discovery and characterization of novel tetrodecamycins · 2017-11-02 · antibiotics are one of...
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Discovery and Characterization of Novel Tetrodecamycins
by
Tomas Antanas Gverzdys
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Biochemistry University of Toronto
© Copyright by Tomas Antanas Gverzdys 2017
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Discovery and Characterization of Novel Tetrodecamycins
Tomas Antanas Gverzdys
Doctor of Philosophy
Department of Biochemistry
University of Toronto
2017
Abstract
Antibiotics are one of the most important groups of drugs ever discovered. In addition to their
clinical use, these small molecules have also been essential for understanding the molecular
details of numerous fundamental biological processes. As a result, the discovery and
characterization of novel small molecules is of extraordinary value to both society and science.
Streptomyces sp. strain WAC04657 is a wild-isolate bacterium that produces an antibiotic
activity against multi-drug resistant Staphylococcus aureus. I purified the responsible molecule
and characterized it as 13-deoxytetrodecamycin, a new antibiotic belonging to the
tetrodecamycin-group of molecules. To study the biosynthesis of this molecule, I sequenced the
genome of WAC04657 and, using bioinformatic methods, identified the biosynthetic gene
cluster. I named this cluster the ted cluster. To confirm that these genes were responsible for
producing 13-deoxytetrodecamycin, I genetically manipulated genes within the cluster with the
goal of making an overproducer strain and a non-producer strain. In addition to affecting the
expression of 13-deoxytetrodecamycin, these mutants show alterations in the expression of four
other molecules. I purified two of these and solved their structures. One I identified as the known
molecule tetrodecamycin, while the second was a novel molecule that I named W5.9. A search of
public genome sequences revealed that the ted cluster was also found in three other bacteria:
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Streptomyces atroolivaceus, Streptomyces globisporus, and Streptomyces sp. LaPpAH-202. By
genetically manipulating the ted cluster in S. atroolivaceus and S. globisporus, I was able to
confirm that these bacteria were also producers of tetrodecamycin –group molecules.
Specifically, they produced tetrodecamycin and dihydrotetrodecamycin. In summary, the work
reported in this thesis describes the discovery of a novel antibiotic as well the biosynthetic gene
cluster responsible for producing it. Future work will focus on assessing the therapeutic potential
of the tetrodecamycin-group molecules and identifying their mechanism of action.
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Dedication
For my parents. While we live in a country where
everyone gets a fair shot, you made sure that I got
to take as many shots as I needed.
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Acknowledgments
Thank you to my grade 6 science teacher, Mr. Calabrese, who asked me “why” during class and
found someone to help me build a radio; to Mrs. Calgary, who let us derive our own spring laws
in grade 11; to Dr. Susan Koval, for her deep passion for teaching microbiology; to Dr. Justin
Nodwell, for letting my mind wander and explore; to Dr. Sheila Elardo, for letting me share my
ideas with you; to Dr. Alex Ensminger, for seeing something in me; and to Dr. Alex Palazzo, for
lending me books from his private library.
Thank you to Leesa Pennell. You are the greatest thing that has ever happened to me. You have
seen me at my best and my worst. You always hug me when I need comfort or tell me to “put on
my big boy pants” when I’m being petty. Every success I have is shared. I love you with all my
heart and soul. Even more than science.
Thank you to my parents. All my early days are shaped by you. You set me free to explore the
world, and I cannot thank you enough. I hope that I have the strength to be as wonderful to my
children as you were to me. To my siblings, you’re all fools and I love you. Thank you for all
your love and abuse. I look forward to many more fights and laughs in the coming years.
To my friends, oh so many of you over the years. You have splashed my life with colour and
excitement. In particular, thank you to April Pawluk. You were my first friend who was my
intellectual equal and also loved microbiology. The department feels empty since you’ve left. To
Fred Ulrich, my best friend. Thank you for doing those ‘mud glove’ experiments with me in
grade five, and for always being excited for my scientific progress (even if you don’t really get
it). I’m gifted to have such a wonderful friend.
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Table of Contents
DEDICATION............................................................................................................................. IV
ACKNOWLEDGMENTS ........................................................................................................... V TABLE OF CONTENTS ........................................................................................................... VI LIST OF TABLES ...................................................................................................................... IX LIST OF FIGURES ..................................................................................................................... X ABBREVIATIONS ................................................................................................................... XII
PUBLICATIONS ..................................................................................................................... XIV CHAPTER 1. INTRODUCTION ................................................................................................ 2 1.1 Why study antibiotics? A brief history. .................................................................................. 2 1.2 The target of antibiotics .......................................................................................................... 4 1.3 Streptomyces are the source of most antibiotics ..................................................................... 6
1.4 Tetronates are a large family of bioactive molecules ............................................................. 7 1.5 History of the TDM-group molecules ................................................................................... 10
1.6 TDM-group molecules are produced by polyketide synthases ............................................. 12 1.7 Tailoring reactions give rise to tetronate ring ....................................................................... 15
1.8 Genetic regulation of antibiotic gene clusters ....................................................................... 18 1.9 Thesis objectives and outline ................................................................................................ 20
CHAPTER 2. DISCOVERY OF 13-DEOXYTETRODECAMYCIN ................................... 22 2.1 Abstract ................................................................................................................................. 23 2.2 Introduction ........................................................................................................................... 24
2.3 Results ................................................................................................................................... 25 2.3.1 Characterization of WAC04657 ................................................................................... 25 2.3.2 Identification of an anti-MRSA activity ....................................................................... 26
2.3.3 Biological activity and structural elucidation of 13-deoxytetrodecamycin .................. 29
2.4 Discussion ............................................................................................................................. 32
CHAPTER 3. THE BIOSYNTHETIC GENE CLUSTER FOR 13-
DEOXYTETRODECAMYCIN ................................................................................................. 36 3.1 Abstract ................................................................................................................................. 37 3.2 Introduction ........................................................................................................................... 38
3.3 Results ................................................................................................................................... 39 3.3.1 454 sequencing was used to identify the partial ted cluster .......................................... 39
3.3.2 Illumina data did not improve the WAC04657 assembly ............................................. 40 3.3.3 The full biosynthetic gene cluster for 13-deoxytetrodecamycin................................... 43 3.3.4 The edges of the ted cluster were identified by comparative genomics ....................... 47
3.4 Discussion ............................................................................................................................. 53
CHAPTER 4. THE TED CLUSTER PRODUCES THE TETRODECAMYCINS .............. 58 4.1 Abstract ................................................................................................................................. 59 4.2 Introduction ........................................................................................................................... 60
4.3 Results ................................................................................................................................... 61 4.3.1 The ted cluster is responsible for producing 13-deoxytetrodecamycin ........................ 61 4.3.2 The ted cluster in WAC0657 produces a novel TDM-group molecule ........................ 63 4.3.3 S. atroolivaceus and S. globisporus are producers of TDM and dhTDM..................... 68
4.4 Discussion ............................................................................................................................. 74
CHAPTER 5. SUMMARY, FUTURE DIRECTIONS, AND CONCLUSIONS ................... 79 5.1 Thesis Summary.................................................................................................................... 79
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5.2 Future Directions .................................................................................................................. 79
5.2.1 Overproducer strain of TDM ........................................................................................ 80 5.2.2 TDM-group molecules as drugs.................................................................................... 81 5.2.3 Mechanism of Action of the TDM-group molecules .................................................... 81
5.2.4 Curious features of the ted cluster ................................................................................ 82 5.3 Conclusion ............................................................................................................................ 86
CHAPTER 6. MATERIALS AND METHODS ....................................................................... 88 6.1 General Experimental Procedures ......................................................................................... 88
6.1.1 Equipment information ................................................................................................. 88
6.1.2 Primers used .................................................................................................................. 88 6.1.3 Strains used ................................................................................................................... 90 6.1.4 General culture methods ............................................................................................... 92 6.1.5 Media recipes ................................................................................................................ 93
6.1.6 General PCR Protocol ................................................................................................... 95 6.2 DNA purification methods .................................................................................................... 96
6.2.1 Plasmids and PAC clone isolation ................................................................................ 96 6.2.2 DNA isolation for phylogenetic tree ............................................................................. 96
6.2.3 DNA isolation for WGS 454 and Illumina Sequencing................................................ 96 6.2.4 DNA isolation for PacBio Sequencing ......................................................................... 96
6.3 Bioassays............................................................................................................................... 97
6.3.1 Colony diffusion assays ................................................................................................ 97 6.3.2 Solid Agar MICs ........................................................................................................... 97
6.4 Molecule purification and HPLC methods ........................................................................... 98 6.4.1 HPLC Methods ............................................................................................................. 98 6.4.2 Small-scale crude extract analysis of Streptomyces...................................................... 99
6.4.3 TLC, bioautography, and initial identification of 13-dTDM ........................................ 99
6.4.4 Purification of 13-dTDM ............................................................................................ 100 6.4.5 Purification of W5.9.................................................................................................... 100 6.4.6 Purification of TDM ................................................................................................... 101
6.4.7 Purification of dhTDM ............................................................................................... 101 6.5 Plasmid assembly and manipulation of Streptomyces ........................................................ 101
6.5.1 tedF1 knock out plasmid ............................................................................................. 101 6.5.2 tedF1 disruption plasmid ............................................................................................ 102
6.5.3 tedR overexpression plasmid ...................................................................................... 103 6.5.4 Streptomyces strain construction ................................................................................. 103
6.6 Genome analysis ................................................................................................................. 103 6.6.1 Phylogenetic tree ......................................................................................................... 103 6.6.2 WAC04657 454 sequencing ....................................................................................... 104
6.6.3 WAC04657 Illumina sequencing ................................................................................ 104 6.6.4 PAC clone screening and sequencing ......................................................................... 104
6.6.5 WAC04657 PacBio sequencing .................................................................................. 104 6.6.6 Accession number for WAC04657 ............................................................................. 105 6.6.7 Identification of the ted genes in other organisms ...................................................... 105
REFERENCES .......................................................................................................................... 106 CHAPTER 7. APPENDIX: NMR SPECTRA ........................................................................ 117 7.1 13-dTDM NMR spectra ...................................................................................................... 117 7.2 W5.9 NMR spectra ............................................................................................................. 124
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7.3 TDM NMR traces ............................................................................................................... 129
7.4 dhTDM NMR spectra ......................................................................................................... 134
CHAPTER 8. APPENDIX: SEQUENCING RECEIPTS ..................................................... 139
COPYRIGHT ACKNOWLEDGEMENTS ............................................................................ 143
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List of Tables
Table 2.1 Colony diffusion assays of WAC04657 against Gram positive bacteria. ......................27 Table 2.2 MIC of 13-dTDM against several pathogens. ...............................................................30
Table 2.3 Physico-chemical properties of 13-dTDM. ...................................................................31 Table 2.4
13C and
1H NMR data for 13-dTDM in CDCl3. .............................................................32
Table 3.1 Summary of high throughput sequencing runs. .............................................................40 Table 3.2 Proposed annotations for WAC04657's biosynthetic gene clusters. .............................46 Table 3.3 Predicted function of ted cluster genes. .........................................................................50
Table 3.4 Comparison of the ted cluster proteins found in S. atroolivaceus, S.
