METABOLISM OF HALOGENATED COMPOUNDS BY RHODOCOCCUS UKMP-5M JAYASUDHA NAGARAJAN A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AND DIPLOMA OF IMPERIAL COLLEGE LONDON DEPARTMENT OF CHEMISTRY FACULTY OF NATURAL SCIENCES IMPERIAL COLLEGE LONDON 2014 DECLARATION I certify that this thesis represents my own work, unless otherwise stated. All external contributions and any information derived from other sources have been acknowledged accordingly. JAYASUDHA NAGARAJAN The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work 2 In living memory of my late granny, Madam Thanalaetchumi Appadurai… Dedicated to Dr. Judit Nagy, an outstanding scientist and the most inspirational woman I have ever met, without whom my PhD journey would have not been possible....may your soul rest in peace… Dedicated to my dad and mom Mr. & Mrs. Nagarajan Sarujuny, the most precious gift in my life. For their endless support, love and encouragement through all my walks of life…. Thank you pa & ma. & To my lovely sister, Jayesree Nagarajan, the little diamond in the family which never fails to shine.. Thank you so much for being there for me whenever I need you.. May you also be motivated to achieve great heights in life… 3 ACKNOWLEDGEMENT First and foremost, I’d like to express my deepest gratitude to Professor Tony Cass for accepting me in his research team and for all his patience and valuable inputs to successfully drive this project. I’d like to also thank my co-supervisor, Professor David Leak for his continued support and guidance throughout the project. Thanks to the Malaysian Ministry of Science, Technology and Innovation for sponsoring my PhD studies in Imperial College London and for giving me an opportunity to be among the best brains in the world! My heartfelt gratitude goes to Dr. Lok Hang Mak for all the guidance, knowledge, ideas and friendship that helped carry me through. To the members, past and present, of Cass Group; Dr. Sanjiv, Dr. Muthu, Dr. Thao, Dr. Chris, Achi, Orada, Sasinee, Ben, Dan, Mahsan, Ascanio and Hannah- thank you for your support encouragement and the ‘giggles’. Sarah, Tami and Nawal – thank you so much girls, you are more like sisters to me and thanks a lot for all the ‘fun’ we had together..I’ll cherish those moments forever~ Very special thanks to my family and friend in London, my best friend Hazeeq. You have been a great support to me in many ways especially during all those time when I was down. You made my life in London, very colourful and I do have really great memories to bring back home! BFF forever. To all my other friends in UK, esp. Raja, Joe, Shahrul, Mus and Roobini - thanks guys for the motivation and enlightening words from all of you especially when I need them the most! Keep in touch all…Not forgetting, friends and colleagues in Malaysia esp. Prof. Latif and Dr. Maegala for all the valuable inputs throughout my PhD. Finally, thank you to my family members, relatives and friends for being there for me. Last but not least, to the Almighty for giving me the strength and perseverance to complete my studies within the stipulated time frame and for keeping me surrounded by these beautiful people! 4 ABSTRACT Members of the genus Rhodococcus are well known for their high metabolic capabilities to degrade wide range of organic compounds ranging from simple hydrocarbons to more recalcitrant compounds such as polychlorinated biphenyls. Their ability to display novel enzymatic capabilities for the transformation of many hazardous contaminants in the environment makes them a potential candidate for bioremediation. Rhodococcus UKMP-5M, an actinomycete isolated in Peninsular Malaysia shows great potential towards degradation of cyanide, hydrocarbons and phenolic compounds. In the present study, the capacity of this strain to degrade halogenated compounds was explored. Preliminary investigations have proven that R. UKMP-5M was not able to utilise any of the halogenated compounds tested as sole carbon and energy source, but the resting cells of R. UKMP-5M was able to dechlorinate several compounds which include chloroalkanes, chloroalcohols and chloroacids and the activity was three fold higher when the cells were grown in the presence of 1-Chlorobutane (1-CB). Therefore, 1-CB was chosen as a substrate to unravel the mechanism of dehalogenation in R. UKMP-5M. In contrast to the classic hydrolytic route for the assimilation of 1-CB in many organisms, R. UKMP-5M was able to metabolise and release chloride from 1-CB, but is unable to use the product from 1-CB metabolism as growth substrate. On comparing the protein profiles of the induced and