ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE TUT FDGH OPERON OF THAUERA AROMATICA STRAIN T1 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 Reena Bhandare November 2007 2 This dissertation titled ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE TUTFDGH OPERON OF THAUERA AROMATICA STRAIN T1 by REENA BHANDARE has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Peter W. Coschigano Associate Professor of Microbiology Benjamin M. Ogles Dean, College of Arts and Sciences 3 ABSTRACT BHANDARE, REENA, Ph.D, November 2007, Biological Sciences ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE TUTFDGH OPERON OF THAUERA AROMATICA STRAIN T1 (134 pp.) Director of Dissertation: Peter W. Coschigano Toluene is an aromatic hydrocarbon that is widely used in our everyday life. It is a major water-soluble constituent of petroleum and can pollute surface as well as ground waters. The toxic nature of toluene is responsible for causing severe health hazards. The study of toluene degrading bacteria has attracted attention because of their potential to clean up spills. Thauera aromatica strain T1 is one such bacterium capable of degrading toluene under anaerobic conditions. The tutE tutFDGH gene cluster is essential for the first step of anaerobic toluene degradation in T. aromatica strain T1. The tutF, tutD and tutG genes are proposed to code for the three subunits of the enzyme benzylsuccinate synthase, which is involved in the initial step of anaerobic toluene degradation pathway. The tutE gene is proposed to code for the enzyme benzylsuccinate synthase activase. The precise role of the tutH gene in toluene degradation is currently unknown, but it is proposed to have an ATP/GTP binding domain and is assumed to be involved in benzylsuccinate synthase complex formation. This is consistent with its proposed role as a chaperone of “ATPases Associated with a Variety of Cellular Activities” (AAA) class. 4 Work presented here demonstrates that the gene tutH is essential for toluene metabolism. A plasmid carrying an in-frame tutH deletion was unable to produce wild- type TutH protein in a tutG chromosomal deletion background (chromosomal deletion in tutG does not result in production of TutH due to a polar effect on downstream genes). The resultant construct was unable to complement a polar tutG chromosomal mutation, indicating the importance of tutH in toluene degradation. Further, site-directed mutagenesis was used to identify amino acids in TutH that are essential for toluene metabolism. The TutH putative ATP/GTP binding domain was disrupted by changing glycine, lysine and serine at positions 52, 53 and 54 to alanine, arginine and alanine respectively. Additionally, other amino acids which are found to be highly conserved across closely related bacteria were also targeted for mutagenesis. Leucine and asparagine at positions 158 and 159 of TutH were changed to serine and alanine, glycine at position 161 was changed to alanine and arginine and phenylalanine at positions 177 and 178 were changed to alanine and serine, respectively. The resultant constructs were unable to complement a polar tutG chromosomal mutation, indicating that these amino acids are essential for toluene metabolism and might play important functional role. Additionally, using ProteoEnrich™ ATP Binders™ Kit (Novagen), it was demonstrated that the wild-type TutH protein can bind ATP, thereby supporting the possibility that it has a role in complex formation of benzylsuccinate synthase. 5 Furthermore, attempts were made to identify amino acids in TutF and TutG proteins that are essential for toluene metabolism. Using site-directed mutagenesis cysteine and alanine at positions 9 and 10 of TutF were changed to tyrosine and serine respectively and cysteine at position 29 of TutG was changed to serine. The resultant mutant constructs were unable to complement relevant chromosomal deletions, indicating that the substituted amino acids are important for toluene metabolism. Approved: Peter W. Coschigano Associate Professor of Microbiology 6 To my parents, for their love and support. 7 ACKNOWLEDGMENTS It is a known fact that “Dissertation = 10% inspiration + 90% perspiration”. I would like to thank all those who have helped me to balance this equation. Firstly, I would like to thank my advisor Dr. Peter Coschigano, for his excellent guidance, immense support and help. This four year journey in science would have been possible without him, but definitely not worth it! I would like to thank my PhD committee members, Dr. Don Holzschu, Dr. Calvin James and Dr. Guy Riefler for their valuable suggestions, help and support. My special thanks go out to Dr. Tomohiko Sugiyama and Dr. Noriko Kantake for their guidance and help with some of my experiments. Further, I would like to thank my current and ex lab members, Bethany Dean-Henderson, Mark Calabro, April Lust and Paul Wiehl, for their much required help and suggestions. I would also like to thank NSF, Ohio University Department of Biological Sciences, Biomedical Sciences, and Graduate Student Senate for financial support. At last but not the least, I would like to thank my family and friends for their love, support and encouragement, which always kept me rolling. 8 TABLE OF CONTENTS Abstract .................................................................................................................. 3 Acknowledgments............................................................................................................... 7 Table of Contents................................................................................................................ 8 List of Tables .................................................................................................................... 12 List of Figures................................................................................................................... 13 Note ..................................................................................................................... 15 1 Introduction................................................................................................... 16 1.1 Objective of this research...................................................................... 16 1.2 Importance of toluene degradation ....................................................... 17 1.3 Aerobic toluene degradation................................................................. 18 1.4 Anaerobic toluene degradation............................................................. 20 1.4.1 Importance of anaerobic toluene degradation................................... 20 1.4.2 Bacteria involved in anaerobic toluene degradation......................... 20 1.4.3 Mechanism of anaerobic toluene degradation .................................. 21 1.5 Anaerobic degradation of other aromatic compounds.......................... 24 1.6 Degradation of halogenated aromatic compounds................................ 26 1.7 Comparison of phylogenetically related toluene degrading bacteria.... 27 1.7.1 Comparison at morphological and physiological levels ................... 27 1.7.2 Comparison at genetic level.............................................................. 28 1.8 Role of tutE tutFDGH operon in toluene degradation.......................... 31 9 1.9 Benzylsuccinate synthase...................................................................... 34 1.9.1 Mechanism........................................................................................ 34 1.9.2 Homology with other enzymes ......................................................... 36 1.9.3 Substrate range for benzylsuccinate synthase................................... 36 1.10 Regulation of toluene degrading pathway in T. aromatica strain T1 ... 37 1.10.1 Regulatory two component system................................................... 37 1.10.2 Regulation by benzylsuccinate......................................................... 38 1.11 β-oxidation pathway ............................................................................. 40 1.12 Benzoyl-CoA pathway.......................................................................... 44 1.12.1 Biochemistry of Benzoyl-CoA pathway........................................... 44 1.12.2 Genes involved in the benzoyl-CoA pathway .................................. 47 2 Materials and methods .................................................................................. 50 2.1 Plasmids and Strains ............................................................................. 50 2.2 Media .................................................................................................... 54 2.3 Anaerobic cultivation............................................................................ 55 2.4 DNA preparation and subcloning ......................................................... 55 2.5 Selection of amino acids for site-directed mutagenesis........................ 56 2.6 Site-directed mutagenesis ..................................................................... 60 2.7 Triparental Mating................................................................................ 64 2.8 Testing for complementation ................................................................ 65 2.8.1 Cell growth and sample preparation ................................................. 65 2.8.2 HPLC analysis