globisporus, and S. sp. LaPpAH-202 against those found in S. str. WAC04657. .........51 Table 3.5 The top 20 most sequenced genera based on unique species name from the
NCBI’s RefSeq database. ..............................................................................................54
Table 4.1 Observed properties of the TDM-group molecule identified in this study. ...................65 Table 4.2
13C and
1H NMR data for W5.9 in CDCl3. ....................................................................68
Table 4.3 13
C and 1H NMR data for TDM and dhTDM. ...............................................................74
Table 6.1 Primers used in this thesis. .............................................................................................88
Table 6.2 Personal strains used in this thesis. ................................................................................90 Table 6.3 Nodwell shared BSL2 strains used in this thesis. ..........................................................92 Table 6.4 Standard PCR reagents. .................................................................................................95
Table 6.5 HPLC Method 1 .............................................................................................................98 Table 6.6 HPLC Method 2 .............................................................................................................98
Table 6.7 HPLC Method 3 .............................................................................................................98 Table 6.8 HPLC Method 4 .............................................................................................................99
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List of Figures
Figure 1.1 The tetronate family of molecules all possess a tetronate ring. ......................................9 Figure 1.2 Tetrodecamycin and dihydrotetrodecamycin were the first discovered
members of the TDM-group molecules. .......................................................................10 Figure 1.3 The proposed mechanism by which the tetrodecamycins could bind covalently
to their target. ................................................................................................................12 Figure 1.4 Type I PKSs act to link carbon monomers into chains. ...............................................14 Figure 1.5 Formation of tetronate rings requires a dedicated set of proteins. ...............................17
Figure 2.1 16S phylogenetic tree indicates that WAC04657 is a strain of Streptomyces. .............26 Figure 2.2 TLC was used to identify the active compound in the crude WAC04657
extract. ...........................................................................................................................28 Figure 2.3 Purification method for 13-dTDM. ..............................................................................29
Figure 2.4 Structure of 13-dTDM. .................................................................................................31 Figure 3.1 Contig 68 represents the partial ted gene cluster. .........................................................40
Figure 3.2 Contig 293 encodes the right arm of the ted cluster. ....................................................42 Figure 3.3 Draft map of the PAC292 clone. ..................................................................................43
Figure 3.4 Location of the biosynthetic gene clusters in the genome of WAC04657. ..................45 Figure 3.5 Position and overlap of PKS clusters, and comparison of the ted cluster as
found in WAC04657, S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202. ........45
Figure 3.6 The ted biosynthetic gene cluster is well conserved in WAC04657 compared
to S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202. .......................................48
Figure 3.7 Proposed biosynthetic gene cluster for 13-dTDM as found in WAC04657. ...............49 Figure 3.8 Number of contigs in each Streptomyces genome assembly divided by
sequencing technology. .................................................................................................55
Figure 4.1 Structures of the TDM-group molecules. .....................................................................60
Figure 4.2 Genetic manipulation of the ted cluster in WAC04657 results in changes in
13-dTDM (W8.0) production as well as altered expression of several other
molecules. ......................................................................................................................62
Figure 4.3 Supporting data for the structural elucidation of molecule W5.9. ...............................67 Figure 4.4 Wild-type S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202 do not
produce 13-dTDM or W5.9. ..........................................................................................70 Figure 4.5 Heterologous overexpression of tedRW in S. atroolivaceus and S. globisporus
stimulates production of TDM-group molecules. .........................................................71 Figure 4.6 Tandem MS-MS data of the molecules eluting at 6.4 and 5.4 minutes suggests
that they are the same molecule. ...................................................................................73 Figure 4.7 Proposed biosynthesis for 13-dTDM, TDM, and dhTDM. ..........................................76 Figure 5.1 Plug n’ Play model for control of a biosynthetic gene cluster with a conserved
peptide signal. ................................................................................................................85 Figure 6.1 Plasmid 231 map. .......................................................................................................102
Figure 6.2 1H NMR spectra for 13-dTDM...................................................................................117
Figure 6.3 13
C DEPTq NMR spectra for 13-dTDM. ...................................................................118 Figure 6.4
1H-
13C HSQC NMR spectra for 13-dTDM. ...............................................................119
Figure 6.5 1H-
13C HSQC-TOCSY NMR spectra for 13-dTDM. .................................................120
Figure 6.6 1H-
1H COSY NMR spectra for 13-dTDM. ................................................................121
Figure 6.7 1H-
13C HMBC NMR spectra for 13-dTDM. ..............................................................122
Figure 6.8 1H-
1H NOESY NMR spectra for 13-dTDM. ..............................................................123
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Figure 6.9 1H NMR spectra of molecule W5.9. ...........................................................................124
Figure 6.10 CRAPT NMR spectra of molecule W5.9. ................................................................125 Figure 6.11 1H-
13C HSQC spectra of molecule W5.9. ................................................................126
Figure 6.12 1H-1H COSY spectra of molecule W5.9. ................................................................127
Figure 6.13 1H-13C HMBC NMR spectra of molecule W5.9. ...................................................128 Figure 6.14
1H NMR spectra of molecule G6.4 (TDM). .............................................................129
Figure 6.15 CRAPT NMR spectra of molecule G6.4 (TDM). ....................................................130 Figure 6.16 1H-13C HSQC NMR spectra of molecule G6.4 (TDM). .........................................131 Figure 6.17 1H-1H COSY spectra of molecule G6.4 (TDM). .....................................................132
Figure 6.18 1H-13C HMBC NMR spectra of molecule G6.4 (TDM).........................................133 Figure 6.19
1H NMR spectra of molecule A5.4 (dhTDM). .........................................................134
Figure 6.20 CRAPT NMR spectra of molecule A5.4 (dhTDM). ................................................135 Figure 6.21 1H-13C HSQC NMR spectra of molecule A5.4 (dhTDM). .....................................136
Figure 6.22 1H-1H COSY spectra of molecule A5.4 (dhTDM). .................................................137 Figure 6.23 1H-13C HMBC NMR spectra of molecule A5.4 (dhTDM).....................................138
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Abbreviations
1,3-BPG 1,3-bisphosphoglycerate
13-dTDM 13-deoxytetrodecamycin
aa amino acid
ABC transporter ATP Binding Cassette transporter
ACP acyl carrier protein
AT acyl transferase
ATCC American Tissue Culture Collection (antiquated name)
ATP adenosine triphosphate
bp base pairs
BSL biosafety level
BTAD bacterial transcriptional activation domain
CAD Canadian dollars
db database
DH dehydratase
dhTDM dihydrotetrodecamycin
ER enoyl reductase
ESI-MS electrospray ionization mass spectrometry
Gbp giga base pairs (i.e. 109 bp)
GCF gene cluster family
HPLC high performance liquid chromatography
KR ketoreductase
KS ketosynthase
LAL large ATP-binding regulator of the LuxR family
Mbp mega base pairs (i.e. 106 bp)
MFS transporter major facilitator superfamily transporter
MIC minimum inhibitory concentration
MRSA multidrug resistant Staphylococcus aureus
MS/MS tandem mass spectrometry-mass spectrometry
NMR nuclear magnetic resonance
NRPS non-ribosomal peptide synthase
ORF open reading frame
PAC phage artificial chromosome
PKS polyketide synthase
Rf retardation factor
SARP Streptomyces antibiotic regulator protein
SMRT single molecule real time sequencing
sp. species (singular)
spp. species (plural)
ssp. subspecies
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TDM tetrodecamycin
TDM-group tetrodecamycin-group
UPLC ultra performance liquid chromatography
USA United States of America
UV-Vis ultra violet-visual light spectrum
WGS whole genome sequencing
wHTH winged helix-turn-helix
Δ deletion
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Publications
All the data reported in this thesis has been published in the following peer-reviewed
publications:
Gverzdys, T., Hart, M.K., Pimentel-Elardo, S., Tranmer, G. & Nodwell, J.R. (2015). “13-Deoxytetrodecamycin, a new tetronate ring-containing antibiotic that is
active against multi-drug-resistant Staphylococcus aureus” J. Antibiot. 68:698-
702. doi: 10.1038/ja.2015.60
Gverzdys, T., & Nodwell, J.R. (2016). “Biosynthetic Genes for the Tetrodecamycin
Family of Antibiotics” J. Bacteriol. 198(14):1965-1973. doi:
10.1128/JB.00140-16
Gverzdys, T., Kramer, G. & Nodwell, J.R. (2016). “Tetrodecamycin: An Unusual and
Interesting Tetronate Antibiotic” Bioorg. Med. Chem. doi:
10.1016/j.bmc.2016.05.028
The only places where I have introduced unpublished figures, tables, and ideas are within the
Discussion section of each chapter and in Chapter 5 (Future Directions).
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“Why do you have a picture of a bee in your thesis?” she asked.
“Because it is my thesis and I thought the pictures were fun,” he
responded with a goofy smile.
“I still think it’s weird, sweetheart.”
