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).
Organisms of the genus Mycobacterium, family Actinomycete, are characteristically aerobic, non-motile, straight or slightly curved rods (5). They may be isolated from water, soil, or warm and cold-blooded animals. Although these cells are not readily stainable by Gram's method, they are considered to be Gram- positive. Their distinct acid-fast and acid-alcohol-fast properties allow easy identification upon acid-fast staining by the Ziehl-Neelsen method although this property is partially or completely lost at some stage of growth. A distinctive characteristic of this genus is the presence of mycolic acids. Colonies on most solid surfaces appear rough, raised and thick, with a wrinkled surface texture and an irregular margin. Species such as M.smegmatis are yellow to orange in colour.
Growth is filamentous or mycelium-like, therefore liquid media require a dispersing agent (such as glycerol). This diverse genus includes obligate parasites, saprophytes, and species varying in nutritional requirements: saprophytic strains grow on very simple substrates; others require a complex medium or supplements for growth; and some species, such as M.leprae, have not been cultivated outside living cells.
Mycobacterial species can be divided into two groups based on growth rate.
Rapid growers, such as M.smegmatis, have a doubling time of 2-4 hours, while the slow growers like M.leprae can take up to two weeks. Some of the slow growers, such as M.tuberculosis and M.leprae, are major human pathogens, causing diseases including tuberculosis (TB), leprosy and other usually chronic, necrotizing, 3 granulomas. World wide statistics indicate that one third of the population is infected with M. tuberculosis and has a 10% lifetime risk of developing TB (6).
Tuberculosis kills 3 million people annually with the majority of these cases centered in the third-world population (7). However, with the emergence of multiple-drug- resistant strains, treatment of the disease is difficult even in the first-world countries.
This has brought about a renewed interest in mycobacterial studies.
II. Thioredoxin
Thiol molecules and disulfide reductases are components of many important biological processes. They play a pivotal role in the management of oxidative stress (radical scavenging, peroxidase, and S-transferase activities) and the maintenance and regulation of intracellular thiol/disulfide redox balance. The intracellular environment is maintained at a highly reduced state by oxido-reductase disulfide systems. Mammalian cells and many aerobic organisms rely mainly on glutathione and glutathione reductase for this function. Most organisms contain millimolar concentrations of glutathione (8). Studies with the gram-positive bacteria
Streptomyces, Staphylococcus, Bacillus, Nocardia, and Mycobacteria have demonstrated that these groups do not contain detectable glutathione or glutathione reductase (9-11). Closer investigation has revealed that the predominant thiol in
S.aureus is coenzyme A and a NADH-dependent disulfide oxido-reductase specific for coenzyme A, which has also been found in B.megaterium (12). The predominant thiol in Actinomycetes (Streptomycetes, Nocardia, and Mycobacteria) is mycothiol
(13-15). It has not been elucidated if the functions of mycothiol are the same or similar to glutathione or if another thiol, such as the thioredoxin system, may compensate for the lack of a glutathione system. The structures of the known low- molecular weight thiols are shown in figure 1.
The thioredoxin system is composed of thioredoxin (TrxA), thioredoxin reductase (TrxB) and NADPH. Thioredoxins are small (8-14 kDa), ubiquitous, heat- stable proteins with a dithiol active site. Prokaryotic thioredoxin reductases are dimeric proteins consisting of two identical FAD-binding subunits of 35 kDa. TrxB belongs to a large family of flavoprotein oxidoreductases which include glutathione reductase, alkyl hydroperoxidase, mercuric reductase, trypanothione reductase, lipoamide dehydrogenase and NADH peroxidase (16). These proteins facilitate the transfer of reducing equivalents from pyridine nucleotides via a flavin moiety to their active cysteine residues where they can proceed to reduce various disulfide substrates. The reduction of TrxA by TrxB is comprised of two half-reactions (figure
2). The FAD prosthetic group of TrxB is first reduced by NADPH and an electron is transferred to the TrxB active-site. Then TrxB reduces the bound, oxidized TrxA where in the reduced form, it can act as a general protein disulfide reductase (17).
The thioredoxin system genes have been studied extensively in several organisms. The genes for TrxA and TrxB of E.coli are located at widely separated sites on the genome as are the genes in most other organisms (18). In contrast, the two genes in the Actinomycete, Streptomyces clavuligerus, are located adjacent to each other (19). TrxA is found in all cells and is structurally conserved in species studied thus far. TrxA contains two redox active cysteine residues in its conserved 5
A B
Figure 1 The structures of the known low-molecular weight thiols. A. Glutathione B. Mycothiol C. Coenzyme A. 6
NADPH + H+ NADP +
FAD Thioredoxin FADH 2 Reductase
Trx-(SH)2 Thioredoxin Trx-S2
-S2 Reductase -(SH)2
Figure 2 The electron pathway from NADPH to ribonucleotide reductase via thioredoxin (TrxA). The reduction of TrxA by thioredoxin reductase (TrxB) is comprised of two half-reactions. The FAD group of TrxB is first reduced by NADPH and an electron is transferred to the TrxB active-site. Then the electron is transferred to the active site of TrxA where now in a reduced form, it can act as a general protein disulfide reductase to substrates such as ribonucleotide reductase. 7 active site (-WCGPC-). These cysteine thiols undergo reversible oxidation to form a disulfide bridge. The TrxB of E.coli is a dimeric protein consisting of two identical 35 kDa subunits (20). These flavoprotein oxidoreductases have three common domains that are essential for their activity as well as a conserved redox active site.
These proteins contain an NADPH binding domain and two domains that are involved in FAD binding. Each TrxB subunit possesses a conserved redox active site containing two cysteine residues (-CATC-) as well as an NADPH binding domain and two domains that are involved in FAD binding. The eukaryotic TrxB differs from the prokaryotic in that it has a higher molecular weight of 116 kDa
(consisting of two identical subunits of 58 kDa) and has a greater similarity with glutathione reductase than with prokaryotic TrxB (21). Mammalian TrxB is also much broader in specificity and reacts with thioredoxins from many species including E.coli as well as with other nondisulfide components.
The thioredoxin system is a general mechanism for reducing a variety of disulfides and was first characterized in E.coli as an in vitro hydrogen donor for ribonucleotide reductase which is involved in the production of deoxyribonucleotides
(22). It has also been implicated in functioning as a soluble cofactor for methionine sulfoxide, sulfate reductases and many other roles; such as an essential subunit of
T7 DNA polymerase (23). In plants, TrxA is involved in the light regulation of photosynthetic enzymes in the chloroplast and is essential for the photosynthetic growth of Anacystis nidulans (24, 25).
In mammalian cells, TrxA has been shown to be involved in additional roles such as the activation of the glucocorticoid receptor to a steroid binding state 8
(26). TrxA has also been shown to interact with transcription factors and regulate gene expression. For example, TrxA has been found to increase the activity of AP-
1, a transcription factor involved in linking extracellular signals with the activation of genes associated with growth, differentiation and cellular stress (27). Thyroid- enriched transcription factors, Pax-8 and Ttf-1, which are involved in the thyroid- specific expression of thyroglobulin gene are redox regulated by TrxA (28). TrxA has also been shown to regulate heat-shock factor-1, which is the transcription activator protein expressed upon exposure to heat shock and reactive oxygen species (29). The transcription factor NF-KB is a protein associated with the inducible expression of a variety of genes involved in the inflammatory and immune responses; it is also involved in the activation of HIV replication. It is thought that
TrxA functions as a regulator of NF-KB since it promotes binding to DNA in the nucleus, but has inhibitory effects on NF-KB in the cytoplasm (27, 30).
It has been discovered that TrxA is involved in a variety of immunological functions. ADF, a factor produced by adult T-cell leukemia-derived cells (caused by
HTLV-1, the human T-lymphocyte virus), has been found to be a human homologue of TrxA and is an inducer of the IL-2 receptor (31, 32). TrxA has been found to be identical to a leukemic cell growth factor responsible for the stimulation of the growth of normal and leukemic B-cell lines (33). TrxA also appears to be involved in the protection of various transformed cell lines against the cytotoxic effects of TNF (34). The ability of TrxA to scavenge free radicals may inhibit the
TNF-induced cytotoxic effects induced by oxygen radicals and hydrogen-peroxide. 9
In addition to inhibiting the cytotoxic effects of reactive oxygen species, nitric oxide, ultraviolet radiation and heavy metals, TrxA has been shown to reverse the damage caused by these oxidative stress factors (35-38).
It is evident that TrxA plays a major role in various biological processes involving redox-regulation and the evidence for its role in immunological signal transduction, gene expression, and cell proliferation is extensive. There is growing evidence that apoptosis or cell death, is a thiol-mediated redox regulatory process involving the oxidative breakdown of the highly reduced intracellular state (39). It appears that the thioredoxin system also plays an important role in cellular protection against various external stress factors. In light of these studies, the thioredoxin system of Mycobacteria, intracellular parasites of mononuclear phagocytes, is of particular interest. Many questions arise with respect to the role that TrxA may play as a redox-regulator within the cell, in an organism lacking glutathione, as well as other possible regulatory roles it may play in response to its environment. Since there is increasing evidence that TrxA may be a multi-faceted protein, the thioredoxin system may be found to be involved in cellular protection during host invasion, intracellular survival and in disease promotion. The first step involved in finding answers to these questions is to proceed with genetic studies of the mycobacterial thioredoxin system.
III. Mycobacterium smegmatis
Given the difficulties in working with M.tuberculosis and other slow-growing mycobacteria due to containment requirements, lack of appropriate genetic tools, 10 and potential health hazards, many workers have adopted M.smegmatis as a model system for a molecular genetic study of mycobacteria. M.smegmatis is categorized as a non-pathogenic species that rarely causes disease in humans. It has been isolated from smegma, soil and water. Although positive cultures have been obtained from the spleens of mice and/or guinea pigs, small inocula are generally not pathogenic for rodents (guinea pigs, mice and hamsters) or chickens (5).
M.smegmatis is an important tool for the manipulation of mycobacterial genetics.
