Identification and Characterization of Two Thauera aromatica Strain T1 Genes Induced by p-Cresol A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Mohor Chatterjee August 2012 © 2012 Mohor Chatterjee. All Rights Reserved. 2 This dissertation titled Identification and Characterization of Two Thauera aromatica Strain T1 Genes Induced by p-Cresol by MOHOR CHATTERJEE has been approved for the Program of Molecular and Cellular Biology and the College of Arts and Sciences by Peter W. Coschigano Associate Professor of Biomedical Sciences Howard Dewald Interim Dean, College of Arts and Sciences 3 ABSTRACT CHATTERJEE, MOHOR, Ph.D., August 2012, Molecular and Cellular Biology Identification and Characterization of Two Thauera aromatica Strain T1 Genes Induced by p-Cresol (109 pp) Director of Dissertation: Peter W. Coschigano p-Cresol is a toxic aromatic compound found in the environment and is a constituent of many disinfectants and preservatives. It may act as a tumor promoter and the US Environmental Protection Agency has listed it as a possible human carcinogen. Thauera aromatica strain T1 is a facultative anaerobic, denitrifying, Gram-negative bacterium that is able to degrade many aromatic compounds including toluene and p- cresol. A proteomics approach was used to identify proteins from T. aromatica strain T1 that have differential expression when cells are induced by p-cresol in comparison to benzoate, a common downstream metabolic intermediate in the degradation of many aromatic compounds. Sequences of peptides from proteins selectively up-regulated by p- cresol in comparison to benzoate were obtained by MS analysis and compared against databases of known proteins from other microorganisms. The sequenced peptides from one of the isolated proteins induced by p-cresol matched a hypothetical protein from a different T. aromatica strain and another matched a hypothetical protein from Azoarcus sp. strain EbN1. Using PCR with degenerate oligonucleotides, the gene fragments corresponding to these proteins were obtained from the T1 genome. Expression analysis also showed differential up- regulation at the RNA level for both these genes when 4 compared to cells induced by benzoate. Using the genome sequence of T. aromatica strain 3CB2, the full-length genes corresponding to these proteins have been cloned from the T. aromatica strain T1 genome. The genes are named pipA and pipB (for p-cresol induced protein) and were determined to reside in a cluster within the T. aromatica strain T1 genome. Bioinformatic analyses of the corresponding gene and protein sequences of pipA and pipB show that these proteins are conserved in many bacterial species involved in aromatic compound biodegradation. PipA is predicted to be an outer membrane transport protein and PipB shows significant homology to a chaperone involved in sorting of lipoproteins in the outer membrane of bacteria. We hypothesize that these proteins may be components of bacterial nanopods that are involved in the uptake of a range of aromatic metabolites from the environment by the aromatic compound degrading bacterial species in the soil and the process may be energy-dependent. Approved: _____________________________________________________________ Peter W. Coschigano Associate Professor of Biomedical Sciences 5 Dedicated to my family and for their love, support and good wishes 6 ACKNOWLEDGMENTS I am thankful to many people for their unending love, support and faith in me during the pursuit of my degree. This journey would not have been possible without them. First and foremost, I would like to thank my advisor Dr. Peter W. Coschigano for the many ways in which he has helped me to carve my future in this field. He is an amazing human being apart from being an amazing scientist. I express my sincere gratitude to him for letting me work with him and for supporting me in every way even during difficult times. I am honored to be a part of his research team and could not have finished this work without his guidance at each and every step. I am extremely thankful to Dr. Chester R. Cooper and Julie M. Chandler of Youngstown State University for their help with proteomics and Dr. Bongkeun Song of the University of North Carolina at Wilmington for kindly providing the sequence of Thauera aromatica strain 3CB2 that helped my research immensely. My special thanks go out to the entire Murphy lab, especially Dr. Erin Murphy, William Broach and Andy Kouse. I am thankful to them for allowing me use their machines and reagents during my research and also for providing an enjoyable atmosphere for work. I have shared lot of good times with the Murphy lab, both in the university and away, especially at conferences we attended together. I also thank Dr. Karen Coschigano and her lab for helping me with the Real Time PCR experiment. Next, I am thankful to all my committee members, Dr. Calvin James, Dr. Xiao Zhuo Chen and Dr. Stefan Gleissberg for their guidance during this journey. Each of 7 them has provided me with valuable insights in my work and I truly believe the dissertation would have been incomplete without their inputs. I am extremely lucky to have such a committee of people to look after me. I am also thankful to Amr Elzawily of the Coschigano lab who was my colleague and a dear friend for his help during the initial days of my research. I wish him all the best in the future. The lab was always an enjoyable place to work with him around. My additional thanks go to Dr. Donald Holzschu, Dr. Joan Cunningham and Dr. Lorie Lapierre with whom I have done TA for more than four years. I consider myself lucky to have learnt the values of teaching from them. I had looked forward to each and every class with them and I know the students of Ohio University are lucky to have them as their instructors. I extend my gratitude to my professors in the Molecular and Cellular Biology Program for their influence in my education and the shaping of my future. I am grateful to Dr. Robert Colvin for being a mentor throughout my stay in this program. I also thank Molecular and Cellular Biology program and Graduate Student Senate for providing financial support to me during my Ph.D. I must also thank my professor Dr. Anjan K. Dasgupta of University of Calcutta who inspired me to embark upon the journey of Ph.D. Special thanks must be extended to my friends both inside and outside the department without whom this journey would have been very difficult. I express my gratitude to my friends Archan, Setu, Andy, Bill, Jian, Yan, Nilesh, Aditya, Aditi and Debarati for the good times spent in Ohio University. I am also thankful to my present and former roommates Gayatri, Sulalita, Koyel, Anupama, Sreerupa and Somali for being 8 such good friends. They have all made Athens a home outside of home and I cherish every good moment spent with them. Lastly, this section is incomplete without extending thanks to my family. My parents Mr. Achintya Kumar Chatterjee and Mrs. Mondira Chatterjee have been a source of perennial strength to me throughout my life. Words cannot express my gratitude towards them. They are the reason for whatever I am today. I also express my thanks to my fiancée Dr. Partha Nandi for providing me impetus to complete my research. He has been a source of strength and I look forward to spending my life with him. I am indebted to each and every people mentioned above and I wish them all best of luck. 9 TABLE OF CONTENTS Abstract ................................................................................................................................3 Dedication ............................................................................................................................5 Acknowledgement ...............................................................................................................6 Table of Contents .................................................................................................................9 List of Tables .....................................................................................................................12 List of Figures ....................................................................................................................13 List of Abbreviations .........................................................................................................16 Chapter 1 Introduction .......................................................................................................17 1.1 Biodegradation of Aromatic Compounds in the Environment ..............................17 1.2 p-Cresol as an Aromatic Compound ......................................................................18 1.3 Biochemistry of Cresol Degradation .....................................................................19 1.3.1 Aerobic Mode of p-Cresol Degradation ..........................................................20 1.3.2 Anaerobic Mode of p-Cresol Degradation .......................................................21 1.4 Benzoate as a Key Intermediate in Anaerobic Degradation ..................................25 1.5 Benzoate Degradation by Anaerobic Bacteria .......................................................27 1.6 Previous Work done with Thauera aromatica Strain T1.......................................32 1.7 Transporter Genes in Anaerobic Biodegradation ..................................................35 1.8 Anaerobic Monocyclic