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Chapter 1. Introduction
1.1 Why study antibiotics? A brief history.
The first use of the word “antibiosis” in scientific literature dates to 1889 when it was defined as
“one creature destroying the life of another in order to sustain its own” (attributed to Vuillemin,
1889 by Waksman, 1947). In the modern sense, the word “antibiotic” was used by Waksman and
colleagues in 1941 to mean “inhibiting the growth or the metabolic activities of bacteria and
other micro-organisms by a chemical substance of microbial origin” (Waksman, 1947). As
knowledge about chemical biology has expanded, the meaning of the term antibiotic has
continued to change. Thus, for the sake of this thesis, I shall define the word antibiotic as
follows: a chemical compound, either of natural or synthetic origin, which interacts with a
specific component of a bacteria cell and results in cessation of growth or cell death.
The ability to inhibit the growth of bacteria has been a topic of great importance to humans for
thousands of years. With ancient origins in food preservation and folk-medicine, and a modern
basis in the study of lysozyme and vaccines, it was during the twentieth century that humans
gained significant control over infectious bacteria (Ridley, 1928; Wainwright, 1989). While
vaccination had given humanity the ability to prevent disease, medicine still lacked methods for
treating acute infections. Indeed, in the early 1900s, the most common causes of death in
Americans were tuberculosis and pneumonia (Centers for Disease Control and Prevention,
1998). What humanity needed was an effective method for treating these infections. Paul
Ehrlich’s work on bacteria-specific dyes had resulted in the development of salvarsan in 1910
(used to treat syphilis), but it was in 1929 that the story of antibiotics began in earnest (Schwartz,
2004; Zaffiri et al., 2012). This was the year that Alexander Fleming first reported on the ability
of the fungus Penicillium notatum to produce a chemical compound capable of inhibiting the
growth of bacteria. The responsible molecule, penicillin, would become one of the most
important chemicals to be used as a drug (Fleming, 1929). While the story of Fleming is a classic
told in every introductory microbiology class, it was the work of Selman Waksman which had a
much more profound effect on the field of antibiotics. Starting in the early 1940s, Waksman and
colleagues began purifying antibiotics from members of the bacterial genus Streptomyces.
Starting with the discovery of streptothricin in 1942 and streptomycin in 1944, the next 40 years
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of research (1940 to 1980) resulted in the discovery of thousands of antibiotics, of which a small
percentage proved to be clinically useful (Baltz, 2006; Metzger et al., 1942; Schatz et al., 1944;
Watve et al., 2001). During the peak of this “golden age of antibiotic discovery”, as many as 70
to 110 new antibiotics were discovered each year, but in the 1980s, the rate of discovery
declined. As of the year 2000, the rate of discovery had trickled to just 20 new antibiotics per
year (Watve et al., 2001).
The reduced productivity following 1980 can be attributed to three primary causes: 1) health
policy changes, 2) the technical difficulty in discovering new antibiotics, and 3) the financial cost
of antibiotic development. In the late 1960s, there was an overall switch in how the United States
of America (USA) approached public health. It was at this time that William H. Stewart, then the
Surgeon General of the United States, was infamously and falsely quoted as declaring that it was
time to “close the book on infectious disease” (Spellberg and Taylor-Blake, 2013). Despite the
misquote, the sentiment at the time was still the same. Advances in the treatment of infectious
diseases had been responsible for a steady decline in the total rate of mortality in the USA from
1900 to 1950 but, from 1950 to 1960, the rate of mortality showed no improvement (Gordon,
1953; Spellberg and Taylor-Blake, 2013). Moreover, the most common causes of death in
Americans had become heart disease and cancer (Centers for Disease Control and Prevention,
1998). As a result, Stewart and the US Public Health Service made a deliberate decision to focus
on the study and treatment of chronic diseases. This marked a new era of reduced interest in
infectious disease and antibiotic discovery (Spellberg and Taylor-Blake, 2013). The second
cause of the slowdown was due to technical difficulties with antibiotic discovery. The
rediscovery of known antibiotics — an issue known as the “dereplication problem” — was
making it harder to discover novel chemical entities among the increasingly large collection of
known molecules. These difficulties, coupled with excitement over new chemical synthesis
methods, drove a paradigm switch in antibiotic screening methods. The “old” method involved
screening Streptomyces fermentations for the ability to inhibit the growth of whole-cell
pathogens, but this method was considered low throughput (~100,000 strains per year). During
the 1990s, this was largely replaced by high throughput methods in which combinatorial
chemical libraries were screened against purified enzymes. Unfortunately, the high throughput
methods proved to be unproductive and failed to discover new antibiotics for the clinic (Baltz,
2006). Finally, the last reason for the reduced rate of antibiotic discovery was related to finances.
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There is anecdotal evidence that developing antibiotics is less profitable than developing other
drugs. This idea focuses on the fact that antibiotics are taken for only short times, while other
drugs (e.g. cholesterol and blood pressure medicines) are consumed for long periods of time.
This makes the latter a better source of revenue. Moreover, developing a new drug has been
estimated to cost anywhere from $1 billion to $5 billion (Herper, 2013). Taken together,
antibiotics are not the most profitable drugs, thus making finances a rate limiting factor in
antibiotic research.
The low rate of antibiotic discovery is problematic because antibiotics, unlike other drugs, have a
finite lifespan in the clinic. The reason for this is the continuous development of antibiotic
resistance in pathogenic bacteria. In the USA, 2 million people per year are infected with
antibiotic-resistant pathogens resulting in 23,000 mortalities. While there are several bacteria
which contribute to this number (e.g. Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa), the most important drug resistant pathogen is multi-drug resistant
Staphyloccocus aureus (MRSA). MRSA is responsible for infecting 80,000 people every year
and killing 11,000 people (14% mortality rate) (Centers for Disease Control and Prevention,
2013). Defined generally, antibiotic resistance is the ability of a bacterium to grow in the
presence of an antibiotic compound. While some resistance is due to fundamental morphological
differences between bacteria (e.g. Gram positive vs Gram negative cells), the more important
method of resistance is due to mutation and the spread of resistance genes. Antibiotic resistance
is not a new phenomenon (e.g. Gots, 1945) and, based on current evidence, resistance to any
antibiotic is inevitable (D’Costa et al., 2006, 2011). None-the-less, there is a lag period between
the clinical introduction of an antibiotic and wide-spread resistance, thus making these molecules
still highly useful (Palumbi, 2001). While there are a number of novel anti-infective therapies
currently under development (e.g. Abdou Mohamed et al., 2016; Chemler et al., 2015; Kalan and
Wright, 2011; Lehar et al., 2015; Lewis, 2013), the best weapon that humanity has against
pathogenic bacteria continues to be antibiotics. Thus, to avoid a return to a pre-Fleming era, it is
imperative that new antibiotics be developed for the clinic.
1.2 The target of antibiotics
After 70 years of antibiotic discovery, it has become apparent that broad-spectrum antibiotics
have limited targets in bacteria. The four general processes that antibiotics target are cell wall
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biosynthesis, DNA/RNA precursor biosynthesis, DNA/RNA biosynthesis, and protein
biosynthesis (Walsh, 2013). Within each of these major groups there are numerous reactions,
steps, or enzymes that can be interfered with. For example, the ribosome is inhibited by
molecules that block the exit tunnel of the nascent peptide (erythromycin), block incoming
tRNAs (tetracycline), interfere with the initiation complex (streptomycin), block assembly of the
70S ribosome (kanamycin), or prevent formation of the peptide bond (linezolid) (Walsh, 2013).
While this may give the impression that there are huge numbers of drugable processes, a survey
revealed that there are only about 40 total target sites which all characterized antibiotics inhibit
(Bumann, 2008). This brings up an interesting question: why are there so few target sites for
antibiotics? Indeed, there are hundreds of essential bacterial genes (Baba et al., 2006; Hutchison
et al., 2016; Xu et al., 2010) yet there are no antibiotics which target processes as fundamental
as, for example, bacterial cell division (den Blaauwen et al., 2014; Jani et al., 2015). An answer
to this question may lie in the idea that nature has already developed antibiotics to all the ‘useful’
broad-spectrum targets; what has been discovered has already gone through the filter of
evolution (Bumann, 2008). Alternatively, the scarcity of targets may be due to sampling bias; the
molecules targeting non-canonical mechanisms may have been discarded as impractical for the
clinic (Lewis, 2013). For example, a screen for antibiotics against Mycobacterium smegmatis
resulted in the discovery of bedaquiline, a synthetic inhibitor that is specific for the ATP
synthase found in mycobacteria (Andries et al., 2005). Examples like this suggest that new
targets may be found by studying narrow-spectrum antibiotics.
Identification of an antibiotic’s molecular target is a non-trivial process. The primary methods
for finding the molecular target can be divided into three general approaches: proteomics,
genetics, and comparative profiling (reviewed thoroughly in Azad and Wright, 2012; and Ziegler
et al., 2013). The proteomic methods are focused on identifying the specific protein which an
antibiotic binds to. The most commonly used proteomic techniques are “affinity methods.” These
methods use the antibiotic to “pull down” the target protein. For example, in affinity
chromatography the compound of interest is bound to a solid matrix and then exposed to a
protein extract. Due to the affinity of the target protein for the antibiotic, the target protein is
retained on the column/beads. After washing off all non-specific proteins, the target protein is
eluted and identified by protein sequencing. Another proteomic method is Target Identification
by Chromatographic Co-elution (TICC). In this method, a protein extract is first mixed with an
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unmodified antibiotic. The protein extract is then finely fractionated under non-denaturing
conditions and each fraction is assayed for the presence of the antibiotic. Proteins which co-elute
with the antibiotic are candidate targets. Genetic methods can also be used to understand the
mechanism of action. These methods rely on alterations in gene expression or gene content to
provide insight into the mechanism of action. A powerful method that is commonly associated
with yeast genetics but has counterparts in bacteria is “gene dosage”. These experiments rely on
building ordered libraries of a model organism which have, for example, each gene in the
genome overexpressed. This overexpression library is then treated with an antibiotic and those
strains that show decreased sensitivity to the antibiotic are candidate targets. The theory behind
this type of library is that by increasing the copy number of the target protein you prevent the
antibiotic from saturating the target protein pool, thus providing the bacterium with a method to
escape the antibiotic’s activity. In addition to overexpression libraries, it is also possible to build
knockout libraries and knockdown libraries in bacteria. Another genetic method is whole
genome sequencing of resistant mutants. First, cells are passaged in increasing concentrations of
an antibiotic to generate resistant mutants. The mutants are sequenced and, by comparing to the
parental strain, all the mutations in the resistant strain are identified. The genes which are
mutated are candidates for the molecular target. Finally, the use of comparative methods can also
be used to inform mechanism of action. These rely on comparing the phenotype caused by the
antibiotic of interest to the phenotypes caused by other antibiotics or mutations. For example,
treating cells with an antibiotic causes a characteristic change to cell morphology. Through the
use of microscopy to visualize patterns in the position/accumulation of DNA, lipids, and cell
wall, a phenotypic ‘fingerprint’ can be assigned to the antibiotic. By searching for molecules or
mutations that have a similar fingerprint, it can be proposed that the antibiotic of interest is
targeting the same protein as the known antibiotic with the matching fingerprint. Similarly, it is
also possible to use transcriptome sequencing to generate a fingerprint. Regardless of the method
used, it is important to note that there are many ways to identify candidate molecular targets and
there is no recognized best method.