Genetic systems have been developed for M.smegmatis such as well characterized phages, plasmids, shuttle vectors, and an electroporatable strain, mc2155 (40).
Rapid growth within days instead of weeks, and the well-developed genetic system are attractive attributes that make M.smegmatis an important model for genetic studies of mycobacterial species (41).
VI. Objectives
The objective of this study was to characterize the M.smegmatis thioredoxin genes, trxA and trxB by determining the nucleotide sequence and the organization of the two genes and to provide the information necessary for a more detailed analysis of the thioredoxin system in mycobacteria. 11
MATERIALS AND METHODS
I. Bacterial Strains and Plasmids
Mycobacterium smegmatis mc26 was used for the preparation of chromosomal DNA, gridded plasmid libraries, and all protein purification studies.
Streptomyces clavuligerus DSM 738 (ATCC 27064; NRRL 3585) was the positive control template in colony PCR assays. Escherichia coli DH5aF' and INVaF'
(Invitrogen) for the cloning host. M.smegmatis strains mc2155, ATCC 607 and
ATCC 19420, and M.phlei and M.aurum were employed to prepare cell free extracts for Western hybridizations. Plasmids pTZ18R, pBluescript II SK, and pCRII
(Invitrogen Corp.) were used as the cloning vectors for creating DNA libraries and cloning PCR products.
II. Growth and Maintenance of Bacteria
M.smegmatis, mc26, was grown in Middlebrook 7H9 (Difco) supplemented with Tween 80 (0.5 g/800 ml) and glycerol (2 ml/800 ml) at 37°C for 48 h. E.coli containing plasmids were grown in Luria Broth (Difco) containing 100 ug/ml of ampicillin at 37°C for 24 h M.smegmatis spp., M.phlei and M.aurum were grown in tryptic soy broth (Difco) supplemented with Tween 80 at 37°C for 48 h for the preparation of cell free extracts for Western hybridization. 12
III. Polymerase Chain Reaction (PCR)
PCR in a MJ Research minicycler was performed for gene amplification (for sequencing), gene detection and cloning, and probe preparation. Taq DNA polymerase was purchased from Bethesda Research Laboratories (BRL). Direct
PCR from colonies was performed for the purposes of cloning PCR products and gene detection in clones. Reaction conditions for colony PCR included a lysing
step of 95°C for 15 min in 50 |al dH20; 5 jil of the lysate was added to PCR buffer
(20 mM Tris-HCl (pH 8.3), 1.5 mM MgCI2, 25 mM KCI, 0.05% Tween 20, 0.1 mg/ml gelatin), dNTP (250 ^iM each), 100 pm each primer and Taq polymerase). Reaction conditions for the amplification reaction were: 1) 2 min at 95°C. 2) 35 cycles of
95°C for 20 s, 56°C for 1 min, and 72°C for 2 min. 3) 72°C for an additional 7 min.
12 fil of the reaction mixture was electrophoresed at 80 v for 1 h in a 1.25% agarose gel, and amplification products were visualized by ethidium bromide staining.
IV. Cloning and Gene Manipulations
M.smegmatis genomic DNA was prepared by harvesting cells by centrifugation after 48 h of incubation at 37°C as described by Davis et al (42). Cell pellets were washed in 30 ml TES (50 mM Tris-HCl (pH 8), 10 mM EDTA, and 0.3 M sucrose) and resuspended in 12 ml of TES (2 ml/g wet cell weight) containing 2 mg/ml of lysozyme and 2mg/ml of lipase and incubated for 60 min at 37°C. Four volumes of 6 M guanidinium chloride, 1% Sarkosyl and 20 mM EDTA were added and the mixture incubated for 2 h at room temperature. The reaction mixture was 13 extracted with an equal volume of chloroform, then precipitated with absolute ethanol at -20°C overnight. The pellet was redissolved in 800 ul of TEX buffer (50 mM Tris-HCl (pH 8), 10 mM EDTA, 0.5% SDS, 0.5 ml proteinase K /ml and 4000
Units RNAse T1) and incubated for 3 h at 37°C . A final phenol/chloroform extraction was performed and the DNA recovered by ethanol precipitation.
Synthetic oligodeoxynucleotides were prepared at the Nucleic Acid and
Protein Sequencing (NAPS) Unit (University of British Columbia). Analysis of restriction digest products and PCR products was carried out on 0.7% agarose gels
(for genomic digests) or 1.25% (for PCR) as described (43).
The cloning procedure for thioredoxin reductase involved creating a limited size-specific DNA library of M.smegmatis. Genomic DNA of M.smegmatis was digested with BamHI or Pstl restriction endonucleases. The digests were run on a
0.7% gel and various sized fragments were excised (2.0-2.5, 2.5-3.0, 3-4 Kb, etc.) and cleaned using QIAquick gel extraction kit (Qiagen). PCR, using primers
MSTRXB1 and MSTRXB3, was performed on samples containing different sized digested DNA fragments to confirm the presence of the partial-toB . BamHI digested fragments of approximate size 5-7 Kb and Pstl fragments of size 2.0-2.5
Kb were cloned into pTZ18R. Competent E.coli cells were prepared using the CaCI2 procedure (43). Electrocompetent cells were prepared and transformed with a Gene
Pulser apparatus according to the manufacturer's protocol (BioRad Inc.). Potential clones were identified initially by colony hybridization as described below and by
PCR with primers MSTRXB1 and MSTRXB3. 14
V. PCR Subcloning
The PCR-amplified products were isolated and recovered from the PCR reaction mixture using the QIAquick-spin PCR purification kit (Qiagen). The isolated product was cloned into the cloning vector, pCRII. Ligation and transformation reactions were performed according to the manufacturers recommendation
(Invitrogen Corp.).
VI. DNA Labeling and Hybridization
DNA probes were labeled using the digoxigenin-11-dUTP (DIG) random priming method by PCR (Boehringer-Mannheim). The PCR mixture was run through a low melting point agarose gel (0.7%) and DIG-incorporated products were excised. The incorporation of DIG-labeled dUTP increased the molecular weight of the products differentiating them from products with no or little DIG-labeled dUTP incorporation. The excised gel fragment was melted at 60°C for 10 min and was used directly for the estimation of the probe concentration by dot blotting using a
DIG-labeled pUC19R DNA as a standard (Boehringer Mannheim).
Southern hybridization was carried out by digesting samples of chromosomal
DNA (1 u.g) with one of the following restricion endonucleases: BamHI, Pstl, Kpnl,
Hindlll, Xbal, EcoRI, and Ndel. DNA was transferred to a positively charged nylon membrane (Boehringer-Mannheim) using the alkaline transfer method (0.4 M NaOH,
0.6 M NaCl). Hybridization for the detection of a thioredoxin reductase gene in
M.smegmatis genomic DNA was performed at 42°C using 100 ng/ml probe DNA. A 15 luminescent detection kit (Boehringer-Mannheim) was used to detect the hybrid on
X-ray film. Colony hybridizations were performed on Hybond N nylon membranes
(Amersham) under high stringency conditions at 65°C with 5 ng/ml probe DNA.
A high-density gridded M.smegmatis mc26 genomic library on a 22 cm x 22 cm membrane was supplied by Glaxo Wellcome (44). Robotics technology allowed the single membrane to contain over 36,000 clones. The library was constructed by cloning partially digested Sau 3A1 1-5 Kb fragments of M.smegmatis genomic DNA into pBluescript II SK and introducing the recombinant plasmids into an E.coli host.
Each clone was represented twice in a square of 16 colonies to ensure reproducible results. The conditions employed were as described above for colony hybridizations.
VII. DNA Sequencing
Nucleotide sequencing was performed by the dideoxy chain termination procedure by direct sequencing from double stranded plasmid DNA using an ABI
373A automated DNA sequencer. The ABI PRISM DNA sequencing terminator kit
(Perkin Elmer) was used according to the manufacturer's protocol. Subcloned PCR products were sequenced using M13 universal primers. Sequences of the plasmid library clones were obtained using both universal and gene-specific primers.
Sequence analysis was performed using ABI software programs; ABI 373 DNA sequencer data analysis program version 1.2.1, and SeqEd version 1.0.3. VIII. SDS-PAGE
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of 15% acrylamide (30% acrylamide: 0.8% N,N'-methylene bisacrylamide) were prepared according to the method described by Laemmli for the analysis of protein samples (45). The gels were stained by a modified Rabilloud procedure for silver staining (Millipore). Protein concentrations were determined using the bicinchoninic acid protein method (Sigma) or a protein assay kit (Biorad) (46). Bovine serum albumin was used as a standard. Prestained low molecular weight protein standard
(BRL) was run simultaneously with samples.
IX. Isolation and Partial Purification of Thioredoxin and Thioredoxin
Reductase
Cultures of M. smegmatis were harvested by centrifugation and washed with
50 mM Tris HCI buffer pH 8.0. The cell pellet was resuspended in four volumes of
TE containing proteinase inhibitors (aprotinin, pepstatin and leupeptin). Disruption of the cells was performed by passing the cells though a Power Laboratory Press at
15,000 ppi (American Instrument Co. Inc.) 3 times and the cell extract centrifuged for 15 min at 14,000 x g. Streptomycin sulfate was added to the cell-free extract to a final concentration of 1% (w/v), centrifuged for 15 min at 14,000 x g, and the pellet discarded. Solid ammonium sulfate was added in a two-step precipitation procedure and the 40-80% precipitate was taken and redissolved in TE buffer (2 ml/g wet weight of cells). This sample was applied to a Superose-12 HR 10/30 gel filtration column of bed size 24 ml (Pharmacia) using a FPLC system (Pharmacia). The 17 column was equilibrated previously with TE buffer at a flow rate of 0.5 ml/min and the fractions assayed for activity using DTNB as described below.
A. Thioredoxin Reductase
The active column fractions were combined, washed and concentrated by
Centricon-10 filtration (Amicon). The sample (2 ml) was applied to a MonoQ HR 5/5 column of bed size 1 ml (Pharmacia) that had been pre-equilibrated with TE buffer and the column was eluted with a linear gradient of 0.0 - 0.6 M NaCl in TE buffer.
