UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Evidence of a chemiosmotic model for halorespiration in desulfomonile tiedjei DCB-1 Louie, Tai Man 1998

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1998-345831.pdf [ 8.12MB ]
Metadata
JSON: 831-1.0099356.json
JSON-LD: 831-1.0099356-ld.json
RDF/XML (Pretty): 831-1.0099356-rdf.xml
RDF/JSON: 831-1.0099356-rdf.json
Turtle: 831-1.0099356-turtle.txt
N-Triples: 831-1.0099356-rdf-ntriples.txt
Original Record: 831-1.0099356-source.json
Full Text
831-1.0099356-fulltext.txt
Citation
831-1.0099356.ris

Full Text

E V I D E N C E OF A C H E M I O S M O T 1 C M O D E L F O R H A L O R E S P I R A T I O N I N DESULFOMONILE TIEDJEIDCBA by T A I M A N L O U I E B . S c , The University of British Columbia, 1992 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A 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. A T P sulfurylase, A P S 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. A n 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 N H 2 - 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 N H 2 - 81 DNA sequence of the small subunit of the reductive dehalogenase Fig. 22 Inverse PCR products containing the entire sequence of the reductive 82 dehalogenase small subunit gene Fig. 23 Putative gene sequence of the reductive dehalogenase small subunit gene 83 Fig. 24 Hydropathy analysis of the reductive dehalogenase small subunit 85 Fig. 25 Comparison of the physical map of the reductive dehalogenase small subunit 86 gene with that of the inducible cytochrome c gene Fig. 26 A tentative chemiosmotic model of halorespiration in D. tiedjei 107 Fig. 27 A tentative chemiosmotic model of sulfate reduction in D. tiedjei 108 vi LIST OF TABLES Table I Common halogenated environmental pollutants which are biotransformed by reductivedehalogenation Table II Specific quantities of cytochromes in different cellular fractions of D. tiedjei cells grown under different metabolic conditions Table in Specific quantities of sulfate-reducing enzymes in the cytoplasmic fractions of D. tiedjei cells grown under different metabolic conditions Table IV Specific hydrogenase activities in different cellular fractions of D. tiedjei cells grown under different metabolic conditions Table V Specific formate dehydrogenase activities in different cellular fractions of D. tiedjei cells grown under different metabolic conditions Table VI Specific reductive dehalogenase activities in whole cell extracts of D. tiedjei vii ABBREVIATIONS AND SYMBOLS AMP APS ATP bp 3CB Ci dATP dCTP DDT dGTP , DNA dNTP dTTP Dv. EDTA FAD FMN FPLC g h HPLC 2-heptyl-4-hydroxyquinoline N-oxide kb kDa L M mg min mL mM MPa MWCO NADH NADPH nm nmole ORF PAGE PCBs PCE PCP adenosine S'-monophosphate adenosine phosphosulfate adenosine 5'-triphosphate base-pair 3-chlorobenzoate Curie deoxvadenosine 5'-triphosphate deoxvcvtidine 5'-triphoshphate 1,1,1 -trichloro-2,2-bis(/?-chlorophenyl)ethane deoxveuanosine 5'-triphosphate deoxyribonucleicacid deoxyribonucleotides deoxvthvmidine 5'-triphosphate Desulfovibrio ethylenediaminetetraacetate flavin adenine dinucleotide flavinmononucleotide fast protein liquid chromatography gram hour(s) high performance liquid chromatography HQNO kilobase-pair kilodaltbns liter molar milligram minute(s) milliliter millimolar megapascal molecular weight cutoff reducednicotinamide-adeninedinucleotide reduced nicotinamide-adenine dinucleotide phosphate nanometer nanomole open reading frame polyacrylamide gel electrophoresis polychlorinated biphenyls perchloroethene(tetrachloroethene) pentachl orophenol viii PCR polymerase chain reaction RNase ribonuclease SDS sodium dodecyl sulfate TCE trichloroethene uCi microcurie UL microliter Um micrometer UM micromolar Limole micromole UV ultraviolet ix ACKNOWLEDGMENTS I would like to thank Dr. William Mohn for his excellent supervision, and my committee members, Dr. Tom Beatty, Dr. Barbara Dill and Dr. William Ramey, for their guidance and advice. I am also grateful to Dr. Grant Mauk, Dean Hildebrand and Federico Rosell for helpful discussions. Many thanks to Dr. Shuisong Ni and Dr. Luying Xun for their collaboration in this project. Last but not least, I would like to thank the members of the Mohn lab, for their support and their tolerance to the odor of D. tiedjei cultures. INTRODUCTION 1. Definition of reductive dehalogenation Reductive dehalogenation is defined as the removal of a halogen substituent from a molecule with concurrent addition of two electrons to the molecule. Reductive dehalogenation can be classified into two types of reactions (Fig. 1). Vicinal reduction involves the replacement of two adjacent halogen substituents from adjacent carbon atoms and results in the formation of a double bond between the two carbon atoms. This type of reductive dehalogenation can only occur with alkyl halides. Hydrogenolysis is the second type of reductive dehalogenation, which involves the replacement of a halogen substituent from a molecule with a hydrogen atom. Both alkyl and aryl halides can be transformed by hydrogenolysis. Both types of reactions require an electron donor. Accordingly, aryl reductive dehalogenation refers to the replacement of a halogen atom from a haloaromatic compound, by hydrogenolysis, with a hydrogen atom. The replaced halogen is released in the form of a halide ion. 2. Significance of reductive dehalogenation Uncontrolled applications and discharges of substantial quantities of toxic xenobiotic chemicals to the environment, and the recalcitrance and bioaccumulation of these chemicals in the environment have raised public concern over their possible effects on the quality of life. Among these environmental pollutants, halogenated organic chemicals constitute one of the largest groups because of their widespread use as herbicides, insecticides, fungicides, solvents, hydraulic and heat transfer fluids, and chemical synthesis intermediates. Halogenated organic compounds are also produced from chlorination processes like drinking water purification and 1 pulp-bleaching. The common names, the chemical names, and the functions of some of these chemicals are listed in Table I. The biological recalcitrance of halogenated chemicals is related to several factors. First of all, the recalcitrance of halogenated chemicals can be attributed to their general toxic properties, preventing the growth of microorganisms which may have the necessary mechanisms to degrade these halogenated chemicals. For examples, monofluoroacetate is a potent inhibitor of the TCA cycle because fluorocitrate synthesized from monofluoroacetate and oxaloacetate inhibits the TCA cycle enzyme aconitase (Quastel 1963), and PCP is an uncoupler of oxidative phosphorylation (Slater 1963). The toxicity of these chemicals can also be attributed to their hydrophobicity. Most of these halogenated chemicals are more or less hydrophobic, and they can interact and accumulate with the cytoplasmic membrane of microorganisms. As a consequence, the membrane loses its integrity, increases in permeability to protons and ions, and the proton-motive force for energy generation is dissipated (Sikkema et al. 1995). In addition to toxicity, the number, type, and position of the halogen substituents also affect the biodegradability of halogenated chemicals. As a general observation, the greater the number of halogen substituents per molecule, the more difficult it is for microorganisms to degrade it. For example, the presence of multiple electron-withdrawing halogen substituents on a benzene ring interferes with the action of the ring-cleavage di oxygenase (Wood 1982). Despite of the biological recalcitrance of halogenated chemicals, we now know that many halogenated compounds can be completely mineralized by aerobic bacteria. However, many of these biodegradable halogenated compounds are relatively less-halogenated. Meanwhile, reductive dehalogenation, which is mainly known to occur under anaerobic conditions, is the only known mechanism for biotransformation of certain highly-halogenated pollutants such as some PCBs, PCP and PCE. Reductive 2 Fig. 1 Examples of reductive dehalogenation. (A) Aryl hydrogenolysis of 3-chlorobenzoate to benzoate; (B) alkyl hydrogenolysis of tetrachloroethene to trichloroethene; (C) vicinal reduction of 1,2-dichloroethane to ethene. Table I. Common names, chemical names and functions of common halogenated environmental pollutants which are biotransformed by reductive dehalogenation. Common Chemical names Functions Dehaloge- References names nation catalysts a Carbon Tetrachloromethane Solvent Co-factor (Assaf-Anid etal. 1994 ; tetrachloride Gantzer and Wackett 1991 ; Krone et al. 1989 ; Krone et al. 1991 ; Picardal et al. 1993 ; Stromeyer et al. 1992) CFC-11 Trichlorofluoromethane Heat- Pure (Krone and Thauer 1992) transfer fluid Co-factor (Krone etal. 1991) Chloroform Trichloromethane Solvent Pure (Mikesell and Boyd 1990) Co-factor (Krone et al. 1989) DDT l,l,l-Trichloro-2,2- Pesticide Co-factor (Baxter 1990 ; Berry and bis(p- Stotter 1977 ; Castro et al. chlorophenyl)ethane 1985 ; French and Hoopingarner 1970 ; Miskus et al. 1965 ; Zoro et al. 1974) Heptachlor 1,4,5,6,7,8,8- Insecticide Undefined (Miles etal. 1971) Heptachloro-3a,5,7,7a-tetrahydro-4,7- Co-factor (Baxter 1990) methanoindene Lindane y-hexachlorocyclo- Insecticide Pure (Jagnow et al. 1977 ; Ohisa hexane et al. 1982 ; Ohisa et al. 1980) Mirex Dodecachlorooctahydro- Insecticide Undefined (Andrade and Wheeler 1,3,4-metheno-2//- 1974) cyclobuta(cd) pentalene Co-factor (Baxter 1990) PCE/ T C E Tetrachloroethene/ Solvents Pure (Cole etal. 1995 ; Gerritse Trichloroethene et al. 1996 ; Holliger et al. 1993 ; Maymo-Gatell et al. 1997 ; Miller et al. 1997b ; Scholz-Muramatsu etal. 1995 ; Sharma and McCarty 1996 ; Townsend and Suflita 1996 ; Wild et al. 1996) Undefined (Wild et al. 1995) Co-factor (Burrisero/. 1996 ; Gantzer and Wackett 1991 ; Jablonski and Ferry 1992 ; Terzenbach and Blaut 1994) 4 Table I. continued Dicamba HCB PCB 3,6-dichloro-2-methoxybenzoic acid Hexachlorobenzene Polychlorinated biphenyls Herbicide Intermediates in chemicals synthesis Transformers, capacitors, hydraulic and heat-transfer fluid Undefined (Milligan and Haggblom 1996 ; Taraban et al. 1993) Undefined (Bushart et al. 1995 ; Fathepure et al. 1988 ; Liang and Grbic-Galic 1991) Co-factor (Assaf-Anid et al. 1992 ; Gantzer and Wackett 1991) Undefined (Boyle et al. 1993 ; Morris et al. 1992 ; Quensen et al. 1990 ; Quensen et al. 1988 ; Van Dort and Bedard 1991 ; Ye et al. 1995) PCDD PCP Picloram 2,4,5-T Polychlorinated dibenzo-/7-dioxins Pentachlorophenol 3,5,6-Trichloro-4-amino-2-pyridinecarboxylic acid 2,4,5-Trichlorophenoxy-acetic acid Chemicals synthesis byproducts Fungicide Herbicide Herbicide Co-factor (Assaf-Anid, et al. 1992) Undefined (Adriaens et al. 1995 ; Toussaint et al. 1992) Pure (Apajalahti et al. 1986 ; Bouchard et al. 1996 ; McAllister et al. 1996 ; Mohn and Kennedy 1992 : Saber and Crawford 1985) Co-factor (Gantzer and Wackett 1991 ; Smith and Woods 1994) Undefined (Ramanand et al. 1993) Pure (Golovleva et al. 1990) Undefined (Gibson and Suflita 1990 ; Kuhn and Suflita 1989 ; Mikesell and Boyd 1985) Pure and undefined mean pure cultures and undefined microbial communities are responsible for the observed reductive dehalogenation activities, respectively, and the dehalogenation activities can be catabolic or fortuitous. Co-factor indicates the observed reductive dehalogenation activities have been proved to be fortuitous and are catalyzed by enzyme co-factors. 5 dehalogenation is also involved in degradation of certain highly-halogenated compounds under aerobic conditions (Apajalahti etal. 1986 ; Golovleva et al. 1990 ; Romanov and Hausinger 1996 ; Saber and Crawford 1985 ; Van den Tweel etal. 1987). The resulting, less-halogenated products are generally less toxic and more amenable to subsequent degradation. As a consequence, reductive dehalogenation is of particular interest because of its involvement in the environmental fate of some of the most recalcitrant pollutants, and its potential application in the bioremediation industry. In addition to pollutant biodegradation, reductive dehalogenation and the dehalogenases which catalyze the dehalogenation reaction might have biotechnological applications. Reductive dehalogenases might be used as industrial biocatalysts for synthesizing valuable chemicals. Enzyme biocatalysts possess chiral specificities that are difficult and expensive to achieve by traditional chemical catalysts. A chiral-specific 2-haloalkanoic acid hydrolytic dehalogenase has been used to produce both enantiomers of lactic acid from racemic mixture of 2-chloropropionic acid (Motosugi etal. 1983 ; Motosugi et al. 1984). Reductive dehalogenases which transform herbicides may have agricultural value. Transgenic crops expressing bacterial reductive dehalogenases responsible for herbicide degradation would be valuable for weed control since the activities of many herbicides are not specific. Reductive dehalogenation is also interesting to study from a fundamental scientific point of view and contributes to areas of study such as physiology and biochemistry. A group of anaerobic bacteria are capable of conserving energy for growth via reductive dehalogenation of alkyl and aryl halides (Christiansen and Ahring 1996 ; Cole et al. 1994 ; Dolfing 1991 ; Gerritse et al. 1996 ; Holligerand Schumacher 1994 ; Maymo-Gatell et al. 1997 ; Miller et al. 1997 ; Mohn and Tiedje 1991; Sanford et al. 1996). These bacteria use the 6 alkyl and aryl halides as their terminal electron acceptors and energy is probably conserved by a chemiosmotic mechanism. This type of chemiosmotic mechanism represents a new area of bacterial energetics previously unknown to microbiologists. In addition, by studying the purified reductive dehalogenases, or enzyme co-factors with reductive-dehalogenating activities, we are beginning to understand the different mechanisms of reductive dehalogenation reaction. Studies of reductive dehalogenation also contribute to the area of bacterial phylogeny. In the early nineties, Desulfomonile tiedjei D C B - 1 was the only pure culture available for studying aryl reductive dehalogenation. A s the study of reductive dehalogenation continued, our understanding of this process broadened and many new isolates capable of reductively dehalogenating different aryl halides and P C E were discovered in the last three years. 3. Reductive dehalogenation by pure cultures In bacterial catabolism, organic compounds can serve as electron donors or as terminal electron acceptors in anaerobic respiration. In these two cases, energy is conserved for growth. In contrast; bacteria can also metabolize organic compounds via co-metabolic reactions. Co-metabolic reactions are fortuitous transformations of the organic compounds by enzymes or co-factors which catalyze other reactions but no nutritional benefits are provided from co-metabolism. The bacterial reductive dehalogenation processes can also be classified as co-metabolic or catabolic reductive dehalogenation. 3.1 Co-metabolic reductive dehalogenation by pure cultures Many studies report alkyl reductive dehalogenation by diverse groups of bacteria ranging from strict anaerobes such as methanogens and homoacetogens to facultative 7 anaerobes such as Escherichia coli. The diversity of bacteria with the capability to reductively dehalogenate alkyl halides indicates that this reaction is general and common for many bacteria. There is strong evidence indicating that transition metal coenzymes in these pure cultures fortuitously catalyze alkyl reductive dehalogenations. The transfer of electrons to alkyl halides by transition metal coenzymes would not benefit the bacteria, since it bypasses ATP generation resulting from transfer of the electrons to the natural terminal electron acceptors. Studies of alkyl reductive dehalogenation already indicated that iron (II) porphyrins possessed dehalogenation activity (Zoro etal. 1974). Purified cytochrome P-450C A M from P. putida has been shown to dehalogenate polychlorinated ethane and polychlorinated methane (Castro et al. 1985 ; Logan et al. 1993). Furthermore, reductive dehalogenation of tetrachloromethane by Shewanella putrefaciens, an obligate respiratory bacterium, is believed to be catalyzed by cytochromes c as tetrachloromethane transformation rate increased with specific heme c content (Baxter 1990 ; Gantzer and Wackett 1991 ; Picardal et al. 1993). Cobalt-containing coenzymes such as vitamin B 1 2 , carbon monoxide dehydrogenase purified from methanogens, and corrinoids in homoacetogen cell extracts also catalyze alkyl reductive dehalogenation of haloalkanes and haloalkenes (Assaf-Anid et al. 1994 ; Burris et al. 1996 ; Gantzer and Wackett 1991 ; Jablonski and Ferry 1992 ; Krone et al. 1991 ; Terzenbach and Blaut 1994). Methanogens also possess the transition metal coenzyme F 4 3 0 , a nickel-containing porphyrin unique to methanogens. Purified F 4 3 0 has been shown to reductively dehalogenate tetrachloromethane, chloroform and 1,2-dichloroethane (Holligere/ al. 1992 ; Krone et al. 1989). Bacterial co-factors other than transition metal coenzymes also catalyze alkyl reductive dehalogenation. Membrane fractions isolated from E. coli actively dehalogenated DDT to 1,1 -diehloro-2,2-bis(/?-chlorophenyl)ethane, and the reaction was strongly stimulated by FAD (French and Hoopingarner 1970). Similar 8 stimulation of the reductive dehalogenation of DDT by Aerobacter aerogenes cell extracts, with addition of FMN under light illumination, was observed by Wedemyer (1966). Transition metal coenzymes also catalyze fortuitous aryl reductive dehalogenation, although this is very rare. Pentachlorophenol and hexachlorobenzene have been shown to be reductively dehalogenated by vitamin B 1 2 and coenzyme F 4 3 0 (Gantzer and Wackett 1991). Generally, the relative rate of aryl reductive dehalogenation by transition metal coenzymes is much slower than that of alkyl reductive dehalogenation. 3.2 Catabolic reductive dehalogenation by pure cultures Although most pure cultures, or coenzymes purified from pure cultures are reported to catalyze alkyl reductive dehalogenation via co-metabolism, some bacterial isolates have been shown to couple reductive dehalogenation of aryl halides or PCE to energy conservation. No pure culture has been isolated so far which is capable of coupling reductive dehalogenation of alkyl halides, other than PCE, to energy conservation. Some of these bacterial isolates are aerobes and use the aryl halides as electron donors. Whereas, other isolates are capable of using either aryl halides or PCE as terminal electron acceptors in anaerobic respiration. In addition, evidence suggests that reductive dehalogenation in these isolates is catalyzed by specific enzymes. I will first review pure cultures which use aryl halides as electron donors, in which reductive dehalogenation is involved in catabolizing the aryl halides. 3.2.1 Pure cultures which catabolize aryl halides as electron donors via reductive dehalogenation Sphingomonas chlorophenolica ATCC 39723, formerly known as Flavobacterium sp. strain ATCC 39723 (Ederer etal. 1997 ; Karlson etal. 1995) is a well studied PCP-degrading 9 bacterium. This bacterium was isolated from PCP-contaminated soils (Saber and Crawford 1985) and was capable of using P C P as a sole carbon source at 100-200 ppm concentration (Orser and Lange 1994 ; Topp etal. 1988 ; Topp and Hanson 1990). Mutants blocked for P C P degradation could not grow on P C P . Accumulated metabolites suggested that P C P was first converted to 2,3,5,6-tetrachloro-p-hydroquinone by a flavoprotein monooxygenase and then it was sequentially transformed to 2,3,6-trichloro-/?-hydroquinone and 2,6-dichloro-/?-hydroquinone by reductive dehalogenation (Steiert and Crawford 1986). P C P 4-monooxygenase, the flavoprotein that converted P C P to 2,3,5,6-tetrachloro-/?-hydroquinone (Xun and Orser 1991 ; X u n et al. 1992a), and tetrachloro-/?-hydroquinone reductive dehalogenase, the enzyme that dehalogenated 2,3,5,6-tetrachloro-/?-hydroquinone to 2,6-dichloro-/?-hydroquinone through 2,3,6-trichloro-/?-hydroquinone (Xun et al. 1992), were later purified. P C P 4-monooxygenase is an inducible enzyme while 2,3,5,6-tetrachloro-/?-hydroquinone reductive dehalogenase is constitutively expressed (Orser and Lange 1994). The 2,3,5,6-tetrachloro-/?-hydroquinone reductive dehalogenase uses the reduced form of glutathione as reductant. It is the first bacterial aryl reductive dehalogenase purified and has limited sequence similarity with members of the glutathione S-transferase family (Orser et al. 1993), which mainly function in eukaryotes as detoxification enzymes (Jakoby 1978). Although glutathione S-transferase has been reported to catalyze dehalogenation of dichloromethane (La Roche and Leisinger 1990 ; Leisinger and Kohler-Staub 1990), the mechanism is hydrolytic dehalogenation rather than reductive dehalogenation. Mycobacterium chlorophenolicum PCP-1 , formerly known as Rhodococcus chlorophenolicusVCP-l (Brigliaet al. 1994 ; Haggblom et al. 1994), is another bacterium which reductively dehalogenatesPCP (Apajalahti etal. 1986). M. chlorophenolicum PCP-1 initiates P C P degradation to 2,3,5,6-tetrachloro-/?-hydroquinone by a membrane-bound 10 cytochrome P-450 enzyme (Uotila etal. 1992). Another chlorine substituent is believed to be removed from 2,3,5,6-tetrachloro-/?-hydroquinone by a second hydrolytic dehalogenase, although the corresponding enzyme has not been purified (Apajalahti and Salkinoja-Salonen 1987). Then, the trichlorotrihydroxybenzene is reductively dehalogenated to trihydroxybenzene (Apajalahti and Salkinoja-Salonen 1987 ; Uotila et al. 1995). Once again, the corresponding reductive dehalogenase has not been purified, but analysis of M chlorophenolicum PCP-1 cell extract suggests that this reductive dehalogenase is different from that of S. chlorophenolica. First, the two reductive dehalogenases use different substrates. In addition, M. chlorophenolicum reductive dehalogenase activity has no requirement for a specific form of reducing power. Ascorbic acid, cysteine, glutathione, N A D H and N A D P H can be used as reductants. N A D P H is the most efficient reductant, but the specific dehalogenation activity has no correlation with the redox potential of these reductants. On the other hand, reductive dehalogenase of S. chlorophenolica only uses reduced glutathione as reductant. Reductive dehalogenase activity of M. chlorophenolicum appears to be inducible, but the S. chlorophenolica reductive dehalogenase is constitutively expressed. Finally, reductive dehalogenase activity of M. chlorophenolicum is oxygen-tolerant, but that of S. chlorophenolica is not. Corynebacterium sepedonicum K Z - 4 is a pure culture which can grow on 2,4-dichlorobenzoate or 4-chlorobenzoate as sole carbon and energy source (Zaitsev and Karasevich 1985). A related Coryneform bacterium strain N T B - 1 (formerly named Alcaligenes denitrificans NTB-1 ) was reported to convert 2,4-dichlorobenzoate to 4-chlorobenzoate by reductive dehalogenation reaction (Van den Tweel et al. 1987). Experiments using cell extracts of these two strains showed that 2,4-dichlorobenzoate was converted to 2,4-dichlorobenzyol C o - A in a M g 2 + , A T P and coenzyme A-dependent 11 reaction. 2,4-dichlorobenzyol Co-A was shown to be dehalogenated to 4-chlorobenzyol Co-A by a novel NADPH-dependent reductive dehalogenase (Romanov and Hausinger 1996). Specific reductive dehalogenase activity with reduced glutathione as reductant was 0% and 14% of that of the NADPH catalyzed reaction, in cell extracts of C. sepedonicum KZ-4 and strain NTB-1, respectively. Furthermore, glutathione ^ -transferase and glutathione reductase activities were not detected in C. sepedonicum KZ-4 cell extract. Therefore, it seems unlikely that C. sepedonicum KZ-4 reductive dehalogenase is a glutathione ^-transferase, like the dehalogenase of S. chlorophenolica. The possible relationship between C. sepedonicum KZ-4 andM chlorophenolicum PCP-1 reductive dehalogenases is not known. 