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Molecular Analysis of Halorespiration in Desulfitobacterium Spp

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Molecular Analysis of Halorespiration in Desulfitobacterium Spp Molecular analysis of halorespiration in Desulfitobacterium spp. − CATALYSIS AND TRANSCRIPTIONAL REGULATION Promotoren Prof. dr. W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Prof. dr. J. van der Oost Hoogleraar Microbiologie en Biochemie Wageningen Universiteit Co-promotor Dr. H. Smidt Universitair docent Laboratorium voor Microbiologie Wageningen Universiteit Leden van de Dr. D. Leys promotiecommissie Manchester Interdisciplinary Biocentre, UK Dr. R. J. M. van Spanning Vrije Universiteit, Amsterdam Prof. dr. I. M. C. M. Rietjens Wageningen Universiteit Prof. dr. S. C. de Vries Wageningen Universiteit Krisztina Gábor Molecular analysis of halorespiration in Desulfitobacterium spp. − catalysis and transcriptional regulation Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M. J. Kropff, in het openbaar te verdedigen op woensdag 1 november 2006 des namiddags te vier uur in de Aula Gábor, Krisztina − Molecular analysis of halorespiration in Desulfitobacterium spp.: catalysis and transcriptional regulation Ph.D. thesis Wageningen University, Wageningen, The Netherlands (2006) 152 p., with summary in Dutch ISBN 90-8504-529-0 Szüleimnek ABSTRACT Gábor, K. (2006). Molecular analysis of halorespiration in Desulfitobacterium spp.: catalysis and transcriptional regulation. PhD thesis. Laboratory of Microbiology, Wageningen University, The Netherlands. Soil and ground water contamination by halogenated organic compounds mainly used as biocides in agriculture or solvents and cleaning agents in industry has been a long-standing problem. The main barrier in the chemical degradation of organohalides is the presence of the halogen group in the molecule, which also contributes to the toxic nature of these compounds. However, a remarkable group of microorganisms, the halorespiring bacteria, are able to reductively dehalogenate organohalides under anaerobic conditions and use the energy generated via a proton gradient pump for bacterial growth (halorespiration). Due to their versatile dehalogenating capacity, halorespiring bacteria have a large potential in the clean-up of contaminated sites (bioremediation). The work described in this thesis aimed to gain knowledge on two aspects of the molecular basis of halorespiration: (i) the reaction mechanism of dehalogenation, catalyzed by the B12/iron-sulphur containing reductive dehalogenases, and (ii) the regulatory mechanism which enables transcriptional activation of genes involved in halorespiration. As model organisms, two Desulfitobacterium spp. were chosen, capable of ortho and/or meta dechlorination of chlorophenols and hydroxylated polychlorinated biphenyls. Unravelling of the novel reaction mechanism of reductive dehalogenases was so far hampered by difficulties in functional overproduction of these enzymes. We developed a protocol which involves co-expression of molecular chaperons to aid functional synthesis of the metalloenzymes in Escherichia coli. Next, multiple potential transcriptional activators were characterised from D. hafniense by promoter fusions and in vitro DNA-binding assays. We found that CprK1 and CprK2 activated transcription in the presence of an ortho-chlorophenol (CHPA) but not with its dechlorinated derivative (HPA), while meta-chlorophenols proved to be effectors for CprK4. All CprK paralogues recognized a conserved motif (dehalobox) in halorespiration-inducible promoters. Site-directed mutagenesis of CprK1 provided further insight on the role of conserved residues in the DNA-recognition α-helix and on redox regulation of the protein. Crystal structures of the CHPA-bound CprK1 and the effector-free form of the closely related CprK of D. dehalogenans revealed a possible mechanism by which the regulators distinguish between CHPA and the non-effector HPA (“pKa interrogation” theory). Native mass spectrometry of protein-DNA complexes confirmed these results and − together with limited proteolysis experiments − also contributed to the fundamental knowledge of ligand-induced changes in the conformation and dynamics of CprK-related transcriptional regulations. Keywords: iron-sulphur proteins, protein-DNA interaction, allosteric regulation, redox regulation, gene redundancy, chlorophenols, bioremediation CONTENTS OF THE THESIS Preface 1 Chapter 1 General introduction 3 Chapter 2 Characterisation of CprK1, a CRP/FNR-type transcriptional regulator of 27 halorespiration from Desulfitobacterium hafniense Chapter 3 CprK crystal structures reveal mechanism for transcriptional control of 49 halorespiration Chapter 4 Transcriptional activation by CprK1 is regulated by protein conformational 67 changes induced by effector binding