The fractions collected were tested for DTNB reduction (see below). The active fractions were combined and applied to a 2'5'-ADP-Sepharose column (1x10 cm) pre-equilibrated in TE buffer; a linear gradient of 0.0 -1.5 M NaCl was applied to the column at a flow rate of 1 ml/min. Proteins were monitored on SDS-PAGE gels detected by silver staining.
B. Thioredoxin
The active fractions were combined, desalted and concentrated by Centricon-
3 filtration (Amicon). The concentrated sample was heated for 10 min at 90°C and centrifuged for 2 min at 14,000 x g and the supernatant applied to a MonoQ HR 5/5 anion exchange column (Pharmacia) that had been pre-equilibrated with TE buffer.
The column was eluted with a linear gradient of 0.0 - 0.3 M NaCl in TE buffer at a flow rate of 1 ml/min. The collected fractions were tested for DTNB reduction.
Proteins were monitored on silver stained/SDS-PAGE gels. 18
X. DTNB Assay
Thioredoxin and thioredoxin reductase activities were assayed using 5,5'- dithiobis 1-nitrobenzoic acid (DTNB), as described by Holmgren (47). Activity was
determined by measuring the increase in A412 at 25°C over a 5 min reaction period.
The reaction mixture (volume 1 ml) contained 50 mM Tris HCI pH 8.0, 1 mM EDTA,
0.2 mM NADPH and 0.02 mM DTNB. Various amounts of thioredoxin or thioredoxin reductase containing-fractions obtained by chromatography were added to the reaction depending upon which component was being assayed. The reaction was initiated by adding an excess of the required component (thioredoxin or reductase).
Controls included reactions omitting one of the following: one of the two protein components or NADPH.
XI. Western Blot
Cell-free extracts for Western hybridization experiments were prepared by sonicating cells in an ultrasonic processor SonicatorXL (Misonix) at setting 2 for 2 x
30 s in TE containing the above-mentioned proteinase inhibitors. DTT (2 mM) was added and the samples run on SDS-PAGE gels for analysis. E.coli thioredoxin was used as the control (Sigma). Gels were soaked in buffer (96 mM glycine, 10 mM
Tris base, 10% methanol) and the proteins were transferred to an Immobilon-P polyscreen PVDF membrane (Millipore), using an Investigator 2D system semi-dry graphite blotter (Millipore). Incubation of the membrane with antisera was performed as described previously by Abou-Zeid et al. (48). Affinity purified 19 polyclonal sheep antiserum directed against E.coli thioredoxin (IMCO) was used at a
1:2000 dilution. Horse radish peroxidase labeled rabbit, anti-sheep immunoglobulin
(Pierce) were used at a 1:5000 dilution. Detection was performed on Kodak X-
OMAT scientific imaging X-ray film using an enhanced chemiluminescence (ECL) detection kit (Pierce).
Xll. Sequence Alignments
Protein sequences were accessed from the PIR or Swiss-Prot data banks using the NCBI search engines. Sequence analysis was performed with PC-GENE
(IntelliGenetics Inc.), and NCBI (National Center for Biotechnology Information)
BLAST (basic local alignment search tool)programs (49). Protein sequence alignments were carried out using PCGENE or FASTA programs, or the CLUSTALV algorithm in the Genetic Data Environment software package, version 2.2 (S.Smith,
University of Illinois and Harvard University). The CLUSTALV program was used with PAM 250 weighting matrix with fixed and floating gap penalties of 10. Unrooted phylogenetic trees were constructed using DeSoete tree fit with no distance corrections. 20
RESULTS AND DISCUSSION
I. Identification of the M.smegmatis Thioredoxin System Genes by PCR
A. Identification of the Thioredoxin and Thioredoxin Reductase Genes by PCR
Using Degenerate Primers
To identify the thioredoxin system genes, thioredoxin (trxA) and thioredoxin reductase (trxB) in M.smegmatis, degenerate PCR primers were created to consensus regions of both genes of S.clavuligerus using the codon preference of
Actinomycetes (table 1, figure 3). The primers for trxB corresponded to the redox site (SCTRXB1) and to an FAD 11 domain (SCTRXB3) of S.clavuligerus trxB. Using these primers on S.clavuligerus genomic DNA would produce a PCR product of about 470 bp.
To determine if the two genes were located in close proximity, two primers were constructed to the redox site of the S.clavuligerus trxA gene; SCTRXA1, the minus strand primer, and SCTRXA2, the plus strand primer. If the two genes in
M.smegmatis were arranged similarly to the S.clavuligerus genes with trxB first and trxA downstream and both genes oriented in the same direction, PCR using the minus strand primer, SCTRXA1 with SCTRXB1 would produce a PCR product. If the genes were positioned in the opposite order, with trxA first and trxB downstream and oriented in the same direction, then SCTRXA2, the plus strand primer, used with SCTRXB3 would produce a PCR product. Different combinations of the four primers would also detect the genes oriented in different directions as long as they 21
PRIMER SEQUENCE POSITION NAME trxB Primers
SCTRXB1 (+) 5'-TGGTG(TC)GCIACITGCCA(TC)GG 403-425 (AGCT)TT-3' SCTRXB3 (-) 5'-GTGAT(CG)GC(TC)TGICGGTA(CG) 843-855 GT(AG)TG(AG)TC-3' trxA Primers
SCTRXA1 (-) 5'-TGICG(AG)CA(CG)GGICC(AG)CAC 97-113 CACTC-3' SCTRXA2 (+) 5'-C(CG)GAGTGGTG(CT)GGICC(CG)TG 89-110 (CT)CG-3' SCTRXA3 (-) 5'-GGATIGACATIACICCGTACT-3' 107-227
Table 1 Degenerate primers constructed to consensus regions of the thioredoxin reductase (trxB) and thioredoxin (trxA) gene sequences of Streptomyces clavuligerus. Codon preference for Streptomycetes was used to determine the degenerate sequences. The name, sequence and strand orientation of each primer is listed along with the position of the each primer relative to the nucleotide sequence of either gene. 22
SCTRXB1 SCTRXA2
1 trxB I r i»^pr s*: • frx>-}
SCTRXB3 SCTRXA1 SCTRXA3
Figure 3 Degenerate primer (see table 1) positions relative to the thioredoxin system gene cluster, thioredoxin reductase (trxB) and thioredoxin (trxA), of Streptomyces clavuligerus. 23 were located in close proximity to one another. The expected sizes of the products would depend on the size of the genes and the number of nucleotides separating the genes. PCR was performed on M.smegmatis mc26 cell-free extracts as the source of template DNA using the degenerate primers in various combinations.
Either no amplicon or multiple products were observed from M.smegmatis
DNA using the degenerate PCR primers to the trxA gene (data not shown).
However, the two degenerate primers to the trxB gene (SCTRXB1 and SCTRXB3) produced a 468 bp product (figure 4). This product was cloned into a pCRII vector and sequenced. The predicted peptide from the sequenced M.smegmatis PCR product had 65% identity and 80% similarity, respectively to the corresponding amino acid sequence of S.clavuligerus TrxB (figure 5).
B. Identification of the Thioredoxin Gene by PCR Using Degenerate Primers
Primers to M.smegmatis, MSTRXB1 and MSTRXB3, were constructed to the
468 bp nucleotide sequence obtained from the PCR product described above (table
4, page 46). A third degenerate primer, SCTRXA3, was designed using the codon preference of Streptomycetes, based on a conserved region of the thioredoxin genes of gram positive organisms (table 1). PCR was performed on M.smegmatis mc26 genomic DNA using the M.smegmatis-PCR primers, MSTRXB1 and
MSTRXB3, and the degenerate trxA primers, in various combinations (figure 6, 7 and 8).
PCR reactions using SCTRXA1 primer are shown in figure 6. Products of various sizes were observed in the reaction using MSTRXB3 as the second primer 24
2072 -
1500 -
1000 -
600 - 500 - 400 -
Figure 4 Agarose gel electrophoresis of the 468 bp PCR amplified product using degenerate primers SCTRXB1 and SCTRXB3 to the trxB gene. M.smegmatis mc26 genomic DNA was used for the template DNA.
Lane 1 100 bp Standard 2 negative control (no template DNA) 3 PCR reaction using SCTRXB1 and SCTRXB3 25
S.clavuligerus VSDVRNVIIIGSGPAGYTAALYTARASLQPLVFEGAVTAGGALMNTTDVE 50
S.clavuligerus NFPGFRDGIMGPDLMDNMRAQAERFGAELIPDDVVSVDLTGDIKTVTDSA 100
M.smegmatis \>j| 16
S.clavuligerus YTVHRAKAVIVTTGSQHRKLGLPREDALSGRGVSW| ATCDGFFFKDQDIV 150
M.smegmatis yVGGGIjJSAIjlEEA'?^ 66
S.clavuligerus VVGGGDTAMEEATFLSRFAKSVTIVHRRDSLRASKAMQDRAFADPKISFA 200
M.smegmatis TyT|iTQj:EyDpyvyyvRyyy|V|yyESKyDV|yvyvAiyyi|)pysyyvRy 116
S.clavuligerus WNSEVATIHGEQKLTGLTLRDTKTGETRELAATGLFIAVGHDPRTELFKG 250
M.smegmatis CjVEyyyyGyvyyQGRT | sn^DG;yy'ij^Dy)Lyij)ijiyy^cp^y^ 15 6 S.clavuligerus QLDLDDEGYLKVASPSTRTNLTGVFAAGDVVDHTYRQAITAAGTGCSAAL 300
S.clavuligerus DAERYLAALADSEQIAEPAPAV 322
Figure 5 Sequence alignment of the predicted amino acid sequence of the M.smegmatis 468 bp PCR product with the thioredoxin reductase amino acid sequence (TrxB) of S.clavuligerus (EMBL Z21946). Identical residues are designated by the character' I' and similar residues are designated by the character '+'. The -CATC- redox active site residues are boxed and the NADPH-binding domain is underlined. 26
Bp.