3.2.2 Pure cultures which utilize aryl halides and PCE as electron acceptors Some anaerobic pure cultures are able to use aryl halides and PCE as respiratory electron acceptors and couple reductive dehalogenation to ATP synthesis, probably via chemiosmotic mechanisms. This type of anaerobic respiration is named halorespiration. Several species of a new genus, Desulfitobacterium, are capable of growing on non-fermentable substrates only in the presence of aryl halides or PCE as electron acceptors. Members of the genus Desulfitobacterium are Gram-positive, rod-shaped bacteria. They are closely related to the genus Desulfotomaculum, a Gram-positive genus of spore-forming sulfate-reducing bacteria, according to a 16S rRNA gene sequence analysis (Utkin et al. 1994). However, Desulfitobacterium sp. only use sulfite and thiosulfate, but not sulfate as terminal electron acceptors. In addition, only some members of the genus of Desulfitobacterium are spore-formers. Desulfitobacterium dehalogenans JW/IU-DC-1 is the first isolate of this genus. The optical density of this culture concurrently increased as 3-chloro-4-hydroxy phenyl acetate was reductively dehalogenated when it was grown with pyruvate as 12 the electron donor with 0.1% yeast extract, although it also fermented pyruvate. However, it grew in the 0.1% yeast extract medium with formate or hydrogen as electron donors only in the presence of electron acceptors such as 3-chloro-4-hydroxyphenylacetate. These observations suggest the bacterium may gain energy via halorespiration. The reductive dehalogenation activity of D. dehalogenans JW/IU-DC1 is orf/jo-specific, and it can also reductively dehalogenatehighly chlorinated phenols such as P C P , 2,3,4,5-, 2,3,4,6-, 2,3,5,6-tetrachlorophenols; 2,3,4-, 2,3,6-, 2,4,6-trichlorophenols; and 2,3-, 2,4-, 2,6-dichlorophenols (Utkin etal. 1995). A related strain Desulfitobacteriumhajhiense D C B - 2 has been shown to grow by coupling 3-chloro-4-hydroxyphenylacetate reductive dehalogenation to pyruvate oxidation (Christiansen and Ahring 1996 ; Madsen and Licht 1992). The bacterial biomass, measured in cellular protein, increased by approximately 50% when equal molar of 3-chloro-4-hydroxyphenylacetate and pyruvate were used for growth, compared to growth under pyruvate-fermenting conditions. Desulfitobacterium hajhiense D C B - 2 also has a broader substrate range than D. dehalogenans JW/ IU-DC1, since it reductively dehalogenates 3,5-dichlorophenol to 3-chlorophenol and 2,4,5-trichlorophenol to 3,4-chlorophenol, in contrast to D. dehalogenans JW/IU-DC1 which does not dehalogenate these chlorophenol isomers with /weta-substituents. Desulfitobacterium sp. strain PCE-1 (Gerritse etal. 1996) has also been suggested to grow via halorespiration. This strain grew on lactate only in the presence of external electron acceptors such as P C E and 3-chloro-4-hydroxyphenylacetate. Growth with formate as electron donor and 3-chloro-4-hydroxyphenylacetate as electron acceptor was also demonstrated, and biomass, measured in cellular protein, increased linearly with increasing amounts of 3-chloro-4-hydroxyphenylacetate being dehalogenated. Desulfitobacterium sp. PCE-1 is therefore the first organism shown to be capable of growing by reductive 13 dehalogenation of both P C E and aryl halides. Recently, another strain, Desulfitobacterium sp. strain P C E - S was isolated (Miller et al. 1997b). Growth by halorespiration has not yet been shown with strain P C E - S . This strain is unique in that it does not use aryl halides like 3-chloro-4-hydroxyphenylacetate, P C P and 2,4,6-trichlorophenol as electron acceptors. A t this time, P C E and T C E are the only known electron acceptors on which it can grow. Similarly, growth of Desulfitobacterium chlororespirans Co23 possibly by halorespiration was supported by the fact that using formate, hydrogen, or lactate were used by D. chlororespirans Co23 as electron donors only in the presence of 3-chloro-4-hydroxybenzoate or other electron acceptors (Sanford et al. 1996). Reductive dehalogenation activity of D. chlororespirans Co23 has been studied in vitro with cell extracts (Loffler et al. 1996). The reductive dehalogenase activity is inducible, membrane-associated and oxygen-insensitive. Interestingly, dehalogenation activity was detected in cell extracts and washed membrane fractions derived from induced cells even in the absence of the exogenous electron donor methyl viologen, although methyl viologen enhanced the dehalogenase activity. This observation suggests that the in vivo electron donor for the reductive dehalogenase is also membrane-bound and is oxygen-tolerant. Preliminary data suggests that this electron donor is co-induced with the reductive dehalogenase activity. Desulfitobacterium frappier PCP-1 is a PCP-dehalogenating strain (Bouchard et al. 1996). A 16S r R N A gene analysis showed that D. frappier PCP-1 exhibited 95% homology with D. dehalogenans J W / I U - D C 1 . However, growth by halorespiration has not been proved with D. frappier PCP-1 . D. frappier PCP-1 is an unique member of the genus of Desulfitobacterium. It reductively dehalogenates P C P to 3-chlorophenol through 2,3,4,5-tetrachlorophenol, 3,4,5-trichlorophenol, and 3,5-dichlorophenol. Hence, it is the first bacterial isolate that possesses ortho-, meta, and para-reductivedehalogenation activities. P C P was rapidly dehalogenatedto 3,4,5-trichlorophenol. 14 But then there was a 3-day lag period before 3,4,5-trichlorophenol was further dehalogenated to 3,5-chlorophenol and 3-chlorophenol. These results suggest at least two dehalogenase systems are present. One dehalogenase system appears to be or//;o-specific, while a second system appears to be responsible for the meta- and /?crra-dehalogenation activities. Bacteria other than members of the genus Desulfitobacterium are also able to grow by halorespiration. A strain named 2CP-1, which has a 16S rRNA gene sequence most similar to that of Mycobacteria, is capable of reductively dehalogenating ort/w-chlorophenol, and growing in a defined medium with acetate and 2-chlorophenol (Cole etal. 1994). No growth was detected when either acetate or 2-chlorophenol was left out of the medium. The protein yield was about double when 2,6-dichlorophenol was used as electron acceptor instead of 2-chlorophenol. It is therefore probable that strain 2CP-1 gains energy by halorespiration, although there is no direct evidence to further support the conclusion. Other organisms are reported to dehalogenate PCE and probably conserve energy by halorespiration. Dehalobacterrestrictus (Holliger et al. 1993 ;Holliger and Schumacher 1994) and Dehalospirilium multivorans (Scholz-Muramatsu et al. 1995), both isolated with PCE as electron acceptors, are two halorespiratory eubacteria. PCE is dehalogenated to C/J-1,2-dichloroethene by both organisms. Dehalobacterrestrictus has a very narrow substrate range. Hydrogen is the only electron donor and PCE and TCE are the only electron acceptors which D. restrictus is known to grow on. Fermentative growth was not observed. The hydrogenase of D. restrictus was shown to be membrane-bound, facing the periplasmic side of the cytoplasmic membrane, while the PCE dehalogenase is located on the inner side of the cytoplasmic membrane (Schumacher and Holliger 1996). Recently, the PCE reductive dehalogenase was purified from the cytoplasmic membrane of D. restrictus (Schumacher et al. 1997). The reductive dehalogenase has an apparent molecular mass of 60 kDa according to 15 S D S - P A G E . Each monomeric unit contains one cobalamin and two [4Fe-4S] clusters. The P C E reductive dehalogenase can be reduced in vitro from the Co(II)- to Co(I)-state by sodium dithionite, and has a measured midpoint potential o f - 3 5 0 mV. Incubation of the Co(I) form of P C E dehalogenase with P C E resulted in the conversion to the Co(II) form. Together with the previous observation that P C E dehalogenase activity in D. restrictus cell suspensions was inhibited by propyl iodide, a corrinoid inhibitor (Schumacher and Holliger 1996), these observations indicate that the P C E reductive dehalogenase contains a cobalamin prosthetic group which is necessary for the enzyme activity. Tests for the presence of cytochromes in the cell extract were not conclusive, but menaquinone was detected in the membrane fraction ofD. restrictus cells by UV-spectroscopy. The menaquinone was reduced by hydrogen and re-oxidized by P C E , indicating menaquinone could be involved in the electron transfer to the P C E dehalogenase (Schumacher and Holliger 1996). However, menaquinone appears not to be the direct electron donor for the P C E dehalogenase, since a menaquinone analogue 2,3-dimethyl-l,4-naphthoquinone failed to directly react with the purified dehalogenase. A strain named T E A was recently isolated, and the 16S r R N A gene sequence of strain T E A is 99.7% identical to that of D. restrictus (Wild etal. 1996). Physiological characteristics of strain T E A are identical to that of D. restrictus. Therefore, the two organisms are likely to be different strains belonging to the same species. Whereas D. restrictus is restricted to hydrogen and P C E or T C E as growth substrates, Dehalospirillum multivorans is able to utilize a variety of electron donors such as pyruvate, lactate, formate, ethanol and glycerol, and other electron acceptors like fumarate or nitrate (Scholz-Muramatsu et al. 1995). D. multivorans also ferments pyruvate. Menaquinone, cytochrome b and cytochrome c were detected in D. multivorans grown with pyruvate and fumarate, but whether the same electron carriers are present in cells grown with hydrogen and 16 P C E is not known. In addition, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) , a respiratory quinone inhibitor, did not inhibit P C E reductive dehalogenation activity in D. multivorans cell suspensions and cell extracts (Miller et al. 1997a). Therefore, the involvement of a menaquinone as an electron carrier in transferring electrons to the reductive dehalogenase is questionable. The P C E reductive dehalogenase of D. multivorans is located in the soluble fraction of ultracentrifuged cell extract, and in the pelleted spheroplast preparation, indicating that the enzyme is most probably located in the cytoplasm (Neumann et al. 1994). Propyl iodide completely inhibited the P C E dehalogenase activity in D. multivorans cell suspensions (Neumann et al. 1994) and in cell extracts only when the cell extracts were pre-reduced (Neumann et al. 1995). These observations suggest the P C E dehalogenase of D. multivorans contains a corrinoid co-factor, which is active in the Co(I)-state. This hypothesis is further supported by studies of the P C E dehalogenase purified from the cell extracts (Neumann et al. 1996). The purified P C E dehalogenase was inhibited by propyl iodide only i f it was pre-reduced by titanium (III) citrate. In addition, the cobalt content was determined to be 1 mole of cobalt per mole of enzyme. The apparent mass of the native enzyme was about 58 kDa, determined by gel-permeation chromatography. S D S - P A G E revealed a single band of 57 kDa, indicating the native dehalogenase is a monomer. These data suggest the presence of 1 corrinoid per dehalogenase molecule. Besides the corrinoid, iron-sulfur clusters are probably present. For 1 mole of P C E dehalogenase, 9.8 moles of iron and 8.0 moles of acid-labile sulfur were detected, which allows for 2 [4Fe-4S] clusters. A s a consequence, the P C E dehalogenase of Dehalospirilium multivorans appears to be very similar to that of Dehalobacter restrictus, except the former is cytoplasmic while the latter is membrane-bound and facing the cytoplasm. 17 A facultative aerobic bacterium, strain MS-1, was isolated and shown to transform PCE to cis- 1,2-dichloroethene, although PCE reductive dehalogenation occurred only anaerobically (Sharma and McCarty 1996). Metabolic characterization, fatty acid composition analysis, and partial 16S rRNA gene sequence analysis showed that strain MS-1 belonged to the family Enterobacteriaceae, but it cannot be assigned to a particular genus currently. PCE dehalogenation was shown to happen in medium with non-fermentable carbon sources such as formate or acetate, or with a complex carbon source like yeast extract. In addition, the bacterial cell count was ten times higher in PCE-dehalogenating cultures than cultures growing fermentatively, and therefore, these findings are consistent with the hypothesis that strain MS-1 can derive energy by a respiratory mechanism when PCE is dehalogenated. The above isolates only dehalogenate PCE to c/'s-1,2-dichloroethene. But strain 195, tentatively named Dehalococcoides ethenogenes, is a eubacterium capable of completely transforming PCE to ethene by reductive dehalogenation and possible conserves energy by a respiratory mechanism (Maymo-Gatell et al. 1997). This is the first bacterial isolate that completely dehalogenates PCE. Hydrogen and PCE are the only known respective electron donors and electron acceptors on which it can grow. Strain 195 also requires cell extracts of a mixed microbial culture from which it was isolated as a growth supplement. The identity of the required growth factor is not known currently. 3.3 Reductive dehalogenation by Desulfomonile tiedjei DCB-1 Desulfomonile tiedjei DCB-1 is the first anaerobic bacterial isolate that was demonstrated to be capable of coupling reductive dehalogenation to ATP synthesis via halorespiration. D. tiedjei was isolated from an anaerobic sludge community which 18 reductively dehalogenated 3-chlorobenzoate to benzoate (Shelton and Tiedje 1984). Nine different organisms were isolated and at least four were necessary for the conversion of 3-chlorobenzoate to methane and carbon dioxide: (1) the dehalogenating strain DCB-1 that dehalogenated 3-chlorobenzoate to benzoate; (2) the benzoate fermenting rod BZ-2 that produced hydrogen and acetate; (3) a hydrogen-consuming methanogen Methanospirillum sp.; and (4) the acetate-consuming Me thanothrix sp. Initially, this consortium was cultured in an undefined medium including rumen fluid as a nutrient supplement. Later, Dolfing and Tiedje (1986) were able to maintain the consortium with the first three strains of bacteria in a defined medium with 3-chlorobenzoate as the sole energy source. In this syntrophic consortium, D. tiedjei dehalogenated 3-chlorobenzoate to benzoate, which was fermented by strain BZ-2. Hydrogen produced by strain BZ-2 was used as an electron donor for dehalogenation by D. tiedjei, and for methanogensis by the Methanospirillum sp. Cultivation of pure cultures of dehalogenating/3. tiedjei in defined medium was finally successful after discovery of the requirements of the vitamins thiamine, nicotinamide and 1,4-naphthoquinone for growth (DeWeerd et al. 1990). This pure culture of/), tiedjei offers a great opportunity to study this organism and the biochemistry of reductive dehalogenation. D. tiedjei is a Gram-negative, straight, non-motile rod. It is a very slow growing, non-spore-forming, obligate anaerobe. It has a unique "collar-like" morphological feature surrounding the cell (Mohn etal. 1990). Stevens et al. (1988) first characterizedD. tiedjei as a sulfidogen. In that study, various sulfoxy anions were added as electron acceptors to D tiedjei growing on pyruvate as carbon and energy source plus rumen fluid as growth supplement. Thiosulfate and sulfite stimulated the growth of D. tiedjei and sulfide was produced. The bacterium also grew with sulfate as electron acceptor; although, the specific growth rate was slower compared to that of thiosulfate-reducing cultures. Desulfoviridin, the bisulfite 19 reductase, was detected in crude cell extract. A 16S rRNA gene sequence analysis was done by DeWeerd et al. (1990), indicating that D. tiedjei represents a new genus which belongs to• the delta subdivision of the class of Proteobacteria of the eubacterial kingdom, which includes sulfate- and sulfur-reducing eubacteria (Woese 1987). Reductive dehalogenation by D. tiedjei expresses specificity towards /weta-substituted benzoates or benzamides (Shelton and Tiedje 1984) and chlorophenols (Mohn and Kennedy 1992). This bacterium also dehalogenates PCE (Fathepure et al. 1987). Bromo and iodo substituents at the ortho- and /?ara-positions are sometimes dehalogenated at a slow rate (DeWeerd et al. 1986). Moreover, reductive dehalogenation activity \nD. tiedjei is inducible by /weta-substituted benzoates, benzamides and benzyl alcohols (Cole and Tiedje 1990). However, some of the inducers might not be substrates for reductive dehalogenation. For example, /weto-substituted benzyl alcohols and 3-fluorobenzoates are inducers which are not dehalogenated. On the other hand, some substrates for reductive dehalogenation by D. tiedjei are not inducers for the activity. For example, it was previously observed that the dehalogenation activities of chlorophenols and PCE were co-induced with that of 3-chlorobenzoate (Cole etal. 1995 ; Mohn and Kennedy 1992 ; Townsend and Suflita 1996). Non-3-chlorobenzoate-induced cells did not dehalogenate chlorophenols or PCE, and chlorophenols or PCE did not induce the corresponding dehalogenation activities. Like the 3-chlorobenzoate reductive dehalogenation activity, the chlorophenols- and PCE-dehalogenation activities were heat-sensitive. PCE dehalogenation was inhibited by 3-chlorobenzoate but not by 2- or 4-chlorobenzoates, which are not dehalogenation substrates of D. tiedjei. These findings suggest that D. tiedjei chlorophenol and PCE dehalogenation are catalyzed by the 3CB reductive dehalogenase, due to relaxed specificity of the enzyme. 20 It has been observed that D. tiedjei could obtain energy for growth by coupling reductive dehalogenation of 3-chlorobenzoate to oxidation of formate and hydrogen (DeWeerd etal. 1991 ; Dolfing 1990 ; Mohn and Tiedje 1990b). These cultures had higher yields in terms of protein and optical density, relative to cultures with benzoate replacing 3-chlorobenzoate. Formate and hydrogen are not known to support substrate-level phosphorylation and so use of these electron donors indirectly suggests that energy is conserved by oxidative phosphorylation, involving an electron transport chain. Later, Mohn and Tiedje (1991) demonstrated that D. tiedjei was capable of coupling reductive dehalogenation with A T P synthesis via a proton-motive force-driven ATPase. In that study, addition of 3-chlorobenzoate to a pre-induced cell suspension increased the A T P pool within the cells. Uncouplers and ionophores which dissipated the proton-motive force reduced the A T P pool relative to the rate of dehalogenation. Hence, the dehalogenation-dependent A T P synthesis is less efficient as the proton-motive force is dissipated and is in agreement with the hypothesis that A T P synthesis is effected by a chemiosmotic mechanism. In addition, low concentrations of A^'-dicyclohexylcarbodiimide, the proton-driven ATPase inhibitor, also inhibited the dehalogenati on-dependent A T P synthesis. This observation is consistent with the presence of a proton-driven ATPase in D. tiedjei, and rules out the possibility that reductive dehalogenation supports substrate-level phosphorylation and forms a proton-motive force via an ATPase functioning reversibly. Thus, these findings suggest that reductive dehalogenation supports the formation of a proton-motive force which in turn drives A T P synthesis via a proton-motive force-driven ATPase. Other evidence also suggests that reductive dehalogenation of D. tiedjei involves an uncharacterized electron transport chain. First of all, Apajalahti etal. (1989) discovered that reductive dehalogenation of D. tiedjei in defined media required rumen fluid or filtered-culture 21 fluid of Propionibacterium sp. as a nutrient supplement. The essential factor in the culture fluid was believed to be a quinoid compound. Later, DeWeerd et al. (1990) showed that a vitamin mixture including 1,4-naphthoquinone or menadione could replace the Propionibacterium culture fluid for growing/), tiedjei in a defined medium. The structure of 1,4-naphthoquinone is very similar to the aromatic nucleus of menaquinone, a membrane-bound electron carrier commonly found in other sulfur- and sulfate-reducing bacteria (Collins and Widdel 1986). Therefore, it seems likely that a menaquinone is involved in the electron transport chain coupled to reductive dehalogenation, and 1,4-naphthoquinone might be a possible menaquinone precursor. Recently, the/), tiedjei reductive dehalogenase was purified from the membrane fractions of the bacterium (Ni et al. 1995). The purified reductive dehalogenase was a heterodimer, consisting of 2 subunits with molecular mass of 64 kDa and 37 kDa. The enzyme was inducible and contained an iron-cofactor, although the nature of this iron-cofactor was not clear. It was speculated that this reductive dehalogenase could function as the terminal reductase of the electron-transport chain involved in halorespiration. Inhibitory effects of sulfoxy anions on D. tiedjei reductive dehalogenase activity also indirectly support the hypothesis that electron carriers are involved in reductive dehalogenation. DeWeerd and Suflita (1990) showed that reductive dehalogenation in D. tiedjei crude extract was inhibited by sulfite and thiosulfate, but not sulfate. In a related study using washed, cell suspensions of D. tiedjei, the presence of sulfite and thiosulfate also inhibited reductive dehalogenation, while sulfate had no effect (DeWeerd et al. 1991). However, a similar study by Linkfield and Tiedje (1990) demonstrated that sulfate, thiosulfate, and sulfite inhibited reductive dehalogenation of 3-chlorobenzoate. SinceD. tiedjei is a sulfidogen, the inhibitory effect of sulfoxy anions on reductive dehalogenation was hypothesized to be due to competition between sulfoxy anions and aryl halides for electron 22 carriers or reducing equivalents. Recently, Townsend and Suflita (1997) demonstrated that sulfite and thiosulfate inhibited in vitro reductive dehalogenation activity of membrane fractions isolated from pre-induced D. tiedjei ceils. Enzymes responsible for sulfite or thiosulfate reduction are cytoplasmic enzymes and should not be present in the membrane fractions. Therefore, this inhibitory effect was interpreted as a direct interaction of the sulfoxy anions with the reductive dehalogenase, and did not necessarily involve competition between the two respiratory processes for electron carriers or reducing equivalents. 23 THESIS OBJECTIVES A l l of the above findings clearly demonstrate that the respiratory components of D. tiedjei play a significant role in reductive dehalogenation. Further understanding of the mechanism of reductive dehalogenation wi l l come from identification and localization o f these respiratory components relative to the cytoplasmic membrane. Unfortunately, details of the chemiosmotic mechanism of D. tiedjei, including its electron transport chain, are poorly understood. A s a consequence, the objectives of my thesis are: (1) to identify the respiratory components, including electron carriers and respiratory enzymes, involved in reductive dehalogenation and sulfoxy anion respiration in D. tiedjei, (2) to locate these respiratory components with respect to the cytoplasmic membrane, and (3) to quantify these respiratory components in cells catalyzing different metabolic processes. This research wi l l increase our understanding of the biochemical mechanism of reductive dehalogenation, and wi l l provide insight into the relationship between sulfoxy anion respiration and reductive dehalogenation in D. tiedjei. 24 MATERIALS AND METHODS 1. Organism and growth conditions Desulfomonile tiedjei D C B - 1 ( A T C C 49306) was grown on reduced anaerobic medium as previously described by Mohn and Tiedje (1991) with the following modifications and additions: 20 m M pyruvate was used as electron donor, 0.1 m M titanium citrate was the only reductant, the phosphate concentration was raised from 4 m M to 10 m M . Except in cultures used for testing the effect of individual vitamin components on reductive dehalogenation, 2 g yeast extract/L plus 2 gtryptone/L were added as nutrient supplements. The vitamin solution was changed to the vitamin mixture reported by W o l i n et al. (1963) plus 200 p:g 1,4-naphthoquinone/L. The gas phase was N 2 / C 0 2 (95:5, v/v), and the p H was adjusted to p H 7.5. When the cells were grown under dehalogenatingconditions, 500 p :M of 3-chlorobenzoate (3CB) was added from a 100 m M anoxic filter-sterilized stock. When depleted, 3 C B was replenished. When the cells were grown under fermenting conditions, no 3 C B was added. When the cells were grown under sulfate-respiring conditions, 20 m M sodium formate plus 5 m M sodium sulfate were added instead of pyruvate and 3-chlorobenzoate. Inocula were 10%, and cultures were incubated stationary at 37 °C. 2. Cell suspension experiments D. tiedjei cells were harvested by centrifugation at 10,000 x g for 20 min in centrifuge tubes degassed with N 2 or stored overnight in anaerobic glove box. Cel l pellets were washed with sterile, anaerobic 10 m M H E P E S plus 10 m M potassium phosphate buffer (pH 7.5) reduced with 0.1 m M titanium (III) citrate. The cells were again harvested by centrifugation, and suspended, concentrated 5- to 10-fold, in sterile anaerobic buffer. The cell suspensions 25 were transferred into sterile 25-mL serum bottles with N 2 in the headspace. Depending on the experiment, different electron donors and metabolic inhibitors were added from sterile, anoxic stocks. 3 C B was added to the cell suspensions from a 100-mM anoxic filter-sterilized stock solution. The cell suspensions were incubated without agitation at 37 °C. Reductive dehalogenation activity was analyzed by measuring benzoate formation in the cell suspensions with a high performance liquid chromatography ( H P L C ) system. 3. Cell fractionation Late exponential-phase cultures were harvested either by centrifugation at 10,000 x g for 20 min or by a bench-scale cross-flow filtration unit equipped with a 0.3-pm pore size microfiltration membrane (Filtron). Cells were suspended in 10 m M Tr i s -HCl buffer (pH 7.7) and were broken by passing the cell suspensions four times through a French pressure cell at a cell pressure of 103.4 M P a . Cell lysates were then centrifuged at 12,000 x g for 20 min. The pelleted, unbroken cells and debris were suspended in 10 m M Tr i s -HCl buffer (pH 7.7), and passed though the French pressure cell four more times and centrifuged as described above. The combined supernatants were centrifuged for 2 h at 180,000 x g. The pellets were washed and suspended in the Tris buffer, and centrifuged at 180,000 x g for another 2 h. The pellets were considered to be membrane fractions and were stored at -20 °C. The supernatants of the two ultracentrifugation preparations were combined and considered as soluble fractions. To isolate the periplasmic fraction, cells were extracted three times with 50 m M Tris-H C l , 50 m M E D T A , and 170 m M N a 2 C 0 3 buffer (pH 9.0), with continuous shaking, at 35 °C for 30 min. This alkaline buffer was previously used for extracting periplasmic fractions from other sulfate-reducing bacteria (Badziong and Thauer 1980). The extracts were pooled and dialyzed against 10 m M Tr is -HCl buffer (pH 7.7) in dialysis tubing with a M W C O of 26 1,000 (Spectrum Medical Industries Inc). The dialyzed samples were then concentrated by covering the dialysis tubing with Aquacide II (Calbiochem), a polyethylene glycol-like material, and considered to be periplasmic fractions. 