Characterization of the Thioredoxin System Genes of Mycobacterium Smegmatis
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Characterization of the Thioredoxin System Genes of Mycobacterium smegmatis by Rumi Lynn Asano B.Sc, The University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1997 © Rumi Lynn Asano, 1997 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 DE-6 (2/88) ABSTRACT The thioredoxin system is composed of thioredoxin (TrxA), thioredoxin reductase (TrxB) and NADPH. Degenerate oligonucleotide primers were designed to detect, by polymerase chain reaction (PCR), trxA and trxB genes in M.smegmatis mc26. The complete nucleotide sequence of the Mycobacterium smegmatis thioredoxin system genes were obtained and were found to be organized similarly to the trxA and trxB gene cluster of Streptomyces clavuligerus, M.tuberculosis and M.leprae. A 14 kDa protein in M.smegmatis lysates was identified by Western blot analysis using antiserum to E.coli TrxA. Analysis of the M.smegmatis trxA and trxB gene sequences by BLAST revealed a high identity with other thioredoxin system genes. Sequence alignment with the M.tuberculosis and M.leprae genes showed that the M.smegmatis trxA and trxB deduced amino acid sequences have a very high degree of similarity; 72.7% identity and 81.5% similarity to the 49 kDa fusion protein of M.leprae, and 76.6% identity and 85.4% similarity to M.tuberculosis, respectively. Sequence alignments and phylogenetic tree analysis of known TrxA's and TrxB's clearly identify the two genes of M.smegmatis as members of the thioredoxin system genes grouped with other Actinomycetes and more specifically, within a mycobacterial branch. iii TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS x INTRODUCTION 1 I. Mycobacteria 1 II. Thioredoxin 3 III. Mycobacterium smegmatis 9 IV. Objectives 10 MATERIALS AND METHODS 11 I. Bacterial Strains and Plasmids 11 II Growth and Maintenance of Bacteria 11 III. Polymerase Chain Reaction 12 IV. Cloning and Gene Manipulations 12 V. PCR Subcloning 14 VI. DNA Labeling and Hybridization 14 VII. DNA Sequencing 15 VIII. SDS-PAGE 16 XI. Isolation and Partial Purification of Thioredoxin and Thioredoxin Reductase 16 A. Thioredoxin Reductase 17 B. Thioredoxin 17 X. DTNB Assay 18 XI. Western Blot 18 XII. Sequence Alignments 19 RESULTS AND DISCUSSION 20 I. Identification of the M.smegmatis Thioredoxin System Genes byPCR 20 A. Identification of the Thioredoxin and Thioredoxin Reductase Genes by PCR Using Degenerate Primers 20 B. Identification of the Thioredoxin Gene by PCR Using Degenerate Primers 23 C. Cloning the M.smegmatis Thioredoxin Reductase Gene (Attempt) 32 II. Partial Purification of M.smegmatis Thioredoxin and Thioredoxin Reductase -35 A. Partial Purification of Thioredoxin Reductase (TrxB) 37 B. Partial Purification of Thioredoxin (TrxA) .37 III Identification and Sequencing of Thioredoxin and Thioredoxin Reductase Clones from a M.smegmatis Plasmid Library 39 IV. The Thioredoxin System Gene Cluster of M.smegmatis 39 V. Western Blot Studies 49 V VI. Multiple Sequence Alignment of Thioredoxins and Thioredoxin Reductases 52 A. Multiple Sequence Alignment of Thioredoxin Reductases . 52 B. Multiple Sequence Alignment of Thioredoxins 57 LITERATURE CITED 63 vi LIST OF TABLES Table Page 1 List of degenerate primers constructed to consensus regions of S.clavuligerus trxB and trxA genes 21 2 List of clones containing PCR products and significant BLAST homologies to sequences obtained 31 3 List of M.smegmatis genomic DNA library clones which hybridized to a DIG-labeled PCR probe 45 4 List of primers used for sequencing the thioredoxin (trxA) and thioredoxin reductase (trxB) genes of M.smegmatis 46 vii LIST OF FIGURES Figure Page 1 The structures of the known low-molecular weight thiols: glutathione, mycothiol, and coenzyme A 5 2 The electron pathway from NADPH to ribonucleotide reductase via thioredoxin reductase and thioredoxin 6 3 Schematic diagram of the S.clavuligerus thioredoxin genes, trxA and trxB and the relative positions of the degenerate primers .... 22 4 Agarose gel of PCR amplified products using degenerate primers to the trxB gene 24 5 Sequence alignment of the predicted protein sequence of the PCR product of SCTRXB1/SCTRXB3 to thioredoxin reductase (TrxB) of S.clavuligerus 25 6 Agarose gel electrophoresis of PCR products using degenerate primer SCTRXA1 to the redox site of S.clavuligerus trxA gene ... 26 7 Agarose gel electrophoresis of PCR products using degenerate primer SCTRXA2 to the redox site of S.clavuligerus trxA gene ... 