CHARACTERIZATION OF 1, 2-DCA DEGRADING Ancylobacter aquaticus STRAINS ISOLATED IN SOUTH AFRICA BY Thiloshini Pillay Submitted in fulfilment of the academic requirements for the degree of Master of Science (MSc) in the Discipline of Microbiology, School of Biochemistry, Genetics and Microbiology, Faculty of Science and Agriculture at the University of KwaZulu-Natal (Westville Campus). As the candidate’s supervisor, I have approved this dissertation for submission. Signed: Name: Date: PREFACE The experimental work described in this dissertation was carried out in the School of Biochemistry, Genetics and Microbiology; University of KwaZulu-Natal (Westville Campus), Durban, South Africa from May 2007 to April 2010, under the supervision of Professor B. Pillay and the co-supervision of Dr. A. O. Olaniran. These studies represent original work by the author and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where use has been made of the work of others it is duly acknowledged in the text. FACULTY OF SCIENCE AND AGRICULTURE DECLARATION 1 – PLAGIARISM I, ……………………………………….……………………………………………………...., declare that 1. The research reported in this dissertation, except where otherwise indicated, is my original research. 2. This dissertation has not been submitted for any degree or examination at any other university. 3. This dissertation does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This dissertation does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed …………………………………………………………………………… Declaration Plagiarism 22/05/08 FHDR Approved FACULTY OF SCIENCE AND AGRICULTURE DECLARATION 2 – PUBLICATIONS DETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and/or include research presented in this dissertation (include publications in preparation, submitted, in press and published and give details of the contributions of each author to the experimental work and writing of each publication) Publication 1 NOT APPLICABLE Publication 2 Publication 3 Etc. Signed: …………………………………………………………………………… Declaration Publications FHDR 22/05/08 Approved ACKNOWLEDGEMENTS The author records her appreciation to the following people: Prof B. Pillay for his supervision during the course of this study; Dr. A. Govender for his guidance and contribution throughout the study; My dearest friends (Desiree Alvika Rajpal, Dr. R. Shaik, Charlene Singh) for their assistance, guidance, constant support, words of encouragement and much appreciated advice in times of despair; Dr. A. O. Olaniran for his support and assistance; The staff and postgraduate students at the Discipline of Microbiology, University of KwaZulu-Natal (Westville Campus) for their support and assistance; LIFElab and DAAD for financial assistance; My cherished friends Ammeshnie Govender, Dhevashnee Pillay and their families for the constant support and words of encouragement provided throughout the course of this study; Most importantly, my family for their immense patience, support and encouragement throughout this study. i CHARACTERIZATION OF 1, 2-DCA DEGRADING Ancylobacter aquaticus STRAINS ISOLATED IN SOUTH AFRICA THILOSHINI PILLAY 2011 TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS i ABSTRACT ii LIST OF FIGURES iv LIST OF TABLES vi CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1 1. 1 INTRODUCTION 1 1.2 CHLORINATED ALIPHATIC HYDROCARBONS (CAHS) 2 1.2.1 Properties and uses of CAHs 2 1.2.2 Presence and persistence of CAHs in the environment 2 1.2.3 Biodegradation of CAHs 4 1.3 DEHALOGENASES: TYPES AND MECHANISMS 6 1.3.1 Hydrolytic dehalogenation 6 1.3.2 Oxygenolytic dehalogenation 7 1.3.3 Thiolytic dehalogenation 7 1.3.4 Reductive dehalogenation 8 1.3.5 Haloalcohol dehalogenation 10 1.3.6 Dehydrohalogenation 10 1.3.7 Hydration 11 1.4 1, 2-DICHLOROETHANE (1,2-DCA) 12 1.4.1 Properties and uses of 1,2-dichloroethane 12 1.4.2 Impact of 1,2-DCA on the environment 12 1.4.3 Impact of 1,2-DCA on human health 13 1.4.4 Microbial degradation of 1,2-DCA 13 1.4.4.1 Anaerobic conditions 15 1.4.4.2 Aerobic conditions 15 1.5 ENZYMES INVOLVED IN THE 1,2-DCA DEGRADATION PATHWAY 17 1.5.1 Haloalkane dehalogenases 17 1.5.1.1 Types of haloalkane dehalogenases 17 1.5.1.2 Mechanism and structure of haloalkane dehalogenases 20 1.5.2 Alcohol dehydrogenase 22 1.5.3 Chloroacetaldehyde (CAA) dehydrogenase 22 1.5.4 Haloacid (HA) dehalogenases 23 1.5.4.1 Types of 2-haloacid dehalogenases 23 1.5.4.2 Mechanism and structure of the 2-haloacid dehalogenase 23 1.6 