and reducing conditions Chapter 5 Divergence of multiple CprK-paralogues in Desulfitobacterium hafniense 95 Chapter 6 Advances in overproduction and purification of active reductive 115 dehalogenases from Desulfitobacterium spp. Chapter 7 Summary and concluding remarks 125 Chapter 8 Samenvatting en conclusies 131 Acknowledgements 137 About the author 139 List of publications 141 PREFACE Dioxins, DDT, and polychlorinated biphenyls (PCBs) are compounds with a negative connotation that belong to the group of persistent organic pollutants. They are suspected to be responsible for birth defects, immune system dysfunction, and reproductive problems in wildlife, deserving the name “dirty dozen” together with nine other organic compounds. They are not only toxic, but also very persistent against chemical degradation, due to the high degree of halogen substitution within the molecules. Therefore the discovery that anaerobic bacteria can degrade halogenated organic compounds in a respiratory-type mechanism was of utmost significance. These bacteria can replace the halogen atom(s) in the molecule with hydrogen, which often renders a less toxic or more bioavailable end-product, and conserve energy from the reaction via electron transport-coupled phosphorylation in a process termed halorespiration. The genus Desulfitobacterium constitutes a prominent group of halorespiring bacteria, including several isolates that can couple dehalogenation of ortho- and/or meta-chlorophenols and hydroxylated PCBs to energy generation and growth. The dechlorination step is catalyzed by cobalamin/iron- sulphur containing enzymes, the reductive dehalogenases, which are almost exclusively produced when their halogenated substrates are present in the environment. The aim of the research described in this thesis was to gain knowledge on the molecular basis of halorespiration. By recognizing the most important factors involved in catalysis and activation of reductive dehalogenase gene expression, it is anticipated that the biodegradation capacity of halorespiring bacteria can be efficiently enhanced. We focused on two main topics: (i) to unravel the reaction mechanism that underlies the carbon-halide bond cleavage by CprA, an ortho- chlorophenol reductive dehalogenase from Desulfitobacterium dehalogenans; and (ii) on the characterisation and structure-function analysis of CprK1, a putative transcriptional regulator of halorespiration in Desulfitobacterium hafniense. Outline of the content of the thesis: • Chapter 1 gives a general overview on halorespiration and related fields, including the production of halogenated organic compounds, the diversity of halorespiring bacteria, the isolation and characterisation of reductive dehalogenases and the CRP-FNR superfamily of transcriptional regulators, with special attention on structural features that influence the biological function of these regulators. • Chapter 2 describes the characterisation of CprK1, a new member of the CRP-FNR family. Promoter fusion and in vitro DNA-binding experiments were used to find the effector molecules that activate CprK1 and enable it to bind to specific DNA sequences in halorespiration inducible promoters. Site-directed mutagenesis of CprK1 revealed details on the specific recognition of its DNA target and on putative redox regulation of the protein. • Chapter 3 provides further details on the allosteric effects of chlorophenol binding to CprK1 by describing the crystal structure of the effector-bound CprK1 and that of the closely related CprK from D. dehalogenans in its effector-free form. • Chapter 4 reports on the structural dynamics and ligand-induced conformational changes of CprK1 using macromolecular mass spectrometry, limited proteolysis coupled to mass spectrometry and in vitro DNA-binding assays in the presence of several potential effector molecules. • In Chapter 5, four cprK gene homologues are described that are present in the almost complete genome sequence of D. hafniense, additionally to the CprK1-encoding gene. To determine whether their redundancy causes merely a dosage effect or their function is diverged and specialized, we overproduced three of the CprK paralogues in E. coli and studied transcriptional regulation mediated by the heterologously-produced proteins in vivo and their DNA-binding properties in vitro. • Chapter 6 focuses on the catalytic basis of halorespiration, and aims to provide details of the yet-unknown reaction mechanism. The first step towards this aim is the overproduction of functional reductive dehalogenases in genetic model systems, for which we selected E. coli. Our attempts included the improvement of anaerobic culturing conditions and the co-expression of molecular chaperons and a trigger factor to aid correct protein folding. • Finally, in Chapter 7 (in English) and in Chapter
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