pathways these organismspossess .T ogai n this knowledge, a study was initiated thataime d to (i) identify and characterize the key components of thehalorespirator y system at the molecular level (Chapter 2-5),(ii )unrave lth eregulator y circuitstha tcontro lhalorespiratio n activity (Chapter 3, 5),an d (iii) develop molecular tools for the further exploitation of halorespiring bacteria as dedicated degraders inbiologica l in situ remediation processes (Chapter 4,6 ,7) . Throughout a major part of this study, the versatile strictly anaerobic ort/io-chlorophenol respiring
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