BIOREMEDIATION 25 1.6.1 Advantages of bioremediation 25 1.6.2 Disadvantages of bioremediation 26 1.7 PURPOSE OF STUDY 26 1.8 HYPOTHESIS 28 1.9 OBJECTIVES 28 1.10 AIMS 29 CHAPTER TWO: MATERIALS AND METHODS 30 2.1 GROWTH AND MAINTENANCE OF BACTERIAL CULTURES 30 2.2 STANDARDIZATION OF BACTERIAL STRAINS FOR INOCULATION INTO GROWTH MEDIA 30 2.3 GROWTH MEASUREMENT STUDIES OF Ancylobacter aquaticus ISOLATES ON VARIOUS HALOGENATED COMPOUNDS 31 2.4 DETERMINATION OF FREE HALIDE RELEASED DURING DEGRADATION OF HALOGENATED COMPOUNDS 32 2.5 MEASUREMENT OF DEHALOGENASE ENZYME ACTIVITIES 32 2.5.1 Preparation of crude extracts 32 2.5.2 Dehalogenase enzyme assays 32 2.6 ISOLATION OF TOTAL DNA 34 2.7 RESTRICTION ENZYME DIGESTION 34 2.8 PCR AMPLIFICATION OF ALDEHYDE DEHYDROGENASE (aldB) AND HALOACID DEHALOGENASE (dhlB) ENCODING GENES PRESENT IN Xanthobacter autotrophicus GJ10 35 2.9 SOUTHERN HYBRIDIZATION 36 2.10 CLONING AND SEQUENCING OF THE aldB AND dhlB GENES 36 2.10.1 Purification of DNA fragments 36 2.10.2 Preparation of electrocompetent E.coli cells 37 2.10.3 Isolation and restriction of pBluescript II SK 36 2.10.4 Ligation of inserts to plasmid vector 38 2.10.5 Transformation of E. coli DH5α’F 38 2.10.6 Plasmid DNA isolation 38 CHAPTER THREE: RESULTS 3.1 DEGRADATION OF HALOGENATED ALIPHATIC HYDROCARBONS 40 3.1.1 Aerobic utilization of 1,2-DCA 40 3.1.1.1 Growth pattern of Ancylobacter strains in minimal media supplemented with either yeast extract or vitamins 40 3.1.1.2 Chloride release during growth of the bacterial isolates in 1,2-DCA 43 3.1.2 Aerobic utilization of MCA by the bacterial isolates monitored in minimal media supplemented with vitamins 45 3.1.2.1 Growth pattern of bacterial isolates on MCA 45 3.1.2.2 Chloride release during growth in MCA 47 3.1.3 Growth of Ancylobacter aquaticus strains on other halogenated aliphatic compounds 50 3.2 DEHALOGENASE ACTIVITY OF BACTERIAL ISOLATES 51 3.2.1 Effect of pH and temperature on dehalogenase activity 51 3.2.2 Dehalogenase activities of crude extract on various halogenated compounds 54 3.3 DETECTION OF HALOACID DEHALOGENASE AND CHLOROACETALDEHYDE DEHYDROGENASE ENCODING GENES IN Ancylobacter ISOLATES 55 3.3.1 PCR amplification of aldB and dhlB encoding genes from Xanthobacter autotrophicus GJ10 55 3.3.2 Detection of the haloacid dehalogenase (HA) encoding genes in Ancylobacter species 55 3.3.3 Detection of CAA dehydrogenase encoding genes in Ancylobacter strains 59 3.4 CLONING OF THE HA DEHALOGENASE AND CAA DEHYDROGENASE GENES 62 CHAPTER FOUR: DISCUSSION 63 CHAPTER FIVE: CONCLUDING REMARKS 75 5.1 THE RESEARCH IN PERSPECTIVE 75 5.2 POTENTIAL FOR FUTURE DEVELOPMENT OF THE STUDY 79 REFERENCES 81 APPENDIX I 94 LIST OF FIGURES PAGE Figure 1.1: Hydrolytic cleavage of the carbon-chlorine by hydrolytic dehalogenases (Van Pee and Unversucht, 2003) 7 Figure 1.2: Thiolytic dehalogenation with glutathione S-transferase (van Pee and Unversucht, 2003) 8 Figure 1.3: Reductive dehalogenation of tetrachlorohydroquinone under aerobic conditions catalyzed by a glutathione transferase (van Pee and Unversucht, 2003) 9 Figure 1.4: Reductive dehalogenation of PCE under anaerobic conditions (van Pee and Unversucht, 2003) 9 Figure 1.5: Thiolytic nucleophilic substitution of halogen catalyzed haloalcohol dehalogenase (Van Pee and Unversucht, 2003) 10 Figure 1.6: Dehalogenation of lindane catalyzed by dehydrohalogenases (van Pee and Unversucht, 2003) 11 Figure 1.7: Dehalogenation by the addition of a water molecule (Van Pee and Unversucht, 2003) 11 Figure 1.8: Catabolic route for 1,2-dichloroethane of Xanthobacter autotrophicus GJ10 and some Ancylobacter aquaticus species (Janssen et al., 1989) 16 Figure 1.9: Substrates of haloalkane dehalogenases from (a) Xanthobacter autotrophicus (DhlA), (b) Rhodococcus erythropolis (DhaA), and (c) Spingomonas paucimobilis (LinB) (Negri et al., 2007) 19 Figure 1.10: Mechanism of haloalkane dehalogenase (Copley, 1996) 20 Figure 1.11: General topology of the haloalkane dehalogenase (Janssen, 2004) 21 Figure 1.12: Reactions catalyzed by 2-haloacid dehalogenases. (A) L-2-Haloacid dehalogenase (B) D-2 haloacid dehalogenase. R indicates an alkyl group (Kurihara et al., 2000) 24 Figure 3.1: Growth pattern of bacterial isolates using 1,2-DCA as sole carbon source supplemented with yeast extract 41 Figure 3.2: Growth pattern of bacterial isolates using 1,2-DCA as sole carbon source supplemented with vitamins 42 Figure 3.3: Chloride concentration