4. Quantification of cytochromes Cytochrome contents were determined in periplasmic, cytoplasmic and membrane fractions of D. tiedjei by analyses of reduced minus oxidized absorption spectra. Air-oxidized and sodium dithionite-reduced absorption spectra were collected separately with a Cary IE spectrophotometer to generate the difference spectra. The oc-peaks of maximal absorbance of the difference spectra were used for preliminary identification of the cytochromes in different cellular fractions. Difference spectra of known concentration of horse-heart cytochrome c (Sigma) were used as standards for quantification of cytochromes and had an experimentally determined extinction coefficient (£419) of 40,000 M ' 1 cm"1. 5. Heme-staining of SDS-PAGE gels SDS-PAGE was performed according to the method of Laemmli (1970) using 5% stacking-10% separating acrylamide gels. The gels were stained with Coomassie Brilliant Blue R250. Heme-staining of SDS-PAGE gels was performed using dimethoxybenzidine hydrochloride (Sigma) according to the method of Francis and Becker (1984), except that protein samples were incubated at 50 °C for 15 min prior to electrophoresis. 6. Purification of a 50-kDa inducible cytochrome c Unless indicated otherwise, the entire purification procedure was carried out at room temperature without protection from oxygen. Cell membrane fractions were thawed and 27 suspended in 10 m M Tr i s -HCl p H 7.7 (about 13 mg protein/mL). Ammonium sulfate was added to reach 35% saturation and the mixture was shaken for 30 min. The mixture was then centrifuged at 12,000 x g for 10 min, and the supernatant was collected. The ammonium sulfate membrane extract was concentrated by Centriplus-30 (Amicon Inc) to 2.5 mg protein/mL. The concentrate was divided into three 3.5-mL aliquots. Each aliquot was loaded independently onto a fast protein liquid chromatography ( F P L C ) Phenyl Superose H 5/5 (1 mL) column (Pharmacia Biotech) equilibrated with 10 m M Tr i s -HCl buffer (pH 7.7) plus ammonium sulfate at 35% saturation. The column was first eluted with a decreasing linear gradient (10 mL) from 35 to 25% saturation of ammonium sulfate in the Tris buffer followed by successive 7-mL steps of 25, 15 and 0% saturation of ammonium sulfate in the same buffer. The flow rate was kept at 0.25 mL/min. The inducible cytochrome was eluted at 25% saturation of ammonium sulfate. Fractions containing the inducible cytochrome were combined, dialyzed against 10 m M Tris -HCl (pH 7.7) overnight at 4 °C in dialysis tubing with a M W C O of 6,000 to 8,000 (Spectrum Medical Industries Inc), and then concentrated by Centricon-10 (Amicon Inc) to 1.3 m g o f protein in 1 mL. The concentrate was loaded onto an F P L C Mono Q H 5/5 (1 mL) column (Pharmacia Biotech) equilibrated with 10 m M Tr i s -HCl buffer (pH 7.7). The inducible cytochrome did not bind to the Mono Q column and was eluted with the same buffer. Unbound fraction from the M o n o Q column was exchanged into 10 m M sodium phosphate buffer (pH 6.5) by dialysis. About 507 ug of protein was recovered and loaded onto a 5 m L hydroxyapatite column (Bio-Rad) equilibrated with 10 m M sodium phosphate buffer (pH 6.5). The inducible cytochrome was eluted at 300 m M sodium phosphate during an increasing linear gradient (50 mL) from 10 m M to 500 m M sodium phosphate (pH 6.5) at 1 mL/min. Fractions containing the inducible cytochrome were 28 pooled, concentrated by Centricon-10 (Amicon Inc) and stored at 4 °C for subsequent analyses. 7. Redox and p H titrations of the inducible cytochrome The midpoint potential of the inducible cytochrome was determined by two methods. A chemical titration was performed under a nitrogen atmosphere by Dutton's method of redox titration (1978), using a cuvette sealed with a butyl rubber stopper. N.N.N'.N'-Tetramethyl phenylenediamine, hexamineruthenium (III) chloride, pyocyanine, 2-hydroxyl-1,4-naphthoquinone, anthaquinone-2-sulfonate, benzyl viologen and methyl viologen in 100 m M sodium phosphate buffer (pH 6.5) were used as redox mediators. Anoxic , freshly dissolved sodium dithionite was used as reductant. Concentrations of purified inducible cytochrome and individual redox mediators were 1.5 \iM and 0.3 p:M, respectively. The redox potential of the mixture was measured by a miniature P t - A g / A g C l combination redox electrode (Microelectrodes Inc) calibrated against freshly prepared p H 4.0 and 7.0 buffers saturated with quinhydrone. The absorbances of the inducible cytochrome at 399 and 555 nm were monitored as a function of the measured redox potential. The midpoint potential of the inducible cytochrome was also determined using equilibrium reaction with 8-hydroxyriboflavin, which has a midpoint potential of -332 m V (Muller 1983). A solution of 1.6 pJVI of inducible cytochrome, 50 pJVI 8-hydroxyriboflavin and 10 m M E D T A in 100 m M sodium phosphate buffer p H 7.0 were sealed in a cuvette with a butyl rubber stopper and kept in the dark. The mixture was made anoxic by flushing with oxygen-free nitrogen and then was gradually photoreduced by direct exposure to sunlight. Progress of reduction of the inducible cytochrome and 8-hydroxyriboflavin was monitored spectrophotometrically after each period of irradiation. 8-hydroxyriboflavin reduction was measured at 475 nm, which was 29 an isosbectic point of the oxidized and reduced inducible cytochrome. Reduction of the inducible cytochrome was measured at 384 nm and 555 nm. A p H titration of the inducible cytochrome was done under an air atmosphere. Small aliquots of sodium hydroxide solution (1 M ) were added to a 1.5 p M solution of purified inducible cytochrome. UV-vis ib le absorption spectra were recorded and p H in the solution was monitored by a miniature P t -Ag /AgCl combination p H electrode (Microelectodes Inc). A n Orion 290A p H / m V meter was used to measure the acidity during the p H titration and redox potential during the chemical redox titration. 8. NH2-terminal protein sequence analysis of the inducible cytochrome The purified inducible cytochrome was first resolved by S D S - P A G E and the gel was soaked in transfer buffer [10 m M C A P S with 0.5 m M dithiothreitol (pH 9.0)]. The protein was electroblotted to a polyvinylidene difluoride membrane (Millipore) by a B io -Rad Trans-Blot cell a t 4 ° C . The blotting voltage was 200 V f o r l h . Then, the polyvinylidene difluoride membrane was stained with Coomassie Brilliant Blue R250 in 50% methanol. After destaining with 50% methanol, the membrane was air-dried. The protein band was then excised from the membrane and sequenced. The NH 2-terminal sequencing was done by the Nucleic Acid-Protein Service Unit at the University of British Columbia. 9. Genomic DNA isolation About 5 g (wet weight) of cells were washed three times with 15 m L of 50 m M Tris-H C l , 50 m M E D T A and 170 m M sodium carbonate buffer (pH 9.0), with continuous shaking, at 35 °C for 30 min. The washed cells were centrifuged and suspended in 30 m L 10 m M T r i s - H C l , 1 m M E D T A buffer (pH 8.0). Genomic D N A was extracted from the cell 30 suspension as previously described by Ausubel et al. (1992), with the following modifications. The final concentration of SDS was 0.33%, 3 mg self-digested Pronase E / m L (Sigma) was used instead of proteinase K and the suspension was incubated at 56 °C for 4 h. The SDS-Pronase E lysis procedure, followed by freezing at -20 °C, was repeated 3 times to achieve adequate cell lysis. Cell lysates were incubated in a solution of 0.7 M sodium chloride and 10% cetyltrimethylammonium bromide (Sigma)at 65 °C for 1 h, dissolved nucleic acids were treated with 2 mg RNase A / m L (Sigma) at 37 °C for 1 h, and the RNase A was inactivated by 0.1 mg protease K / m L at 37 °C for 1 h. 10. DNA manipulation Restriction enzymes used were purchased from Gibco B R L and N e w England Biolabs. Taq D N A polymerase and Vent D N A polymerase for P C R reactions were purchased from Gibco B R L and New EnglandBiolab, respectively. Southern blot analyses were done using Nytran Nylon membranes (Schleicher and Schuell) according to the alkaline transfer method of Ausubel et al. (1992), except sodium chloride-sodium dihydrogen phosphate-E D T A (SSPE) buffer instead of sodium chloride-sodium citrate (SSC) buffer was used for prehybridization, hybridization and washing. D N A probes for the Southern analyses were synthesized by P C R reactions. If the D N A probes were end-labeled, 100 pmoles of the forward and the reverse primers were incubated with 700 u C i y - 3 2 P - A T P (7000 Ci/mmole) and 10 units T4 polynucleotide kinase in kinase buffer (Gibco B R L ) separately at 37 °C for 1 h. After that, the two kinase reaction mixtures were heat-killed at 65 °C for 10 min and mixed with the P C R reaction mixture containing 10 ng plasmid D N A template, 200 u M of each dNTP, 1.5 m M M g C l 2 and 0.5 U Taq D N A polymerase in 100 p L of polymerase buffer. The P C R reaction was performed as follows: (i) denaturation at 95 °C for 3 min; (ii) 0.5 min at 95 31 °C, 0.5 min at 35 °C, and 0.5 min at 72 °C for 30 cycles; (iii) a final incubation at 72 °C for 10 min. The end-labeled P C R product was purified with a spin column kit (Qiagen Inc.) and was stored in 10 m M Tr i s -HCl buffer with 1 m M E D T A (pH 8.0) at 4 °C. Some o f the radio-labeled D N A probes were synthesized by direct incorporation of a - 3 2 P - d C T P into the P C R product. In this case, the P C R mixture contained 10 ng plasmid D N A template, 50 pmole of forward and reverse primers, 2 m M M g C l 2 , 4 u M of d A T P , dGTP and dTTP, 3.2 u M of dCTP with 125 u C i a - 3 2 P - d C T P (3000 Ci/mmole) and 0.5 U Taq D N A polymerase in 50 u L of polymerase buffer. The P C R reaction was performed as follows: (i) denaturation at 95 °C for 3 min; (ii) 0.5 min at 95 °C, 1.0 min at 45 °C, and 2.0 min at 72 °C for 35 cycles; (iii) a final incubation at 72 °C for 10 min. The radio-labeled P C R product was precipitated in 100% ethanol at -70 °C for 1 h, and was stored in 10 m M Tr i s -HCl buffer with 1 m M E D T A (pH 8.0) at 4 °C. 11. Inverse PCR and cloning of the inverse PCR product D. tiedjei genomic D N A was completely digested by an appropriate restriction enzyme, self-ligated and used for inverse P C R as previously described by Y o u et al. (1996) with the following modifications. Five pig D N A / m L was self-ligated by 200 U T4 D N A ligase (New England Biolabs) at 16 °C, and the inverse P C R reaction contained 1 p:g of self-ligated D N A , 0.5 \iM of each primer, and 1 m M M g C l 2 in P C R buffer provided with the Taq or Vent D N A polymerases. Inverse P C R was performed as follows: (i) denaturation at 95 °C for 3 min; (ii) 0.5 min at 95 °C, 1 min at 45 °C, and 3.5 min at 72 °C for 5 cycles; (iii) 0.5 min at 95 °C, 1 min at 40 °C, and 3.5 min at 72 °C for 30 cycles; (iv) a final incubation at 72 °C for 10 min to complete any unfinished single-stranded products. Fresh inverse P C R products were ligated to p C R II vector in T A P C R cloning kit (Invitrogen Inc), under conditions 32 recommended by the manufacturer. Competent E. coli DH5oc was transformed with ligation mixtures by standard procedure (Ausubel et al. 1992). The primers used were custom synthesized by the Nucleic Acid - Protein Service Unit at the University of British Columbia. 12. D N A sequencing Plasmid DNA or gel-purified PCR product were used as templates for double-stranded DNA sequencing, using the dye-terminator cycle sequencing kit (Perkin-Elmer). DNA sequencing reactions were performed according to the protocol developed by the Nucleic Acid-Protein Service Unit at the University of British Columbia. 13. Analysis of respiratory quinones Menaquinones were extracted overnight with a mixture of chloroform-methanol (2:1, v/v) from lyophilized Desulfovibrio gigas cells (Collins and Widdel 1986). The extract was then filtered to remove cell debris and evaporated to dryness at 37 °C in a rotary-evaporator. The dried extract was suspended in a small volume of acetone and loaded onto a Kieselgel 60F254 thin-layer chromatography plate (Merck). The thin-layer chromatography plate was developed with a mixture of hexane-diethyl ether (85:15 , v/v). Menaquinones separated on the thin-layer chromatography plate were revealed by brief irradiation with U V light (254 nm) and eluted from the silica gel with ethanol. After that, the ethanol eluant was further purified by HPLC with an ODS hypersil column (Hewlett Packard 5 p:m, 250 x 4 mm). Menaquinones were monitored at 270 nm and eluted with methanol-isopropyl ether (85:15, v/v) at 1 mL/min. The "quinoid" isolated from lyophilized D. tiedjei cells was extracted and separated from other lipids by thin-layer chromatography as described above. The silica gel eluant containing the "quinoid" was further purified by the same HPLC column, with the 33 mobile phase changed to a mixture of methanol-water (70:30 from 0 to 4 min; then, a linear gradient of increasing methanol proportion from 70:30 to 90:10, from 4 to 11 min; holds at 90:10 from 11 to 19 min; returns to 70:30 re-equilibrate from 19 to 25 min). Absorbance at 280 nm, rather than 270 nm was monitored Air-oxidized and sodium-borohydride-reduced U V absorption spectra, between 190 to 340 nm, of the HPLC-purif ied menaquinone isolated from D. gigas, the HPLC-pur i f ied "quinoid" isolated from D. tiedjei, and a mixture of ubiquinones extracted from activated sludge (a gift from Dr. H . Satoh, University of Tokyo, Japan) were measured with a Cary I E spectrophotometer. A n electron impact mass spectrum of the HPLC-purif ied "quinoid" was recorded by the Mass Spectrometry Lab at the Department of Chemistry of the University of British Columbia, following the conditions for analyzing menaquinones reported by Coll ins and Widdel(1986). 1 4 . Hydrogenase activity assay Hydrogenase activity was assayed by measuring the hydrogen-dependent reduction of methyl viologen at 37 °C. The assay was performed under anaerobic conditions in glass cuvettes sealed with butyl rubber stoppers. The 1-mL reaction mixture contained 100 m M Tr i s -HCl buffer (pH 8.8) and 5 m M methyl viologen. The headspace of the cuvette was hydrogen. Anaerobic conditions were obtained by the injection of a few drops of freshly prepared 0.5 m M sodium dithionite in water until the methyl viologen was slightly reduced and turned the reaction mixture pale blue. The reaction was started by injection of cellular fractions or whole cells into the cuvette. Methyl viologen reduction was followed at 582 nm spectrophotometrically (e 5 8 2 = 9,600 M ' 1 cm' 1). One unit of hydrogenase activity was defined 34 as the amount of enzyme catalyzing the reduction of 2 umole methyl viologen with hydrogen per min under these conditions. 15. Formate dehydrogenase assay Formate dehydrogenase was assayed by measuring the formate-dependent reduction of methyl viologen at 37 °C. The assay was performed under anaerobic conditions in glass cuvettes sealed with butyl rubber stoppers. The 1-mL reaction mixture contained 50 m M Tr i s -HCl buffer (pH 8.0), 5.0 m M methyl viologen and whole cells or cellular fractions. The headspace of the cuvette was nitrogen. Anaerobic conditions were obtained by the injection of a few drops of P-mercaptoethanol until the methyl viologen was slightly reduced and turned the reaction mixture pale blue. The reaction was started by injection of 0.1 m L of sodium formate solution into the cuvette from a 10X (100 m M ) anoxic stock. Methyl viologen reduction was followed at 582 nm (e 5 8 2 = 9,600 M ' 1 cm"1). One unit of formate dehydrogenase activity was defined as the amount of enzyme catalyzing the reduction of 2 Umole methyl viologen with formate per min under these conditions. 16. A T P sulfurylase activity assay A T P sulfurylase activity was assayed by measuring adenosine phosphosulfate (APS) - and pyrophosphate (PPj)-dependent A T P production in a coupled spectrophotometric test. A T P production was measured with a kit which measured A T P production (Sigma Chem. Co.) via the phosphorylation of 3-phosphoglycerate and subsequent reduction of 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate by N A D H . The A P S and sodium pyrophosphate were also purchased from Sigma. The assay was performed under air in a quartz cuvette. The 1.02 m L reaction mixture contained 0.5 m L 3-35 phosphoglycerate buffered solution with 0.15 mg N A D H , 0.125 m L of 2 m M stock A P S solution, 0.125 m L 2 m M stock sodium pyrophosphate solution, 0.02 m L phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase mixture, and 0.25 m L of D. tiedjei cell fractions. Oxidation of N A D H was followed as decrease in absorbance at 340 nm (e 3 4 0 = 6220 M^cm" 1 ) . One unit of A T P sulfurylase activity was defined as the oxidation of 1 pimole of N A D H per minute under these conditions. 17. A P S reductase activity assay A P S reductase activity was assayed under aerobic conditions according to Odom and Peck (1981), by measuring sulfite- and AMP-dependent reduction of ferricyanide at 420 nm ( £ 4 2 0 = 990 M^cm" 1 ) . The 1-mL reaction mixture contained 20 p L 1.5 M sodium sulfite, 20 u L 2 M A M P , 10 u L of 100 m M K 3 [Fe (CN) 6 ] , 450 u L 100 m M Tr i s -HCl buffer (pH 7.7) and 500 p L of a D. tiedjei cell fraction. One unit of A P S reductase activity was defined as the reduction of 2 pimole of ferricyanide per minute under these conditions, after correction for non-enzymatic reduction of ferricyanide. 18. Desulfoviridin quantification Desulfoviridin was spectrophotometrically identified \nD. tiedjei cellular fractions by its unique absorption at 630 nm and was quantified according to the method of Badziong and Thauer (1980). 19. Reductive dehalogenase activity assay Reductive dehalogenase activity mD. tiedjei cell extracts was assayed according to the method of N i et al. (1995). The 100-p i reaction mixture contained 50 m M potassium 36 phosphate buffer (pH 7.8), 0.5 m M 3 C B , 1 m M dithiothreitol, 0.1 m M methyl viologen, 0.3 m M 2,4-dichlorobenzoate as an internal standard and the cell extracts (1.6 mg protein). The reaction mixture was mixed in a miniature glass tube placed in an 1.5-mL H P L C sample vial. The H P L C sample vial was in turn placed in a 10-mL serum bottle, with a crimp-sealed butyl rubber stopper, inside an anaerobic glove box. The serum bottle was then taken out of the anaerobic glove box, and the headspace was exchanged with hydrogen. Hydrogenase present in the cell extracts then reduced the methyl viologen, which functioned as an artificial electron donor for the reductive dehalogenase. The reaction mixture was incubated at 37 °C in darkness. Mult iple reaction mixtures were prepared for sampling at different time points. The reaction was stopped by precipitating the proteins with 1% ice-cold trichloroacetic acid. The supernatant was then analyzed for benzoate production by H P L C . 20 . A n a l y t i c a l me thods Reductive dehalogenation of 3 C B to benzoate was analyzed by an H P L C equipped with an O D S hypersil column (Hewlett Packard 5 pm, 150 x 4 mm), according to the method of M o h n and Tiedje (1990a). Sulfate consumption in sulfate-respiring cultures was analyzed by an H P L C equipped with an anion chromatography column (Hewlett Packard, 125 x 4 mm) operated under manufacturer-suggested conditions. Formate consumption was analyzed with an H P L C equipped with an A m i n e x H P X - 8 7 H column (Bio-Rad, 300 x 7.8 mm). Formate was monitored at 220 nm and eluted with 0.018 M H 2 S 0 4 , at 65 °C, at 1 mL/min. Protein was determined by the bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin as the standard. 37 21. Chemicals A l l reagent grade chemicals, and sodium salts of A M P , A P S , PP ; , methyl viologen, potassium ferricyanide, propyl iodide, and 2-heptyl-4-hydroxyquinoline N-oxide were purchased from Sigma. 3 C B and 2,4-dichlorobenzoate were purchased from Aldr ich Chemical. A l l o f the organic solvents were purchased from Fisher Scientific Co. 38 RESULTS C H A P T E R O N E Cytochromes of D. tiedjei 1. Introduction Cytochromes c with a variable number of heme groups are electron carriers commonly found in sulfate-reducing bacteria. Moreover cytochromes b have also been detected in Desulfotomaculum sp., and those Desulfovibrio sp. which are capable of using fumarate as alternate terminal electron acceptor. A s a consequence, it is reasonable to expect that some cytochromes function as electron carriers in the D. tiedjei respiratory electron transport system. Stevens etal. (1988) previously detected cytochrome c in D. tiedjei cells grown with thiosulfate as terminal electron acceptor, and DeWeerd et al. (1990) reported purifying a tetraheme cytochrome c 3 from D. tiedjei. Despite the numerous reports that bacterial respiratory cytochrome patterns change with the growth conditions, no one had compared the cytochrome profde of D. tiedjei cells grown under different metabolic conditions or investigated whether specific cytochromes were co-induced with reductive dehalogenation. Therefore, I investigated these questions more closely. 2 . Cytochromes in different cellular fractions Oxidized and reduced visible absorption spectra of soluble and membrane fractions of D. tiedjei cells grown under reductive-dehalogenating, pyruvate-fermenting and sulfate-respiring conditions were measured. The reduced-minus-oxidized spectra o f soluble and membrane fractions isolated from cells grown under different conditions were very similar to those of cytochromes c, with a , (3 and y peaks of absorption at 552, 522 and 419 nm, 39 respectively (Fig. 2). In addition, cell extracts of the three types of cultures were extracted with acidified acetone, and the heme groups of the cytochromes were non-extractable, in agreement with their identification as c-type, rather than 6-type, cytochromes. From the reduced-minus-oxidized absorption spectra, specific quantities of cytochromes in different fractions ofZ). tiedjei cells grown under different metabolic conditions were estimated (Table II). Membrane fractions of dehalogenating and fermenting cells had 3 to 4 times more cytochromes than that of sulfate-respiring cells. In addition, membrane fractions o f cells of the three types of cultures had significantly more cytochromes than the soluble fractions. However, the visible absorption spectra did not clearly indicate whether different cytochromes c were present in cells grown under different conditions. Therefore, cellular fractions of D. tiedjei cells grown under the three conditions were loaded onto S D S - P A G E gels and heme-stained directly. Different cytochromes c appear as different bands on heme-stained S D S - P A G E gels. Two D. tiedjei cytochromes were detected in membrane fractions solubilized by 1% SDS. A cytochrome with apparent molecular mass of 50 kDa, was present only in the membrane fractions of D. tiedjei cells induced for reductive dehalogenation (Fig. 3A) . A more abundant cytochrome of apparent molecular mass 17 kDa was present in membrane fractions of cells grown under reductive-dehalogenating, pyruvate-fermenting and sulfate-respiring conditions. This 17-kDa cytochrome was also the only type of cytochrome stained positively in the cytoplasmic (Fig. 3B) and periplasmic fractions of cells grown under these growth conditions. 3. Purification of the 50-kDa inducible cytochrome A majority of membrane cytochromes were extracted from membrane fractions of dehalogenating cells by 10 m M Tris -HCl buffer (pH 7.7) containing 35% saturation of 40 0.05 | -0.05 -0.1 --0.15 -0.2 400 440 480 520 560 600 Wavelength (nm) 400 440 480 520 560 600 Wavelength (nm) Fig . 2 Reduced-minus-oxidized absorption spectra of cytochromes in (A) soluble fractions, and (B) membrane fractions of D. tiedjei cells grown under reductive-dehalogenating (—), pyruvate-fermenting (—), and sulfate-respiring {—) conditions. Table II. Specific quantities of cytochromes in different cellular fractions o f D. tiedjei cells grown under reductive-dehalogenating, pyruvate-fermenting, and sulfate-respiring conditions (ug cytochrome c/mg total protein, based on £ 4 1 9 = 40 mJVf'cm"1). reductive-dehalogenating conditions pyruvate-fermenting conditions sulfate-respiring conditions Periplasmic fraction 2.85 1.96 1.99 Cytoplasmic fraction 4.55 8.00 4.22 Membrane fraction 26.42 37.27 8.94 41 I 2 3 97.4 — 66.2 — 45.0 — 31.0 — B 97.4 66.2 45.0 31.0 21.5 14.4 Fig . 