1.3 Streptomyces are the source of most antibiotics
The majority of antibiotics are produced by soil-dwelling bacteria from the genus Streptomyces
and other closely related genera (Watve et al., 2001). In addition to antibiotics, Streptomyces are
also a source of anti-cancer drugs, anti-fungal drugs, immunomodulators, ionophores, and
7
herbicides and numerous other molecules (Katz and Baltz, 2016). Collectively, these molecules
are referred to as secondary metabolites and are synthesized by clusters of genes called
biosynthetic gene clusters. While it has long been established that Streptomyces are extremely
gifted in the number of bioactive molecules they produce, a study of genome sequences has
confirmed their place as a figurative gold-mine for bioactive molecules. Starting in 2002 with the
model organism, genome sequencing has revealed that the typical Streptomyces species encodes
anywhere from 20 to 70 different biosynthetic gene clusters (Bentley et al., 2002; Doroghazi and
Metcalf, 2013; Nett et al., 2009). With the exception of some closely related genera (i.e. Frankia,
Micromonospora, Salinispora, Verrucosispora, Mycobacterium, Rhodococcus), most bacterial
genomes possess fewer than 10 biosynthetic gene clusters. This makes Streptomyces the most
plentiful source of bioactive molecules in the entire domain of Bacteria (Doroghazi and Metcalf,
2013).
As will be seen with the gene cluster that I report in this thesis, not all biosynthetic gene clusters
are unique. Rather, there are some biosynthetic gene clusters which are found in many
Streptomyces spp., while others appear to be unique to just one strain (Baltz, 2006; Doroghazi et
al., 2014). Interestingly, this seems to follow the same pattern as described by Koonin and
colleagues for gene content in bacteria, namely the core-shell-cloud model (Koonin and Wolf,
2012). In this model, there are a few core genes (or, in this case, biosynthetic gene clusters)
which are conserved in many organisms, followed by shell genes which are found in greater
numbers but less ubiquitously, followed by cloud genes which are found in extremely large
numbers but are unique to single organisms (Koonin and Wolf, 2012). There are two valuable
implications to this observation. First, this suggests that the overwhelming majority of molecules
remain to be discovered (a controversial idea originally proposed by Watve et al., 2001). Thus,
Streptomyces and other closely related bacteria are likely to be a rich source of future molecules.
The second implication is that because these clusters are found in more than one organism, it is
possible to compare those clusters and identify which genes are conserved. This is useful for
identifying which genes are part of a gene cluster (Doroghazi and Metcalf, 2013).
1.4 Tetronates are a large family of bioactive molecules
During the course of this thesis, I will focus on a group of molecules called the tetrodecamycin-
group (TDM-group) molecules. These molecules are members of the tetronate family. The
8
tetronate molecules are a large family of secondary metabolites that are characterized by having
a unique, five-membered lactone ring called a tetronate ring (Figure 1.1A). Molecules in the
family have bioactivities ranging from antiviral to antibacterial to antitumor (Bister et al., 2004;
Morimoto et al., 1982; Roggo et al., 1994). The family can be divided into three subfamilies: the
linear tetronates, the spirotetronates, and the miscellaneous tetronates (Vieweg et al., 2014). The
linear tetronates are epitomized by molecules such as agglomerin A, acaterin, didehydro-
acaterin, RK-682, pesthetoxin, and tetronomycin (Figure 1.1B) (Hatano et al., 2007; Keller-
Juslén et al., 1982; Kimura et al., 1998; Naganuma et al., 1992; Roggo et al., 1994; Shoji et al.,
1989). These molecules are characterized by a tetronate ring modified with a carbon chain
attached at either the C-3 or C-5 position. The attached carbon chains, synthesized as either a
fatty acid or a polyketide, may be saturated or unsaturated and may include additional small rings
(e.g. tetronomycin). The spirotetronate molecules (reviewed in Lacoske and Theodorakis, 2015)
are characterized by a spirotetronate ring system (in which a six-membered ring and the tetronate
ring are linked to form a spirane) and a macrocyclic ring. This group is further divided into three
groups on the basis of the size of their macrocyclic ring: the small spirotetronates have a
macrocyclic ring of 11 carbons (e.g. maklamicin, nomimicin, abyssomicin C); the medium
spirotetronates have a macrocyclic ring of 13 carbons (e.g. chlorothricin, kijanimicin, tetrocarcin,
decatromicin B); and the large spirotetronates have more than 13 carbons in their macrocyclic
ring (e.g. quartromicin, versipelostatin, tetronothiodin) (Figure 1.1B) (Bister et al., 2004; Igarashi
et al., 2011, 2012; Keller-Schierlein et al., 1969; Momose et al., 1999; Ohtsuka et al., 1992; Park
et al., 2002; Tomita et al., 1980; Tsunakawa et al., 1992; Waitz et al., 1981). Finally, the last
group of tetronate molecules is the miscellaneous tetronates. As the name suggests, these are
tetronate molecules which do not fit into either of the above categories. This group includes
members like the artapetalins (excluded from the linear tetronate group because they are
alkylated, not acylated, at the C-3 position), picrodendrin B (which possess an unusual
spirotetronate system), and, my specific focus, the TDM-group molecules (Figure 1.1B and
Figure 1.2) (Ozoe et al., 1998; Wong and Brown, 2002). Prior to my thesis, there were two
reported TDM-group molecules: tetrodecamycin (TDM) and dihydrotetrodecamycin (dhTDM)
(Tsuchida et al., 1995a).
9
Figure 1.1 The tetronate family of molecules all possess a tetronate ring.
A) The tetronate ring is a 5-membered lactone ring. Modifications to the ring are commonly
found at R1 and R2. B) The tetronate family can be divided into three general subfamilies: the
linear tetronates, the spirotetronates, and the miscellaneous tetronates. Representing the linear
tetronates are agglomerin A, acaterin, tetronomycin, didehydro-acaterin, pesthetoxin, and RK-
10
682. Representing the small spirotetronates are maklamicin, nomimicin, and abyssomicin C.
Representing the medium spirotetronates are chlorothricin, tetrocarcin A, kijanimicin, and
decatromicin B. Representing the large spirotetronates are quartromicin D3, tetronothiodin, and
versipelostatin. Representing the miscellaneous tetronates are picrodendrin B, artapetalin A and,
as shown in Figure 1.2, tetrodecamycin and dihydrotetrodecamycin.
Figure 1.2 Tetrodecamycin and dihydrotetrodecamycin were the first discovered members
of the TDM-group molecules.
1.5 History of the TDM-group molecules
TDM was first reported in 1994 in a brief letter to the editor (Tsuchida et al., 1994). This was
followed a year later by a detailed description of the producing strain, method of isolation, and
the structural elucidation of TDM and dhTDM (Figure 1.2) (Tsuchida et al., 1995a, 1995b). The
organism that produces both of these molecules, Streptomyces nashvillensis MJ885-mF8, was
isolated from a soil sample collected in Suginami-ku, Tokyo, Japan. Studies into this wild-isolate
streptomycete were commenced with the observation that it produced anti-bacterial activity
against Photobacterium damselae ssp. piscicida (formerly described as Pasteurella piscicida)
(Thyssen et al., 1998). P. damselae ssp. piscicida is the causative agent of pseudotuberculosis in
fish (this disease is also called pasteurellosis or photobacteriosis). Indeed, the name piscicida is
derived from the Latin words “fish” (piscus) and “to kill” (-cidus) (Janssen and Surgalla, 1968).
The disease presents as a bacterial septicemia followed by the development of tubercles on
internal organs that can ultimately kill the fish (Romalde, 2002). At the time of TDM’s
discovery, cultured yellowtail (Seriola quinqueradiata, also called Japanese amberjack)
accounted for 56% of the farmed fish produced in Japan and losses due to pseudotuberculosis
had significant economic impact (Kusuda and Kawai, 1998). Antibiotics were in use to treat
infections but, mirroring the same problem observed in human health, the widespread use of
11
antibiotics was accompanied by the reciprocal development of antibiotic resistance (Kim and
Aoki, 1993). This led to the need for new antibiotics.
TDM and dhTDM were isolated from ethyl acetate extracts of S. nashvillensis in liquid culture.
The structures of TDM, dhTDM, and 14-O-acyltetrodecamycin (a semi-synthetic derivative of
TDM) were determined by NMR (Figure 1.2). The structure revealed a tetracyclic molecule with
a 6,6,7,5-membered ring system. The pair of six-membered rings are arranged in a decalin
system, the seven-membered ring is a heterocycle containing an oxygen, and the five-membered
ring is the characteristic tetronate ring (Tsuchida et al., 1995a, 1995b). Another notable feature of
TDM is the presence of an exo-methylene attached to the tetronate ring at position C-4/C-5
(Figure 1.2). In contrast, dhTDM possesses a methyl group at this same position. Bioassays
performed with TDM confirmed that it had strong antibiotic activity against P. damselae ssp.
piscicida (1.56 to 6.25 μg mL-1
, 4.67 to 18.7 μM) as well as antibiotic activity against a number
of strains of Staphylococcus aureus, MRSA, Micrococcus luteus, and Bacillus subtilis (6.25 to
12.5 μg mL-1
, 18.7 to 37.4 μM). Curiously, while TDM has activity against P. damselae ssp.
piscicida (a Gram negative organism), it has no antibiotic activity against other Gram negative
organisms tested. In contrast to TDM’s potent antibiotic activity, dhTDM has no appreciable
activity against either Gram positive or Gram negative organisms (Tsuchida et al., 1995a).