2072 — 1500 —
1000 —
600 — 500 — 400 —
Figure 6 Agarose gel electrophoresis of PCR products using degenerate primer SCTRXA1 to the trxA gene on S.clavuligerus. M.smegmatis mc26 genomic DNA was used for the template DNA.
Lane 1 100 bp Standard 2 negative control (no template DNA) 3 PCR reaction using SCTRXA1 alone 4 PCR reaction using SCTRXA1 and MSTRXB1 5 PCR reaction using SCTRXA1 and MSTRXB3 6 PCR reaction using SCTRXA1 and SCTRXA3 27
2072 —
1500 —
1000 —
600 —
400 — 300 _
Figure 7 Agarose gel electrophoresis of PCR products using degenerate primer SCTRXA2, to the redox site of S.clavuligerus trxA gene. M.smegmatis mc26 genomic DNA was used for the template DNA.
Lane 1 100 bp Standard 2 negative control (no template DNA) 3 PCR reaction using SCTRXA2 alone 4 PCR reaction using SCTRXA2 and MSTRXB1 5 PCR reaction using SCTRXA2 and MSTRXB3 6 PCR reaction using SCTRXA2 and SCTRXA3 28
Figure 8 Agarose gel electrophoresis of PCR products using degenerate primer SCTRXA3, to the trxA gene of S.clavuligerus.
Lane 1 100 bp Standard 2 negative control (no template DNA) 3 PCR reaction using SCTRXA3 alone 4 PCR reaction using SCTRXA3 and MSTRXB1 5 PCR reaction using SCTRXA3 and MSTRXB3 (figure 6, lane 5). The products of this reaction were cloned into a pCRII cloning vector. A representative number of clones were sequenced to see if any fragments of other flavo-oxidoreductase genes had been identified by the use of primers specific to a redox active site and to a FAD-binding site. Comparison with the sequence database using BLASTX detected no significant identity to other genes.
Clone S5C displayed a similarity score of 67 to a heat shock protein 70 (HSP70) of
Staphylococcus aureus (table 2).
All PCR reactions using primer SCTRXA2 showed multiple bands as seen in figure 7. If the two degenerate primers, SCTRXA2 and SCTRXA3, were to amplify a
DNA fragment from a thioredoxin gene, the predicted product size would be about
130 bp. Since the cloning vector pCRII preferentially inserts smaller-sized products, a 130 bp PCR product may occur more frequently than clones containing larger
PCR fragments. The PCR reaction products (figure 7, lane 6) were shotgun cloned and several of these clones were sequenced. There were no clones obtained which had sequence homology to thioredoxin sequences. One clone, S6A, showed significant sequence homology to an acetyl CoA transferase of Clostridium acetobutylicum (table 2, figure 9). No white colonies containing inserts less than
300 bp were identified. It was discovered later that clones containing small sized inserts produce blue colonies which were indistinguishable from clones containing the vector alone.
The PCR products of primers SCTRXA2 and MSTRXB1 (figure 7, lane 4) were also shotgun cloned and several clones sequenced. A clone containing an insert of about 950 bp, S8B, showed a DNA fragment with significant similarity of 30
S8B putative transposase for insertion sequence element IS986 [MycoJbacteriuzn tuberculosis] Length 278
Score = 125, Identities = 24/42 (57%), Positives = 30/42 (71s!
M. smegmatis 125 jI^RyAGSGjYjTAjYFGKTPMLAGLRPSIGiyGDALyyAyCE j1 250
M. tuberculosis 241 IHHTCRGSQYTSIRFSERLAEAGIQPSVGAVGSSYDNALAET 282
S8C acyltransferase (putative)
[Mycobacterium leprae] Length = 499
Score = 246, Identities = 47/76 (61%), Positives = 57/76 (75%)
, M. smegmatis 4 WAGRtj.Tyyi jERGSEjJAPySGDDED| GyS|Lyy|Gy|'AQRMTTTAQV 50
M.leprae 21 FGGTNAHVVIEQGPELTPVTECSSNTAVSTLVVTGKTASRVAAMAGM 77 ,M.smegmatis 51 yyy|MyypyyyyTvyij)yyy|'vyyyTYyQA 79
M.leprae 78 LADWVEGPGAEVALADVAHTLNHHRSRHA 96
S6A acetyl coenzyme A acetyltransferase (thiolase) [Clostridium acetobutylicum] Length = 392
Score = 124, Identities = 26/48 (54%), Positives = 33/48 (68%)
M. smegmatis 68 iyyQ|yyyvyiMTGj|yyy^Ij)RWGVRR^NRyyyG^yNyFyERyjTPyTL 115
C. acetobutylicum 157 MGITAENIAERWNISREEQDEFALASQKKAEEAIKSGQFKDEIVPVVI 204
Score = 50, Identities = 9/18 (50%), Positives = 13/18 (72%)
M. smegmatis 1 |AGEGIj>AF jSAGVETVSR 18
C.acetobutylicum 103 KAGDADVIIAGGMENMSR 120
Figure 9 BLASTX results of significant homologies to the nucleotide sequences of the cloned PCR products. The clone names; S8B, S8C and S6A, are in bold and underlined. BLAST scores are listed with identical residues designated by the character' I' and similar residues represented with the character'+'. 31
PRIMERS: SIZE BLAST HOMOLGY BLAST Clone (bp) SCORE MSTRXB3/SCTRXA1
S5C 900 HSP 70 67
S5D 700
S5L 600
MSTRXB1/SCTRXA2
S8B 950 transposase 125
S8C 500 acyltransferase 246
S8F 500
SCTRXA2/SCTRXA3
S6A 400 acetyl CoA acetyltransferase 124
S6B 400
S6C 360
Table 2 List of clones containing PCR products of mixed primers. The PCR reaction mixture was shotgun cloned into a cloning vector, pCRII and sequenced. The primers used are underlined with the clone names listed below. The size of the PCR product inserts, significant BLAST homologies found to the nucleotide sequences obtained and their scores are listed for each clone. 32
BLASTX score of 125 to a transposase of M.tuberculosis and another, S8C with a score of 246, to a putative acyltransferase of M.leprae (table 2, figure 9).
The results obtained from the PCR experiments indicated that M.smegmatis possessed a thioredoxin reductase-like gene, but no clear evidence of a thioredoxin gene was obtained. The degenerate primers constructed to the S.clavuligerus trxA gene did not generate a product of the expected size. In addition, if the correct PCR product had been made and inserted into the pCRII cloning vector, the clone would not have been identified using the blue/white screening method due to the small size of the insert. PCR data obtained using primers from both thioredoxin system genes were also inconclusive in providing evidence for the presence of a thioredoxin gene. It should be noted that three of the primers used were constructed to a redox active site; MSTRXB1 to the trxB redox site, and SCTRXA1 and SCTRXA2 to the trxA redox site. The degeneracy of thefrxA-primers may have provided some primer sequences which bind MSTRXB1 thus creating primer-dimers. No product bands were seen in the PCR reaction using MSTRXB1 with SCTRXA1 (figure 6, lane 4). The PCR reaction using MSTRXB1 with SCTRXA2 had fewer and fainter products than the other reactions using SCTRXA2 (figure 7). If primer-dimers were formed, the concentration of available primers for PCR would be reduced, which could lead to a reduction in product formation.
C. Cloning the M.smegmatis Thioredoxin Reductase Gene (Attempt)
A nucleotide probe for the thioredoxin reductase gene of M.smegmatis was created by PCR with primers, MSTRXB1 and MSTRXB3, using a DIG-labeling 33 system. M.smegmatis mc26 genomic DNA was digested with various restriction endonucleases and probed by Southern hybridization. The probe hybridized to an area which contained 6 Kb fragments in the BamHI digestion lane and a 2.3 Kb fragment in the Pstl digestion lane (figure 10).
A limited DNA library of M.smegmatis was created by excising size-specific fragments digested with BamHI or Pstl. PCR amplification using primers MSTRXB1 and MSTRXB3 was performed on various sized fragments excised from both BamHI and Pstl digestions to confirm the Southern hybridization results. BamHI-digested
DNA fragments of 5-7 Kb and Pstl-digested fragments of 2.0-2.5 Kb were ligating into pTZ18R and transformed into E.coli (DH5a). Clones obtained were screened by colony hybridization using the DIG-labeled PCR probe and by PCR using primers
MSTRXB1 and MSTRXB3. Colony hybridization, even under high stringency conditions, proved to be an ineffective screening procedure because E coli contains a trxB gene that is 60% homologous at the nucleotide level to the actinomycete probe. Therefore colony hybridization was used as a method for primary screening and positive clones were further screened by PCR using the primers MSTRXB1 and
MSTRXB3. The PCR products of M.smegmatis and E.coli were distinguishable by a size difference of 24 bp which was detectable on 1.25% agarose gel electrophoresis. 34
Figure 10 Southern hybridization of M.smegmatis mc26 genomic DNA digested with various restriction endonucleases. Gel A: Agarose gel of M.smegmatis mc26 genomic DNA digested with BamHI and Pstl. Gel B: Autoradiogram of Southern blot probed with DIG-labeled PCR product.
Gel A: Lane 1 1 Kb standards 2 BamHI digested M.smegmatis mc26 genomic DNA 3 Pstl digested M.smegmatis mc26 genomic DNA
Gel B: Lane 1 BamHI digest lane 2 Pstl digest lane 35
II. Partial Purification of M.smegmatis Thioredoxin and Thioredoxin Reductase
It was clear from the PCR results that M.smegmatis possessed a gene that displayed sequence similarity to known thioredoxin reductases, but no clear evidence was obtained for a thioredoxin gene in this organism. PCR experiments using degenerate primers to consensus regions of S.clavuligerus trxA and trxB suggested that trxA and trxB in M.smegmatis were not linked. Therefore an alternative strategy was developed to isolate the thioredoxin gene of M.smegmatis.
Purification of the protein and the subsequent N-terminal sequencing should provide additional protein sequence information to derive a trxA nucleic acid probe or to generate PCR primers that could be used to identify a genomic fragment containing the trxA gene. The purification strategy employed to isolate the S.clavuligerus proteins was used as a guideline for the isolation of the M.smegmatis thioredoxin system proteins (50).