3. Heme-stained S D S - P A G E gels of (A) membrane fractions and (B) cytoplasmic fractions of D. tiedjei cells grown under the following conditions: (1) sulfate-respiring; (2) pyruvate-fermenting and (3) reductive-dehalogenating. Cytochrome contents in these fractions were estimated by the absorbance of the y-peaks of the reduced-minus-oxidized spectra and calibrated against horseheart cytochrome c. The amount of cellular fractions loaded onto the S D S - P A G E gels were adjusted such that each lane was loaded with equal amount of cytochromes (equivalent to about 11 pig of horseheart cytochrome c). The numbers at the left are molecular size markers in kDa. 42 ammonium sulfate. Left-over membrane materials were solubilized with 1% SDS, loaded onto S D S - P A G E gels and heme-stained to check the extraction efficiency. Only a small amount of the 17-kDa cytochrome, and none of the 50-kDa inducible cytochrome remained in membranes after the extraction. The inducible cytochrome was sequentially purified to electrophoretic homogeneity by phenyl superose hydrophobic interaction chromatography, Mono Q anionic exchange,and hydroxyapatite chromatography (Fig. 4). About 105 pg of purified protein was recovered from 20 g wet cells. During S D S - P A G E , the purified protein migrated as a single band corresponding to an apparent molecular mass of 50 kDa (Fig. 4). When the purified protein was chromatographed by an F P L C equipped with a Superose-6 gel filtration column, the apparent molecular mass of the inducible cytochrome was determined to be 155 kDa. 4. Visible absorption spectra of the inducible cytochrome The inducible cytochrome displayed high-spin absorption spectra. The purified inducible cytochrome was in the oxidized form with a broad Soret peak at 399 nm (Fig. 5A). Reduction of this cytochrome by sodium dithionite resulted in a shift of the Soret peak to 423 nm. The shape of the Soret peak also changed. A shoulder at 430 nm appeared. In addition, a low, broad a-peak at 555 nm appeared upon reduction. These spectral characteristics are similar to those of high-spin cytochromes c' (Bartsch and Kamen 1960 ; Imaie/a / . 1969 ; Saraivae/a/. 1995 ; Yamanakaand Imai 1972) which have been shown to display two major spectral changes at p H 10 and 12 (Imai etal. 1969 ; Monkara et al. 1992). Therefore, a p H titration was performed to compare the purified inducible cytochrome to cytochromes c'. N o significant changes in absorption spectra occurred until the p H was raised above 12 (Fig. 5B). When the p H reached 13.2, the inducible cytochrome had an oxidized 43 absorption spectrum similar to that of low-spin eukaryotic ferricytochrome c, with a narrow and symmetric Soret peak at 413 nm plus a broad oc-peak at 540 nm. When reduced by sodium dithionite at p H 13.2, the spectrum changed to that of a reduced typical low-spin cytochrome c, with the Soret peak shifted to 416 nm. Distinctive a and (3 peaks at 549 and 521 nm, respectively, also appeared. Hence, the D. tiedjei inducible cytochrome only displayed one pH-dependent spectral change at p H 13.2 and thus is different from cytochromes c'; although, it is a high-spin cytochrome. 5. Midpoint potential determination for the inducible cytochrome Since the 50-kDa c-type cytochrome was co-induced with reductive dehalogenation, it would be important to determine whether it is the direct electron donor to the reductive dehalogenase in vivo. Therefore, I decided to measure the midpoint potential of this inducible cytochrome, as the midpoint potential would suggest its relative position among other electron carriers in the respiratory electron transport chain. The purified inducible cytochrome was first titrated chemically with sodium dithionite as reductant over a wide range of redox potentials and the midpoint potential was estimated to be -340 m V (Fig 6). Then, photoreducing an equilibrated mixture of inducible cytochrome and 8-hydroxyriboflavin with E D T A , gradual reduction of the inducible cytochrome was controlled more precisely and the midpoint potential was calculated to be -342 m V at p H 7.0 (Fig. 7). 6. NH 2-terminal sequence of the inducible cytochrome The NH 2 -terminal amino acid sequence of the purified inducible cytochrome was determined to be E S K K V P S S Y S P V V I T E P F D S M T R M K A A K P . Based on this amino acid sequence, two forward degenerate P C R primers (50N1 and 50N2) and one reverse degenerate 44 1 2 3 4 5 97.4 — 31.0 — 21.5 — 14.4 — Fig . 4. S D S - P A G E of the successive purification steps of the D. tiedjei inducible cytochrome. Lane 1: crude membrane fraction solubilized by 1.0% SDS (10 pg protein); lane 2: 35% ( N F L ; ) 2 S 0 4 membrane extract (3.0 pg protein); lane 3: Phenyl Superose 25% eluant (3.0 pg protein); lane 4: Mono Q unbound fraction (3.4 pg protein); lane 5: Hydroxyapatite 300 m M sodium phosphate buffer eluant (1.0 pg protein). The numbers at the left are molecular size markers in kDa. 45 Fig . 5. (A) Oxidized and reduced absorption spectra o f 0.75 u M purified D. tiedjei inducible cytochrome in 100 m M sodium phosphate buffer (pH 6.5). (B) Gradual spectral changes o f 1.5 u M purified D. tiedjei inducible cytochrome being titrated by 1 M sodium hydroxide solution. 46 400 500 600 700 Wavelength (nm) Fig . 6. Spectral changes of the D. tiedjei inducible cytochrome when reduced by freshly prepared sodium dithionite solution. The number associated with each absorption spectrum is the measured redox potential in mV. 47 Fig . 7. Spectral changes observed from 350 to 650 nm during photoreduction, by 8-hydroxyriboflavin, o f D. tiedjei inducible cytochrome. The arrows show the direction o f changes in regions o f the absorption spectrum, as the inducible cytochrome (A and C) and the 8-hydroxyriboflavin (B) were reduced. Insert: Nernstian plot o f the redox titration. The experimental points are denoted by solid squares. The straight line is drawn according to a 59 , f o x d ^ theoretical Nernst equation, Eh = Emj +—log n red with n = 2. 48 P C R primer (50C) were designed from both ends of the NH 2 -terminal protein sequence (Fig. 8). A D N A sequence encoding the NH 2-terminal protein sequence was successfully amplified from D. tiedjei genomic D N A by P C R , with primer pairs 50N2 and 50C (Fig. 9), and the expected 89-bp P C R product was purified from the agarose gel and cloned. Two clones with inserts of the correct size were isolated, and those inserts were sequenced. In both cases, the encoded protein sequences of the inserts matched the NH 2 -terminal protein sequence. 7. Putative gene sequence of the inducible cytochrome A Southern blot analysis of D. tiedjeiDNA cleaved with various restriction enzymes was probed with the above 89-bp cloned fragment. The end-labeled 89-bp probe hybridized to a singleSstI fragment of 1.2 kb in size (Fig. 10). However, attempts to clone this SstI fragment from a genomic cosmid library and a partial genomic library of SM-digested D N A of 1 to 2 kb in size, by colony hybridization with the same 89-bp probe were unsuccessful. Therefore, the gene and flanking D N A sequences were cloned by inverse P C R . D. tiedjei genomic D N A was cleaved by SstI and self-ligated. The circularized D N A was then used as template for inverse P C R with primers TA2F2 and T A 2 R 2 . A linear inverse P C R product of the expected 1.2 kb size was cloned and named ISST18 (Fig. 11). D N A sequencing of clone ISST18 revealed a 1151-bp insert flanked by SstI sites (Fig. 12). The D N A sequence encoding the NH 2 - terminal amino acid sequence of the inducible cytochrome was identified in the insert. Directly 5' to the NH 2-terminal D N A sequence was a D N A sequence encoding a putative 26-amino acid signal peptide, which began with a putative G U G start codon. A putative ribosome binding site was identified about 10 bp 5' to the start codon. In addition, a c-type heme-binding site, C F D C H , was identified. N o stop codon was identified within this O R F . Also , the encoded product of this inverse P C R product was smaller than the apparent 49 molecular mass of the inducible cytochrome determined from S D S - P A G E . Therefore, the 3' end of the gene was not present in clone ISST18. A second D N A fragment containing the entire sequence of the inducible cytochrome gene was cloned by a second inverse PCR. A 719-bp P C R product amplified with primers 18a and 18b (Fig. 12) from pISST18 was labeled as a probe for another Southern blot analysis. This probe hybridized to a single 2.2-kb MwwI-cleaved genomic D N A fragment (Fig. 13). A second inverse P C R with self-ligatedMw«I-cleaved D N A as template and primers 18a and 18c yielded an expected 2.2-kb inverse P C R product and another major non-specific product of 2.7 kb in size (Fig. 14). Shotgun cloning of this mixture of inverse P C R products yielded two recombinant clones, I M U N 3 and I M U N 2 2 , with the desired product. The insert of p I M U N 3 was sequenced and contained the D N A sequence identified previously from the insert of clone ISST18, plus 1183 bp of sequence 3' to the SstI site within the gene encoding the inducible cytochrome (Fig. 12). A second c-type heme-binding domain, C A V C H , and a U A A stop codon were identified. The complete O R F was therefore 1398 bp long, encoding a 466-amino acid product. The molecular mass of the encoded product, excludingthe putative signal peptide, was calculated to be 49,722 D a and closely matched the apparent molecular mass of 50 kDa determined by S D S - P A G E . The gene and protein sequences were not found to have substantial similarity to any sequences in GenBank. The NH 2 - terminal half of the protein sequence was also compared to GenBank sequences independently. It was also not similar to any Genbank sequences. N o other conserved redox-center-bindingdomain could be identified within the complete protein sequence. In addition, hydropathy analysis did not identify any apparent transmembrane domain within the protein sequence (Fig. 15). The gene of this inducible cytochrome was named hsc. 50 A 335-bp D N A sequence with significant sequence similarity to ferric uptake regulator (FUR) protein sequences in GenBank was identified about 275 bp beyond the 3' end of the hsc gene. This suggests the presence of another O R F encoding a F U R - l i k e protein directly downstream of the hsc gene. 51 GAR TCN AAR AAR GTN CC (50N2) G7AR AGY 7A7AR AAR GTN CC (50N1) • G l u S e r L y s L y s V a l P ro Ser Ser T y r Se r P ro V a l V a l l i e Thr G l u P r o Phe A s p Se r l i e Met Thr A r g Met L y s A l a A l a L y s P r o 4 TAC TTY CGN CGN TTY GG ( 50C) Fig . 8. The NH2-terminal protein sequence of the D. tiedjei inducible cytochrome. Forward degenerate P C R primers 50N1 and 50N2, and reverse degenerate P C R primer 50C were designed according to the protein sequence and were used to PCR-amplify the corresponding D N A sequence from D. tiedjei genome. (N: A or T or G or C; R: A or G ; Y : T or C) 52 1 2 3 Fig . 9. A n 89-bp P C R product which encoded the NH 2-terminal protein sequence o f the D. tiedjei inducible cytochrome. Lane 1: molecular size marker; lane 2: mixture of P C R products amplified from D. tiedjei genomic D N A with degenerate primers 50N2 and 50C; lane 3: the purified 89-bp P C R product which was cloned and sequenced. 53 1 2 3 4 5 6 7 12.0-10.0 — 8.0-6 .0 -5 .0-4 . 0 -3 . 0 -2 . 0 -1.6-1.0-Fig . 10. Southern blot analysis of D. tiedjeiDNA digested with restriction enzymes and probed with the end-labeled 89-bp P C R product which encoded the NH 2 -terminus of the inducible cytochrome. Each lane contained 1 pg of restriction-digested genomic D N A . Lane 1: BamHl; lane 2: Hindlll; lane 3: EcoRl; lane4 Kpnl; lane 5 Stul, lane 6 Sstl and lane 7: Smal. The numbers at the left are molecular size markers in kb. 1 2 Fig . 11. A 1.2-kb inverse P C R product amplified from &fl-digested, self-ligated D. tiedjei genomic D N A with primers TA2F2 and TA2R2. This inverse P C R product contains a partial putative gene sequence of the inducible cytochrome c. Lane 1: molecular size markers in kb and lane 2: the inverse P C R product. 5 4 Fig . 12A 1 CATCTTCCGA AATCCTGGAA GAGAGGGCAT TAGTAACTAA CCCGACAAAC Muni rbs start 51 AATTGGAAAA GGAGGTACTT TAGTGAAATT CGTACGACCC AAGTTAATCT ( M K F V R P K L I 101 TTGTTATGGC CTGCTGCATG ATTGTGGCTC TTGCTGGATT GATCTATGCT F V M A C C M I V A L A G L I Y A ) ^TA2F2 151 GAATCCAAGA AGGTCCCAAG CAGTTATTCT CCGGTTGTCA TCACTGAGCC E S K K V P S S Y S P V V I T E TA2R2 201 CTTTGATTCG ATTATGACTC GGATGAAGGC AGCCAAACCT GAAATCGAGA P F D S I M T R M K A A K P E I E 251 AGAAACATAC CGACCTGCTC AGTTCTCGTT ACGATCTCAG CAATAAGCCG K K H T D L L S S R Y D L S N K P Smal 301 GCCCAGGGCG TTACTATGTC CCGGGGGAAG GCAATACAGG AAGGCGTGCG A Q G V T M S R G K A I Q E G V Hindi I I 18b^ 351 AGTCAAGCTT CCTCAGGGCG GCGTTACATG GGAGCAGCTC GCTGCCTTGA R V K L P Q G G V T W E Q L A A L 401 CACCCGAGCA GATTAAAGAA AAGAATGTAT GGCCTGAAGG CTTCTATCCG T P E Q I K E K N V W P E G F Y P 4 51 CTTCCTCATC CGAATCATCC TGAAGGCGGC ATGGTCTTCC CGAAGACGCA L P H P N H P E G G M V F P K T 501 CATAGAGGAA ATCAAGAAAC AGGAGCAAAG AGACCTCACG CGCTTCGACC H I E E I K K Q E Q R D L T R F D 551 TGGATTTCGA CCTGCCGGAC CACGTTTTGC CGGAATCTCC TGCTGCAATC L D F D L P D H V L P E S P A A I 601 CTTTTGACAA CAAGACCGGA CCTTGGGGAC GTTTCCAAGG GTAAACTGGT L L T T R P D L G D V S K G K L 651 GACCATCGAC AATTACTTCG AGCTATTTAA CGGAATTCTT AACCCCAAAC V T I D N Y F E L F N G I L N P K 7 01 AGCTTGAGGG TCTCAGACTT CTTGTTACTC CATTTCCTCA GCAGCAGTTC Q L E G L R L L V T P F P Q Q Q F 55 Fig . 12A continued 7 51 A A C C A G A C G G A C G A T C G T C G T T C C G A A A A G C C T T C T C G T G G A G T C A C C T G N Q T . D D R R S E K P S R G V T 801 T T T T G A T T G T C A C G C A A A C G G G C A T A C G A A C G G G G C T A C T C A C C T G G T G G C F D C H A N G H T N G A T H L V 851 G T G A C A T T C G A C C C C A G G A A T T T C G C C A T C G A C T C G A C A C T C C T A C A C T G G D I R P Q E F R H R L D T P T L 901 C G A G G A G T C A A C A T T C A G C G T T T G T T C G G T T C T C A A C G C G C C C T G A A G A G R G V N I Q R L F G S Q R A L K 951 T G T C G A G G A T T T C A C T G A A T T C G A A C A G C G T G C G G C T T A T T T C G A C G G C G S V E D F T E F E Q R A A Y F D G 1001 A T C C T G T C A T A G C A A C C A A G A A A G G C G T C A A C G T T C T G G A GCGGGGCAGT D P V I A T K K G V N V L E R G S ^ 18a 18c ^ -4 • 1051 C A G G T G C A T T T C A T G G C T G A A T T C C A G G A G C T T C T T G A C T T T C C A C C T G C Q V H F M A E F Q E L L D F P P S s t I 1101 C C C C A A G T T G G A T A T C T A T G G G A A A C T C G A T C C C C A A A A G G C A T C C G A G C A P K L D I Y G K L D P Q K A S E 1151 T C G A A C T A A A GGGTCAGGAA G T G T T T T T C G G A A A A G C C A A A T G C G C A G T G L E L K G Q E V F F G K A K C A V 12 01 T G T C A T C C T G C C C C T T A C T A T A C G G A T A A C C T C A T G C A C A A T C T G A A A G C C H P A P Y Y T D N L M H N L K 1251 C G A G C G A T T C T T C A A A C C G A A A A T G A T A A A C G G A A G A A T G G C T T C C G C T G A E R F F K P K M I N G R M A S A 1301 A C G G A C C G A T C A A G A C C T T T C C C C T C A G A G G C A T C A A A G A G T C G C C T C C G D G P I K T F P L R G I K E S P P 1351 T A T C T T C A T G A C G G C A G G T T G A T C A C A C T T G A A G A C A C A G T A G A A T T T T T Y L H D G R L I T L E D T V E F 14 01 C A A C C T C A T T C A G G G T C T C A A C T T G A A T G C A G A C G A G A A A A A G G C T C T C G F N L I Q G L N L N A D E K K A L s t o p 1451 T T G C C T T C A T GAGGGCGCTG T A A A C C C A T A A A C G T T C G G C A G G A T C G G T G V A F M R A L 56 Fig. 12A. continued 1501 CCAACCTGCT GTCACAGCCC 1551 TGAAGGATCT TCATTTCGAA 1601 AATTGAGGCT ACAGAACGTG 1651 AGTCCAGAAA TCAAAAAATT 17 01 GCTATCAAAG TTACGCCTCA 1751 CTGCCAACGA TCATCCCTCG 1801 AGACTGCCTA CTGTTTCACT 1851 TGATGAGCAA GGATTGATCG 1901 GGTTTGACCC GAATACTCAG 1951 GGCAGTATTA CGGATTTCGT 2001 TTCCGAAGTA GAGGGATGGG 2051 GTGGAGTATG CTCACGCTGT 2101 GATGCTGATA CCAGTCCCAA 2151 ATTAGACAGA TATTGTTGCT 2201 AAAAAGGACG GTCATTTCAG 2251 TTATACGAAT TGTCCGAAAG 2301 CCGGCCTCCA TGTCGGCGGA ATCATCCTTC AAAGGTCGTT TGAACACCTC CTGCCGGGGG AACCAGGAAA CTTTTCGCGA AAGTACTTAC AAGGATTTCT TCAGTGAAAC GAGATGTTTC GCACTGTTTG TAAAGAGCAC GCGCCTTGAG ATTTTCCCTT GAAGTTATTT GCTGAAGAGA TATTCAGACG TGTACAAAAA CGATACGGTG TACAGAACGC TCAGCACGTT CCAAAGTTCA TTTTCTTGAT GACAAAACGC CAACAT CAT C ACATGAGCTG TATCAAATGC ATGGCCGGAA ATTGACGCGA TGTCCCTCCC GTAAAGTGAG CGACCGACAC GTGCTCGTTC GCGGATAAAA TCGATCGCGG AGACGGCAAA CGATTCATAA ATTGCGTACA GATATTGTTG CCATTATTGC CGGCCTGCAA GCCGATTACA GTAAGTAGTT TTCTTCAATA TTCCTTAGTC AGTGCCGATT GACAAAAGCA TTGGTAGGTG TGTTCAAAAT AATC Fig. 12B. Sstl Muni Sstl Muni =m rbs II Heme 1 fl Heme 2 Signal peptide NHs-terminal sequence Fig. 12. The putative nucleotide sequence (A) and a physical map (B) of the D. tiedjei inducible cytochrome c gene (hsc). The deduced protein sequence is shown using the one-letter amino acid code below the gene sequence. Nucleotide numbering starts at the last C deoxynucleotide of an Sstl site and proceeds to the left through a Muni site, a Smal site, a HindUl site, another Sstl site, and ends with the first C deoxynucleotide of a Muni site. Sequence between position 1-1151 represents the insert of inverse PCR clone ISST18. Sequence between position 51-2334 represents the insert of inverse PCR clones IMUN3 and IMUN22. The ORF was identified between position 73-1470. The putative ribosome binding site (rbs), the putative start codon, and an U A A stop codon are underlined. TA2F2 and TA2R2 are inverse PCR primers used to obtain the inverse PCR clone ISST18, while 18a and 18c are inverse PCR primers used to obtain inverse PCR clones IMUN3 and IMUN22. A putative 26-amino acid signal peptide is in parentheses. The NH2-terminal protein sequence is bold. Two c-type heme-binding domains, at position 798-813 and position 1192-1206, are bold and underlined. 57 1 2 3 4 5 6 7 8 9 10 12.0-10.0-8.0-6.0-5.0-4.0-3.0-2.0-1.6-12.0 -10.0 — 8 . 0 -6.0 5.0 4.0 3 . 0 -2 . 0 -1.6-I 1.0-s. Fig. 13. Southern blot analysis of D. tiedjei DNA digested with restriction enzymes and probed with the 719-bp PCR product amplified from clone ISST18 with primer pairs 18a and 18b. Each lane contained 1 pig of restriction-digested genomic DNA. Lane l.BamHl, lane 2: StuI, lanes 3 & 10: Smal, lane 4: BamHl + Smal, lane 5: BamHl + Kpnl, lane 6: BamHl + Stul, lane 7: Smal + Kpnl, lane 8: Muni, lane 9: Hindlll. Fig. 14. A 2.2-kb inverse PCR product amplified from MwwI-digested, self-ligated D. tiedjei genomic DNA with primer pairs 18a and 18c. The 2.7-kb band is a non-specific PCR product. Lane 1: molecular size marker in kb and lane 2: the inverse PCR product. 5 8 Fig. 15. Hydropathy analysis of the deduced protein sequence of the D. tiedjei inducible cytochrome. The hydrophobicity scale increases from least hydrophobic (negative number) to most hydrophobic (positive number). 59 8. Summary Only c-type cytochromes were present in the soluble and membrane fractions of D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation and sulfate reduction. However, specific amounts of cytochrome c varied with growth conditions. Membrane fractions of reductive-dehalogenating and pyruvate-fermenting cultures had 3 to 4 times more cytochrome than that of sulfate-respiratory cultures. Also , much more cytochrome was detected in the membrane fractions than the soluble fractions. A 17-kDa cytochrome c was found in the periplasmic, cytoplasmic and membrane fractions of D. tiedjei cells grown under the three metabolic conditions, but an inducible 50 kDa cytochrome c was present only in the membrane fractions of reductive-dehalogenatingZ). tiedjei cells. Although this inducible cytochrome displayed a high-spin absorption spectrum, it is not a cytochrome c' according to a p H titration experiment. The midpoint potential of the inducible cytochrome at p H 7.0 was -342 mV. The NH 2-terminal amino acid sequence of the inducible cytochrome was determined and was used to obtain inverse P C R products containing the putative sequence of the gene encoding the inducible cytochrome. The inducible cytochrome is a diheme cytochrome because two c-type heme-binding domains were identified in the C O O H -terminal half of the protein. The gene and protein sequences were not found to have substantial similarity to any other sequences in GenBank, suggesting this cytochrome c is substantially different from previously characterized ones. 60 C H A P T E R T W O A putative respiratory quinone of D. tiedjei 1. Introduction Quinone molecules are common electron carriers found in eukaryotic and prokaryotic respiratory electron transport chains. Collins and Widdel (1986) examined 45 strains of Gram positive and Gram negative sulfate-reducing bacteria, and 5 strains of sulfur-reducing bacteria. Their results showed that all strains possessed menaquinones with different numbers of isoprenoid subunits. N o ubiquinones were detected. A s D. tiedjei is also a member of the group of sulfate-reducing bacteria, it is reasonable to suspect D. tiedjei possesses a menaquinone as an electron carrier. In addition, Apajalahti et al. (1989) discovered that reductive dehalogenation of D. tiedjei in defined media required rumen fluid or filtered-culture fluid of Propionibacterium sp. as a nutrient supplement. The essential factor in the culture fluid was believed to be a quinoid compound. Later, DeWeerd et al. (1990) showed that a vitamin mixture including 1,4-naphthoquinone or menadione could replace the Propionibacterium culture fluid for culturingZ). tiedjei in defined media, although the effect of 1,4-naphthoquinone on reductive dehalogenation was not tested. 1,4-naphthoquinone and menadione are very similar in structure to the aromatic nucleus of menaquinones. This finding leads to the hypothesis that 1,4-naphthoquinone may be used as a precursor for synthesizing a menaquinone, which functions as an electron carrier coupled to reductive dehalogenation. However, no one had extracted a respiratory quinone directly from D. tiedjei. A s a consequence, I performed different experiments to test the involvement o f a respiratory quinone coupled to reductive dehalogenation in the D. tiedjei electron transport chain. 61 2. Effects of individual vitamins on reductive dehalogenation Reductive dehalogenation was dependent on 1,4-naphthoquinone whereas thiamine stimulated growth and reductive dehalogenation. D. tiedjei cultures were grown and transferred five times in defined medium supplemented with a vitamin solution containing thiamine, 1,4-naphthoquinone, lipoic acid and nicotinamide, or with a vitamin solution deficient in a specific component. Serial transfer of these cultures should have minimized the effects of vitamins carried over from the original inocula. Lipoic acid deficiency had no effect on growth or reductive dehalogenation (Fig. 16). 1,4-naphthoquinone-deficient cultures grew slower than the control and had no dehalogenation activity. The dependency of reductive dehalogenation on 1,4-naphthoquinone was therefore confirmed. The growth rate of thiamine-deficient culture was comparable to that of 1,4-naphthoquinone, but slower than the control. In addition, reductive dehalogenation activity was partially inhibited in the thiamine-deficient culture. So, thiamine has a stimulatory effect on growth and reductive dehalogenation of D. tiedjei. 3. Effect of 2-heptyl-4-hydroxyquinoline /V-oxide (HQNO) on reductive dehalogenation M y data suggested that a respiratory quinone is coupled to reductive dehalogenation in D. tiedjei. H Q N O is a common respiratory quinone inhibitor. In this study, washed cell suspensions of D. tiedjei were incubated under a N 2 atmosphere at 37 °C for 2 days without any electron donor. The purpose of this treatment was to deplete any endogenous energy source and reducing power. Then, 2 m M sodium formate plus H Q N O were added to the cell suspensions, and reductive dehalogenation of 3CB was measured. A t a concentration of 150 nmole of H Q N O per mg of protein reductive dehalogenation was inhibited by about 50% 62 0.160 B Length of incubation (days) Fig. 16. Growth curves (A) and reductive dehalogenation activity (B) of D. tiedjei after five transfers in medium deficient in a specific vitamin component. (•) culture with no vitamin deficiency, (X) culture deficient in lipoic acid, (A) culture deficient in thiamine, and ( • ) culture deficient in 1,4-naphthoquinone. 63 (Fig. 17). Ten times more H Q N O completely inhibited reductive dehalogenation. 4. Purification of a putative respiratory quinone A quinone-like molecule, which was different from common respiratory quinones in absorption spectra, was purified from D. tiedjei. Neither menaquinone nor ubiquinone were extracted from D. tiedjei. The amount of lyophilized cells used for extraction and the extraction time were varied, but neither menaquinone nor ubiquinone were detected. Instead, an UV-absorbing, quinone-like molecule, or quinoid, was purified. Approximately two fold more quinoid was extracted from cells grown under reductive-dehalogenatingand pyruvate-fermenting conditions, than from sulfate-reducing cells. The oxidized and reduced U V absorption spectra of this quinoid were very different from those of menaquinone purified from Desulfovibriogigas (Fig. 18). The quinoid was purified in the oxidized form and had a peak of maximal absorbance at 280 nm. This oxidized UV-absorption spectrum was similar to that of oxidized ubiquinone, which had a peak of maximal absorbance at 275 nm. When the quinoid was reduced with sodium borohydride, the peak of maximal absorbance decreased in size and shifted to about 283 nm, and another peak of maximal absorbance appeared at about 308 nm. However, when ubiquinone was reduced with sodium borohydride, the peak of maximal absorbance decreased in size and shifted to 280 nm, but no new peak appeared. Therefore, the reduced U V spectrum of the quinoid is only weakly similar to that of ubiquinone. 5. Mass spectrometric analysis of the quinoid The quinoid was different from common bacterial respiratory quinones in chemical structure. From a mass spectrometric analysis, the molecular ion of the quinoid was identified 64 Length of incubation (hours) Fig. 17. Effect of 2-heptyl-4-hydroxyquinoline iV-oxide (HQNO) on reductive dehalogenation activity of D. tiedjei cell suspensions. (•) no HQNO, ( X ) 150 nmole HQNO/mg protein, (A) 1.5 pmole HQNO/mg protein, ( • ) boiled cell suspensions. Data are means of triplicates with standard errors (bars). 