EVIDENCE OF A CHEMIOSMOT1C MODEL FOR HALORESPIRATION IN DESULFOMONILE TIEDJEIDCBA by TAI MAN LOUIE B.Sc, The University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1998 © Tai Man Louie, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date 11. 1^8 DE-6 (2/88) ABSTRACT Desulfomonile tiedjeiDCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized anaerobic respiratory process. Different electron carriers and respiratory enzymes of D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation and sulfate respiration were therefore examined quantitatively. Only cytochromes c were detected in the soluble and membrane fractions of cells grown under the three conditions. These cytochromes include a constitutively expressed 17-kDa cytochrome c, which was detected in cells grown under all three conditions, and a unique diheme cytochrome c with an apparent molecular mass of 50 kDa, which was present only in the membrane fractions of dehalogenatingcells. This inducible cytochrome c had a very negative midpoint potential of --342 mV. Absorption spectra and the putative gene sequence suggest that the inducible cytochrome c is substantially different from previously characterized cytochromes. Reductive dehalogenation activity of D. tiedjei-was shown to be dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinone. Moreover, cell suspension experiments indicated that reductive dehalogenation of D. tiedjei was inhibited by the respiratory quinone inhibitor, 2-heptyl-4-hydroxyquinoline JV-oxide, suggesting a respiratory quinone is involved in the electron transport chain coupled to reductive dehalogenation. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, an UV-absorbing, quinone-like molecule or quinoid, was extracted. The oxidized and reduced UV-absorption spectra of the quinoid were similar in some ways to those of ubiquinones and pyrrolo-quinoline quinones, respectively. But the quinoid was different from these common respiratory quinones in chemical structure according to mass spectrometric analysis. ATP sulfurylase, APS reductase and desulfoviridin, the enzymes involved in sulfate-reduction, appeared to be constitutively expressed in the cytoplasm of D. tiedjei cells grown under the three metabolic conditions. An ii inducible, periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. An inducible, membrane-bound, periplasm-oriented formate dehydrogenase was active only in cells grown with formate as electron donor; while, a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with D. tiedjei cell suspensions suggest the membrane-bound reductive dehalogenaseis facing the cytoplasm . The inducible cytochrome c, or the quinoid, alone or in combination, failed to replace reduced methyl viologen as the electron donor for the reductive dehalogenase in vitro. The putative gene sequence of the reductive dehalogenase small subunit was determined from inverse PCR products amplified from genomic DNA, but the sequence did not have substantial similarity to any sequences in GenBank. These data clearly demonstrate that D. tiedjei possesses elements necessary for producing protons directiy in the periplasm, generating a proton-motive force across the cytoplasmic membrane. However, the data did not exclude the existence of additional transmembrane proton translocation mechanisms, which would further enhance the proton- motive force. iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vi LIST OF TABLES vii ABBREVIATIONS AND SYMBOLS viii ACKNOWLEDGMENTS x INTRODUCTION 1 1. Definition of reductive dehalogenation 1 2. Significance ofreductive dehalogenation 1 3. Reductive dehalogenation by pure cultures 7 3.1 Co-metabolic reductive dehalogenation by pure cultures 7 3.2 Catabolic reductive dehalogenation by pure culture 9 3.21 Pure cultures which catabolize aryl halides as electron donor via 9 reductive dehalogenation 3.22 Pure cultures which utilize aryl halides and PCE as electron acceptors 12 3.3 Reductive dehalogenation by Desulfomonile tiedjei DCB-l 18 THESIS OBJECTIVES 24 MATERIALS AND METHODS 25 1. Organism and growth conditions 25 2. Cell suspension experiments 25 3. Cell fractionation 26 4. Quantification of cytochromes 27 5. Heme-staining of SDS-PAGE gels 27 