The initial step in the purification of the thioredoxin system proteins involved ammonium sulfate precipitations. DTNB assays revealed that most of the activity resided in the 40-80% ammonium sulfate precipitate; the redissolved pellet was fractionated by gel filtration separation using a fast phase liquid chromatography system (FPLC) and the fractions containing thioredoxin (TrxA) and thioredoxin reductase (TrxB) activity were identified (figure 11). The fractions exhibiting disulfide reducing activity (TrxA-active) co-eluted with the salts and lower molecular weight proteins. The disulfide reductase active fractions (TrxB-active) eluted with 36
Figure 11 FPLC profile of M.smegmatis 40-80% ammonium sulfate fraction applied to a Superose-12 HR 30/10 gel filtration column. Fractions containing thioredoxin (TrxA) activity are indicated by the character '*' (fractions 11 and 12) and those fractions displaying thioredoxin reductase (TrxB) activity are indicated by the character'+' (fractions 2, 3 and 4). The relative absorbance levels at 280 nm are displayed on the Y-axis and the volume of wash buffer is displayed on the X-axis. The fractions collected are numbered above the X-axis. The conductivity level is indicated by the dashed line (- — -). The TrxA-active fractions fall under the salt peak in fractions 11 to 13. 37 proteins of higher molecular weight. The TrxB-active fractions were combined for further purification using the method employed for the isolation of S.clavuligerus
TrxB.
A. Partial Purification of Thioredoxin Reductase (TrxB)
The first step in purifying thioredoxin reductase involved application of the gel-filtrated sample to an anion exchange column (figure 12). The active fractions were identified and pooled and applied to a 2'5'ADP-Sepharose column. The activity was found in the flowthrough, indicating that TrxB did not bind to the column.
2'5' ADP is known to bind to proteins such as TrxB, that contain an NADPH binding domain. The 468 bp PCR product that was isolated showing sequence similarity to other TrxB's indicated the presence of a NADPH-binding domain (figure 5). If, in fact, this fragment coded for a portion of the M.smegmatis trxB gene, it could only be concluded that the NADPH-binding domain of a TrxB was blocked and therefore did not bind to the column. Since the purification of the TrxB did not appear to be straightforward, more emphasis was put on cloning efforts to obtain the
M.smegmatis trxB gene.
B. Partial Purification of Thioredoxin (TrxA)
The purification of M.smegmatis TrxA was continued by collecting the TrxA- active, fractions from gel filtration chromatography. Because ammonium sulfate co- eluted in these fractions, a desalting step as well as a concentration step was required before proceeding. The second step of the purification process involved 38
Figure 12 FPLC profile of M.smegmatis gel-filtrated fractions containing thioredoxin reductase (TrxB) activity applied to a MonoQ HR 5/5, an anion exchange column. Fractions displaying TrxB activity are indicated by the character; '-' indicating low levels of activity, '+' indicating moderate levels of activity, and '*' indicating high levels of activity. The TrxB-active fractions are 27 to 34. The relative absorbance levels at 280 nm are displayed on the Y-axis and the volume of wash buffer is displayed on the X-axis. The fractions collected are numbered above the X-axis. The fractions collected are numbered above the X-axis. The dashed line (- - -) indicates the salt gradient employed (0 - 40% of 1.5 M NaCl over a 35 ml volume) while the second dashed line (- — -), which shadows the salt gradient, indicates the actual conductivity level. 39 heating the sample to eliminate heat labile proteins; TrxA's are known to be very heat stable (51). This sample was then applied to an anion exchange column and the active fractions collected (figure 13). At this point the protein purification procedures were discontinued due to the identification of positive clones in a
M.smegmatis DNA library as described below.
III. Identification and Sequencing of Thioredoxin and Thioredoxin Reductase
Clones from a M.smegmatis Plasmid Library
Hybridization using the DIG-labeled PCR probe was performed on a high- density gridded M.smegmatis genomic DNA library which was kindly provided by
Glaxo Wellcome (figure 14). Eight clones were identified and the nucleotide sequences of the inserts obtained first using universal primers to the M13 sequence of pCRII (table 3, figure 15). Additional primers were created in order to obtain the complete sequence of both genes (table 4). The entire nucleotide sequences of the
M.smegmatis trxA and trxB gene cluster were obtained and shown in figure 16, along with the predicted protein sequences.
IV. The Thioredoxin System Gene Cluster of M.smegmatis
The complete nucleotide sequence of M.smegmatis trxA and trxB revealed that the thioredoxin system genes are arranged similarly to those found in
S.clavuligerus. The M.smegmatis trxA and trxB genes are oriented in the same direction with trxA downstream of trxB with a 33 nucleotide separation. Both genes have ATG as their translational initiation codons and contain ribosomal binding site- 40
Figure 13 FPLC profile of M.smegmatis gel-filtrated fractions containing thioredoxin (TrxA) activity applied to a MonoQ HR 5/5, an anion exchange column. Fractions displaying TrxA activity are indicated by the character; '-' indicating low levels of activity, '+' indicating moderate levels of activity, and '*' indicating high levels of activity. The TrxA-active fractions are 14 to 16. The relative absorbance level at 280 nm is displayed on the Y-axis and the volume of wash buffer is displayed on the X-axis. The fractions collected are numbered above the X-axis. The dashed line (—) indicates the salt gradient employed (0 -12% of 1.5 M NaCl over a 30 ml volume) while the second dashed line (- — -), which shadows the salt gradient, indicates the actual conductivity level. 41
Figure 14 Colony hybridization of a high density grid of M.smegmatis genomic DNA library using a DIG-labeled PCR probe to trxB. The positive clones are at the centers of the gray shaded circles. Each clone is placed twice within a 4 by 4 grid square, therefore a positive clone will appear as a doublet within a square of 16. The two clones in the lower right hand corner, that appear singly, are not positive clones. 42
trxB \fxAi
Clone Insert DNA
Y9 Y10 Y11 Y12 Y13 Y14 Y15 Y16
Figure 15 Schematic diagram the region of DNA containing the thioredoxin reductase (trxB) and thioredoxin {trxA) of M.smegmatis. Below are listed the clones from table 3, and their insert DNA fragment relative to the trxB and trxA genes. Clones Y13 and Y16 did not contain sequence to trxB or trxA. One inch is approximately equivalent tolOOO bp. -81 tcccacaccttcqqcqqqaacaqcqcaqcataqqctggcgtttatqacggtgccggqaqa -20 tttccggcggaaaggcccatATGTCCACTTCACAGACGGTTCACGACGTCATCATCATCG MSTSQTVHDVIII 41 GTTCCGGCCCGGCCGGGTACACCGCGGCGATCTATGCGGCCCGCGCTCAGCTCAAGCCCC GSGPAGYTAAIYAARAQLKP 101 TGGTGTTCGAGGGCACACAGTTCGGCGGCGCGTTGATGACCACCACCGAGGTGGAGAACT LVFEGTQFGGALMTTTEVEN 161 ACCCGGGCTTCCGTGAGGGCATCACGGGTCCGGAGCTGATGGATCAGATGCGCGAGCAGG YPGFREGITGPELMDQMREQ 221 CGCTGCGGTTCCGTGCGGACCTGCGCATGGAAGACGTCGACGCAGTACAGCTCGAAGGCC ALRFRADLRMEDVDAVQLEG 281 CCGTCAAGACCGTCGTGGTCGGCGACGAGACCCACCAGGCCCGCGCGGTCATCCTGGCGA PVKTVVVGDETHQARAVILA 341 TGGGCGCGGCCGCACGCCACCTCGGCGTGCCCGGCGAGGAAGCGTTGACCGGCATGGGTG MGAAARHLGVPGEEALTGMG 4 01 TGAGCACCTGCGCGACGTGTGACGGCTTCTTCTTCCGCGACCAGGACATCGTGGTGGTCG VST | C AT C | DGFFFRDQDIVVV 4 61 GCGGCGGCGACTCGGCGATGGAGGAAGCGACGTTCCTCACACGCTTTGCCCGCAGTGTGA GGGDSAMEEATFLTRFARSV 521 CGTTGATCCACCGTCGGGACGAGTTCCGCGCGTCCAAGATCATGCTGGAGCGCGCCCGCG TLIHRRDEFRAS-KIMLERAR 581 CCAACGAGAAGATCACGTTCCTCACCAACACCGAGATCACCCAGATCGAGGGTGACCCGA ANEKITFLTNTEITQIEGDP 641 AGGTCACCGGTGTGCGCCTGCGTGACACCGTGACGGGCGAGGAGTCCAAGCTCGACGTCA KVTGVRLRDTVTGEESKLDV 7 01 CCGGCGTGTTCGTCGCGATCGGCCACGACCCGCGATCGGAGCTCGTGCGCGGTCAGGTCG TGVFVAIGHDPRSELVRGQV 7 61 AACTCGACGACGAGGGCTACGTCAAGGTCCAGGGCCGCACCACATACACGTCGCTCGACG ELDDEGYVKVQGRTTYTSLD
821 GGGTGTTCGCGGCAGGCGATCTGGTGGACCACACTTACCGCCAGGCCATCACGGCCGCGG GVFAAGDLVDHTYRQAI TAA