65 230 270 310 350 390 230 270 310 350 390 230 250 270 290 310 330 Wavelength (nm) Wavelength (nm) Wavelength (nm) Fig. 18. Oxidized (—) and reduced (—) UV absorption spectra of (A) menaquinone, (B) ubiquinone and (C) the quinoid extracted from D. tiedjei. 66 as the peak with M / z value of 340 (Fig. 19). This molecular weight is different from the molecularweightsofmenaquinonesor ubiquinones with any number of isoprenoid subunits. Moreover, the mass spectra of ubiquinones or menaquinones have a characteristic fragmentation pattern, with major peaks differing by 68 mass units, due to successive fragmentation of the isoprenoid sidechain. This fragmentation pattern was not observed in the mass spectrum of the quinoid, suggesting that the quinoid does not possess an isoprenoid sidechain in contrast to other respiratory quinones. In addition, the aromatic nuclear fragments of ubiquinones and menaquinones result in major peaks with M / z values of 235 and 225, respectively. But these peaks were not observed in the mass spectrum of the quinoid. Therefore, the quinoid extracted from D. tiedjei is very different from respiratory quinones in structure. 67 361 ,1.,,, J50 mm 177 mm ; j'I Ullillllljj , ,UI) 228 200 401 419 435 441 493 450 550 400 455 650 340 Fig. 19. Electron impact mass spectrum of the quinoid purified from D. tiedjei. 6 8 6. Summary Vitamin deficient D. tiedjei cultures demonstrated that lipoic acid was not required for growth and reductive dehalogenation, whereas thiamine had a stimulatory effect on growth and reductive dehalogenation, and 1,4-naphthoquinone was required for D. tiedjei reductive dehalogenation activity. Moreover, a respiratory quinone is probably involved in the electron transport chain coupled to reductive dehalogenation, since reductive dehalogenation of D. tiedjei cell suspensions were inhibited by H Q N O , a common respiratory quinone inhibitor. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, a different UV-absorbing molecule was extracted. The oxidized UV-ab sorption spectrum of this molecule was similar but not identical to ubiquinone. This quinone-like molecule, or quinoid, is different from other common respiratory quinones in chemical structure, according to an electron impact mass spectrometric analysis. 69 C H A P T E R T H R E E Quantification and localization of respiratory enzymes in D. tiedjei 1. Introduction D. tiedjei was shown to grow with hydrogen or formate as electron donors, and 3 C B or sulfoxy anions as electron acceptors (DeWeerd et al. 1991 ; Dolfing 1990 ; M o h n and Tiedje 1990a& 1990b). Energy conservation by metabolism of the above substrates can be explained only by electron transport phosphorylation, as fermentative growth is unlikely with hydrogen or formate. Later, a chemiosmotic mechanism of energy conservation was confirmed in D. tiedjei (Mohn and Tiedje 1991). However, details of the chemiosmotic mechanism, especially the topology of different respiratory enzymes, are unknown. The 3 C B reductive dehalogenase, which is likely to function as the terminal reductase, has been shown to be an integral membrane protein (Ni etal. 1995), although which side o f the cytoplasmic membrane its active site faces is unclear. Similarly, the locations of enzymes which catalyze reduction of sulfoxy anions have not been confirmed. Primary dehydrogenases like hydrogenase and formate dehydrogenase can also be located on either side of the cytoplasmic membrane. Knowing the topology of these respiratory enzymes w i l l improve our understanding of how D. tiedjei generates a proton-motive force during halorespiration and sulfoxy anion respiration. Therefore, I quantitatively investigated the distribution of these enzymes in different cellular fractions of D. tiedjei cells grown under different metabolic conditions. 70 2. Distribution of enzymes involved in sulfate reduction Enzymes involved in sulfoxy anion reduction were constitutively expressed at similar levels in the cytoplasm of D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation and sulfate reduction (Table III). The other three types of bisulfite reductases commonly found in sulfate-reducing bacteria (LeGall and Fauque 1988) were not detected. A P S reductase activity and desulfoviridin were detected only in the cytoplasmic fractions of cells grown in the three types of medium, and not in the periplasmic and membrane fractions. N o A T P sulfurylase activity was detected in the periplasmic fractions. However, the membrane fractions had strong NADH-producing enzyme activities which interfered with the A T P sulfurylase assay. But since no A P S reductase activity and desulfoviridin were present in the membrane fractions, it is very likely that A T P sulfurylase is also cytoplasmically located in D. tiedjei, as in other sulfate-reducing bacteria. These data also indicate that the periplasmic and membrane fractions are free of cytoplasmic contaminants. 3. Distribution of hydrogenase D. tiedjei appears to possess an inducible, periplasmic hydrogenase in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. The hydrogenase activities in soluble (periplasmic plus cytoplasmic) fractions of cells grown under reductive-dehalogenating and pyruvate-fermenting conditions were hundreds of times higher than the hydrogenase activities in the soluble fractions of sulfate-reducing cells (Table IV). This finding suggests the presence of a hydrogenase system induced in cells grown under reductive-dehalogenatingand pyruvate-fermenting conditions but not induced in cells grown with formate plus sulfate. Hydrogenase activity measured in whole cells grown under 71 reductive-dehalogenating conditions was about 50% of the hydrogenase activity measured in the soluble fraction. Since methyl viologen, the artificial electron acceptor used for the hydrogenase activity assay, has been shown to be inefficient in permeating the cytoplasmic membrane of E. coli when oxidized or reduced (Jones and Garland 1977), my results suggest that there is a soluble hydrogenase is located in the periplasm of cells grown under reductive-dehalogenating conditions. I f the hydrogenase were cytoplasmic rather than periplasmic, hydrogenase activity measured with whole cells should be much lower than that measured in cell extracts, since methyl viologen could not diffuse into the cytoplasm. Furthermore, whole cell hydrogenase activity was completely inhibited by C u 2 + ions (Table IV) , the membrane-impermeable hydrogenase inhibitor (Cypionka and Di l l ing 1986; Fernandez et al. 1989 ; Fitz and Cypionka 1989). These observations are in agreement with the conclusion that the soluble hydrogenase is a periplasmic enzyme. The soluble hydrogenase observed in D. tiedjei grown under pyruvate-fermenting conditions is probably also a periplasmic enzyme, since cells grown under reductive-dehalogenatingconditions used pyruvate as the electron donor and pyruvate fermentation is believed to be happening in dehalogenating cultures. 4. Distribution of formate dehydrogenase D. tiedjei appears to have two formate dehydrogenases, one cytoplasmic and induced during pyruvate fermentation, and the other one membrane-associated, facing the periplasm and induced during growth on formate. Whole cells grown under reductive-dehalogenating conditions had no measurable formate dehydrogenase activity with methyl viologen as the artificial electron acceptor (Table V ) . When the cells were lysed, formate dehydrogenase activity was detected in the soluble fraction, whereas formate dehydrogenase activity in the membrane fraction was 10% of that detected in the soluble fraction. A s explained above, 72 Table HI. Specific quantities of sulfate-reducing enzymes in the cytoplasmic fractions of D. tiedjei cells grown under different metabolic conditions. Growth conditions A T P sulfurylase A P S reductase Desulfoviridin (U/mg total protein) (U/mg total protein) (ug/mg total protein) Reductive- 0.34 0.14 1.40 dehalogenating Pyruvate-fermenting 0.31 0.17 1.47 Formate/sulfate- 0.24 0.18 1.77 reducing Table I V . Specific hydrogenase activities in different cellular fractions of D. tiedjei cells grown under different metabolic conditions. Cultures Cellular fractions Specific hydrogenase activity (mU/mg total protein) Formate/sulfate-reducing Membrane 1.0 Soluble 3.0 Pyruvate-fermenting Membrane 2.8 soluble 673.0 Reductive-dehalogenating Membrane 9.1 Soluble 1358.6 Wholecell 730.0 Whole cell + 3 m M C u C l 2 0.0 Cell extract 1900.0 Cell extract + 3 m M C u C l 2 0.0 73 methyl viologen is not efficient in permeating the cytoplasmic membrane and therefore this soluble formate dehydrogenase is probably a cytoplasmic enzyme. A similar situation was observed in pyruvate-fermenting D. tiedjei cells, with higher formate dehydrogenase activity in the soluble fraction than the membrane fraction. In cells grown with formate plus sulfate, the situation was different. Soluble formate dehydrogenase activity was at background level, whereas a membrane-bound formate dehydrogenase was active. When whole cells grown on formate plus sulfate were used in the activity assay, formate dehydrogenase activity was detected and was six times higher than that in the membrane fraction. The data, therefore, suggest that an inducible, membrane-bound formate dehydrogenase in formate-grown cells faces the periplasm. The higher specific activity detected with whole cells could be due to an underestimation of total protein in whole cells as well as to partial loss of enzyme activity during cell fractionation. 5. Reductive dehalogenase Specific reductive dehalogenation activity in cell extracts was calculated to be 1.1 nmole benzoate formed per hour per mg of protein, with reduced methyl viologen as the artificial electron donor (Table VI) . Also, activity of theD. tiedjei reductive dehalogenase was not inhibited by propyl iodide, an agent which interferes in corrinoid-dependent processes by alkylating cobalamins, in vitro. It has been demonstrated that the P C E reductive dehalogenases of Dehalobacter restrictus and Dehalospirillum multivorans had corrinoid cofactors and the dehalogenase activities were inhibited by propyl iodide (Neumann et al. 1995 ; Neumann et al. 1996 ; Schumacher and Holliger 1996 ; Schumachers al. 1997). This observation suggests the D. tiedjei reductive dehalogenase apparently does not contain any corrinoid cofactor, as do the P C E dehalogenases. H Q N O at a concentration of 150 nmole/mg 74 protein had no inhibitory effect on reductive dehalogenation activity with D. tiedjei cell extracts (Table VI) . The identical concentration of H Q N O , however, inhibited reductive dehalogenation in whole cells (Fig. 17). The finding suggests that H Q N O has no inhibitory effect on the reductive dehalogenase directly, and further supports the conclusion that the observed inhibitory effect of H Q N O with whole cells is on a respiratory quinone in the electron transport chain, whose function is substituted by reduced methyl viologen in the dehalogenase activity assays using cell extracts. The inducible cytochrome and the quinoid failed to function as electron donors in the in vitro dehalogenase assay. A variety of potential physiological electron donors were previously tested for functioning as the electron donor for reductive dehalogenation of 3 C B (DeWeerd and Suflita 1990). None of these compounds could replace reduced methyl viologen. Since I had purified a 50 kDa cytochrome c, which was co-induced with reductive dehalogenation, and a quinoid from D. tiedjei, I tested whether these two electron carriers could replace reduced methyl viologen in the reductive dehalogenase assay. The inducible cytochrome, the quinoid, or both of them were added to the reaction mixture with no methyl viologen. However, no reductive dehalogenation activity was detected (Table VI) . Pre-reducing these electron carriers with 2 m M titanium (III) citrate right before adding them to the reaction mixture did not improve the result. The low potential 8-hydroxyriboflavin also failed to replace methyl viologen as the electron donor for the reductive dehalogenase. 6. Localization of the reductive dehalogenase Although the D. tiedjei reductive dehalogenase is an integral membrane protein (Ni et al. 1995), the orientation of its active site is not clear. Washed cell suspensions were starved for electron donor for two days before 2 m M methyl viologen was added from an anoxic 75 Table V . Specific formate dehydrogenase activities in different cellular fractions of D. tiedjei cells grown under different metabolic conditions. Cultures Cellularfractions Specific formate dehydrogenase activity (mU/mg total protein) Reducti ve-dehal ogenati ng Whol e eel 1 0.0 Membrane 3.1 Soluble 37.4 Pyruvate-fermenting Membrane 1.3 soluble 18.3 Formate/sulfate-reducing Wholecells 74.1 Membrane 13.0 Soluble 2.3 Table V I . Specific reductive dehalogenase activities in whole cell extracts of D. tiedjei cells grown under reductive dehalogenation conditions. Reaction mixture Speci fi cdehal ogenase activity (nmole benzoate formed per mg protein per hour) Cel l extract + H 2 + M V 2 + 1.1 Boiled cell extract + H 2 + M V 2 + 0.0 Cel l extract + N 2 + M V 2 + 0.0 Cel l extract + H 2 0.0 Cel l extract + H 2 + M V 2 + + 250 u M Propyl iodide 1.2 Cel l extract + H 2 + M V 2 + + 150 nmole H Q N O per mg protein 1.0 Cel l extract + H 2 + 50 kDa inducible cytochrome 0.0 Cel l extract + H 2 + quinoid 0.0 Cel l extract + H 2 + 50 kDa inducible cytochrome + quinoid 0.0 Cel l extract + H 2 + 8-hydroxyriboflavin 0.0 76 stock, which was automatically reduced by the medium. The control cell suspensions showed some dehalogenation of 3CB due to endogenous reducing power, but reductive dehalogenation in cell suspensions with methyl viologen was completely inhibited (Fig. 20). Since reduced methyl viologen functioned as an artificial electron donor for the dehalogenase in the in vitro enzyme assay, and reduced methyl viologen does not permeate the cytoplasmic membrane efficiently, the cell suspensions with methyl viologen should have higher dehalogenation activity i f the active site of the reductive dehalogenase faced the periplasmic side of the membrane. Although the origin of the inhibition is not clear, this result is not consistent with a reductive dehalogenase whose active site faces the periplasm. 7. Putative gene sequence of the reductive dehalogenase small subunit The putative gene sequence of the small subunit of the heterodimeric reductive dehalogenase was determined from 2 inverse P C R products amplified from the genome of D. tiedjei. A n E. coli recombinant clone, containing a 66-bp insert which encoded the N H 2 -terminus of the small subunit of the dehalogenase was provided by our collaborators N i and Xun . This 66-bp insert was labeled by P C R amplification from this clone with oc- 3 2 P-dCTP and was used as a probe for a Southern blot analysis. This probe hybridized to a single 2.0-kb HindlE fragment, and a single 2.8-kb Muni fragment (Fig. 21). A 2.0-kb linear product was amplified from ///wfiffll-digested, self-ligated genomic D N A by inverse P C R , with primers designed from the NH 2 -terminal D N A sequence of the dehalogenase small subunit (Fig. 22). This result agreed with that of the Southern blot analysis. This 2.0-kb inverse P C R product was purified from agarose gel and sequenced directly (Fig. 23). D N A sequence coding for the dehalogenase small subunit NH 2 -terminal protein sequence was 554 bp away from the 3' end of the inverse P C R product. Directly 5' to the NH 2 -terminal D N A sequence was a putative 77 900 500 " ' ' ' ' 1 0 10 20 30 40 50 Time (hours) Fig . 20. Reductive dehalogenation activity o f starved D. tiedjei cell suspensions without any electron donor ( ) or with 2 m M reduced methyl viologen ( • ) as electron donor. Data are means o f triplicates with standard errors (bars). 78 NH 2 - terminal peptide, which began with an A T G start codon. In addition, a putative ribosome binding site was identified 18 bp away from the start codon. Since this inverse P C R product only contained part of the gene, another inverse P C R was performed with new P C R primers DehS-5 and DehS-6, and withMwwI-digested, self-ligated genomic D N A as template. A 2.8-kb linear inverse P C R product was formed (Fig. 22), and this product was purified from the agarose gel for direct D N A sequencing. In this 2.8-kb inverse P C R product, the complete gene sequence encoding the dehalogenase small subunit was found, including the partial sequence found in the 2.0-kb inverse P C R product amplified from ////ftflll-digested, self-ligated D N A template. A U G A stop codon was identified, and the complete O R F was 987 bp. The molecular mass of the deduced protein sequence, excludingthat of the putative signal peptide, was 34.3 kDa which is close to the S D S - P A G E predicted molecular mass of 37 kDa. The D N A sequence and the deduced protein sequence were not found to have substantial similarity to any other sequences in GenBank. Also, no conserved redox-center-binding domain could be identified from the deduced protein sequence. Hydropathy analysis of the deduced protein sequence predicted a transmembrane domain at the NH 2 - terminal end of the protein (Fig. 24), further supporting the conclusion that the reductive dehalogenase is an integral membrane protein. This is the first ever known gene sequence encoding a reductive dehalogenase from any halorespiratory bacterium, and it was named dehS. Southern blot analyses showed that the dehS gene and the hsc gene are not physically linked and probably not arranged in the same operon (Fig. 25). The hsc gene was about 750 bp from the 5' end of a 12.0-kb BamHl fragment (Fig. 10, lane 1). On the other hand, the dehS gene was on another BamHl fragment, which was larger than 12.0 kb in size (Fig. 21, lane 8). The dehS gene was located on a 4.0-kb Smal fragment (Fig. 21, lane 3) and this fragment could not be directiy 5' to the hsc gene, since the Smal fragment 5' to the hsc gene 79 was over 12.0 kb in size (Fig. 10, lane 7). In addition, the hsc gene was located on a Kpnl fragment over 12.0 kb in size (Fig. 10, lane 4) while the dehS gene was on a 10.5-kb Kpnl fragment (Fig. 21, lane 2). Therefore, the two genes are at least 12.0 kb apart from each other and unlikely to be arranged in the same operon, even though the two genes are co-induced. 80 1 2 3 4 5 6 7 8 9 12.0. 10.0-8.0-6.0-5.0-4.0-3.0-2.0. 1.6' 1.0-Fig . 21. Southern blot analysis of D. tiedjei D N A digested with restriction enzymes and probed with the 3 2P-labeled 66-bp P C R product which encoded the NH 2 -terminus of the reductive dehalogenase small subunit. Each lane contained 1 pg of restriction-digested genomic D N A . Lane 1: Pstl; lane 2: Kpril; lane 3: Smal; lane 4 Muni; lane 5 Sstl; lane 6 Hindlll and lane 7: EcoRl, lane 8: BamHl and lane 9: AatU. The numbers at the left are molecular size markers in kb. 81 Fig . 22. Inverse P C R products of the putative reductive dehalogenase small subunit gene, amplified from //wcflll-digested, self-ligated D N A (lane 2), and M/wI-digested, self-ligated D N A (lane 4). Lane 1 and 3 are molecular size markers in kb. 82 Fig . 23 1 AATTTCGAAA GGGAAGTCCT GAGAGACATT CCCGGAATTG TAACTATTTT rbs start 51 GAAGGACATT GAAGGGGTGA ATCAATGAAG TCGGACAATT CTGTTTTGCA (M K S D N S V L IjehS-IR 101 TGGCCTCAGA AACGAGAGAG GTTCCGCAGC GTACTCCTTT CTCGGATTTG H G L R N E R G S ) A A Y S F L 6 F DehS-IF^ 151 CGATTGTCGC TGTCTGCATT ATTGCAGTAA TCTTTGTCTT CCCCTGGAAC A I V A V C I I A V I F V F P W N 201 CAGATAGGTG GTGAAAGCCC TAAAGATGCA AACTATCTTT CCGGAATGGA Q I G G E S P K D A N Y L S G M 251 TGCCCTGAAG CAAAAGCAGT ACAATGAAGC AATCGCTTAC TTTGATAAAT D A L K Q K Q Y N E A I A Y F D K ^DehS-5 301 CAATCCAGGC GAATCCCAAC GGAGCGGCTT TCCTGGGTAA GGCAAAGGCA S I Q A N P N G A A F L G K A K A DehS-6^ • 351 GACATGGCAC TCGGCAATAT CGATAAAGCA CTTCAGGACG CAACTGCCGC D M A L G N I D K A L Q D A T A 401 CATAGAGAAG AATGCCGGCG CCGAGGCGTA CGGGCAGCGG GGTGTCATTT A I E K N A G A E A Y G Q R G V I 451 ACAAGATCCA GGATAAGACG GATCAAGCTC TCAAGGATTT CAACGAAGCC Y K I Q D K T D Q A L K D F N E A 501 ATCAAAAAAG ACAGTAGATA CGCTTGGGCC TTGGCGCAGA GAGCAGACCT I K K D S R Y A W A L A Q R A D 551 CTTCAATAAG CAGAAGAATT ATGAAAAAGC TCTCGATGAT GCGAATAGAG L F N K Q K N Y E K A L D D A N R 601 CCGTGTCGGC CAAAAAGGAC TTCGTAGAAG CATATAGACT GAGAGGATCG A V S A K K D F V E A Y R L R G S H i n d i I I 651 ATTCTGACCC GTATGGGTAA GTGCAAGGAA GCTTCTGCTG ATTTTATCGC I L T R M G K C K E A S A D F I 83 Fig . 23. Continued 7 01 CGTTCAGAAG ATGAAACCGG ATGATCCTGC AGCTATTCAG GATACCGCTT A V Q K M K P D D P A A I Q D T A 751 GGTTCCTGCT CACCTGTCCC GACGAGAAAC TCCAGGACTC CGCCAAAGCA W F L L T C P D E K L Q D S A K A 801 ATGGAACTTG CAAAGAAAGC CGTTGACATG AGCGGCGGGA TGAACAGCGC M E L A K K A V D M S G G M N S 851 CGCCCAGGAG ACATTGGCCG AGGCGTATTT CCGTCAAGGC GATGCTTTGA A A Q E T L A E A Y F R Q G D A L 901 AAGCAGTCGA GCACCAGAAA AAAGCTATAG AGCTTGGATC GCAGAATTGT K A V E H Q K K A I E L G S Q N C 951 CCTGACGGCT CATGTGTGAA GGATATGCAA CAGAGATTGC AGAAGTATGA P D G S C V K D M Q Q R L Q K Y 1001 ACTCGCAGGA AGACAGGAAA TCAGAACCGG GTACGAAATA TTGCCGTTGA E L A G R Q E I R T G Y E I L P L stop 1051 ACAGCAGTCT CTAGATACTT TCAGCATACA GCGGCAGAAT ACATTTTCAT N S S L -Fig . 23. Putative nucleotide sequence o f the D. tiedjei reductive dehalogenase small subunit gene (dehS). The deduced protein sequence is shown using the one-letter amino acid code, below the gene sequence. The O R F was identified between position 75-1061. The putative ribosome binding site, the putative A T G start codon and a U A G stop codon are underlined. A putative 17-amino acid signal peptide is in parentheses and the NH2-terminal protein sequence is bold. Primers DehS-IR and DehS-IF are used for inverse P C R with the template produced by Hindlll digestion; while, primers DehS-5 and DehS-6 are used for inverse P C R with the template produced by Muni digestion. 84 50 100 150 Position 200 250 300 Fig. 24. Hydropathy analysis of the deduced protein sequence of the D. tiedjei reductive dehalogenase small subunit. The hydrophobicity scale increases from least hydrophobic (negative number) to most hydrophobic (positive number). A membrane-spanning region is predicted at the NH2-terminal end of the deduced protein sequence. 85 12 kb Muni Muni Smal B a m m L Smal Smal Bamm -lh \ —\\f* I Sstl Sstl Hindm Hindm i h Hindm 4.0 kb Hindlll Hindlll Smal Muni Muni Smal -zzzz- -IF 1 kb F ig . 25. Physical maps of the hsc gene (above) and the dehS gene (below). 86 8. Summary A T P sulfurylase, A P S reductase and desulfoviridin, three enzymes which are involved in sulfate reduction in Desulfovibrio sp., were constitutively expressed in the cytoplasm of D. tiedjei cells grown under reductive-dehalogenating, pyruvate-fermenting and sulfate-reducing conditions. A n inducible hydrogenase activity was detected in whole cells and soluble fractions of cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. This soluble hydrogenase is probably located outside of the cytoplasmic membrane, according to hydrogenase activity assays using whole cells. Two formate dehydrogenase systems were present in D. tiedjei. A n inducible, membrane-bound, periplasm-oriented formate dehydrogenase was present in cells grown with formate as electron donor; while, a cytoplasmic formate dehydrogenase was detected in reductively dehalogenatingand pyruvate-fermenting cells. The active site of the reductive dehalogenase appears to face the cytoplasm, according to a reductive dehalogenation assay with whole cell suspensions of D. tiedjei. The 50-kDa inducible cytochrome, 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. N o conserved redox-center-binding domain could be identified in the deduced protein sequence, which was found to lack substantial similarity to any sequence in GenBank, suggesting that the D. tiedjei reductive dehalogenase is substantially different from the terminal reductases of other respiratory systems. Southern blot analyses indicated that the gene of the dehalogenase small subunit and the gene of the inducible cytochrome are 87 separated by at least 12 kb and therefore probably not located in the same transcriptional unit. 88 DISCUSSION 1. Characteristics of cytochromes in D. tiedjei and their potential roles in the halorespiratory electron transport chain Only c-type, and no 6-type cytochromes were detected in D. tiedjei cells grown under reductive-dehalogenating,pyruvate-fermenting and sulfate-reducing conditions (Table II and Fig . 