The only difference between these two molecules is the presence of an exo-methylene in TDM
and a methyl at the same position in dhTDM (Figure 1.2). As a result, this suggests that the exo-
methylene is involved in the mechanism of action for TDM. Notably, the exo-methylene forms
part of a possible Michael Acceptor which extends from the carbonyls attached to C-1 and C-6
through the conjugated system running through C-2 to C-5. This suggests that C-5 can act as an
electrophile in a nucleophilic addition. The consequence of this reaction would be the formation
of a covalent bond with the molecular target (Figure 1.3). dhTDM also contains a Michael
acceptor with the electrophilic carbon being at C-3, but, given the lack of activity, this position is
likely sterically hindered (Gverzdys et al., 2016).
12
Figure 1.3 The proposed mechanism by which the tetrodecamycins could bind covalently to
their target.
The conjugated system composed of C-1/C-6, C-2, C-3, C-4, and C-5 forms a possible Michael
acceptor. Due to the presence of the electronegative carbonyls, C-5 is able to act as an
electrophile in a nucleophilic addition reaction, potentially with a proteinaceous target. This
results in the formation of a covalent bond with the target molecule. “X” denotes the nucleophile
from the target biomolecule.
In regard to its clinical potential, TDM has been reported to have no toxicity in mice when tested
up to 100 mg kg-1
by intraperitoneal injection, though due to a lack of materials and methods
pertaining to this result, this information should be considered with care (Tsuchida et al., 1995a).
In vitro cytotoxicity studies were never conducted with the full molecule, but partial structures
from synthesis efforts are toxic in cell culture (Paintner et al., 2003a). Follow-up studies have
been performed to improve the bioactivity of TDM. The study focused on making modifications
to the 14-OH position and the exo-methylene (Tsuchida et al., 1995c) and were moderately
successful. A patent was filed for TDM and its derivatives in 1996 (patent number JPH0892256),
though there is no evidence that the molecule was ever used in the clinic or for aquaculture.
Following the patent, no further work on the molecule was reported until 2000, at which time a
number of groups attempted to synthesize the molecule (He et al., 2006; Paintner et al., 2000,
2002, 2003a, 2003b; Warrington and Barriault, 2005). These efforts culminated with the
successful total synthesis of TDM in 2006 (Tatsuta et al., 2006).
1.6 TDM-group molecules are produced by polyketide synthases
The biosynthesis of polyketide antibiotics follows a two-step process. In the first step, the
antibiotic’s “backbone” is synthesized. In the second step, this linear molecule is cyclized and
tailored to form the final structure. Biosynthesis of the backbone can be performed by several
different enzymatic mechanisms, though one of the most common is via polyketide synthase
(PKS) proteins. More specifically, the backbone of the TDM-group molecules is produced by a
Type I PKS system. Detailed knowledge of how these systems function is not required to
13
understand the content of this thesis, though a cursory understanding is beneficial. A thorough
review on the topic is recommended to the interested reader (Fischbach and Walsh, 2006).
Type I PKS systems are large, multi-domain proteins which act to link carbon-based monomers
together into a chain. These large proteins are referred to as megasynthases. Each megasynthases
is composed of ‘modules’, which in turn are composed of several protein domains (Figure 1.4A).
At minimum, each module must possess an acyltransferase domain (AT), an acyl carrier protein
domain (ACP), and a ketosynthase domain (KS). Additional domains that may also be included
are ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) domains. Each module is
responsible for adding a single monomer to the growing carbon chain in an assembly line-
fashion (Figure 1.4B). Thus, the growing carbon chain is passed from the first module to the
second to the third, etc. getting larger at each step. A number of different carbon-based
monomers can be added to the growing chain. The most common are acetyl (loading domain
only), malonyl, or methylmalonyl. Monomer selection is performed by the AT domain and can
be predicted based on the amino acid sequence of this domain. A type I PKS in its most simple
form (modules that posses only KS, AT, ACP domains) will generate a chain of ketones (see
final step of Figure 1.4B), but the inclusion of KR, DH, and ER domains will result in the
reduction of the β-ketone generated by each module. The KR domain will reduce a ketone to an
alcohol; the DH domain will reduce the alcohol to an alkene; and the ER domain will reduce the
alkene to an alkane (Figure 1.4C).
14
Figure 1.4 Type I PKSs act to link carbon monomers into chains.
A) Type I PKSs are large proteins which are composed of different “modules”, each of which is
15
formed by several domains. Each module must possess at least a KS, AT, and ACP domain, but
may additionally have a KR, DH, and ER domain. B) Each module is responsible for adding a
new carbon monomer into the growing chain. The name below the module number indicates the
type of monomer added. C) In its most basic form, each module possesses only a KS, AT, and
ACP domain. This generates a chain of ketones. If a KR domain is present, the β carbon will be
reduced to a hydroxyl. If a KR and DH domain is present, the β carbon will be reduced to an
enoyl. If a KR, DH, and ER domain are present, the β carbon will be reduced to an alkane.
Abbreviations: PKS, polyketide synthase. Ac, acetyl. Mal, malonyl. Mmal, methylmalonyl. KS,
ketosynthase. AT, acyl transferase. ACP, acyl carrier protein. KR, ketoreductase. DH,
dehydrogenase. ER, enoyl reductase.
Each module in a PKS acts independently of the previous. Thus, Type I PKS systems are able to
generate linear chains with tremendous diversity. Each module integrates a different carbon-
based monomer and reduces the β-carbon from a ketone to a hydroxyl, alkene, or alkane.
Multiple megasynthases can additionally be linked together via docking domains without
interrupting the biosynthesis. The result is a longer carbon chain. When the growing chain
reaches the last module, it is released and tailored into the final molecule. Since the discrete
function of each domain can be predicted from amino acid sequence, it is possible to predict the
structure of the final carbon chain based on the protein sequence of the megasynthase. As a
result, genome sequencing data can be used to predict the type of linear carbon molecule that a
Type I PKS will produce.
1.7 Tailoring reactions give rise to tetronate ring
Following the production of the linear carbon chain, tailoring reactions are responsible for
bringing the polyketide molecule to its final structure. There is a wide diversity of different
tailoring reactions that can take place (e.g. cyclization, glycosylation, and halogenation). In the
case of tetronate molecules, the defining tailoring step is the formation of the tetronate ring. This
takes place in two steps: first, formation of the five-membered lactone ring; and second,
formation of the exo-methylene. Tetronate ring biosynthesis has been studied in great detail in
for tetronomycin, RK-682, agglomerin, and quartromicin (He et al., 2012; Kanchanabanca et al.,
2013; Sun et al., 2008, 2010; Wu et al., 2014).
To synthesize a tetronate ring, 1,3-bisphosphoglycerate (1,3-BPG) is taken from glycolysis and
cyclized with the linear carbon backbone of the antibiotic. The result is an immature tetronate
ring (Figure 1.5A). There are three enzymes which are involved in this process: an FkbH-like
protein (specifically a Tmn16 homolog), a dedicated ACP, and a FabH-like protein. The FkbH-
16
like protein is responsible for binding to 1,3-BPG, di-dephosphorylating it, and then transferring
the subsequent glyceryl moiety to the dedicated ACP. The FabH-like protein then mediates the
cyclization of the glyceryl-ACP with the polyketide backbone to generate the immature tetronate
ring. Interestingly, FkbH-like proteins can be divided into two groups. Those involved in
tetronate ring biosynthesis have an additional 200 aa N-terminal extension that is essential for the
formation of the tetronate ring. These proteins are referred to as Tmn16 homologs (Sun et al.,
2008). As a result of this unique extension, Tmn16 proteins can be used as a “flag” to identify
biosynthetic gene clusters which produce tetronate molecules. FkbH-like enzymes lacking the N-
terminal extension are found in PKS biosynthetic gene clusters which do not produce tetronate
molecules. In these clusters, the FkbH-like enzyme is involved in making atypical carbon
monomers for use by the PKS machinery (Motamedi and Shafiee, 1998; Wenzel et al., 2006).
17
Figure 1.5 Formation of tetronate rings requires a dedicated set of proteins.
A) Formation of the immature tetronate ring is performed by three enzymes. A Tmn16 homolog
takes 1,3-BPG, dephosphorylates it, and transfers it to a dedicated ACP. A FabH-like protein
then catalyzes the cyclization of the glyceryl-linked ACP with the backbone of the PKS. B)
Formation of the mature tetronate ring requires removing the hydroxyl attached to C-5. This is
performed in two steps. First, an Agg4-like protein acetylates the hydroxyl. Second, an Agg5-
like protein removes the acetylated hydroxyl as acetic acid. This process is called acetylation-
elimination. Abbreviations: 1,3-BPG, 1,3-bisphosphoglycerate. ACP, acyl carrier protein. PKS,
polyketide synthase. KS, ketosynthase. AT, acyltransferase. SCoA, Coenzyme A.
The next step in tetronate ring biosynthesis involves formation of the exo-methylene (Figure
1.5B). After formation of the immature tetronate ring, the hydroxyl attached to C-5 must be
removed. Dehydration of this position yields the exo-methylene. This is accomplished by an
18
acylation-elimination reaction. First, the hydroxyl is acylated by an acyl transferase (Agg4
homolog). Next, the hydroxyl and the attached acyl are removed as acetic acid through the
actions of an α/β hydrolase (Agg5 homolog). The end result is the formation of the mature
tetronate ring (Kanchanabanca et al., 2013).