881 GCAGCGGCTGTGCGGCATCCATCGACGCCGAGCGCTGGCTCGCCGAACAAGACTGAtaqa GSGCAAS I DAERWLAEQD- 941 caataqttttcqactgacaggagacagccATGAGTGAGGACAGCGCGACCGTAGCGGTCA MSEDSATVAV 1001 CCGACGATTCGTTCTCCACCGACGTGCTGGGAAGCAGCAAGCCGGTGCTGGTCGATTTCT TDDSFSTDVLGSSKPVLVDF 1061 GGGCCACCTGGTGCGGCCCGTGCAAGATGGTCGCCCCGGTGCTCGAGGAGATCGCCGCCG WAT | W C G P C -|K MVAPVLEEIAA 1121 AGAAGGGTGACCAACTCACCGTCGCCAAGATCGACGTCGACGTCGACGCCAACCCGGCCA EKGDQLTVAKI DVDVDANPA
1181 CGGCGCGCGATTTCCAGGTCGTGTCGATCCCCACCATGATCCTGTTCAAGGATGGCGCGC TARDFQVVSIPTMILFKDGA 1241 CGGTGAAACGCATCGTCGGCGCCAAGGGCAAGGCCGCCCTGCTGCGCGAGCTCTCCGACG PVKRIVGAKGKAALLRELSD 1301 CCCTCTGA A L - 44
Figure 16 Nucleotide and predicted protein sequence of thioredoxin reductase (trxB) and thioredoxin (trxA) of M.smegmatis (GenBank AF023161). Nucleotides are numbered in ascending order at the left margin from the ATG start codon (the first A is position 1) of the trxB gene. The first nucleotide of the ATG start codon of the trxA gene is at position 970. Underlined nucleotide sequences show the positions of putative ribosome-binding sites upstream of each gene. Double underlined nucleotide sequences denote the positions of putative -10 promoter elements upstream of each gene. The cysteine dithiol redox-active regions of both proteins are shaded. 45
CLONE INSERT SIZE GENES (KB) Y9 trxB/trxA Y10 4.0 trxB (partial) Y11 5.0 trxB/trxA Y12 (Y15) 1.1 trxB (partial) Y13 1.2 unknown sequence Y14 3.0 trxB/trxA (partial) Y15 (Y12) 1.1 trxB (partial) Y16 2.8 unknown sequence
Table 3 List of clones from the M.smegmatis genomic DNA library which hybridized to the DIG-labeled trxB probe. The insert DNA size of each clone is also listed. Not all clones contained sequence to trxB. Clones Y12 and Y15 were found to contain identical insert fragments. 46
NAME SEQUENCE POSITION (STRAND) trxB Primers
MSTRXB1 (+) 5'-TGTGCGACGTGTGACGGCTTCTT-3' 408-429 MSTRXB3 (-) 5'-GATCGCCTGGCGGTAAGTGTGGTC-3' 846-869 MSTRXB4 (-) 5'-ATGTCCTGGTCGCGGATGAAGAA-3' 426-448 MSTRXB5 (+) 5'-GGCAGACGATCTGGTGGACCAC-3' 830-852 . MSTRXB6 (+) 5'-ACGTTCCTCACACGCTTTGCCCGCAG-3' 489-514 MSTRXB7 (-) 5'-TGGTGCGGCCCTGGACCTTGACGTA-3' 777-801 MSTRXB8 (+) 5'-CTGCGTGACACCGTGACGGGCGAGGAG 657-685 TCC-3' MSTRXB9 (+) 5'-CACACTTACCGCCAGCCATCACGGCCGC-3' 849-877 trxA Primers
MSTRXA6 (+) 5'-ACAGGAGACAGCCATGAGTGAGGACAG 56-84 CG-3' MSTRXA7 (-) 5'-GCACGGGGCGACATCTTGCACTGGC-3' 156-170
Table 4 List of primers used to sequence thioredoxin reductase (trxB) and thioredoxin (trxA) genes of M.smegmatis. The primer strand orientations, sequences, and positions are listed beside the primer names. 47 like sequences in the -10 region upstream; -GAAAGG- upstream of trxB and
-AGGAGA- upstream of trxA. The trxB gene consists of 936 nucleotides, encoding a protein of 311 amino acids, with an estimated molecular weight of 33,722. The consensus redox-active site of-CATC- as well as the NADPH and FAD binding domains are present and found in the same location as those of other trxB genes.
The trxA gene consists 339 nucleotides, encoding a protein of 112 amino acids, with an estimated molecular weight of 11,948. The conserved sequence -WCGPC-, containing the two redox-active cysteine residues is found within the protein sequence. Sequence alignment with the M.tuberculosis and M.leprae genes clearly shows that the M.smegmatis TrxA and TrxB deduced amino acid sequences have a very high degree of similarity (figure 17). M.smegmatis TrxA and TrxB have a
67.8% identity at a nucleotide level and 72.7% identity and 81.5% similarity to the 49 kDa fusion protein of M.leprae, respectively. The M.smegmatis genes show a greater similarity to the M.tuberculosis trxA and trxB with a nucleotide identity of
72.7% and a 76.6% identity and 85.4% similarity, respectively at the primary amino acid level.
In addition to the Mycobacterium spp. mentioned above, clustering of the genes for the thioredoxin system genes has been observed for other Actinomycete spp., S.clavuligerus and S.coelicolor. Eubacterium acidaminophilum, belonging to the Propionibacteriaceae, and Clostridium litorale, from the family Bacillaceae, are the only other organisms that possess the clustered gene organization (52). Both bacteria are Gram positive, anaerobic rods that belong to families closely related to
Actinomycetaceae. 48
M. smegmatis MSTS QTVHDV—IIIGSGPAGYTAAIYAAPvAQLKPLVFEGTQFGGA 44 M. tuberculosis MTAPPVHDRAHHPVRDVIVGSGPAGYTAALYAARAQLAPLVFEGTSFGGA 50 M. leprae MNTTPSAHETIHEV—IVIGSGPAGYTTALYAARAQLTPLVFEGTSFGGA 4 8
* t t t . * * g ******** * ******* ******* * * * *
M. smegmatis . LMTTTEVENYPGFRNGITGPELMDQMREQALRFRADLRMEDVDAVQLEGP 94 M.tuberculosis LMTT-EVENYPGFRNGITGPELMDEMREQALRFGADLRMEDVESVSLHGP 99 M.leprae LMTTTEVENYPGFRNGITGPELMDDMREQALRFGAELRTEDVESVSLRGP 98 **** ********* ********* ******** * ** *** * * **
M. smegmatis VKTVVVGD-ETHQARAVILAMGAAARHLGVPGEEA-LTGMGVSTCATCDG 142 M.tuberculosis LKSVVTADGQTHRARAVILAMGARARYLQVPGEEQELLGRGVSSCATCDG 14 9 M.leprae IKSVVTAEGQTYQARAVILAMGTSVRYLQIPGE-QELLGRGVSACATCDG 147 . * . * *** * * * ** ******
M. smegmatis FFFRDQDIVVVGGGDSAMEEATFLTRFARSVTLIHRRDEFRASKIMLERA 192 M. tuberculosis FFFRDQDIAMIGGGDSAMEEATFLTRFARSVTLVHRRDEFRASKIMLDRA 199 M. leprae SFFRGQDIAVIGGGDSAMEEALFLTRFARSVTLVHRRDEFRASKIMLGRA 197
* * *<* * * t * * * * * * * *** *********** ************* **
M.smegmatis RANEKITFLTNTEITQIEGDPKVTGVRLRDTVTGEESKLDVTGVFVAIGH 242 M-. tuberculosis RNNDKIRFLTNHTVCAVDGDTTVTGLRYRDTNTGAETTLPVTGVFVAIGH 24 9 M.leprae RNNDKIKFITNHTVVAVNGYTTVTGLRLRNTTTGEETTLVVTGVFVAIGH 247 * ^ * ^ * * * * * * ********* ***********
M. smegmatis DPRSELVRGQVELDDEGYVKVQGRTTYTSLDGVFAAGDLVDHTYRQAITA 292 M.tuberculosis EPRSGLVREAIDVDPDGYVLVQGRTTSTSLPGVFAAGDLVDRTYRQAVTA 299 M.leprae EPRSSLVSDVVDIDPDGYVLVKGRTTSTSMDGVFAAGDLVDRTYRQAITA 297
* * *^* * t ^ * ** * * * * * * * * ********** * * * * * * *
M. smegmatis AGSGCAASIDAERWLAEQ DTIVFDQETAMSED 324 M. tuberculosis AGSGCAAAIDAERWLAEHAA TGEADSTDALIGAQRMTDSED 340 M.leprae AGSGCAAAIDAERWLAEHAGSKANETTEETGDVDSTDTTDWSTAMTDAKN 347 ******* ********* *
M.smegmatis SA-TVAVTDDSFSTDVLGSSKPVLVDFWATWCGPCKMVAPVLEEIAAEKG 373 M. tuberculosis SA-TIKVTDASFATDVLSSNKPVLVDFWATWCGPCKMVAPVLEEIATERA 38 9 M.leprae AGVTIEVTDASFFPDVLSSNKPVLVDFWATWCGPCKMVAPVLEEIASEQR 397 •k -k-k-k * * * k k k-k-k-k-k-k-k-k-k-k-k-k-k-k k *-k-k-k-k-k-k-k-k -k
M. smegmatis DQLTVAKIDVDVDANPATARDFQVVSIPTMILFKDGAPVKRIVGAKGfCAA 423 M.tuberculosis TDLTVAKLDVD—TNPETARNFQVVSIPTLILFKDGQPVKRIVGAKGKAA 437 M. leprae NQLTVAKLDVD—TNPEMAREFQVVSIPTMILFQGGQPVKRIVGAKGKAA 445
t^*****t*** * * * * ********* * * * * *************
M.smegmatis LL-RELSDAL 432 M. tuberculosis LL-RELSDVVPNLN 450 M.leprae LLLRDLSDVVPNLN 459 ** * ***
Figure 17 Multiple alignment of the primary amino acid sequences of the thioredoxin system proteins of Mycobacterium spp.; M.smegmatis, M.tuberculosis (Accession no. P52229.P52214), and M.leprae (Accession no. P46843). The TrxB- TrxA fusion protein sequence of M.leprae is displayed while the sequences between TrxB and TrxA are included for M.smegmatis and M.tuberculosis. Conserved amino acids are designated with the character '*' and similar amino acids are designated with the character'.' . Alignment was performed using PCGENE amino acid cluster program. 49
The similarity in the organization of M.smegmatis trxA and trxB to the genes of S.clavuligerus may suggest a similarity in gene regulation as well. M.smegmatis possesses -10 like promoter regions upstream of trxA, -TAGACA-, in addition to two upstream of trxB, -TAGGCT- and -TATGAC-. The promoter region upstream of trxA is located in the C-terminal encoding region of trxB. It has been shown that
S.clavuligerus contains two different-sized RNA transcripts corresponding to those expected for a trxB-trxA mRNA and for a trxA mRNA alone (53). Promoter like sequences are located upstream of trxB and upstream of trxA, which lies within the open reading frame of trxB. These studies suggest that more than one site for transcription initiation may exist in the trxA-trxB gene cluster of S.clavuligerus. In
Streptomyces, several genes such as gal-p2 and nshR-P within multi-gene operons have been shown to initiate transcription within the open reading frames of other genes upstream (54). A temporal regulation of gene expression has been suggested as a function for these promoters. Thus it may be that the trxA-trxB gene clusters in M.smegmatis and other mycobacterial spp. studied to date have similar patterns of gene regulation to the Streptomycetes.