2). On the one hand, cytochromes b are present only in a few sulfate-reducing bacteria. Cytochrome b was the only type of cytochrome found in the genus Desidfotomaculum, a Gram-positive genus of the sulfate-reducing bacteria (LeGall and Fauque 1988), and this type of cytochrome was found in Desulfovibrio gigas and seemed to relate to fumarate respiration (Hatchikian and LeGal l 1972). On the other hand, a variety of cytochromes c, with variable number of hemes per protein molecule, have been purified from sulfate-reducing bacteria. The best known of these is the 13-kDa tetraheme cytochrome c 3 . DeWeerd et al. (1990) reported purifying a major cytochrome c with apparent molecular mass of 13 k D a from D. tiedjei. They calculated the cytochrome had 3.5 hemes per protein by pyridine hemochromogen assays and these characteristics closely resembled those of tetraheme cytochrome c 3 . However, heme-stained S D S - P A G E (Fig. 3) clearly demonstrated that the major cytochrome c in D. tiedjei was a protein with an apparent molecular mass of 17 kDa. I had performed pyridine hemochromogen assays with the purified 17-kDa cytochrome c but the results were not consistent. Therefore, the answer to the question of whether this 17-kDa cytochrome c is a member of the tetraheme cytochrome c 3 family awaits cloning the gene encoding this cytochrome, and analysis of its protein sequence for c-type heme binding domains. The 17-kDa cytochrome c is probably a periplasmic protein and associated with the membrane, although it was detected by heme-staining in the cytoplasmic, periplasmic and 89 membrane fractions of D. tiedjei cells grown under sulfate-respiring, pyruvate-fermenting and reductive dechlorinating conditions (Fig. 3). Most bacterial cytochromes function in photosynthetic electron transport or in respiration, and they are either membrane-associated or in the periplasmic space (Thony-Meyer 1997). Moreover, it is relatively well established that binding of hemes to cytochrome c apoproteins is catalyzed by a heme lyase, and this reaction occurs on the periplasmic side of the membrane. Although a prokaryotic heme lyase has not yet been isolated, several bacterial cytochrome c-negative mutants with mutations that map to genes speculated to function in heme ligation (Grove et al. 1996 ; Lang et al. 1996 ; Thony-Meyer etal. 1995). This 17-kDa cytochrome c is probably a periplasmic protein which interacts with factors exposed on the periplasmic face of the cytoplasmic membrane. Detection of this protein in the cytoplasmic fraction is probably due to inefficient release of the periplasmic content, which results in mixing part of the periplasmic material with the cytoplasmic material. The 17-kDa cytochrome c may function as an electron carrier between the cytoplasmic pyruvate-oxidizing system and the periplasmic hydrogenase. Generally, about 4-fold more cytochromes are present in pyruvate-fermenting or reductive-dehalogenatingcells in which pyruvate fermentation is a major mode of metabolism, than in cells grown under conditions for sulfate reduction (Table II). Since the 17-kDa cytochrome c is the major cytochrome in D. tiedjei according to heme-stained P A G E gels (Fig. 3), the 17-kDa cytochrome c appears to be induced under these two growth conditions, and probably involved in pyruvate fermentation. D. tiedjei ferments pyruvate to acetate only in the presence of carbon dioxide, and the enzymes involved are believed to be cytoplasmic (see below). However, protons could also function as an electron sink for pyruvate fermentation, and hydrogen could be produced by a hydrogenase. M y data indicate that D. tiedjei has a 9 0 hydrogenase on the periplasmic side of the membrane. Therefore, the 17-kDa cytochrome c may have a role in transferring electrons between the membrane-separated pyruvate-oxidizing system and the hydrogenase. I also purified a membrane-associated diheme cytochrome c from D. tiedjei, which was co-induced with reductive dehalogenation activity (Figs. 3 and 4). This cytochrome seems to be a peripheral membrane protein, as it was released from the membrane by buffer with high concentration of salt, and no obvious transmembrane domain was identified within the protein sequence by hydropathy analysis (Fig. 15). The molecular mass of the protein was about 50 kDa, according to the deduced protein sequence and S D S - P A G E (Fig. 3 and 12). The native molecular mass determined by gel-permeation chromatography was 155 kDa, suggesting that the native inducible cytochrome was a homotrimer. However, this observation is probably an artifact, depending on the chromatographic and electrophoretic conditions. Most of the purified protein aggregated and stayed in the 5% stacking gel o f S D S - P A G E , when the protein was heated at 50 °C for 15 min (heme-staining conditions), indicating that the apparent molecular mass was greater than 200 kDa. However, all of the protein converted to monomelic form after being heated at 95 °C for 5 min. Thus, it appears that the inducible cytochrome forms aggregates of variable size under different conditions. This inducible cytochrome c displayed high-spin UV-vis ib le absorption spectra (Fig. 5A) similar to those of cytochromes c', which are found in purple photosynthetic, denitrifying and nitrogen-fixing bacteria (Bartsch and Kamen 1960 ; Imai etal. 1969 ; Saraiva etal. 1995 ; Yamanaka and Imai 1972). The physiological function of cytochromes c' in these bacteria, however, has not been clearly established: Despite the similarity in absorption spectra, the D. tiedjei inducible cytochrome differs from cytochromes c' in several aspects. First, all except one cytochromes c', are homodimers with a monomeric molecular weight of 91 14,000. A single c-type heme-binding domain is located at the COOH-terminus of the monomer (Moore and Pettigrew 1990). Secondly, cytochromes c' shows two significant p H -dependent spectral changes at p H 10 and 12 (Imai etal. 1969 ; Monkara etal. 1992). In contrast, the D. tiedjei inducible cytochrome did not exhibit any spectral change within p H 6.5 to 11. The inducible cytochrome underwent a spectral transition to a predominantly low-spin form at p H 13 (Fig. 5B). Furthermore, amino acid residues conserved in cytochromes c' (Moore and Pettigrew 1990) are absent from the D. tiedjei inducible cytochrome. The p H titration experiment (Fig. 5B) showed that both hemes were high-spin at neutral p H and then switched to low-spin at alkaline pH, suggesting the two hemes might have similar spectroscopic properties. But amino acid sequences around the two heme-binding domains of the D. tiedjei inducible cytochrome did not show any significant sequence similarity, indicating that the two heme-binding domains do not arise from gene duplication or gene fusion, as in some diheme cytochromes (Ambler et al. 1984). A l l of the data above suggest that the inducible cytochrome of D. tiedjei is a novel high-spin cytochrome c. The midpoint redox potential of the inducible cytochrome at p H 7.0 was calculated to be -342 m V . The results fits very well to a theoretical Nernstian curve for a 2-electron transfer reaction (n=2) (Fig. 7). Despite the fact that the inducible cytochrome is a diheme protein, a theoretical Nernstian curve for a 1-electron transfer reaction (n=l) is expected, i f the two heme centers are identical and independent. It has been demonstrated with a polymer of vinylhydroquinone, that the redox titration of a macromolecule containing many independent but identical redox centers results in an n value identical to that obtained for a single redox center (Cassidy 1949). On the other hand, the Nernstian curve should resolve into two one-electron transfer reactions i f the two heme centers have different midpoint potentials. One possible explanation for the discrepancy between my result and the expected 92 result is that the two heme centers are not independent and there are interactions between them. Le i tche /a / . (1985) pointed out that positive cooperativity between two heme centers of a diheme cytochrome could result in a Nernstian curve with an n value greater than 1. Moreover, both positive and negative cooperativity were demonstrated between the 4 heme centers of cytochrome c 3 isolated from several species of Desulfovibrio (Benosman et al. 1989 ; Nilci etal. 1984 ; Santos etal. 1984). A n alternative explanation is that the two heme centers vary in contributions to spectral changes when the inducible cytochrome is reduced. Under simulated conditions, the slope of a Nernstian plot of two independent redox centers with different midpoint potential varied, depending on the contribution of each redox center to the total change in absorbance (Kristensen et al. 1991). Whether or not there are interactions between the two heme centers, the results indicate that the D. tiedjei inducible cytochrome has a very low midpoint potential, compared to an E0' o f +297 m V for the 3-chlorobenzoate/benzoate redox couple (Dolfing and Harrison 1992). The low redox potential suggests that this inducible cytochrome is not likely to be the direct electron donor for the reductive dehalogenase in vivo. Its co-induction with reductive dehalogenation suggests it does function in the halorespiration process. Although it failed to substitute for methyl viologen for transferring electrons to the dehalogenase (Table VI) , its potential role as an electron carrier of the halorespiratory electron transport system in D. tiedjei could not be discounted, since it is possible that another electron carrier or a redox center of the reductive dehalogenase is irreversibly damaged after air-exposure and hinders efficient electron transfer between the inducible cytochrome and the reductive dehalogenase in the in vitro dehalogenase assays. 93 2. The presence of a putative quinoid in D. tiedjei electron transport chain The presence of a respiratory quinone in the halorespiratory electron transport system of D. tiedjei is supported by the fact that reductive dehalogenation was dependent on 1,4-naphthoquinone and was inhibited by H Q N O (Fig. 16 and 17). A menaquinone appears to function as an electron carrier, between a hydrogenase and the P C E reductive dehalogenase, in the PCE-dehalogenatingbacterium Dehalobacter restrictus. 1,4-Naphthoquinone is very similar in structure to the aromatic nucleus of menaquinones and therefore leads to the hypothesis that 1,4-naphthoquinone possibly functions as a precursor for menaquinone biosynthesis in D. tiedjei. The menaquinone biosynthetic pathway has been well studied in E. coli (Meganathan 1996). The menaquinone nucleus was derived from isochorismate and a -ketoglutarate in the presence of thiamine pyrophosphate. l,4-Dihydroxy-2-naphtholic acid was formed after a series of enzyme-catalyzed reactions. Then, the l,4-dihydroxy-2-naphtholic acid was decarboxylated and prenylated, and demethylmenaquinone was formed. Finally, methylation at the menaquinone nucleus completed the biosynthesis of menaquinone (Meganathan 1996). The prenylation and decarboxylation reactions probably occur in concert since symmetry experiments excluded any symmetric intermediates including 1,4-naphthoquinone (Baldwin et al. 1974). However, a few bacteria like Mycobacterium phlei, Bacteroidesmelaninogenicusand"Aerobacteraerogenes" strain 170-44 utilized 1 4 C-labeled 1,4-naphthoquinone for menaquinone biosynthesis (Bentley and Meganathan 1982). Therefore, it is possible that some bacteria use different pathways for synthesizing menaquinone. The situation of D. tiedjei becomes complicated, as I could not extract any menaquinones or ubiquinones from D. tiedjei. A similar study by Loftier and Tiedje (1995) also failed to extract any menaquinones from D. tiedjei. However, a possible quinone-like molecule, or quinoid was purified from D. tiedjei extracts by methods used for purification of 94 menaquinones or ubiquinones. The oxidized UV-absorption spectrum o f this quinoid was similar to that of a ubiquinone, except that it absorbed at a slightly longer wavelength (Fig. 18). In addition, the mass spectrum indicated that the quinoid had no isoprenoid sidechain (Fig. 19), as do menaquinones or ubiquinones. Moreover, the M / z value of the major fragment in the quinoid mass spectrum, which presumably arose from the quinoid nucleus, did not resemble those of menaquinones or ubiquinones. Hence the quinoid was different from menaquinones or ubiquinones in chemical structure. The reduced UV-spectrum of the quinoid is in some ways similar to that of pyrrolo-quinoline quinone or methoxatin. Methoxatin is a different type of quinone with no isoprenoid sidechain and it functions as the prosthetic group of the periplasmic methanol dehydrogenases of methylotrophs (Duine et al. 1980), the membrane-bound alcohol and aldehyde dehydrogenases of acetic acid bacteria (Anthony 1992) and the membrane-bound glucose dehydrogenase of many bacteria including E. coli and Acinetobacter calcoaceticus (Cleton-Jansen et al. 1990 ; Cleton-Jansen et al. 1988). Methoxatin is tightly, but not covalently bound in these enzymes and is buried deeply inside these proteins (Anthony et al. 1994), and coupled to the electron transport chain by different electron acceptors. For instance, the periplasmic cytochrome c L accepts electrons from the methoxatin of the methanol dehydrogenase of methylotrophs while the methoxatin of membrane-bound glucose dehydrogenase of Acinetobacter calcoaceticus passes electrons directly to a ubiquinone (Beardmore-Gray and Anthony 1986 ; Cox etal. 1992). The oxidized UV-absorption spectra of methoxatin has a peak of maximum at 255 nm, a shoulder at 275 nm and a low broad peak of maximum around 350 nm, and therefore is not very similar to the D. tiedjei quinoid. When it is reduced, the 255 nm peak decreases in size significantly, the 275 nm shoulder disappears, and absorption at 350 nm increases significantly (Duine and Frank 1980 ; Duine et al. 1978). 95 These types of changes in the reduced absorption spectrum are similar to those observed when the quinoid was reduced. On the other hand, the molecular weight of methoxatin is 330, and its electron-impact mass spectrum is different from the that of the quinoid (Buffoni et al. 1992). So, the D. tiedjei quinoid is not likely to be methoxatin, unless it is significantly modified during the extraction and the spectrophotometric properties are thus altered. However, its absorption spectral features support its identity as a quinone type of molecule. 1,4-naphthoquinone may be used as a precursor for synthesizing this quinoid in D. tiedjei. Inhibition of reductive dehalogenation by H Q N O confirms the presence of a respiratory quinone coupled to the halorespiratory electron transport system of D. tiedjei (Fig. 17). DeWeerd etal. (1990) reported that 200 p M of H Q N O has no inhibitory effect on the growth of D. tiedjeicells under reductive-dehalogenatingconditions. However, the effect of H Q N O on reductive dehalogenation was not addressed in that study. H Q N O is a general quinone inhibitor and is effective against both ubiquinone- and menaquinone-dependent electron transport chains. So, it appears that the quinoid is the site of inhibition by H Q N O , and hence the quinoid is possibly an electron carrier of the halorespiratory electron transport system. The quinoid and the 50-kDa inducible cytochrome c failed to function as electron donors for the dehalogenase in vitro. In order to elucidate the mechanism of halorespiration, it is necessary to determine the physiological electron donor for the reductive dehalogenase in the organism. 3 C B reductive dehalogenase activity of D. tiedjei is routinely measured with reduced methyl viologen as the artificial electron donor. Different common physiological electron donors, and tetraheme cytochrome c 3 and desulfoviridin isolated from D. tiedjei failed to replace methyl viologen as the artificial electron donor (DeWeerd and Suflita 1990). The inducible, 50-kDa inducible cytochrome c and the quinoid purified from D. tiedjei in my 96 study also did not replace methyl viologen in this aspect (Table VI) . This finding does not necessarily exclude these two electron carriers as components of the electron transport system couple to the reductive dehalogenase in vivo, since it is possible that another electron carrier or a redox center of the reductive dehalogenase is irreversibly damaged after air-exposure and hinders electron transfer between the reductive dehalogenase and the inducible cytochrome or the quinoid in the in vitro experiments. L ipoic acid deficiency had no inhibitory effect on growth and reductive dehalogenation in D. tiedjei suggesting D. tiedjei can synthesize lipoic acid de novo. However, thiamine had a stimulatory effect on growth and reductive dehalogenation. Thiamine deficiency slightly reduced the growth rate of D. tiedjei, and reduced reductive dehalogenation activity by about 50% (Fig. 16). These results on growth are similar to those reported by DeWeerd et al. (1990), although, they did not report the effect of vitamin deficiency on reductive dehalogenation. Thiamine, in the form of thiamine pyrophosphate, is a cofactor of enzyme complexes which catalyze decarboxylation reactions, such as the pyruvate decarboxylase of the pyruvate dehydrogenase complex, the a-ketoglutarate dehydrogenase complex and an cc-ketoglutarate dehydrogenase-independent decarboxylase activity involved in menaquinone synthesis (Mathews and Van Holde 1990 ; Meganathan 1996). The thiazole moiety of thiamine pyrophosphate attacks the carbonyl carbon of a-keto acids like pyruvate or cc-ketoglutarate, and leads to the decarboxylation of the carboxylic acid group. If thiamine deficiency interfered with the pyruvate metabolism of D. tiedjei, the growth of the culture should be reduced in proportion with the reductive dehalogenation activity. Whether thiamine deficiency wi l l affect the biosynthesis of the quinoid is not clear at this moment because the structure and the biosynthetic pathway of the quinoid are not clarified, and it is not known whether thiamine-pyrophosphate is required for synthesizing the quinoid. 97 3 . Topology of different respiratory enzymes in D. tiedjei The findings presented here suggest a spatial organization of respiratory enzymes in D. tiedjei. Despite the fact that D. tiedjei has been shown to be a sulfate-reducing bacterium, the terminal reductases involved in sulfate reduction i n D . tiedjei were not examined before. M y results demonstrated that D. tiedjei used the same enzyme system for sulfate reduction as other sulfate-reducing bacteria (Table HI). These enzymes were located in the cytoplasm, as in other sulfate-reducing bacteria (Kobayashi etal. 1975). In addition, these enzymes are constitutively expressed at similar levels in D. tiedjei cells grown under different conditions. This finding is consistent with previous immunoelectron microscopy study with Desulfovibrio (Dv.) gigas and Dv. vulgaris (Kremer et al. 1988). The expression of these proteins in D. tiedjei, when grown in the absence of sulfoxy anions, suggests that D. tiedjei wi l l use these electron acceptors whenever they are available to the bacterium. This may be beneficial to the bacterium energetically as sulfoxy anions are relatively more abundant than halogenated xenobiotic compounds in natural environments. In addition, this finding can partly explain the general observation that reductive dehalogenation by D. tiedjei is inhibited by sulfoxy anions, as these electron acceptors could be used immediately and drain away the reducing power. M y data clearly showed that a periplasmic hydrogenase is induced in D. tiedjei cells grown under pyruvate-fermenting conditions and reductive-dehalogenating conditions, in which pyruvate fermentation would still be the major mode of energy metabolism. This hydrogenase was not induced when D. tiedjei was grown with formate and sulfate as energy source (Table TV). Induction of hydrogenase activity has not been widely reported in other sulfate-reducing bacteria before. Hydrogenase activity in Dv. vulgaris strain Groningen cell extracts was ten times higher when cells were grown on hydrogen plus sulfate, than on lactate 98 plus sulfate. Immunocytolocalization in ultrathin frozen sections also showed more periplasmic hydrogenase produced in cells grown on hydrogen than on lactate (Hatchikian et al. 1995). Expression of a membrane-bound hydrogenase in Methanosarcina mazei G o l also depended on whether the cells were grown on hydrogen plus carbon dioxide or on acetate (Deppenmeier et al. 1995). Therefore, it is possible that the hydrogenase system in D. tiedjei is genetically regulated, and the genes are not expressed when the cells are grown under conditions when hydrogenase activity is not required. This type of regulation should be beneficial to D. tiedjei as the metabolism of this bacterium is not expected to be very efficient in energy conservation. The relationship between the D. tiedjei periplasmic hydrogenase and hydrogenases found in other sulfate-reducing bacteria, is not clear at present. Three common types of hydrogenases have been identified in Desulfovibrio species and they have been termed [Fe], [NiFe] and [NiFeSe] hydrogenases on the basis of the metal contents of the cofactors, amino acid sequences and sensitivity to inhibitors. The genes encoding these three main types of hydrogenases have been cloned and the gene sequences of individual types o f hydrogenases are strongly conserved (Voordouw et al. 1990). A fourth type of NADP-reducing [Fe] hydrogenase, which is very similar to the hydrogenase I of Clostridium pasteurianum has been identified in cytoplasm of Dv. fructosovorans (Malki etal. 1995). Different species of Desulfovibrio could have variable numbers and types of hydrogenases in different cellular compartments. The [NiFe] hydrogenase genes are found and the proteins are expressed in 22 Desulfovibrio strains tested (Voordouw, etal. 1990). For example, Dv. gigas (Niviere et al. 1991) and Dv. vulgaris strain Groningen (Hatchikian et al. 1995) only possessed the [NiFe] hydrogenase in their periplasmic space. On the other hand, Dv. vulgaris strain Fhldenborough was found to possess a periplasmic [Fe] hydrogenase (Voordouw and Brenner 1985 ; 99 Voordouwef ar/. 1985) and a membrane-associated, periplasm-facing [NiFe] hydrogenase and another membrane-associated, cytoplasm-facing [NiFeSe] hydrogenase (Lissolo et al. 1986 ; Rohdee/ al. 1990). Thus, the identity of the D. tiedjei periplasmic hydrogenase has to wait for purification of the enzyme, and cloning and analysis of the gene encoding this enzyme. The presence of a periplasmic hydrogenase when D. tiedjei was grown on a fermentable organic substrate like pyruvate is consistent with a theory that the periplasmic hydrogenase functions for hydrogen evolution, with proton acting as an electron acceptor, as in some Desulfovibrio species growing under fermentative conditions. Badziong and Thauer (1980) first proposed direct hydrogen oxidation by a hydrogenase in the periplasm, and generation of a proton gradient across the cytoplasmic membrane in Dv. vulgaris strain Marburg. Electrons were translocated across the cytoplasmic membrane to the cytoplasmically located terminal reductase for sulfate reduction. This bioenergetic model is similar to that of fumarate reduction in Wolinella succinogens except that the fumarate reductase is membrane-bound (Kroger 1978). With the discovery of cytoplasmically located, or cytoplasm-facing membrane-bound hydrogenases in some Desulfovibrio species, the above model was further elaborated and the intracellular hydrogen cycling model was proposed. The intracellular hydrogen cycling model requires two distinct hydrogenases. Organic substrates like pyruvate are oxidized by cytoplasmic enzymes. The reducing equivalents produced are used for hydrogen production by the cytoplasmic hydrogenase. Hydrogen then diffuses across the cytoplasmic membrane and is in turn oxidized by the periplasmic hydrogenase. Two protons are therefore produced directly in the periplasm, generating a proton-motive force. The two electrons are translocated across the membrane for sulfoxy anions reduction in the cytoplasm. When there is no external electron acceptor available, the intracellular hydrogen cycling wi l l continue to function, as long as the hydrogen partial pressure is kept 100 low enough by a mechanism like interspecies hydrogen transfer to a hydrogen-consuming organism. The lack of a cytoplasmic hydrogenase in strains MkeDv. gigas and Dv. vulgaris strain Groningen, which are able to grow fermentatively, strongly suggests that the periplasmic hydrogenase accepts electrons for hydrogen evolution v ia transmembrane electron transfer. Pyruvate fermentation by D. tiedjei is possibly similar to fermentation by Dv. gigas and Dv. vulgaris strain Groningen. However, hydrogen was rarely detected as a fermentation product of D. tiedjei, and this observation is inconsistent with the hypothesis mentioned above. Acetate and lactate were the only fermentation products of D. tiedjei cells grown under pyruvate-fermenting conditions in a complex medium supplemented with rumen fluid (Stevens etal. 1988 ; Stevens and Tiedje 1988). In these studies, the carbon recovery was close to 100% but the redox balance was poor. Later, Mohn and Tiedje (1990a) showed that acetate was the only fermentation product in D. tiedjei cells grown fermentatively on pyruvate in a defined medium with carbon dioxide as an exogenous electron acceptor. Both the carbon and redox balance was very close to 100%, and no hydrogen was detected. Moreover, M o h n and Tiedje (1990a) showed that Methanospirillm sp. P M - 1 , a hydrogen-consuming bacterium which wi l l enhance hydrogen evolution thermodynamically, failed to grow in co-culture with D. tiedjei on a bicarbonate-buffered medium with pyruvate, suggesting/), tiedjei do not produce hydrogen even under favorable conditions. However, a small amount of hydrogen was detected in D. tiedjei resting cell suspensions fed with pyruvate with no exogenous electron acceptor (DeWeerd etal. 1991). Furthermore, previous study showed that Dv. gigas grew fermentatively on lactate by interspecies hydrogen transfer only with Methanospirillum hungatii, but not Methanosarcina hakeri, probably because Methanospirillum hakeri has a lower affinity for hydrogen compared with that of 101 Methanosarcina hungatii'(Hatchikian et al. 1995). Thus, the potential role o f this inducible hydrogenase in hydrogen evolution by D. tiedjei growing fermentatively is unclear. The same periplasmic hydrogenase could oxidize hydrogen when hydrogen is used by D. tiedjei as an electron donor with sulfate or 3 C B as electron acceptors, resulting in proton production in the periplasm and generation of a proton-motive force. The situation would again be similar to Dv. gigas and Dv. vulgaris strain Groningen, which possess a single