6. Purification of a 50-kDa inducible cytochrome c 27 7. Redox and pH titrations of the inducible cytochrome 29 8. NH2-terminal protein sequence analysis of the inducible cytochrome 30 9. Genomic DNA isolation 30 10. DNA mani pul ati on 31 11. Inverse PCR and cloning of the inverse PCR product 32 12. DNA sequencing 33 13. Analysis of respiratory quinones 33 14. Hydrogenase activity assay 34 15. Formate dehydrogenase assay 35 16. ATP sulfurylase activity assay 35 iv 17. APS reductase activity assay 36 18. Desulfoviridin quantification 36 19. Reductive dehalogenase activity assay 36 20. Analytical methods 37 21. Chermicals 38 RESULTS 39 CHAPTER ONE Cytochromes of D. tiedjei 39 1. Introduction 39 2. Cytochromes in different cellular fractions 39 3. Purification of the 50-kDa inducible cytochrome 40 4. Visible absorption spectra of the inducible cytochrome 43 5. Midpoint potential determination for the inducible cytochrome 44 6. NH2-terminal sequence of the inducible cytochrome 44 7. Putative gene sequence of the inducible cytochrome 49 8. Summary 60 CHAPTER TWO A putative respiratory quinone of D. tiedjei 61 1. Introduction 61 2. Effects of individual vitamins on reductive dehalogenation 62 3. Effect of 2-heptyl-4-hydroxyquinoline Af-oxide (HQNO) on reductive 62 dehalogenation 4. Purification of a putative respiratory quinone 64 5. Mass spectroscopic analysis of the quinoid 64 6. Summary 69 CHAPTER THREE Quantification and localization of respiratory enzymes in D. tiedjei 70 1. Introduction 70 2. Distribution of enzymes involved in sulfate-reduction 71 3. Distribution of hydrogenase 71 4. Distribution of formate dehydrogenase 72 5. Reductive dehalogenase 74 6. Localization of the reductive dehalogenase 75 7. Putative gene sequence of the reductive dehalogenase small subunit 77 8. Summary 87 DISCUSSION 89 1. Characteristics of cytochromes in D. tiedjei and their potential roles in the 89 halorespiratory electron transport chain 2. The presence of a putative quinoid in D. tiedjei electron transport chain 94 3. Topology of different respiratory enzymes in D. tiedjei 98 4. Chemiosmotic model for D. tiedjei halorespiration and sulfate reduction 105 REFERENCES 111 v LIST OF FIGURES Fig. 1 Examples of reductive dehalogenation 3 Fig. 2 Reduced-minus-oxidized absorption spectra of cytochromes in different 41 cellular fractions of D. tiedjei Fig. 3 Ffeme-stained SDS-PAGE gels of different/), tiedjei cellular fractions 42 Fig. 4 SDS-PAGE of the successive purification steps of the inducible cytochrome c 45 Fig. 5 Absorption spectra of the inducible cytochrome c at different pH 46 Fig- 6 Chemical redox titration of the inducible cytochrome c by sodium dithionite 47 Fig. 7 Redox titration of the inducible cytochrome c by 8-hydroxyribflavin 48 Fig. 8 The NH2-terminal protein sequence of the inducible cytochrome c 52 Fig. 9 PCR amplification of the DNA sequence encoding the NH2-terminal protein 53 sequence of the inducible cytochrome c Fig. 10 A Southern blot analysis of D. tiedjei DNA, probed with the 89-bp NH2- 54 DNA sequence of the inducible cytochrome c Fig. 11 An inverse PCR product containing partial gene sequence of the inducible 54 cytochrome c Fig. 12 Putative gene sequence and a physical map of the inducible cytochrome c gene 55 Fig. 13 A Southern blot analysis of D. tiedjei DNA, probed with a 719-bp DNA 58 sequence of the inducible cytochrome c Fig. 14 An inverse PCR product containing the entire sequence of the inducible 58 cytochrome c gene Fig- 15 Hydropathy analysis of the inducible cytochrome c 59 Fig. 16 Growth curves and dehalogenation activity of D. tiedjei cultures deficient in 63 specific vitamin components Fig. 17 Effect of 2-A/-heptyl-4-hydroxyquinoline A'-oxide on dehalogenation activity 65 of D. tiedjei cell suspensions Fig. 18 UV absorption spectra of the quinoid extracted from D. tiedjei 66 Fig. 19 Electron impact mass spectrum of the quinoid purified from D. tiedjei 68 Fig. 20 Reductive dehalogenation activity of D. tiedjei cell suspensions with reduced 78 methyl viologen as electron donor Fig. 21 A Southern blot analysis of D. tiedjei DNA, probed with the 66-bp NH2- 81 DNA sequence of the small subunit of the reductive dehalogenase Fig. 22 Inverse