1.8 Genetic regulation of antibiotic gene clusters
Antibiotics are produced by groups of genes which are spatially clustered on the genome into
biosynthetic gene clusters. In addition to possessing all of the enzymatic genes (i.e. PKS and
tailoring genes), they also possess transcriptional regulatory genes required for coordinating
expression of those enzymatic genes. Contained within the average cluster are one or more
transcription factors. These can be divided into two groups. The first group is composed of
transcription factors which act as the master ON-OFF switch for the cluster. These are referred to
as “cluster specific regulators”. These genes are essential for producing the antibiotic and, as a
result, deleting these genes results in an antibiotic-null phenotype. The second group consists of
non-essential regulators. These secondary regulators appear to “tune” biosynthesis of the
antibiotic or activate resistance genes. Regulators found in the cluster, regardless of where they
fall in the hierarchy, are sorted into different families based on the protein domains they possess
(Liu et al., 2013). Found in the biosynthetic gene cluster which I present herein are three
different types of transcriptional regulators: 1) a SARP-family regulator, 2) a TetR-family
regulator, and 3) an LAL-family regulator.
A group of regulators called the small-SARPs (Streptomyces Antibiotic Regulatory Protein) are
an important class of cluster specific regulators. The small SARPs are found in approximately
16% of biosynthetic gene clusters and often act as a master ON-OFF switch for that cluster (Liu
et al., 2013). Small SARPs are typically transcriptional activators (Fernández-Moreno et al.,
1991; Narva and Feitelson, 1990), thus by overexpressing them it is possible to overproduce the
associated molecule (Martín and Liras, 2010; Narva and Feitelson, 1990; Pérez-Llarena et al.,
1997). The small-SARPs are part of a larger family of regulators known as the SARP-family
regulators. SARPs are broadly defined as transcriptional regulators which possess an N-terminal
OmpR-like winged helix-turn-helix (wHTH) domain (also called a “Trans_reg_C” domain) and
a bacterial transcriptional activator domain (BTAD) (Wietzorrek and Bibb, 1997). SARPs are
divided into three groups based on their size, with small-SARPs classified as those that are less
19
than 300 aa and possess only a wHTH and BTAD domains (Liu et al., 2013). For the interested
reader, I would additionally like to bring to your attention that while almost all small SARPs are
positive regulators, the regulator farR4 from Streptomyces lavendulae FRI-5 is a rare example of
a small SARP with repressor activity (Kurniawan et al., 2014).
The second class of transcriptional regulators that I will discuss are the TetR-family regulators
(TFRs). Named after the well-studied TetR protein, these proteins typically act as transcriptional
repressors. After being expressed, a TFR binds as a homodimer to its target operator sequence
and blocks transcription from overlapping promoters. In the presence of its cognate effector
molecule, the molecule binds to the TFR at an allosteric site which allows the TFR to dissociate
from the operator sequence, thus allowing transcription to resume. In the most well-studied
example, the TetR protein binds to two palindromic operator sequences in the intergenic region
between the divergently oriented tetR gene and tetA gene and blocks expression from the two
promoters in this region. When tetracycline is present in the cell, the tetracycline is able to bind
to TetR. This causes a conformational change in TetR that reduces affinity for the operator site
and ultimately relieves repression of tetA (a tetracycline efflux pump) and tetR (Cuthbertson and
Nodwell, 2013). While the majority of TFRs appear to regulate the expression of a gene
divergent to their own (e.g. tetR/tetA), there are also TFRs which act on non-divergent genes or
over long genomic distances (Ahn et al., 2012). Thus, unless there is a neighbouring divergent
gene, it is difficult to predict the target of the TFR.
The last class of regulator that needs an introduction are the LAL-family (Large ATP-binding
regulators of the LuxR family) regulators. The proteins are typically around 900 to 1000 residues
long. At the far N- and C-terminal ends they possess, respectively, an ATP binding domain and a
LuxR-like DNA binding domain. Aside from these domains, sequence conservation is minimal
between LAL-family members (De Schrijver and De Mot, 1999). Allosteric regulation may be
important for the function of these regulators. For example, studies in Escherichia coli have
shown that MalT (an LAL-family regulator) is a transcriptional activator, but only when
complexed with ATP and maltotriose (Boos and Shuman, 1998). LAL-family regulators are
commonly found in Streptomyces (De Schrijver and De Mot, 1999) and studies have shown that
some act as cluster specific regulators (Xie et al., 2015). Taken together, this suggests that LAL
20
regulators can act as positive regulators but may be subject to allosteric regulation via ATP
levels and other effector molecules.
1.9 Thesis objectives and outline
Given the need for new antibiotics, the original objective of my work was to identify novel
antibiotics that could be used in the clinic. Streptomyces sp. strain WAC04657 is a wild-isolate
bacterium that has antibiotic activity against MRSA. Given the medical importance of MRSA, I
decided that studying the responsible antibiotic was a valuable topic of research for my PhD
studies. Thus, the overarching goal of this thesis was to purify the responsible molecule and
further study its capacity to be used in the clinic.
This thesis has been broken down into three data chapters. In the first data chapter, I report on
how I isolated the antibiotic responsible for the anti-MRSA activity. I find that this molecule, 13-
deoxytetrodecamycin (13-dTDM), is produced in prohibitively small quantities. To facilitate
future work on this molecule, I decided that the next best step was to rationally engineer an
overproducer strain. To do this, I needed to identify the biosynthetic gene cluster responsible for
producing 13-dTDM. Thus, the second data chapter reports on the genome sequencing of
WAC04657 and the discovery of the ted gene cluster. Finally, the third data chapter reports on
how I confirmed that the ted gene cluster produces 13-dTDM. In the process of doing this, I also
discovered that the ted cluster produces a series of related molecules. Future experiments will
involve identifying the mechanism of action of 13-dTDM and testing it for activity against a
collection of clinically-isolated MRSA strains.
21
22
Chapter 2. Discovery of 13-Deoxytetrodecamycin
Contributions by others to this work. Early investigations into WAC04657’s antibiotic activity
were initiated by Michael Kamin Hart. The structure of 13-deoxytetrodecamycin was solved by
Dr. Sheila Pimentel-Elardo and Dr. Geoff Tranmer.
23
2.1 Abstract
Antibiotic resistance is one of the most important problems that modern medicine faces. To
combat this challenge, there is a significant need to isolate novel antibiotics with activity against
these “super bugs”. WAC04657 is a wild-isolate Streptomyces that was observed to have
antibiotic activity against MRSA. Using bioactivity-guided fractionation, I isolated the antibiotic
13-deoxytetrodecamycin (13-dTDM) from solid agar extracts of WAC04657. The molecule was
characterized as a novel antibiotic related to the known compound tetrodecamycin (TDM). 13-
dTDM has a molecular of mass of 318 g mol-1
and a molecular formula of C18H22O5. 13-dTDM
was observed to have bioactivity against Gram positive pathogens including MRSA.
24
2.2 Introduction
There is an important need to develop novel antibiotics as drug-resistant pathogens (e.g. MRSA)
become more prevalent in the clinic (Centers for Disease Control and Prevention, 2013). Most of
the antibiotics that are in clinical use were developed in the 30 years following the discovery of
streptomycin (Watve et al., 2001). Since then, the rate of antibiotic discovery has dropped off
precipitously and resistance has been reported to all current antibiotics (Ventola, 2015). The
majority of antibiotics were discovered as natural products produced by the bacterial genus
Streptomyces. Despite having been studied extensively since the 1940s, genome sequencing has
revealed that Streptomyces have the genetic potential to produce thousands of previously
undiscovered molecules. This makes them a likely source of future drugs. While the mecca of
drug discovery is the identification of a novel chemical scaffold (e.g. teixobactin [Ling et al.,
2015]), there is also considerable value in the study of “less novel” compounds.
The rediscovery of known compounds is a common problem in drug discovery, but there is
renewed interested in these previously discovered molecules. Many old molecules were never
developed because of poor pharmacological properties, but thanks to advances in drug delivery
and chemistry, the rediscovery of these “untouchables” means they can be reconsidered for the
modern clinic (Falagas et al., 2008). Further, it is likely that many old molecules were never
conclusively ruled out as possible drugs. Taken together, this means that even old compounds
have the potential to be new drugs. Halfway between known and novel, there is also a
tremendous diversity of unmined wealth in “congeners”. Congeners are variants of molecules
which have different “decorations” on the core molecule (e.g. TDM vs dhTDM). Knowledge
about these is useful because a) it inspires the chemical modification of approved drugs, and b) it
provides a basis for studying the enzymes and reactions which make these modifications. Such
variants of known molecules are extremely important in medicinal chemistry as they allow the
development of new ‘generations’ of drugs with improved properties that, for example, allow
them to circumvent resistance mechanism (Chopra, 2001; Fischbach and Walsh, 2009). External
to drug development, antibiotics and other small molecules also have an extremely important
role as chemical probes of biology. Small molecules have been vital in understanding immune
signaling, the ribosome, and peptidoglycan biosynthesis (Davies, 1964; Liu et al., 1991;
Tiyanont et al., 2006). As a result, even if a molecule is ultimately unsuitable for the clinic, a
detailed understanding of its structure and function is valuable.
25
In this chapter I report on the discovery of a novel antibiotic named 13-deoxytetrodecamycin
(13-dTDM). I begin my research with the observation that an unstudied, wild-isolate
Streptomyces, strain WAC04657, is capable of killing MRSA in co-culture experiments.
Through the use of activity-guided chromatography, I develop a method for purifying the active
antibiotic. Using nuclear magnetic resonance (NMR) and mass spectrometry (MS), we solve the
structure of the antibiotic and I confirm that, in its pure form, 13-dTDM is able to inhibit the
growth of MRSA. The active molecule, 13-dTDM, is a congener of the previously discovered
molecules TDM and dhTDM.
2.3 Results
2.3.1 Characterization of WAC04657
WAC04657 is a wild-isolate from the Wright Actinomycete Collection (wac.mcmasteriidr.ca).