periplasmic hydrogenase, for both hydrogen uptake and evolution. However, the possibility of additional proton translocation from the cytoplasm is not excluded. Dv. desuljuricans strain Essex 6 possesses only an intracellular hydrogenase and no periplasmic hydrogenase, and generates a proton gradient by pumping protons across the cytoplasmic membrane (Fitz and Cypionka 1989). Similarly, such proton translocation mechanism has been reported in Dv. vulgaris strain M K and strain Marburg which had both periplasmic and cytoplasmic hydrogenases (Fitz and Cypionka 1991; Kobayashi etal. 1982). Membrane fractions of cells grown under pyruvate-fermenting conditions and reductive-dehalogenating conditions possess very low hydrogenase activities. The data presented here could not conclusively distinguish whether this minor hydrogenase activity represents a true membrane-bound hydrogenase or is due to periplasmic contamination. The hydrogenase activities in different cellular fractions were measured by hydrogen uptake assay. Although the three types of Desulfovibrio hydrogenases are reversible enzymes, specific enzyme activities in each direction vary between enzymes and strains, and can differ by ten folds (Van der Westen et al. 1978). Moreover, it is possible that there is another hydrogenase, unlike those of Desulfovibrio sp., which is highly oxygen-sensitive and responsible for the low hydrogenase activities measured in membrane fractions. So, the presence of a membrane hydrogenase could not be excluded. This uncertainty could be 102 clarified by Southern blot analysis of the D. tiedjei genome with labeled probes derived from the three types of Desulfovibrio hydrogenase genes. Also, native P A G E of D. tiedjei cell extracts prepared under anaerobic conditions could be stained directly for hydrogenase activity to distinguish possible multiple hydrogenases in D. tiedjei. The data presented here show that D. tiedjei has two different formate dehydrogenase systems. When D. tiedjei cells were grown under pyruvate-fermenting and reductive-dehalogenating conditions, a cytoplasmic formate dehydrogenase activity was induced (Table V ) . A s mentioned earlier, acetate was the major product of pyruvate fermentation by D. tiedjei in the presence of carbon dioxide. Mohn and Tiedje (1990a) also detected carbon monoxide dehydrogenase activity, and Stevens (1987) detected no ribulose bisphosphate carboxylase activity \r\D. tiedjei. Mohn and Tiedje (1990a) concluded that 20% of the acetate was produced by carbon dioxide fixation via the acetyl C o A pathway which is found in homoacetogens and some sulfate-reducing bacteria (Jansen et al. 1984 ; Lange etal. 1989 ; Schauder et al. 1989). Therefore, the cytoplasmic formate dehydrogenase detected in pyruvate-fermenting or reductive-dehalogenatingD. tiedjei cells is probably involved in carbon dioxide fixation, as formate dehydrogenase and carbon monoxide dehydrogenase are the first enzymes in the reductive acetyl C o A pathway. In contrast, this cytoplasmic formate dehydrogenase is not active in D. tiedjei cells grown on formate plus sulfate as the energy sources, and a membrane-bound, periplasm-oriented formate dehydrogenase was instead induced under these conditions (Table V ) . A n inducible, membrane-associated, periplasm-facing formate dehydrogenase was detected in Dv. gigas cells when the carbon and energy source was switched from lactate to formate (Odom and Peck 1981). A formate dehydrogenase was purified from the periplasmic fraction of Dv. vulgaris strain Hildenborough cells (Sebban et al. 1995). The D. tiedjei membrane-bound, 103 periplasm-facing formate dehydrogenase therefore would oxidize formate and produce protons directly in the periplasm, generating a proton-motive force, and suggesting that formate-sulfate or formate-3CB oxidation-reduction couples in D. tiedjei are transmembraneous. Again, this scheme for generation of a proton-motive force does not exclude the possibility of additional proton translocation mechanisms in which protons are transferred across the cytoplasmic membrane. The data presented in my thesis suggest that the active site of the reductive dehalogenase faces the cytoplasm. The reductive dehalogenase was previously shown to be an integral membrane protein (Ni etal. 1995), but the orientation of the dehalogenase was not demonstrated. The membrane-impermeable methyl viologen failed to function as the electron donor for the reductive dehalogenase in a cell suspension assay, but worked with cell extracts (Fig. 20 and Table VI) . This observation is not consistent with a periplasm-oriented dehalogenase. Furthermore, Mohn and Tiedje (1991) failed to completely uncouple A T P synthesis from reductive dehalogenation of 3 C B . This finding could be explained by an energy-driven uptake system for transporting the 3CB to a cytoplasm-facing reductive dehalogenase or by an energy-consuming activation step. The existence o f such an uptake system might be proven with 1 4C-labeled 3 C B in an uptake assay. The requirement for an activation step for sulfate reduction is well known, and such a step was also proposed for methanogensis (Mountfort 1978). The putative gene and deduced protein sequences of the small subunit of the reductive dehalogenase show no significant similarity to GenBank sequences, and the nature of this protein's iron-cofactor is still unclear. The purified D. tiedjei reductive dehalogenase, which is a heterodimer, was shown to contain an iron cofactor by atomic absorption spectroscopy (Ni etal. 1995). Oxidized and reduced visible absorption spectra of the purified dehalogenase 104 were similar to those of b- and c-type cytochromes. However, these absorption spectra are very "noisy", and this "noise" could be due to a contaminating cytochrome in the dehalogenase preparation. Moreover, the yellow chromophore of the dehalogenase was found to associate with the small dehalogenase subunit, when the two dehalogenase subunits were separated by reverse phase H P L C . However, no conserved b- or c-type heme binding domains could be identified from the deduced protein sequence of the dehS gene (Fig. 23). Four cysteine residues are present in the COOH-terminal half of the dehalogenase small subunit, and might be involved in coordination with iron in an iron-sulfur cluster. The answer to the question of whether or not the D. tiedjei reductive dehalogenase contains any heme cofactor w i l l have to await purification of a larger amount of the reductive dehalogenase for further analyses, or cloning and analysis of the gene encoding the large subunit of the dehalogenase. 4. Chemiosmotic models for D. tiedjei halorespiration and sulfate reduction A tentative model of electron transport in reductive-dehalogenating D. tiedjei is presented in Fig . 26. The formate-induced, membrane-bound, periplasm-facing formate dehydrogenase, and the periplasmic hydrogenase are proposed to be the primary dehydrogenases of this respiratory system. The membrane-bound, cytoplasm-oriented 3 C B reductive dehalogenase is thought to function as a terminal reductase. In this model, the formate dehydrogenase or the hydrogenase oxidize formate or hydrogen, respectively, and release two protons directly in the periplasm, generating a proton-motive force. Meanwhile, the two electrons produced are hypothesized to be transported across the membrane, via the inducible cytochrome c and the quinoid, to the dehalogenase which reductively dehalogenates 3 C B . This scenario results in consumption of one proton and production of one chloride ion 105 in the cytoplasm, and further enhances the electrochemical gradient across the membrane. This chemiosmotic model is also valid for sulfate respiration, since the sulfate-reducing enzymes are also cytoplasmic enzymes (Fig. 27). The resulting proton-motive force could be used for A T P synthesis, and at least 1 molecule of A T P could be formed per 3 C B dehalogenated or per sulfate reduced, based on the assumption that three protons are required for the synthesis one A T P molecule. The A T P yield would be higher, i f there were additional proton translocation via mechanisms, such as the proposed redox loop involving the quinoid (Figs. 26 and 27), and i f the reductive dehalogenase functions as a proton pump, as does the mitochondrial terminal oxidase complex. M y data cannot exclude the possibility of such proton-translocation mechanisms existing in D. tiedjei. However, M o h n and Tiedje (1991) reported medium acidification by D. tiedjei cell suspensions with hydrogen as electron donor, when pulses of 3 C B were added to the suspension. They calculated a FT73CB ratio of 2.1. This ratio agrees with the formation of only two protons during hydrogen oxidation, and could be interpreted as evidence against extra proton translocation. Moreover, the molar growth yield of D. tiedjei grown with formate or hydrogen as electron donor and 3 C B as electron acceptor was about 2 to 3 g of protein per mole of 3 C B dehalogenated (Mohn and Tiedje 1990b). The protein content was estimated to be 49% of the dry weight, and hence the molar growth yield was estimated to be about 4 to 6 gram dry weight per mole of 3 C B dehalogenated in that study. This molar growth yield is slightly lower than that of Dv. vulgaris strain Marburg grown with hydrogen plus sulfate, which had a calculated A T P yield of 1 A T P per sulfate (Badziong and Thauer 1978). It is possible that D. tiedjei also has an A T P yield in this range when grown with 3 C B as electron acceptor, which would be further evidence against extra proton translocation. 106 Fig . 26. A tentative chemiosmotic model o f halorespiration in D. tiedjei. The inducible hydrogenase (Ffcasei) and the 50-kDa inducible cytochrome c (cytj) are periplasmic proteins. The inducible formate dehydrogenase (Fdhj) is membrane-bound but periplasm-oriented. The inducible reductive dehalogenase (DClasei) is membrane-bound and facing the cytoplasm, and dehalogenates 3-chlorobenzoate (3CB) to benzene (Bz). The quinoid is depicted as " Q " in the membrane. 107 Fig . 27. A tentative chemiosmotic model o f sulfate reduction in D. tiedjei. The inducible hydrogenase (FfcaseO is a periplasmic protein. The inducible formate dehydrogenase (Fdhj) is membrane-bound but periplasm-oriented. The constitutively expressed A T P sulfurylase, A P S reductase and desulfoviridin, plus the pyrophosphatase (PP;ase) are cytoplasmic enzymes. The quinoid is depicted as " Q " in the membrane. X and Y are two probable electron carriers, whose identities are currently unknown. 108 The physiological electron donor of the 3 C B reductive dehalogenase is still unknown. The 50-kDa cytochrome c, although co-induced with reductive dehalogenation, is not likely to be the direct electron donor because of its low midpoint potential. Also , it is only a peripheral membrane protein; while, the reductive dehalogenase is an integral membrane protein. Therefore, the direct electron donor is probably located in the cytoplasmic membrane. The quinoid is therefore a good candidate for the electron donor of the 3 C B reductive dehalogenase. The redox potential of menaquinone, methoxatin and ubiquinone are -75 m V , +90 m V and +110 mV, respectively. Although my data indicated the quinoid is none of these three quinones, its spectral properties to some extent are similar to those of ubiquinone and methoxatin. Therefore it is possible that the midpoint potential of the quinoid falls in the range of the three respiratory quinones. This midpoint potential would be consistent with its potential role as the electron donor for the dehalogenase to catalyze 3 C B reduction (E0' = +297 mV) . For sulfate reduction with formate or hydrogen as electron donors, electron carriers located in the cytoplasmic membrane should be involved, since the primary dehydrogenase and the terminal reductases are physically separated. The 50-kDa cytochrome c o f D. tiedjei should not be involved in sulfate-respiration, since it is only induced under reductive-dehalogenatingconditions. In Dv. vulgaris strain Hildenborough, ah operon named the hmc operon, encodes six proteins which have been proposed to form a multi-protein, transmembrane complex which links the periplasmic and cytoplasmic electron transport reactions (Pollock et al. 1991 ; Rossi et al. 1993). Three of the gene products of the hmc operon are highly hydrophobic, integral membrane proteins with limited sequence similarity to integral membrane protein subunits of mitochondrial electron transport complexes. Another O R F encodes a hexadecahemecytochrome c which possibly functions as electron 109 acceptor for the periplasmic hydrogenase. On the other hand, a membrane-bound heterotrimeric cytochrome c was purified in Dv. gigas which can couple oxidation of hydrogen to the reduction of sulfite in vitro (Chen et al. 1993). The existence of an hmc operon or a Dv. gigas-type of heterotrimeric cytochrome c in D. tiedjei were not examined. Heme-staining might not detect a cytochrome similar to the Dv. gigas heterotrimeric cytochrome c, or a hexadecaheme cytochrome c in D. tiedjei, i f the amounts of these proteins are below the detection limit of heme-staining. Respiratory quinones are also possible transmembrane electron carriers. H Q N O has been shown to inhibit sulfate and sulfite reduction (Kremer and Hansen 1989 ; Peck and Lissolo 1988). The quinoid from this study is therefore a conceivable transmembrane electron carrier involved in D. tiedjei sulfate-respiration. To resolve this possibility, it would be useful to test whether H Q N O also inhibits sulfate reduction i n D . tiedjei, as it inhibits 3CB dehalogenation. In conclusion, my data clearly demonstrate that D. tiedjei possesses elements necessary for producing protons directly in the periplasm, thereby generating an electrochemical gradient across the cytoplasmic membrane. Furthermore, potential elements required for additional transmembrane proton translocation are also present. The operation of such extra proton translocation mechanisms requires further verification. 110 REFERENCES Adriaens, P., Q. Fu and D . Grbic-Galic. 1995. Bioavailability and transformation o f highly chlorinated dibenzo-/?-dioxins and dibenzofumas in anaerobic soils and sediments. Environ. Sci. Technol. 29:2252-2260 Ambler, R. P., M . Daniel, K . Mel is and C. D . Stout. 1984. The amino acid sequence of the dihaem cytochrome c 4 from the bacterium Azobacter vinelandii. Biochem. J. 222:217-227 Andrade, P. S. L . and W . B . Wheeler. 1974. Biodegradation of mirex by sewage sludge organism. B u l l . Environ. Contam. Toxicol. 11:415-416 Anthony, C. 1992. The structure of bacterial quinoprotein dehydrogenase. Int. J. Biochem. 24:29-39 Anthony, C , M . Ghosh and C. C. F. Blake. 1994. The structure and function of methanol dehydrogenase and related quinoproteins containing pyrrolo-quinoline quinone. Biochem. J. 304:665-674 Apajalahti, J., J. Cole and J. M . Tiedje. (1989) Characterization of a dechlorination cofactor: an essential activator for 3-chlorobenzoate dechlorination by the bacterium D C B - 1 , abstr. Q-36, p. 336. In Abstr. 89th Annu. Meet. A m . Soc. Microbio l . American Society for Microbiology, Washington, D . C. Apajalahti, J. H . A . , P. Karpanoja and M . S. Salkinoja-Salonen. 1986. Rhodococcus chlorophenolicus sp. nov., a chlorophenol-mineralizing actinomycete. Int. J. Syst. Bacteriol. 36:246-251 Apajalahti, J. H . and M . S. Salkinoja-Salonen. 1987. Complete dechlorination of tetrachlorohydroquinone by cell extracts of pentachlorophenol-induced Rhodococcus chlorophenolicus. J. Bacteriol. 169:5125-5130 Assaf-Anid, N . , K . F . Hayes and T. M . Vogel. 1994. Reductive dechlorination of carbon tetrachloride by cobalamin (II) in the presence of dithiothreitol: mechanistic study, effect of redox potential and pH. Environ. Sci. Technol. 28:246-252 Assaf-Anid, N . , L . Nies and T. M . Vogel. 1992. Reductive dechlorination of a polychlorinated biphenyl congener and hexachlorobenzene by vitamin B 1 2 . App l . Environ. Microbiol . 58:1057-1060 111 Ausubel, F . M . , R. Brent, R. E . Kingston, D. D . Moore, J. G. Seidman, J. A . Smith and K . Struhl. 1992. Short protocols in molecular biology., 2nd ed. John Wiley and Sons, Inc., N e w York Badziong, W . and R. K . Thauer. 1978. Growth yields and growth rates of Desulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate and hydrogen plus thiosulfate as the sole energy sources. Arch. Microbiol . 117:209-214 Badziong, W . and R. K . Thauer. 1980. Vectorial electron transport in Desulfovibrio vulgaris (Marburg) growing in hydrogen plus sulfate as sole energy source. Arch . Microbio l . 125:167-174 Baldwin, R. M . , C. D . Snyder and H . Rapoport. 1974. Biosynthesis o f bacterial menaquinones. Dissymmetry in the naphthalenic intermediates. Biochem. 13: 1523-1530 Bartsch, R. G . and M . D . Kamen. 1960. Isolation and properties of two soluble heme proteins in extracts of the photoanaerobe Chromatium. J. B i o l . Chem. 235.825-831 Baxter, R. M . 1990. Reductive dechlorination of certain chlorinated organic compounds by reduced hematin compared with their behavior in the environment. Chemosphere 21:451-458 Beardmore-Gray, M . and C. Anthony. 1986. The oxidation of glucose by Acinetobacter calcoaceticus: interaction of the quinoprotein glucose dehydrogenase with the electron transport chain. J . Gen. Microbiol . 132:1257-1268 Benosman, H . , M . Asso, P. Bertrand, T. Yagi and J. P. Gayda. 1989. E P R study of the redox interactions in cytochrome c 3 from Desulfovibrio vulgaris Miyazaki . Eur. J. Biochem. 182:51-55 Bentley, R. and R. Meganathan. 1982. Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol . Rev. 46:241-280 Berry, J. D . and D . A . Stotter. 1977. Dechlorination of D D T by vitamin B 1 2 under mild reducing conditions. Chemosphere 6:783-787 Bouchard, B . , R. Beaudet, R. Vi l l emur ,G. McSween, F. Lepine and J. G . Bisail lon. 1996. Isolation and characterization of Desulfitobacterium frappieri sp. nov., an anaerobic bacterium which reductively dechlorinate pentachlorophenol to 3-chlorophenol. Int. J. Syst. Bacteriol. 46:1010-1015 112 Boyle, A . W. , C. K . Blake, W . A . Price, II. and H . D . May. 1993. Effects of polychlorinated biphenyl congener concentration and sediment supplementation on rates of methanogensis and 2,3,6-Trichlorobiphenyl dechlorination in an anaerobic enrichment. App l . Environ. Microbiol . 59:3027-3031 Brigl ia , M . , R. I. L . Eggen,D. J. Van Elsas and W . M . de Vos. 1994. Phylogenetic evidence for transfer of Pentachlorophenol-mineralizing^cWococc^ chlorophenolicus PCP-1 to the germs Mycobacterium. Int. J. Syst. Bacteriol. 44:494-498 Buffoni, F. , S. Cambi and G . Moneti. 1992. Pyrroloquinoline quinone, a method for its isolation and identification by mass spectrometry. Biochim. Biophys. Acta. 1116:297-304 Burris, D . R., C. A . Delcomyn, M . H . Smith and A . L . Roberts. 1996. Reductive dechlorination of tetrachloroethylene and trichloroethylene catalyzed by vitamin B 1 2 in homogenous and heterogeneous systems. Environ. Sci. Technol. 30:3047-3052 Bushart, S. P., C. W . Boylen, R. J. Soracoo, J. M . Rankin and M . M . M c E w e n . 1995. In situ anaerobic microbial dehalogenation of chlorobenzenes from a sulfidogenic, mixed-contaminant site, abstr. Q-78, p. 413. In Abstr. 95th Gen. Meet. A m . Soc. Microbio l . American Society for Microbiology, Washington D . C. Cassidy, H . C. 1949. Electron exchange polymers. J. A m . Chem. Soc. 71:402-410 Castro, C. E . , R. S. Wade andN. O. Belser. 1985. Biodehalogenation: reactions of cytochrome P-450 with polyhalomethanes. Biochem. 24:204-210 Chen, L . , M . Y . L i u and J. LeGal l . 1993. Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas. Arch. Biochem. Biophys. 303:44-50 Christiansen, N . and B . K . Ahring. 1996. Desulfitobacterium hafniense sp. nov., an anaerobic, reductively dechlorinating bacterium. Int. J. Syst. Bacteriol. 46:442-448 Cleton-Jansen, A . M . , N . Goosen, O. Fayet and P. Van de Putte. 1990. Cloning, mapping, and sequencing of the gene encoding Escherichia coli quinoprotein glucose dehydrogenase. J. Bacteriol. 172:6308-6315 Cleton-Jansen, A . M . , N . Goosen, G . Odle and P. Van de Putte. 1988. Nucleotide sequence of the gene coding for quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Nucleic Acids Res. 16:6228 113 Cole, J. R. , A . L . Cascarelli, W . W . Mohn and J. M . Tiedje. 1994. Isolation and characterization of a novel bacterium growing via reductive dehalogenation of 2-chlorophenol. Appl . Environ. Microbiol . 60:3536-3542 Cole, J. R., B . Z . Fathepure and J. M . Tiedje. 1995. Tetrachloroethene and 3-chlorobenzoate dechlorination activities are co-induced in Desulfomonile tiedjei D C B - 1 . Biodegradation 6:167-172 Cole, J. R. and J. M . Tiedje. 1990. Induction of anaerobic dechlorination of chlorobenzoate in strain D C B - 1 , abstr. Q-43, p. 295. In Abstr. 90th Annu. Meet. A m . Soc. Microbio l . American Society for Microbiology, Washington, D . C. Collins, M . D . and F. Widdel. 1986. Respiratory quinones of sulfate-reducing and sulfur-reducing bacteria: a systematic investigation. System. Appl . Microbio l . 8:8-18 Cox, J. M . , D . J. Day and C. Anthony. 1992. The interaction of methanol dehydrogenase and its electron acceptor, cytochrome c L in methylotrophic bacteria. Biochim. Biophys. Acta. 1119:97-106 Cypionka, H . and W . Di l l ing . 1986. Intracellular localization of the hydrogenase in Desulfotomaculum orientis. F E M S Microbiol . Lett. 36:257-260 Deppenmeier, U . , M . Blaut, S. Lentes, C. Herzberg and G . Gottschalk. 1995. Analysis of the V/JOGAC and vhtGAC operons from Methanosarcina mazei strain G o l , both encoding a membrane-bound hydrogenase and a cytochrome b. Eur. J. Biochem. 227:261-269 DeWeerd, K . A . , F. Concannon and J. M . Suflita. 1991. Relationship between hydrogen consumption, dehalogenation and the reduction of sulfur oxyanions by Desulfomonile tiedjei. App l . Environ. Microbiol . 57:1929-1934 DeWeerd, K . A . , L . Mandelco, R. S. Tanner, C. R. Woese and J. M . Suflita. 1990. Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic, dehalogenating, sulfate-reducing bacterium. Arch. Microbiol . 154:23-30 DeWeerd, K . A . and J. M . Suflita. 1990. Anaerobic aryl reductive dehalogenation of halobenzoates by cell extracts of "Desulfomonile tiedjei". App l . Environ. Microbiol . 56:2999-3005 DeWeerd, K . A . , J. M . Suflita, T. Linkfield, J. M . Tiedje and P. H . Pritchard. 1986. The relationship between reductive dehalogenation and other aryl substituent removal reactions catalyzed by anaerobes. F E M S Microbiol . Ecol . 38:331-339 114 Dolting, J. 1990. Reductive dechlorination of 3-chlorobenzoate is coupled to A T P production and growth in anaerobic bacterium, strain D C B - 1 . Arch. Microbiol . 153:264-266 Dolfing, J. and B . K . Harrison. 1992. Gibbs free energy of formation of halogenated aromatic compounds and their potential role as electron acceptors in anaerobic environments. Environ. Sci. Technol. 26:2213-2218 Dolfing, J. and J. M . Tiedje. 1986. Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate. F E M S Microbiol . Ecol . 38:293-298 Duine, J. A . and J. Frank, Jr. 1980. The prosthetic group of methanol dehydrogenase. Purification and some of its properties. Biochem. J. 187:221-226 Duine, J. A . , J. Frank, Jr. and P. E . J. Verwiel . 1980. Structure and activity of the prosthetic group of methanol dehydrogenase. Eur. J. Biochem. 108:187-192 Duine, J. A . , J. Frank, Jr. and J. Westerling. 1978. Purification and properties of methanol dehydrogenase from Hyphomicrobium X . Biochim. Biophys. Acta. 524:277-287 Dutton, P. L . 1978. Redox potentiometry: Determination of midpoint potentials of oxidation-reduction components of biological electron-transfer systems. Methods Enzymol . 54:411-435 Ederer, M . M . , R. L . Crawford, R. P. Herwig and C. S. Orser. 1997. P C P degradation is mediated by closely related strains of the genus Sphingomonas. M o l . Ecol.6:39-49 Fathepure, B . Z . , J. P. Nengu and S. A . Boyd. 1987. Anaerobic bacteria that dechlorinate perchloroethene. Appl . Environ. Microbiol . 53:2671-2674 Fathepure, B . Z . , J. M . Tiedje and S. A . Boyd. 1988. Reductive dechlorination of hexachlorobenzene to h i - an dichlorobenzenes in anaerobic sewage sludge. App l . Environ. Microbiol . 54:327-330 Fernandez, V . M . , M . L . Rua, P. Reyes, R. Cammackand E . C. Hatchikian. 1989. Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions. Eur. J. Biochem. 185:449-454 Fitz, R. M . and H . Cypionka. 1989. A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans Arch. Microbiol . 152:369-376 Fitz, R. M . and H . Cypionka. 1991. Generation of a proton gradient in Desulfovibrio vulgaris. Arch. Microbiol . 155:444-448 115 Francis, R. T., Jr. and R. R. Becker. 1984. Specific indication of hemoproteins in polyacrylamide gels using a double-staining process. Anal . Biochem. 136:509-514 French, A . L . and R. A . Hoopingarner. 1970. Dechlorination of D D T by membranes isolated from Escherichia coli. J. Econ. Entomol. 63:756-759 Gantzer, C. J. and L . P. Wackett. 1991. Reductive dechlorination catalyzed by bacterial transition-metal coenzymes. Environ. Sci. Technol. 25:715-722 Gerritse, J., V . Renard, T. M . P. Gomes, P. A . Lawson, M . D . Collins and J. C . Gottschal. 1996. Desulfitobacterium sp. strain P C E 1 , an anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene or o/Y/jo-chlorinated phenols. Arch. Microbiol . 165:132-140 Gibson, S. A . and J. M . Suflita. 1990. Anaerobic biodegradation of 2,4,5-trichlorophenoxyacetic acid in samples from a methanogenic aquifer: stimulation by short-chain organic acids and alcohols. Appl . Environ. Microbiol . 