non-induced cells of R. UKMP- 5M, two types of monooxygenases were identified in the induced condition, which were not present in the uninduced sample. The strict oxygen requirement for dechlorination of 1-CB and the identification of monooxygenases in the induced protein extract suggests that 1-CB dehalogenation is likely to be catalysed by a monooxygenase. In addition to these monooxygenases, a protein that was later identified as amidohydrolase (Ah) was also found to be induced when the cells were exposed to 1-CB. Therefore, Ah from R. UKMP-5M was cloned and expressed in E. coli to test the ability of the purified Ah to release chloride from 5 1-CB. The heterologous expression of Ah in E. coli resulted in the formation of inclusion bodies and the western blot analyses further confirmed that no soluble form of Ah was present. Multiple attempts to obtain a soluble and functionally active Ah were not successful. Therefore, on-column refolding was carried out to obtain a biologically active Ah. A 3D model based on structural homology was predicted as a preliminary step to characterize this protein. However, when assayed with 1-CB, Ah was found not to catalyze dehalogenation. All results of this thesis suggest that metabolism of 1-CB by R. UKMP-5M is via γ-butyrolactone which acts as a potent intracellular electrophile that covalently modifies proteins and nucleic acids. The findings from this research are important to determine the metabolic capacity of a Malaysian Rhodoccoccus in dehalogenation of halogenated compounds and its potential application in bioremediation. 6 LIST OF FIGURES Figure 1.1 Concept of bioremediation 21 Figure 1.2 The most frequently detected organic compounds in the European ground 24 water reported in Environmental Agency Database Figure 1.3 The common routes of horizontal gene transfer 28 Figure 1.4 Degradability of some halogenated compounds by microorganism 31 Figure 1.5 Reaction of hydrolytic dehalogenation 32 Figure 1.6 Mechanism of thiolytic dehalogenation 33 Figure 1.7 Dehalogenation of halomethane catalysed by methyltransferase 34 Figure 1.8 Mechanism of dehalogenation through hydration 34 Figure 1.9 Mechanism of oxidative dehalogenation 35 Figure 1.10 Dehalogenation of lindane by dehydrohalogenase 36 Figure 1.11 Mechanism of dehalogenation through intramolecular substitution reaction 37 Figure 1.12A Mechanism of reductive dehalogenation of tetrachlorohydroquinone (TCHQ) 38 Figure 1.12B Reductive dehalogenation of tetrachloroethylene (PCE) 39 Figure 1.13 The increasing pattern of number of publications concerned with 40 degradation of nitriles, aromatic and halogenated compounds by Rhodococci Figure 1.14 Different morphological forms of Rhodococcus 42 Figure 2.1 Growth of two Rhodococcus strains monitored spectrophotometrically at 12 56 hours interval for 60 hours Figure 2.2 Growth of R. UKMP-5M in minimal media containing 1-CB supplemented 58 with other primary substrates Figure 2.3 Growth of R. UKMP-5M in minimal media supplemented with 20 mM of 59 various carbon sources Figure 2.4 Calibration curve for chloride assay 60 Figure 2.5 Chloride release from 1-CB by resting cells of R. UKMP-5M grown in 61 different carbon sources 7 Figure 2.6 Chloride release from 1-CB by resting cells of R. UKMP-5M grown in 20 mM 64 glucose and 20 mM glucose supplemented with 1-CB Figure 2.7 Chloride release from 1-CB by resting cells of R. UKMP-5M grown in LB and 65 LB supplemented with 1-CB Figure 2.8 Chloride release from 1-CB by resting cells of R. UKMP-5M grown in 66 glucose and glucose supplemented with each component of LB Figure 2.9 Effect of manipulating physical parameters on the degradation of 1-CB by R. 67 UKMP-5M Figure 2.10A Chloride release from 1-CB by resting cells of R. UKMP-5M monitored in the 71 presence and absence of oxygen Figure 2.10B Cells monitored in the absence of oxygen were aerated and re-incubated 71 with 1-CB for their activity recovery Figure 2.11 Chloride release from 1-CB by resting cells of R. UKMP-5M monitored in the 73 presence of carbon monoxide Figure 2.12A Possible metabolic routes for the dehalogenation of 1-CB in R. NCIMB 74 13064 Figure 2.12B Dehalogenation of 1-CB via internal hydroxylation 75 Figure 2.13 Cell density of R. UKMP-5M grown on several potential intermediates of 1- 76 CB degradation Figure 2.14 Growth recovery patterns of R. UKMP-5M unexposed and exposed to 1-CB 77 Figure 3.1 The schematic workflow of two-dimensional gel electrophoresis 80 Figure 3.2 Calibration curve for protein assay 91 Figure 3.3 The non-reproducibility of 2D GE 92 Figure 3.4 1D GE of protein samples prepared from R.