Early in my PhD I also referred to this strain as Ja2b and Streptomyces cinereus. These names
are obsolete. During genetic engineering experiments, I observed that WAC04657 was capable
of producing a strong antibiotic activity against several antibiotic-resistant pathogens (discussed
further below). Given the need for new antibiotics, I decided to further characterize WAC04657
and its antibiotic activity. Based on the colony morphology of the strain, WAC04657 appeared to
be a member of the genus Streptomyces. I sequenced the partial 16S rDNA of WAC04657 and
compared it to several well-characterized Streptomyces and members of the closely related
genera Streptacidiphilus and Catenulispora. After building a maximum likelihood tree, I found
that WAC04657 clustered with members of the genus Streptomyces (Figure 2.1). This confirmed
that WAC04657 is a member of the genus Streptomyces.
26
Figure 2.1 16S phylogenetic tree indicates that WAC04657 is a strain of Streptomyces.
A maximum likelihood tree of 16S rRNA sequences shows that WAC04657 clusters within the
genus Streptomyces and not with the closely related genera of Streptacidiphilus or
Catenulispora. Kribella flavida was used as an outgroup.
WAC04657 grows very rapidly compared to Streptomyces coelicolor, progressing through its
life cycle within 2 to 3 days at 30°C. When individual colonies are grown on MYM or MS agar,
they appear after 12 to 16 h as beige, bald colonies which stain the media with a brown pigment.
By the second and third days, the colonies will produce a white aerial mycelium followed by
ash-grey spores. Colonies are typically circular and dome-shaped, though they can sometimes
take on the appearance of “castles”, with crenellations forming at the outer edges of a colony
with a sunken middle. Colonies typically adhere poorly to MYM agar, but adhere well to MS and
ISP4 agar. WAC04657 can additionally be grown at 37 °C, which results in faster progression
through its life cycle (sporulation within one day of growth). When grown in high density (e.g. in
lawns), WAC04657 will progresses through its life cycle more quickly and visually appears to
produce more of the brown pigment. Fully developed lawns of WAC04657 are primarily ash-
grey with white spots and sometimes accumulate small light brown droplets of fluid on their
surface.
2.3.2 Identification of an anti-MRSA activity
To characterize the antibiotic potential of WAC04657, I performed co-culture experiments. A
droplet of spores was pipetted onto an agar plate and incubated for 48 h. An indicator strain
suspended in molten agar was then added to the WAC04657 plate as an overlay. The plate was
then incubated for an additional 16 h and examined for inhibition of the indicator strain.
27
WAC04657 exhibited strong antibiotic activity against a number of Gram positive bacteria,
including MRSA (Table 2.1). No antibiotic activity was observed against Gram negative bacteria
(data not shown).
Table 2.1 Colony diffusion assays of WAC04657 against Gram positive bacteria.
Zone of clearance is the distance from the edge of the WAC04657 colony to the point at which
growth of the indicator organism is visible. S. aureus ATCC BAA-41 is methicillin resistant. S.
aureus ATCC BAA-44 is multidrug-resistant.
Test organism Zone of
Clearance (mm)
Bacillus subtilis 168 4
Micrococcus luteus 3
Staphylococcus aureus ATCC 29213 1.5
S. aureus ATCC BAA-41 5
S. aureus ATCC BAA-44 4
S. saprophyticus ATCC 15305 2
S. epidermidis ATCC 12228 2
Enterococcus faecalis ATCC 29212 2
To isolate the anti-MRSA molecule(s), I performed an activity-guided purification. I grew
WAC04657 on MYM agar for five days on MYM agar and extracted the agar and cells with
ethyl acetate. The extract was filtered and concentrated under vacuum to yield an oily, dark
brown crude extract that smelled strongly of soil. The crude extracted was spotted on a silica
thin-layer chromatography (TLC) plate and developed in 1:9 methanol:chloroform. The plate
was air dried, overlaid with B. subtilis, and incubating for 16 h at 37 °C. Staining the plate with
thiazolyl blue tetrazolium bromide dye (MTT) revealed three zones of inhibition (Figure 2.2A).
The compound with the largest zone migrated through the plate with an Rf of 0.5. From a TLC
plate run in parallel, I used chloroform to extracted the antibiotic out of the silica pertaining to
the Rf of 0.5, dried the extract under vacuum, suspended it in DMSO, and then ran a sample on
an HPLC. This revealed the presence of a primary peak eluting at 8.0 min (Figure 2.2B).
Comparison to a sample of crude extract run on an HPLC under the same conditions revealed a
corresponding peak with the same retention time and UV-Vis absorbance profile (Figure 2.2C). I
tentatively named this molecule W8.0 (‘W’ denoting that the molecule is produced by
WAC04657 and 8.0 denoting its elution time on the HPLC) but, for the sake of clarity, I will
primarily refer to this molecule as 13-dTDM.
28
Figure 2.2 TLC was used to identify the active compound in the crude WAC04657 extract.
A) Crude extract was separated by TLC and the plate was overlaid with B. subtilis to visualize
the active compounds. Active molecules appear as a zone of clearance marked here with white
arrows. B) The molecule corresponding to Rf 0.5 was extracted from the silica of a plate run in
parallel and visualized by HPLC at 220 nm. C) This guided purification of the active molecule
from the crude extract.
To further study 13-dTDM, I developed a large-scale purification method (Figure 2.3). Briefly,
12.8 L of MYM solid agar was inoculated with WAC04657 and grown for 48 h at 30 °C. The
cultures were extracted overnight with ethyl acetate. The extract was then dried under vacuum.
The resulting oily, brown extract (663 mg) was suspended in 2 mL of 50% aqueous acetonitrile
and separated on a Sep-Pak C18 column. The column was washed with 10 mL each of 100%
H2O and 30% aqueous acetonitrile, and the active fraction was eluted with 10 mL of 70%
aqueous acetonitrile. The eluate was dried in a centrifugal evaporator (115 mg), suspended in
50% aqueous acetonitrile, and washed with hexane. Molecule W8.0 was purified by HPLC on a
C18 column and lyophilized to yield a light pink powder (4 mg). This powder was suspended in
DMSO (important note: the antibiotic is unstable in DMSO), separated by HPLC on a PFP
column, and lyophilized (1.2 mg). Finally, the antibiotic was separated from a contaminant by
dissolving in chloroform, pelleting the immiscible contaminant, and then drying under vacuum to
yield the pure molecule (1.1 mg). The final yield of 13-dTDM from the crude extract is 0.17%.
29
Figure 2.3 Purification method for 13-dTDM.
13-dTDM was purified in five steps. Total yields from crude extract to pure compound are
0.17%.
2.3.3 Biological activity and structural elucidation of 13-
deoxytetrodecamycin
To assess the antibiotic activity of 13-dTDM, I performed minimum inhibitory concentration
(MIC) assays using the solid agar dilution method (Table 2.2). The solid agar method was used
because 13-dTDM is insoluble in liquid media. 13-dTDM had antibacterial activity against all
the tested Gram positive organisms in the range of 1 to 8 μg mL-1
. This included methicillin-
resistant S. aureus ATCC BAA-41 and multidrug-resistant S. aureus ATCC BAA-44. 13-dTDM
had no activity against Gram negative organisms.
30
Table 2.2 MIC of 13-dTDM against several pathogens.
MIC was determined using the agar dilution method. S. aureus ATCC BAA-41 is methicillin
resistant. S. aureus ATCC BAA-44 is multidrug-resistant.
MIC (μg mL-1
)
Test organism 13-dTDM Ampicillin Vancomycin
B. subtilis 168 2 <1 <1
M. luteus 8 <1 <1
S. aureus ATCC 29213 8 <1 2
S. aureus ATCC BAA-41 1 16 2
S. aureus ATCC BAA-44 8 32 2
S. saprophyticus ATCC 15305 8 <1 2
S. epidermidis ATCC 12228 8 <1 2
E. faecalis ATCC 29212 4 <1 2
E. coli ATCC 25922 >64 16 >64
The physiochemical properties of 13-dTDM are summarized in Table 2.3. Using high resolution
electrospray ionization mass spectrometry (ESI-MS) by direct injection, the molecular formula
of C18H22O5 was calculated based on the observed mass of m/z 341.1365 [M+Na]+ (calculated
m/z 341.1374 [M+Na]+). This indicated eight degrees of unsaturation. The similarity of the
molecular formula to TDM (C18H22O6) and extensive analysis of the 1D (1H,
13C DEPTQ) and
2D (1H-
13C HSQC,
1H-
1H HSQC-TOCSY,
1H-
1H COSY, and
1H-
13C HMBC) NMR spectra
suggested that 13-dTDM is a new TDM derivative (Figure 2.4) (Tsuchida et al., 1995b). The 1H
and 13
C NMR data is summarized in Table 2.4 and all NMR spectra can be found in the
appendix. The main difference from TDM is that 13-dTDM lacks a hydroxyl at C-13 of the
decalin ring thus motivating the name “13-deoxy” tetrodecamycin. The tertiary carbon at this
position was found at δC 36.8 which indicated a lack of hydroxyl. This is in contrast to δC 69.0
(C-13) as found in TDM which indicates the presence of a hydroxyl (Tsuchida et al., 1995b).
The position of this carbon was further supported by COSY and HMBC.
31
Table 2.3 Physico-chemical properties of 13-dTDM.
Description Details
Appearance white residue
Molecular formula C18H22O5
Molecular mass (g mol-1
) 318
ESI-MS (m/z)
Found [M+Na]+ 341.1365
Calculated [M+Na]+ 341.1374
[α]D (20 °C, CHCl3) -17.6° (c 0.65)
Solubility:
soluble DMSO, CHCl3
insoluble/poorly soluble water, acetonitrile, methanol,
acetone, ethyl acetate, 2-propanol,
n-hexane
UV λmax (logϵ) (CHCl3) 272.6 nm (3.96)
Figure 2.4 Structure of 13-dTDM.
13-dTDM is a tetracyclic molecule produced by WAC04657. It is a novel congener of TDM
which differs at C-13. A Michael acceptor in 13-dTDM is composed of conjugated system
starting at C-1/C-6 through C-2, C-3, C-4, and terminating at C-5.
32
Table 2.4 13
C and 1H NMR data for 13-dTDM in CDCl3.