56:1825-1832 Golovleva, L . A . , R. N . Pertsova, L . I. Evtushenko and B . P. Baskunov. 1990. Degradation of 2,4,5-trichlorophenoxyacetic acid by a Nocardioides simplex culture. Biodegradation 1:263-271 Grove, J., S. Tanapongpipat, G . Thomas, L . Griffiths, H . Crooke and J. Cole. 1996. Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. M o l . Microbiol . 19:467-481 Haggblom, M . M . , L . J. Nohynek, N . J. Palleroni, K . Kronqvist, E . Nurmiaho-Lassila, M . S. Salkinoja-Salonen, S. Klatte and R. M . Kroppenstedt. 1994. Transfer of polychlorophenol-degradingi?/;oiiococcw5chlorophenolicus (Apajalahti etal. 1986) to the genus Mycobacterium as Mycobacterium chlorophenolicum comb. nov. Int. J. Syst. Bacteriol. 44:485-493 Hatchikian, E . C , N . Forget, A . Bernadac, D . Alazard and B . Ollivier. 1995. Involvement of a single periplasmic hydrogenase for both hydrogen uptake and production in some Desulfovibrio species. Res. Microbiol . 146:129-141 Hatchikian, E . C. and J. LeGal l . 1972. Evidence for the presence of a 6-type cytochrome in the sulfate-reducing bacterium Desulfovibrio gigas, and its role in the reduction of fumarate by molecular hydrogen. Biochim. Biophys. Acta. 267:479-484 Holliger, C , G . Scharaa, A . J. M . Stams and A . J. B . Zehnder. 1993. A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth. App l . Environ. Microbiol . 59:2991-2997 116 Holliger, C , G . Schraa, E . Stupperich, A . J. M . Stams and A . J. B . Zehnder. 1992. Evidence for the involvement of corrinoids and factor F 4 3 0 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri. J. Bacteriol. 174:4427-4434 Holliger, C. and W . Schumacher. 1994. Reductive dehalogenation as a respiratory process. Antonie Van Leeuwenhoek J. Microbiol . Serol. 66:239-246 Imai, Y . , K . Imai, R. Sato and T. Horio. 1969. Three spectrally different states of cytochrome cc' and c' and their interconversion. J. Biochem. 65:225-237 Jablonski, P. E . and J. G. Ferry. 1992. Reductive dechlorination of trichloroethylene by the CO-reduced C O dehydrogenase enzyme complex from Methanosarcina thermophila. F E M S Microbiol . Lett. 96:55-60 Jagnow, G . , K . Haider and P. C. Ellwardt. 1977. Anaerobic dechlorination and degradation of hexachlorocyclohexaneisomersby anaerobic and facultative anaerobic bacteria. Arch. Microbio l . 115:285-292 Jakoby, W . B . 1978. The glutathiones-transferases: a group of multifunctional detoxification proteins. Adv . Enzymol. 46:383-414 Jansen, K . , R. K . Thauer, F. Widdel and G. Fuchs. 1984. Carbon assimilation pathways in sulfate reducing bacteria. Formate, carbon dioxide carbon monoxide, and acetate assimilation by Desulfovibrio baarsii. Arch. Microbiol . 138:257-262 Jones, R. W . and P. B . Garland. 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. J. 164:199-211 Karlson, U . , F. Rojo, J. D . VanElsas and E . Moore. 1995. Genetic and serological evidence for the recognition of four pentachlorophenol-degrading bacterial strains as a species of the genus Sphingomonas. Syst. Appl . Microbiol . 18:539-548 Kobayashi, K . , H . Hasegawa, M . Takagi and M . Ishimoto. 1982. Proton translocation associated with sulfite reduction in a sulfate-reducing bacterium, Desulfovibrio vulgaris. F E B S Lett. 142:235-237 Kobayashi, K . , Y . Morisawa, T. Ishituka and M . Ishimoto. 1975. Biochemical studies on sulfate-reducing bacteria. XTV. Enzyme levels of adenylylsulfate reductase, inorganic pyrophosphatase, sulfite reductase, hydrogenase and adenosine triphosphatase in cells grown on sulfate, sulfite and thiosulfate. J. Biochem. (Tokyo) 78:1079-1085 117 Kremer, D . R. and T. A . Hansen. 1989. Demonstration of H O Q N O and antimycin A sensitive coupling of N A D H oxidation and A P S and sulfite reduction in a marine Desulfovibrio strain. F E M S Microbiol . Lett. 58:43-48 Kremer, D . R., M . Veenhuis, G. Fauque, H . D . Peck, Jr., J. LeGa l l , J. J. G . Moura and T. A . Hansen. 1988. Immunocytochemical localization of A P S reductase and bisulfite reductase in three Desulfovibrio strains. Arch. Microbiol . 150:296-301 Kristensen, E . W. , D . H . Igo, R. C. Elder and W. R. Heineman. 1991. Non-ideal behavior of Nernstian plots from spectroelectrochemistry experiments. J Electroanal Chem 309:61-72 Kroger, A . 1978. Fumarate as terminal electron acceptor of phosphorylative electron transport. Biochim. Biophys. Acta. 505:129-145 Krone, U . E . , K . Laufer, R. K . Thauer and H . P. C. Hogenkamp. 1989. Coenzyme F 4 3 o as a possible catalyst for the reductive dehalogenation of chlorinated G hydrocarbons in methanogenic bacteria. Biochem .28:10061 -10065 Krone, U . E . and R. K . Thauer. 1992. Dehalogenation of trichlorofluoromethane (CFC-11) by Methanosarcina barkeri. F E M S Microbiol . Lett. 90:201-204 Krone, U . E . , R. K . Thauer, H . P. C. Hogenkamp and K . Steinbach. 1991. Reductive formation of carbon monoxide from CC1 4 and F R E O N s 11,12, and 13 catalyzed by corrinoids. Biochem. 30:2713-2719 Kuhn, E . P. and J. M . Suflita. 1989 Dehalogenation of pesticides by anaerobic microorganisms in soils and groundwater—a review, p. 111-180. In B . L . Sawhyney and K . Brown (ed.), Reactions and movements of organic chemicals in soils. Soil Science Society of American and American Society of Agronomy, Madison, Wis , L a Roche, S . D . and T. Leisinger. 1990. Sequence analysis and expression o f the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family. J. Bacteriol. 172:164-171 Laemmli , U . K . 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Lang, S. E . , F. E . Jenny, Jr. and F. Daldal. 1996. Rhodobacter capsulatus C y c H : a bipartite gene product with pleiotropic effects on the biogenesis of structurally different c-type cytochromes. J. Bacteriol. 178:5279-5290 118 Lange, S., R. Scholtz and G . Fuchs. 1989. Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum. 1. Characterization and metabolic function of the cellular tetrahydropterin. Arch. Microbio l . 151:77-83 L e G a l l , J. and G . Fauque. 1988. Dissimilatory reduction of sulfur compounds, p.587-639. In A . J. B . Zehnder (ed.), Biology of anaerobic microorganisms. John Wiley & Sons, Inc., N e w York Leisinger, T. and D . Kohler-Staub. 1990. Dichloromethane dehalogenase from Hyphomicrobium D M 2 . Methods Enzymol. 188:355-361 Leitch, F . A . , K . R. Brown and G . W. Pettigrew. 1985. Complexity in the redox titration of diheme cytochrome c 4 . Biochim. Biophys. Acta. 808:213-218 Liang, L . N . and D . Grbic-Galic. 1991. Reductive dechlorination of hexa- to di-chlorobenzenes by anaerobic microcosms and enrichments derived from a creosote-contaminated ground water aquifer, abstr. Q-107, p. 294. In Abstr. 91st Gen. Meet. A m . Soc. Microbio l . American Society for Microbiology, Washington D . C. Linkfield, T. G . and J. M . Tiedje. 1990. Characterization of the requirements and substrates for reductive dehalogenation by strain D C B - 1 . J. Ind. Microbiol . 5:9-16 Lissolo, T., E . S. Choi , J. LeGal l and H . D . Peck, Jr. 1986. The presence of multiple intrinsic membrane nickel containing hydrogenases in Desulfovibrio vulgaris (Hildenborough). Biochem. Biophys. Res. Commun. 139:701-708 Loffler, F. E . and J. M . Tiedje. 1995. Personal communication. Loffler, F. E . , R. A . Sanford and J. M . Tiedje. 1996. Initial characterization o f a reductive dehalogenase from Desulfitobacterium chlororespirans Co23. A p p l . Environ. Microbio l . 62:3809-3813 Logan, M . S. P., L . M . Newman, C. A . Schanke and L . P. Wackett. 1993. Cosubstrate effects in reductive dehalogenation by Pseudomonas putida G786 expressing cytochrome P-450 C A M. Biodegradation 4:39-50 Madsen, T. and D . Licht. 1992. Isolation and characterization of an anaerobic chlorophenol-transforming bacterium. Appl . Environ. Microbiol . 58:2874-2878 M a l k i , S., I. Saimmaime,G. De Luca, M . Rousset, Z . Dermoun and J. P. Belaich. 1995. Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibriofructosovorans. J. Bacteriol. 177:2628-2636 119 Mathews, C. K . and K . E . Van Holde. 1990. Biochemistry. The Benjamin/cummings Publishing Company, Inc., California Maymo-Gatell , X . , Y . Chien, J. M . Gossett and S. H . Zinder. 1997. Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 276:1568-1571 McAll i s te r , K . A . , H . Lee and J. T. Trevors. 1996. Microbial degradation of pentachlorophenol. Biodegradation 7:1-40 Meganathan, R. 1996. Biosynthesis of the isoprenoid quinones menaquinone (vitamin K 2 ) and ubiquinone (coenzyme Q), p. 642-656. In F. C. Neidhardt (ed.), Escherichia coli and Salmonella, cellular and molecular biology. A S M Press, Washington, D . C. Mikesel l , M . D . and S. A . Boyd. 1985. Reductive dechlorination of the pesticides 2,4-D and 2,4,5-T and pentachlorophenol in anaerobic sludges. J. Environ. Qual. 14:337-340 Mikese l l , M . D . and S. A . Boyd. 1990. Dechlorination of chloroform by Methanosarcina strains. Appl . Environ. Microbiol . 56:1198-1201 Mi les , J. R. W. , C. M . Tu and C. R. Harris. 1971. Degradation of heptachlor epoxide and heptachlor by a mixed culture of soil microorganisms. J. Econ. Entomol. 64:839-841 Mi l le r , E . , G . Wohlfarth and G. Diekert. 1997a. Studies on tetrachloroethene respiration in Dehalospirillum multivorans. Arch. Microbiol . 166:379-387 Mil le r , E . , G . Wohlfarth and G . Diekert. 1997b. Comparative studies on tetrachloroethene reductive dechlorination mediated by Desulfitobacterium sp. strain P C E - S . Arch. Microbiol . 168:513-519 Mi l l igan , P. W . and M . M . Haggblom. 1996. Anaerobic transformation of dicamba and chlorsalicylic acids under denitrifying and methanogenic conditions, abstr. Q-410, p. 457. In Abstr. 96th Gen. Meet. A m . Soc. Microbiol . American Society for Microbiology, Washington, D . C. Miskus, R. P., D . P. Blair and J. E . Casida. 1965. Conversion of D D T to D D D by bovine rumen fluid, lake water, and reduced porphyrins. J. Agr i . Food Chem. 13:481-483 Mohn, W . W. , T. G. L inkf ie ld ,H. S. Pankaratz and J. M . Tiedje. 1990. Involvement of a collar structure in polar growth and cell division of strain D C B - 1 . App l . Environ. Microbiol . 56:1206-1211 Mohn, W . W . and K . J. Kennedy. 1992. Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei D C B - 1 . Appl . Environ. Microbiol . 58:1367-1370 120 Mohn, W . W . and J. M . Tiedje. 1990a. Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain D C B - 1 , a reductively dechlorinating anaerobe. J. Bacteriol. 172:2065-2070 Mohn, W . W . and J. M . Tiedje. 1990b. Strain D C B - 1 conserves energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbio l . 153:267-271 Mohn , W . W . and J. M . Tiedje. 1991. Evidence for chemiosmotic coupling of reductive dechlorination and A T P synthesis in Desulfomonile tiedjei. Arch. Microbio l . 157:1-6 Monkara, F. , S. J. Bingham, F. H . A . Kadir, A . G . McEwan , A . J. Thomson, A . G . P. Thurgood and G . R. Moore. 1992. Spectroscopic studies of Rhodobacter capsulatus cytochrome c' in the isolated state and in intact cells. Biochim. Biphys. Acta. 1100:184-188 Moore, G . R. and G . W . Pettigrew. 1990. Cytochromes c: Evolutionary, structural and physiochemical aspects. Springer-Verlag, Berlin Morris , P. J., W . W . Mohn, J. F. Quensen, III., J. M . Tiedje and S. A . Boyd . 1992. Establishment of a polychlorinated biphenyl-degrading enrichment culture with predominantly meta dechlorination. Appl . Environ. Microbiol . 58:3088-3094 Motosugi, K . , N . Esaki and K . Soda. 1983. Purification and properties of 2-haloacid dehalogenase fromPseudomonasputida. Agric. B i o l . Chem. 46:837-838 Motosugi, K . , N . Esaki and K . Soda. 1984. Enzymatic preparation of D - and L-lactic acid from racemic 2-chloropropionic acid. Biotechnol. Bioeng. 26:805-806 Mountfort, D . O. 1978. Evidence for A T P synthesis driven by a proton gradient in Methanosarcina barkeri. Biochem. Biophys. Res. Commun. 85:1346-1351 Muller , F . 1983. The flavin redox-system and its biological function. Top. Curr. Chem. 108:71-107 Neumann, A . , H . Schloz-Muramatsu and G . Diekert. 1994. Tetrachloroethene metabolism of Dehalospirillum multivorans. Arch. Microbiol . 162:295-301 Neumann, A . , G . Wohlfarth and G . Diekert. 1995. Properties of tetrachloroethene and trichloroethene dehalogenase of Dehalospirillum multivorans. Arch . Microbiol . 163:276-281 121 Neumann, A . , G . Wohlfarth and G . Diekert. 1996. Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans. J. B i o l . Chem. 271:16515-16519 N i , S., J. K . Fredrickson and L . Xun. 1995. Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei D C B - 1 . J. Bacteriol. 177:5135-5139 N i k i , K . , Y . Kawasaki, N . Nishimura, Y . Higuchi, N . Yasuoka and M . Kakudo. 1984. Electrochemical and structural studies of tetraheme proteins from Desulfovibrio — standard potentials of the redox sites and heme-heme interactions. J. Electroanal. Chem. 168:275-286 Niviere, V . , A . Bernadac, N . Forget, V . M . Fernandez and E . C. Hatchikian. 1991. Localization of hydrogenase in Desulfovibrio gigas cells. Arch. Microbio l . 155:579-586 Odom, J. M . and H . D . Peck, Jr. 1981. Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas. J. Bacteriol. 147:161-169 Ohisa, N . , N . Kurihara and M . Nakajima. 1982. A T P synthesis associated with the conversion of hexachlorocyclohexanerelated compounds. Arch. Microbio l . 131:330-333 Ohisa, N . , M . Yamaguchi andN. Kurihara. 1980. Lindane degradation by cell-free extracts of Clostridium rectum. Arch. Microbiol . 125:221-225 Orser, C . S., J. Dutton, C. Lange, P. Jablonski, L . X u n and M . Hargis. 1993. Characterization of a Flavobacterium glutathione .^-transferase gene involved in reductive dechlorination. J. Bacteriol. 175:2640-2644 Orser, C. S. and C. C. Lange. 1994. Molecular analysis of pentachlorophenol degradation. Biodegradation 5:277-288 Peck, H . D . , Jr. and T. Lissolo. 1988. Assimilatory and dissimilatory sulphate reduction: enzymology andbioenergetics, p. 99-132.In J. A . Cole and S. J. Ferguson (ed), The nitrogen and sulphur cycles. Cambridge University Press, Cambridge, U . K . Picardal, F . W. , R. G . Arnold, H . Couch, A . M . Little a n d M . E . Smith. 1993. Involvement of cytochromes in the anaerobic biotransformation of tetrachloromethane by Shewanella putrefaciens 200. Appl . Environ. Microbiol . 59:3763-3770 122 Pollock, W . B . R., M . Loutfi, M . Bruschi, B . J. Rapp-Giles, J. D . Wal l and G . Voordouw. 1991. Cloning, sequencing and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 173:220-228 Quastel, J. H . 1963. Inhibitions in the citric acid cycle, p. 473-502. In R. M . Hochster and J. H . Quastel (ed.), Metabolic inhibitors — a comprehensive treatise, vol . n . Academic Press, Inc., New York Quensen, J. F. , III., S. A . Boyd and J. M . Tiedje. 1990. Dechlorination of four commercial polychlorinated biphenyl mixtures (Aroclors) by anaerobic microorganisms from sediments. App l . Environ. Microbiol . 56:2360-2369 Quensen, J. F. , III., J. M . Tiedje and S. A . Boyd. 1988. Reductive dechlorination of polychlorinated biphenyls by anaerobic microorganisms from sediments. Science 242:752-754 Ramanand, K . , A . Nagarajan and J. M . Suflita. 1993. Reductive dechlorination of the nitrogen heterocyclic herbicide picloram. Appl . Environ. Microbiol . 59:2251-2256 Rohde, M . , V . Furstenau, F. Meyer, A . E . Przybyla, H . D . Peck, Jr., J. L e G a l l , E . S. Choi and N . K . Menon. 1990. Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures. Eur. J. Biochem. 191:389-396 Romanov, V . andR. P. Hausinger. 1996. NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and Coryneform bacterium strain N T B - 1 via 2,4-dichlorobenzoyl coenzyme A . J. Bacteriol. 178:2656-2661 Rossi, M . , W . B . R. Pollock, M . W . Reij, R. G . Keon, R. Fu and G . Voordouw. 1993. The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex. J. Bacteriol. 175:4699-4711 Saber, D . L . and R. L . Crawford. 1985. Isolation and characterization of Flavobacteium strains that degrade pentachlorophenol. Appl . Environ. Microbiol . 50:1512-1518 Sanford, R. A . , J. R. Cole, F . E . Ldffler and J. M . Tiedje. 1996. Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to reductive dechlorination of 3-chloro-4-hydroxybenzoate. App l . Environ. Microbiol . 62:3800-3808 123 Santos, H . , J. J. G . Moura, I. Moura, J. LeGal l and A . V . Xavier. 1984. N M R studies of electron transfer mechanisms in a protein with interacting redox centers: D. gigas cytochrome c 3 . Eur. J. Biochem. 141:283-296 Saraiva, L . M . , S. Besson, I. Moura and G. Fauque. 1995. Purification and preliminary characterization of three c-type cytochromes from Pseudomonas nautica strain 617. Biochem. Biophys. Res. Commun. 212:1088-1097 Schauder, R., A . Preuss, M . Jetten and G. Fuchs. 1989. Oxidative and reductive acetyl CoA/carbon monoxide dehydrogenase pathway in Desulfobacterium autotrophicum. 2. Demonstration of the enzymes of the pathway and comparison of carbon monoxide dehydrogenase. Arch. Microbiol . 151:84-89 Scholz-Muramatsu, FL, A . Neumann, M . Mepmer, E . Moore and G . Diekert. 1995. Isolation and characterization of Dehalospirilium multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing, strictly anaerobic bacterium. Arch. Microbio l . 163:48-56 Schumacher, W . and C. Holliger. 1996. The proton/electron ratio of the menaquinone-dependent electron transport from dihydrogen to tetrachloroethene in "Dehalobacter restrictus". J. Bacteriol. 178:2328-2333 Schumacher, W. , C. Holliger, A . J. B . Zehnder and W . R. Hagen. 1997. Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus. F E B S Lett. 409:421-425 Sebban, C , L . Blanchard, M . Bruschi and F. Guerlesquin. 1995. Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough. F E M S Microbiol . Lett. 133:143-149 Sharma, P. K . and P. L . McCarty. 1996. Isolation and characterization of a facultatively aerobic bacterium that reductively dehalogenates tetrachloroethene to cis-1,2-dichloroethene. Appl . Environ. Microbiol . 62:761-765 Shelton, D . R. and J. M . Tiedje. 1984. Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. A p p l . Environ. Microbiol . 48:840-848 Sikkema, J., J. A . M . De Bont and B . Poolman. 1995. Mechanisms of membrane toxicity of hydrocarbons. Microbiol . Rev. 59:201-222 124 Slater, E . C. 1963. Uncouplers and inhibitors of oxidative phosphorylation, p. 503-516. In R. M . Hochster and J. H . Quastel (ed.), Metabolic inhibitors — a comprehensive treatise, vol . II. Academic Press, Inc., New York Smith, M . H . and S. L . Woods. 1994. Regiospecificity of chlorophenol reductive dechlorination by vitamin B i 2 . Appl . Environ. Microbiol . 60:4111-4115 Smith, P. K . , R. I. Krohn, G . T. Hermanson, A . K . Mal l ia , F . H . , Gartner, M . D . Provenzano, E . K . Fujimoto, N . M . Goeke, B . J. Olson and D . C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal . Biochem. 150:76-85 Steiert, J. G . and R. L . Crawford. 1986. Catabolism of pentachlorophenol by a Flavobacterium sp. Biochem. Biophys. Res. Commun. 141:825-830 Stevens, T. O. 1987. M . S . Thesis. Michigan State University, East Lansing. Stevens, T. O., T. G . Linkfield and J. M . Tiedje. 1988. Physiological characterization of strain D C B - 1 , a unique dehalogenating sulfidogenic bacterium. App l . Environ. Microbio l . 54:2938-2943 Stevens, T. O. and J. M . Tiedje. 1988. Carbon dioxide fixation and mixotrophic metabolism by strain D C B - 1 , a dehalogenating anaerobic bacterium. App l . Environ. Microbiol . 54:2944-2948 Stromeyer, S. A . , K . Stumpf, A . M . Cook and T. Leisinger. 1992. Anaerobic degradation of tetrachloromethane by Acetobacteriumwoodii: separation of dechlorinative activities in cell extracts and roles for vitamin B i 2 and other factors. Biodegradation 3:113-123 Taraban, R. H . , D . F. Berry, D . A . Berry and H . L . Walker, Jr. 1993. Degradation of dicamba by an anaerobic consortium enriched from wetland soil. App l . Environ. Microbiol . 59:2332-2334 Terzenbach, D . P. and M . Blaut. 1994. Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria. F E M S Microbiol . Lett. 123:213-218 Thony-Meyer, L . 1997. Biogenesis of respiratory cytochromes in bacteria. Microbio l . M o l . Rev. 61:337-376 Thony-Meyer, L . , F. Fischer, P. Kunzler, D . Ritz and H . Hennecke. 1995. Escherichia coli genes required for cytochrome c maturation. J. Bacteriol. 177:4321-4326 125 Topp, E . , R. L . Crawford and R. S. Hanson. 1988. Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degradingF/avo6acferzw/w sp. App l . Environ. Microbiol . 54:2452-2459 Topp, E . and R. S. Hanson. 1990. Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations. App l . Environ. Microbiol . 56:541-544 Toussaint, M . , C . M . Commandeur, J. R. Parsons, J. E . M . Beurskens and J. de Wolf . 1992. Reductive dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin by a bacterial consortium isolated from Lake Ketelmeer sediment: preliminary results, p. 578-585. In International symposium on soil decontamination using biological processes. D E C H E M A Townsend, G . T. and J. M . Suflita. 1996. Characterization of chloroethylene dehalogenation by cell extracts of Desulfomonile tiedjei and its relationship to chlorobenzoate dehalogenation. Appl . Environ. Microbiol . 62:2850-2853 Townsend, G . T. and J. M . Suflita. 1997. Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei. Appl . Environ. Microbio l . 63:3594-3599 Uotila, J. S., V . H . Kitunen, J. H . A . Apajalahti and M . S. Salkinoja-Salonen. 1992. Environment-dependent mechanism of dehalogenation by Rhodococcus chlorophenolicusPCP-1. Appl . Microbiol . Biotechnol. 38:408-412 Uotila, J. S., V . H . Kitunen, T. Coote, T. Saastamoinen, M . Salkinoja-Salonenand J. H . A . Apajalahti. 1995. Metabolism of halohydroqui nones in Rhodococcus chlorophenolicus PCP-1 . Biodegradation 6:119-126 Utkin, I., D . D . Dalton and J. Wiegel. 1995. Specificity of reductive dehalogenation of substituted orf/io-chlorophenols by Desulfitobacterium dehalogenans J W / I U - D C 1 . App l . Environ. Microbiol . 61:346-351 Utkin, I., C. Woese and J. Wiegel. 1994. Isolation and characterization o f Desulfitobacterium dehalogenans gen. nov., sp. nov., an anaerobic bacterium which reductively dechlorinates chlorophenolic compounds. Int. J. Syst. Bacteriol. 44:612-619 Van den Tweel, W . J. J., J. B . K o k and J. A . M . De Bont. 1987. Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorbenzoate and hydrolytic dehalogenation of 4-chloro, 4-bromo, and 4-iodobenzoate by Alcaligenes denitrificans N T B - 1 . App l . Environ. Microbiol . 53:810-815 126 Van der Westen, H . M , S. G . Mayhew and C. Veeger. 1978. Separation of hydrogenase from intact cells of Desulfovibrio vulgaris. F E B S Lett. 86:122-126 V a n Dort, H . M . and D . L . Bedard. 1991. Reductive ortho and meta dechlorination of a polychlorinated biphenyl congener by anaerobic microorganisms. A p p l . Environ. Microbiol . 57:1576-1578 Voordouw, G . and S. Brenner. 1985. Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough). Eur. J. Biochem. 148:515-520 Voordouw, G. , V . Niviere, F. G . Ferris, P. M . Fedorak and D . W . S. Westlake. 1990. Distribution of hydrogenase genes in Desulfovibrio spp. and their use in identification of species from the oil field environment. Appl . Environ. Microbio l . 56:3748-3754 Voordouw, G. , J. E . Walker and S. Brenner. 1985. Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH 2 - terminal sequence. Eur. J. Biochem. 148:509-514 Wedemeyer, G . 1966. Dechlorination of D D T by Aerobacter aerogenes. Science 152:647 W i l d , A - , R- Hermann and T. Leisinger. 1996. Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene. Biodegradation 7:507-511 W i l d , A . P., W . Winkelbauer and T. Leisinger. 1995. Anaerobic dechlorination of trichloroethene, tetrachloroethene and 1,2-dichloroethane by an acetogenic mixed culture in a fixed-bed reactor. Biodegradation 6:309-318 Wol in , E . A . , M . J. Wol in and R. S. Wolfe. 1963. Formation of methane by bacterial extracts. J. B i o l . Chem. 238:2882-2886 Wood, J. M . 1982. Chlorinated hydrocarbons: oxidation in the biosphere. Environ. Sci. Technol. 16:291A-297A Woese, C. R. 1987. Bacterial evolution. Microbiol . Rev. 51:221-271 X u n , L . and C. S. Orser. 1991. Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain A T C C 39723. J. Bacteriol. 173:4447-4453 127 X u n , L . , E . Topp and C. S. Orser. 1992a. Confirmation of oxidative dehalogenation of pentachlorophenol by a Flavobacterium pentachlorophenol hydroxylase. J. Bacteriol. 174:5745-5747 Xun , L . , E . Topp and C. S. Orser. 1992b. Purification and characterization of a tetrachloro-/?-hydroquinone reductive dehalogenase from a Flavobacterium sp. J. Bacteriol. 174:8003-8007 Yamanaka, T. and S. Imai. 1972. A cytochrome cc'-like haemoprotein isolated from Azobacter vinelandii. Biochem. Biophys. Res. Commun. 46:150-154 Y e , D . , J. F. Quensen, III., J. M . Tiedje and S. A . Boyd. 1995. Evidence for para dechlorination of polychlorobiphenyls by methanogenic bacteria. App l . Environ. Microbiol . 61:2166-2171 Y o u , Y . , S. Elmore, L . L . Colton, C. Mackenzie, J. K . Stoops, G . M . Weinstock and S. J. Norris. 1996. Characterization of the cytoplasmic filament protein gene (cjpK) of Treponema pallidum subsp. pallidum. J. Bacteriol. 178:3177-3187 Zaitsev, G . M . and Y . N . Karasevich. 1985. Preparatory metabolism of 4-chlorobenzoate and 2,4-dichlorobenzoic acids in Corynebacterium sepedonicum. Mikrobiologiya 54:356-359 Zoro, J. A . , J. M . Hunter, G . Eglintonand G. C. Ware. 1974. Degradation of p,p'-DDT in reducing environments. Nature 247:235-237 128 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0099356/manifest

Comment

Related Items