27 8 Agarose gel electrophoresis of PCR products using degenerate primer SCTRXA3 to the conserved region of Gram positive bacteria trxA genes 28 9 BLAST homologies of predicted peptide sequences from 3 clones containing PCR products: S8B, S8C, and S6A 30 10 Southern hybridization using DIG-labeled PCR probe to trxB on restriction endonuclease digested mc26 genomic DNA 34 11 FPLC profile of gel filtration column 36 12 FPLC profile of TrxB-active fractions on an anion exchange column 38 13 FPLC profile of TrxA-active fractions on an anion exchange column 40 viii 14 Colony hybridization of a high density grid of a M.smegmatis genomic DNA library using a DIG-labeled PCR probe to trxB .... 41 15 Schematic diagram of the clone inserts and their relative location to the M.smegmatis thioredoxin (trxA) and thioredoxin reductase (trxB) genes 42 16 The complete nucleotide sequence and predicted protein sequence of M.smegmatis thioredoxin (trxA) and thioredoxin reductase (trxB) genes 43 17 Protein sequence alignment of TrxA and TrxB of M.smegmatis with M.tuberculosis and the fusion protein of M.leprae 48 18 Immunoblot of cellfree extracts using antiserum raised against E.coli thioredoxin 50 19 Multiple alignment of thioredoxin reductases 54 20 Phylogenetic tree based on known thioredoxin reductase protein sequences 56 21 Multiple alignment of thioredoxins 58 22.1 Phylogenetic tree based on known thioredoxin protein sequences . 59 22.2 Eukaryotic branch of thioredoxin phylogenetic tree 60 22.3 Prokaryotic branch of thioredoxin phylogenetic tree 61 ABBREVIATIONS AIDS auto immune deficiency syndrome BLAST basic local alignment search tool DIG digoxigenin-11-dUTP DNA deoxyribonucleic acid DTNB 5,5'-dithiobis 1-nitrobenzoic acid DTT dithiothreitol EDTA ethylenediaminetetraacetic acid FAD(H) flavin adenine dinucleotide (reduced) IL interleukin NAD(H) p-nicotinamide adenine dinucleotide (reduced) NADP(H) (5-nicotinamide adenine dinucleotide phosphate (reduced) NF nuclear factor PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PVDF polyvinylidene fluoride SDS sodium dodecyl sulphate TB tuberculosis TE tris-EDTA TNF tumor necrosis factor TrxA thioredoxin TrxB thioredoxin reductase ACKNOWLEDGEMENTS I would like to thank my supervisor Professor Julian Davies and the Canadian Bacterial Disease Network for the opportunity to pursue a Master's degree, and my committee members, Prof. R.E.W. Hancock, and Prof. David Speert for their guidance and advice. I received invaluable help from all the privileged members of the Davies Laboratory, in particular, Yossi Av-Gay for his computer and thiol knowledge and Kevin Chow's computer expertise. I would also like to thank the office staff at the microbiology department for their patience, reminders, and guidance throughout my years as a student. I would like to give special thanks to my family (Takako, Tarn, Nikki and Tyrin) and Vera Webb, who have been there for support in all respects. I would also like to acknowledge all those people who were my coffee buddies, whine (and wine) buddies, beak recipients, and most importantly, baby sitters. I dedicate this thesis to the memory of my father, Edward Shozo Asano. l INTRODUCTION I. Mycobacteria Mycobacteria are a group of organisms that have plagued the history of mankind. Mycobacterial infections have been traced in stained preparations from mummified bodies and evidence of invasions of bones and joints have been seen in prehistoric skeletons of man (1). Although evidence of tuberculosis exists though history, it was not considered a disease of importance until The Great White Plague' epidemic of the 1600's (2). The social conditions of feudal Europe provided the required environment for person-to-person spread of the airborne pathogen. With the centralization of people, the subsequent increase in population density, and the migration of the Europeans throughout the world, epidemics of tuberculosis followed. By the turn of the 19th century, tuberculosis had claimed lives on all continents, becoming a world-wide problem. In the United States, a national surveillance of tuberculosis cases, initiated in the 1950's reported a downward trend in the occurrence of the disease primarily due to the improvement of social conditions and health care in industrialized countries. With the introduction of antibiotic therapy, the battle against tuberculosis appeared to be won. However, since the 1980's a resurgence of tuberculosis and other non- tuberculosis mycobacterial infections has become a major medical concern (3). Triggered by a new immunologically compromising disease, AIDS, an increase in the homeless, the decay of the health care system, and the development of mycobacterial strains resistant to antibiotics, the number of reported cases of 2 tuberculosis is again on the rise. Once known as the 'greatest killer in history', tuberculosis is again a feared and deadly disease (4).