V. Western Blot Studies
A Western blot was performed using cell-free extracts of M.phlei, M.aurum, and four different isolates of M.smegmatis: mc26, mc2155, ATCC 19420, and ATCC
607 (figure 18). Since thioredoxin is highly conserved, antiserum to E.coli TrxA was employed to screen various species of mycobacteria for cross-reacting proteins. In all four M.smegmatis strains a protein of approximately 14 kDa was 1 2 3 4 5 6 7
Figure 18 Western blot of cell free extracts of Mycobacterium spp. using antiserum raised against E.coli thioredoxin.
Lane 1 E.coli thioredoxin (Sigma) 2 M.smegmatis mc26 3 M. smegmatis mc2155 4 M.smegmatis ATCC 19430 5 M.smegmatis ATCC 607 6 M.aurum 7 M.phlei 51 recognized. Minor cross-reacting proteins of approximate size: 25, 29, and 43 kDa were also recognized. The major protein recognized in M.phlei was about 38 kDa, although proteins of about 14 and 25 kDa were also observed. M.aurum appears to possess a different profile of cross-reaction, although a very faint protein band of size 14 kDa was seen on the autoradiogram (but not in the reproduction in figure
18). The 29 kDa protein may represent a dimer form of the 14 kDa TrxA. The other proteins of sizes 25, 38 and 43 that were recognized are unknown. A Western blot published by Weiles et al., showed the presence of a 14 kDa protein cross-reacting protein in M.smegmatis ATCC 607 lysate and a protein of approximately 12 kDa in lysates of M.tuberculosis and M.leprae (55).
Weiles et al., also performed PCR experiments on a variety of species and strains of both rapid and slow growing mycobacteria using degenerate primers to the redox sites of both trxA and trxB; PCR products were produced by a number of slow-growing mycobacterial spp. including M.leprae and M.tuberculosis. The deduced amino acid sequences derived from the PCR product nucleotide sequence showed identity to the corresponding sequences of S.clavuligerus and E.coli TrxA and TrxB. No PCR product was produced for a number of other mycobacterial spp. including M.smegmatis. From the Western blot and PCR data, it was postulated by
Weiles et al., that the M.smegmatis thioredoxin system genes were quite different from those of M.tuberculosis and M.leprae. However, our nucleotide sequence analysis of M.smegmatis trxA-trxB region has revealed that the thioredoxin system genes are clustered and when compared with those of M.tuberculosis, a similarity of
86% is observed between the deduced protein sequences. 52
However, a discrepancy is found in the estimated molecular weight of TrxA by SDS-PAGE. The TrxA's from M.smegmatis and M.tuberculosis contain approximately the same number of amino acid residues and the predicted molecular weights by computer analysis are also similar. A slight difference in molecular weight can be accounted for by differences in gel electrophoretic mobility, variance in charge due to amino acid differences, and differences in hydrophobicity. A 2 kDa difference may also be accounted for by differences in post-translational modification such as methylation, glycosylation, or adenylation of amino acid residues. But despite the small difference in molecular weight of the TrxA,
M.smegmatis appears to possess a thioredoxin system that is very similar to
M. tuberculosis, and can therefore be used as a simple laboratory model to study the function of this redox control process in the mycobacterial pathogens.
VI. Multiple Sequence Alignment of Thioredoxins and Thioredoxin
Reductases
A. Multiple Sequence Alignment of Thioredoxin Reductases
Thioredoxin and thioredoxin reductase, together with NADPH, are the components that comprise the thioredoxin system. This system transfers reducing equivalents to a broad range of disulfides, thus acting as a general protein disulfide reductase (47). Thioredoxin reductase (TrxB) belonging to the large family of flavoprotein oxidoreductases, facilitates the transfer of electrons from pyridine nucleotides via FAD to their redox-active site; the electrons can then reduce various 53 disulfide substrates such as ribonucleotide reductase. The flavoprotein oxidoreductases possess conserved regions; an NADPH binding domain and two domains that are involved in FAD binding. TrxB is a protein of about 70 kDa consisting of 2 identical subunits of about 35 kDa. The two subunits bind the co- factor FAD which accepts electrons from NADPH which then transfers these to the redox active site of TrxB. Multiple alignment of the primary amino acid sequences of known thioredoxin reductases with the TrxB sequence of M.smegmatis was performed. M.smegmatis TrxB shows identity in the essential domains that are conserved in flavoprotein oxidoreductases and confirms that it is indeed a member of the large thioredoxin reductase family (figure 19).
A phylogenetic tree displaying the relationship of the organisms based on the primary amino acid sequences of known TrxB proteins was constructed (figure 20).
The M.smegmatis TrxB is clustered with the other Mycobacterium spp.,
M.tuberculosis and M.leprae, although it is given a different branch from the slow growing mycobacteria. The Mycobacterium spp. are also grouped with
Streptomyces spp. which are members of the Actinomycete family. It is interesting to note that the other two organisms, E.acidaminophilum and C.litorale, which possess a clustered thioredoxin system gene organization fall within the same larger branch. The exception within this group is Mycoplasma genitalium, from the family
Mycoplasmataceae, which are small bacteria lacking a true cell wall. Recent studies with two other mycoplasma spp., M.pneumoniae and M.capricolum revealed that their thioredoxin system genes, like M.genitalium are not linked (56). 54
TRXB CLOL 1 MENVYD.' TRXB EUBAC 1 MENVYD? TRXB MYCLE 1 MNTTPSAHETIHE| TRXB MYCTU 1 -MTAP PVHDRAHHPVRD:| TRXB MSMG 1 MSTSQTVHD:; TRXB STRCL 1 SDVRN, TRXB STRCO 1 SDVRN TRXB PENCH 1 VHSKl* TRXB NEUCR 1 MHSKJ: TRXB YEAST 1 VHNKi; TRXB ECOLI 1 GTTKHSK: TRXB HAEIN 1 MSDIKHAK TRXB COXBU 1 MNKPQHHS; TRXB_ _MYCGE 1 MLKVNADFLTKDQVIYD
TRXB CLOL 45 PG SVPEAT -KIKVLKGAKG TRXB EUBAC 45 mm PG SVREAT -KIKVIKGEKA TRXB MYCLE 52 PG FRNG TMgE • -PIKSWTAEG TRXB MYCTU 55 PG FRNG T^Ei - PLKS WTADG TRXB MSMG 48 PG FREG T^E -PVKTVWGD- TRXB STRCL 45 PG FRDG MB3D -DIKTVTDSAG TRXB STRCO 4 5 PG • -EIKTVTDTAG TRXB PENCH 4 8 PG SSRPFKMWTEWNDDE TRXB NEUCR 4 8 PG ISARPFKYATEWSPEE TRXB YEAST 48 PG S S KP FKLWTE FNEDA TRXB ECOLI 46 PG ONR--PFRLNGDNGE TRXB HAEIN 4 7 PG |SSR- -PFKLFGDVQN TRXB COXBU 47 PG PR--PFLLQGDNAT TRXB MYCGE 56 PG LN--DTFILYLDNKT
TRXB CLOL 101 EYKAK0 aKELTS GVSBCATCD jEDME FKGGG D TRXB EUBAC 101 EYKAK SQELTB| GVSgCATCD 5--D JEDME F| GGGD TRXB MYCLE 108 —QTYQARH 3QELL1|GVSgCATCD G --S | GQD GGGD TRXB MYCTU HI —QTHRARII 3QELLS GVSgCATCDG --F JDQDF GGGD TRXB MSMG 103 ETHQARH; aEALT* GVsjjCATCDG --F 1DQDI GGGD GVSgCATCDG --F GGGD TRXB STRCL 101 TVHRAKg SDALS1 jDQD TRXB STRCO 101 TVHRAKg; aDALSlg GVsBcATCDG --F VJJGGHD GISSCASCDG AVPI DQD A TRXB PENCH 10 7 GSEPVRTADg NKP Y VIGGGD TRXB G EKYWQN GISBCAKCDG RVPI NEUCR 10 7 YHTAD NKH V VIGGGD TRXB VTTD0 |G ETYWQS G|SBCAQCDG AVPI YEAST 10 7 EP NKP A VIGGGD TRXB EAFKB| GVSSCATCDG --F ECOLI 103 YTCDS NQK A VIGGGN TRXB HAEIN EN S .GVSgCATCD G --F VIGGGN 104 FTCDS NKP G TRXB COXBU 105 YSCDBJ; KPYMS: GVSgCATCDG --F vVGGGN 1 AKK A TRXB MYCGE 112 TVFSKT', EK@DYFY§| GISHCAHCD."- -ALj GKT G VVGGG:
TRXB CLOL 155 QE jAFKKE - - -HlNFMWfflTV^EEf ; TRXB EUBAC 155 QE. AFKNP S'DFMwSs»DFMV\ A EE TRXB MYCLE 163 LC-HARNND- MKFITteTfflVAg TRXB MYCTU 166 LDgARNND- RRFLTBHTAGAI TRXB MSMG 158 *"gARANE- TFLTfflTE TQ TRXB STRCL 156 FADP- j SFAW*SE AT TRXB STRCO 155 FADP- RSFVWSSEJJAEJ TRXB PENCH 167 LLAHP- FKVRFSTVATEB TRXB NEUCR 163 LLNHE- !7TVRFBTVGVE: TRXB YEAST 164 5JAEKNE WjjE I LYfflTVALEAI TRXB ECOLI 156 LMOKVENGN-ilLHT^RT ES TRXB HAEIN 157 IDSLYKKVEEGHIVLHT ;RTSDE^ TRXB COXBU 158 SAQLIKKVEEGH'AIVWSHV EE TRXB MYCGE 166 VEILKKI S—NQVFHLBATBKQHI TRXB CLOL 212 ESAVF NRE GEVTEFVAPEEDGTFS TRXB EUBAC 212 ESAVF NLV GETTEYFANEEDGTFg TRXB MYCLE 220 TG NTT GE ETT TRXB MYCTU 223 TG Y DTN GA ETT TRXB MSMG 215 TG DTV GE ES TRXB STRCL 213 TG T DTK GE TRE TRXB STRCO 212 AG NVK GE LSDjjPVT TRXB PENCH 224 PNGL TH DVL NA EEV ,EAN TRXB NEUCR 220 - -GL SH V DVT GK EETBEAN TRXB YEAST 221 L NA NTKKNE ETDWPVS TRXB ECOLI 216 ---G TG DTQNSDN IESgDVA TRXB HAEIN 217 ---G TG ANTK GE KEEWKLDU TRXB COXBU 218 ---G TG HVKEEK TQDgTIDgl TRXB MYCGE 223 QT ASTVDKS ESE AIDCj BLG
TRXB CLOL 267 N I GVFAAGD EELFAE TRXB EUBAC 267 T N £ GVFAAGD IEANFEE TRXB MYCLE 269 T S E GVFAAGD |AEHAGSKANETT TRXB MYCTU 272 T S E GVFAAGD AEHAATGEAD S T TRXB MSMG 264 T S E GVFAAGD JAEQD TRXB STRCL 262 N 1 GVFAAGD [AALADSEQIAEP TRXB STRCO 260 N 1 GVF|AGD SD-EDKAEP TRXB PENCH 277 T N E GVFASGD .ETET HQE TRXB NEUCR 271 T S E GVFAAGD SEHEETPAEHRD TRXB YEAST 271 T S E GVFAAGD TRXB ECOLI 266 II S E GVFAAGD DGLADAK- TRXB HAEIN 266 KSGLDGNATJ T S E GVFAAGD MJ3 DAQEA--- TRXB COXBU 22668 8 KSGLQGNAT; T N E GVFPlVVigRGQLF l DSLNQA-- TRXB MYCGE 273 T SIB ~BCRS!S: KVRDD -
TRXB CLOL TRXB EUBAC TRXB MYCLE 325 EETGDVDSTDTTDWSTAMTDAKNAGVTIEVTDASFFADVLSSNKPVLVDFWATWCGPCKM TRXB MYCTU 328 DALIGAQR TRXB MSMG TRXB STRCL 318 -APAV TRXB STRCO 315 EKTAV TRXB PENCH 329 AKPVL TRXB NEUCR 327 TSAVQGNL TRXB YEAST TRXB ECOLI TRXB HAEIN TRXB COXBU TRXB MYCGE
TRXB_MYCLE 3 85 VAPVLEEIASEQRNQLTVAKLDVDTNPEMAREFQWSIPTMILFQGGQPVKRIVGAKGKA
TRXB MYCLE 44 5 ALLRDLSDWPNLN
Figure 19 Multiple alignment of known thioredoxin reductase primary amino acid sequences. The alignment was performed using the program CLUSTALV. Conserved amino acids are shaded black and similar amino acids are shaded grey. 56
Eubacterium acidaminophilum
Clostridium litorale
Mycoplasma genitalium
— Mycobacterium tuberculosis
— Mycobacterium leprae
Mycobacterium smegmatis
Streptomyces coelicolor
Streptomyces clavuligerus
Haemophilus influenzae
Escherichia coli
Coxiella burnetii
Neurospora crassa
• Penicillium chrysogenum
Saccharomyces cerevisiae
Figure 20 Phylogenetic tree derived from the primary amino acid sequences of known thioredoxin reductases. The alignment file presented in Figure 19 was used to create a phylogenetic tree using DeSoete tree fit with no distance corrections. 57
B. Multiple Sequence Alignment of Thioredoxins
Thioredoxin, the smaller protein component of the thioredoxin system, is a well-characterized protein which is found in all organisms so far studied (51). The reduced TrxB transfers its electrons to the TrxA active cysteines which in turn can reduce other molecules. TrxA is a well characterized protein which has been found to be involved in a variety of different metabolic processes. Typically, TrxA's have a molecular mass of around 12-14 kDa and possess a conserved -CXXC- redox active site. Multiple alignment was performed for M.smegmatis TrxA with over 45 known thioredoxin amino acid sequences (figure 21). A phylogenetic tree, displaying the relationship of the organisms based on TrxA sequences clearly group these organisms as expected by 16S and 18S rRNA comparisons (figure 22.1) (57).
Eukaryotes are grouped together in mammalian, plant and fungal clusters. The prokaryotes are grouped together although a few are observed in the larger branch closer to the eukaryotes (figures 22.2 and 22.3). Chloroplast TrxA's are clustered with other photosynthetic bacteria. M.smegmatis TrxA is found in a branch containing other Mycobacterium spp. which are a part of a larger group of
Actinomycetes with the exception of Alicyclobacillus acidocaldarius, which is a Gram positive, thermophilic, high %G+C organism and is classified as a Bacillus spp. The lower %G+C Gram positive bacterium Bacillus subtilus, is found closer to
Corynebacterium spp. It is interesting that for TrxA, Mycoplasma genitalium,
E.aciaminophilum and C.litorale are located in the branch closer to eukaryotes. The sequence alignment and phylogenetic data of both thioredoxin system genes, 58
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M U K 12 D QJ B H ro D Ul rtj D X U hi o Ul H £> Ul Ul 53 u to Ul Ul «( h f£ 2 U H w >H Ol >H o EH u o UJ h^ QC r- CO hi hi < to Cu m THIO_CHLL THI1_ANAS0 THI 1_ANANI THI0_SYNY3 THIO_PORYE THIO_PORPU THIO_GRIPA — THIO_CYACA THIM_SPIOL — THIO_MYCTU TRXA_MYCLE M.smegmatis TrxA THIO_ALIAC THI 0_STRCO THIO_STRCL — THIO_ECOLI — THIO_THIFE THIO_CHRVI THIO_RHORU THI2_ANASP — THIO_HAEIN — THI2_C0RNE THIO_RHOSH THI1_CORNE — THIO_BACSU I- THIO_MACMU JL- THIOJHUMAN II THIO_SHEEP r- THIO_RAT L. THIO_MOUSE THIO_RABIT THIO_CHICK THI3_DICDI THI1_DICDI THII TOBAC - THIH_ARATH THI1 CHLRE THI2_YEAST - THI1_YEAST THIO_EMENI - THIO_PENCH THIO DROME THIO_MYCGE THIO_EUBAC THIO CLOU THIO BPT4 THIO CHLPS Figure 22.1 Phylogenetic tree derived from the primary amino acid sequences of known thioredoxins. The alignment file presented in Figure 21 was used to create a phylogenetic tree using DeSoete tree fit with no distance corrections. 60 - Macaca mulatto (rhesus macaque) ™ Homo sapiens (human) I—• Ovis aries (sheep) . • m Rattus norvegicus (rat) ^ Mus musculus (mouse) Oryctolagus cuniculus (rabbit) Gallus gallus (chick) Dictyostelium discoideum (slime mold) ——— Dictyostelium discoideum (slime mold) Nicotiana tabacum (common tabacco) Arabidopsis thaliana (mouse-ear cress) ——— Chlamydomonas reinhardii (green algae) —•• Saccharomyces cerevisiae — Saccharomyces cerevisiae Emericella nidulans ^—^— Penicillium chrysogenum Drosophila melanogaster (fruit fly) Mycoplasma genitalium ^• Eubacterium aciaminophilum ———• Clostridium litorate (Bacterium W6) Bacteriophage T4 Chlamydia psittaci Figure 22.2 The branch of the thioredoxin phylogenetic tree of Figure 22.1 containing eukaryotic organisms. 61 Chlorobium limicola F. SP. Thiosulfatophilum • Anabaena sp.(strain PCC 7119) Anacystis nidulans Synechocystis sp. (strain PCC 6803) i— Porphyra yezoensis (chloroplast) Porphyra purpurea (chloroplast) —— Griffithsia paciflca (chloroplast) — Cyanidium caldarium —••••Spinacia oleracea (chloroplast) — Mycobacterium tuberculosis Mycobacterium leprae — Mycobacterium smegmatis _ Alicyclobacillus acidocaldarius ~~ Streptomyces coelicolor — Streptomyces clavuligerus ~~ Escherichia coli ™ Thiobacillus ferrooxidans ^—^— Chromatium vinosum Rhodospirillum rubrum Anabaena sp. (strain PCC 7120) Haemophilus influenzae Corynebacterium nephridii Rhodobacter sphaeroides Corynebacterium nephridii •" Bacillus subtilis Figure 22.3 The branch of the thioredoxin phylogenetic tree of Figure 22.1 containing prokaryotic organisms. 62 clearly shows that the M.smegmatis can be grouped with other high %G+C Gram positive bacilli from such families as Actinomycetes. This phylogenetic tree also shows that the thioredoxin system genes, trxA and trxB of M.smegmatis are closely related to those of the pathogenic mycobacterial spp., M.tuberculosis and M.leprae. Nucleotide sequencing of the M.smegmatis genes has shown that the gene organization is similar to those found in M. tuberculosis; making M.smegmatis an appropriate candidate to use in further studies of the mycobacterial thioredoxin system. Further investigations may include studies addressing the following questions: Is the regulation of the thioredoxin system genes different in organisms possessing a clustered gene arrangement? What role does the thioredoxin system play in the general redox-regulation and is it greater in organisms such as mycobacteria in which glutathione is absent? Does the thioredoxin system play a role in the regulation of other functions such as gene expression, cell proliferation, cellular protection, and cell death? 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