Position δC δH mult (JH-H)
1 165.0
2 101.3
3 164.6
4 148.5
5 96.5 5.36 d (2.7)
5.26 d (2.7)
6 195
7 53.6
8 40.9 1.23 m
9 25.1 1.72 m
1.15 m
10 27.3 1.57 m
1.17 m
11 25.9 1.73 m
1.05 m
12 32.7 2.3 m
0.96 tdd (13.2, 10.5, 3.6)
13 36.8 1.21 m
14 79.8 3.59 d (4.2)
15 92.1 4.75 dd (2.8, 1.8)
16 34.4 2.13 dq (7.4, 2.9)
17 16.7 1.14 s
18 13.7 1.02 d (7.4)
2.4 Discussion
In this chapter I reported on how I discovered a novel antibiotic molecule. This molecule,
13-dTDM, is produced by the wild-isolate bacterium Streptomyces sp. strain WAC04657. Our
lab’s work with WAC04657 began with a genetic engineering project under the leadership of
another student. When we realized that this strain produced a strong anti-MRSA activity, I
became interested in identifying the responsible molecule. To identify the antibiotic, I performed
an activity-guided screen using TLC. This experiment is specifically called a “bioautography”.
The bioautography allowed me to identify the antibiotic molecule in HPLC traces of the crude
extract which, subsequently, allowed me to develop a method to purify the molecule. Once
purified, I was able to elucidate the structure of the molecule by NMR and determine MICs. 13-
dTDM has antibiotic activity against MRSA as well as several other Gram positive bacteria.
33
13-dTDM is the third discovered member of the TDM-group of tetronate molecules. While the
structure of 13-dTDM is not “super novel”, its discovery is important for numerous reasons. The
first reason is that 13-dTDM is the first TDM-group molecule to be discovered outside of the
original producer. Since these related molecules occur in more than one organism, it suggests
that they are not just “junk” compounds or an evolutionary dead end. Rather, they likely provide
a fitness benefit to the producer and, as a result, were not purged from the genome following
acquisition by horizontal gene transfer. This suggests that the TDM-group molecules are useful
in some unknown condition and supports the idea that they could be useful as a therapeutic. The
second reason that the discovery of 13-dTDM is important is because it marks the rediscovery of
a group of compounds which have strong antibiotic activity against MRSA. Regardless of
novelty, the ultimate goal of this research is to develop new antibiotics for the clinic. 13-dTDM
has activity against S. aureus ATCC BAA-44, a strain of MRSA reported by the ATCC to be
resistant to 19 different antibiotics (ampicillin, amoxicillin/clavulanic acid, ciprofloxacin,
cephalothin, doxycycline, gentamicin, erythromycin, imipenem, methicillin, penicillin,
tetracycline, oxacillin, azithromycin, clindamycin, ceftriaxone, rifampin, amikacin, tobramycin,
and streptomycin) (http://atcc.org/Products/All/BAA-44.aspx#characteristics). This activity
makes 13-dTDM a valuable drug candidate. Interestingly, TDM’s use as an antibiotic has been
patented in Japan. Despite this, there is no evidence that the molecule was used in humans or
otherwise. Was the development of these molecules abandoned with cause? Even if it was,
modern developments in chemistry could mean that this molecule is now a feasible drug. Further
testing of 13-dTDM will be needed to evaluate its utility as a therapeutic.
External to its potential as a drug, 13-dTDM is also exciting because of its possible utility as a
molecular probe. Built into the structure of 13-dTDM is a potential Michael acceptor (recall
Figure 1.3 on page 12). The Michael acceptor is composed of the conjugated system running
from C-1/C-6 through C-2, C-3, C-4, and terminating at C-5. Due to the presence of the
electronegative carbonyls at C-1 and C-6, C-5 is proposed to act as an electrophile in a
nucleophilic addition reaction. The end result would be a covalent bond between 13-dTDM and
its target biomolecule. If this reaction occurs as predicted, it could be possible to “label” a cell
component with 13-dTDM. This idea has precedent with vancomycin. Vancomycin is a potent
antibiotic which binds to newly peptidoglycan precursors. By labelling vancomycin with a
fluorophore, it was possible to generate a probe which could be used to identify the location of
34
newly synthesized peptidoglycan (Tiyanont et al., 2006). 13-dTDM has significantly fewer
locations to tailor, but previous work on TDM has shown that the 14-OH position can be
modified (Tsuchida et al., 1995c). Thus, it is reasonable to think that we could engineer 13-
dTDM into a probe.
Despite the exciting qualities of 13-dTDM, it suffers from one major limitation. The molecule is
produced in very small quantities. Yields from crude extracts of WAC04657 are only 0.17% and
the isolation protocol takes approximately two weeks to complete. Thus, in order to efficiently
study 13-dTDM, it would be of significant value to generate an overproducer strain.
In summary, 13-dTDM is a novel TDM-group molecule which has anti-MRSA activity. Given
the need for new antibiotics against MRSA, this makes 13-dTDM an interesting candidate for
further research. Moreover, even if the molecule does not find utility as a drug, the presence of a
Michael acceptor in the structure suggests that it could be developed into a probe for use in basic
science.
35
36
Chapter 3. The Biosynthetic Gene Cluster for 13-Deoxytetrodecamycin
Contributions by others to this work. Nicholas Waglechner and Fredric Ulrich provided
technical assistance with genome assembly. The PAC clone library was made by BioS&T
(Montreal, Canada). Genome sequencing was performed by Christine King at the Farncombe
Institute (McMaster University, Canada) or Genome Quebec (Montreal, Canada).
37
3.1 Abstract
In the previous chapter, I reported on the discovery of 13-dTDM, a tetronate ring-containing
antibiotic with anti-MRSA activity that is produced by S. sp. strain WAC04657. Given the
therapeutic potential of this molecule, I wanted to study it further. The difficulty in studying this
molecule is that it is produced in prohibitively small quantities. To facilitate future work, I
wanted to generate an overproducer strain of 13-dTDM and thus needed to identify the
biosynthetic gene cluster responsible for producing 13-dTDM — the ted cluster. To find the ted
cluster, I sequenced the genome of WAC04657 by 454 pyrosequencing and assembled the
genome into 513 contigs. By searching the assembly for genes involved in tetronate ring
biosynthesis I was able to discover a partial ted cluster. In an attempt to assemble the full ted
cluster, I attempted to improve the sequencing depth (and thus contiguity) of the genome with
Illumina and also ordered genomic library in which I hoped to capture the full ted cluster. Both
of these techniques failed to improve the assembly. Finally, I sequenced the genome again, but
this time with PacBio, and assembled the genome into 5 contigs with 27 biosynthetic gene
clusters. Analysis of the genome sequence allowed me to identify the full ted cluster. Using the
ted cluster as a query, I was also able to find ted-like clusters in three other organisms. By
comparing the ted clusters from each organism, I was able to map the core genes of the cluster.
The ted cluster has 25 conserved genes, 6 semi-conserved genes, and a single gene found in
WAC04657 which has no homologs in the other clusters.
38
3.2 Introduction
13-dTDM is an antibiotic with activity against MRSA. In the previous chapter, I reported on the
isolation of this molecule from the wild-isolate bacterium Streptomyces sp. strain WAC04657. I
am interested in 13-dTDM and the other TDM-group molecules because of their therapeutic
potential and potential to be developed into a covalent-acting molecular probe. Further, neither
the mechanism of action nor the biosynthetic gene cluster for any of the TDM-group molecules
is known. Given that these molecules are classified as “miscellaneous tetronates”, a more
detailed understanding of these molecules is of benefit to the tetronate field. Unfortunately,
studies into 13-dTDM are limited by the fact that the molecule is produced in prohibitively small
quantities. In order to facilitate future work on this molecule, I have a long-term aim to generate
a strain of WAC04657 which overproduces 13-dTDM. In order to do this, I need to sequence the
genome of WAC04657 and identify the biosynthetic gene cluster which is responsible for
making 13-dTDM.
Once the genome of WAC04657 is sequenced, there are two challenges associated with
identifying the gene cluster. The first challenge is identifying the specific biosynthetic gene
cluster that produces 13-dTDM. Given that the typical streptomycete encodes from 20 to 70
different biosynthetic gene clusters (Doroghazi and Metcalf, 2013), identifying a specific gene
cluster can be challenge. This process is facilitated by looking for genetic features that are unique
to your molecule of interest. 13-dTDM possesses two such distinctive features. The most useful
is its tetronate ring. Previous work on tetronate rings has shown that they are biosynthesized by a
set of five dedicated enzymes (Kanchanabanca et al., 2013; Sun et al., 2008). Of these enzymes,
Tmn16-like proteins are particularly useful as markers since they are unique to tetronate ring
biosynthesis. The second distinctive feature of 13-dTDM is its backbone. Based on the structure
of 13-dTDM, the molecule’s backbone appears to be produced by a PKS with seven modules.
Thus, this can be used as secondary filter to confirm the identity of any identified gene clusters.
The second challenge in identifying a biosynthetic gene cluster is identifying the edges of the
cluster. Traditionally, this has relied on deleting genes near the edges of the cluster. Thanks to
the proliferation of genome sequences, it is now possible find the edges of a cluster
bioinformatically. This is done by searching for the same gene cluster in multiple organisms and
then using comparative genomics to identify which of the genes in this cluster are conserved
across all representatives (Doroghazi and Metcalf, 2013). Those genes which are conserved are
39
part of the “core” biosynthetic gene cluster and those which are unconserved are unlikely to be
members of the cluster.
In this chapter, I report on my work sequencing the genome of WAC04657. After sequencing
with several different technologies, I am able to get a good genome sequence which I then
annotate. Using my knowledge about tetronate ring biosynthesis, I am able to identify the genes
responsible for producing 13-dTDM — the ted gene cluster. Using this cluster as a query, I am
able to find ted-like clusters in three other organisms. Comparison of all the ted clusters allows
me to identify the conserved genes.
3.3 Results
3.3.1 454 sequencing was used to identify the partial ted cluster
To identify the biosynthetic gene cluster for 13-dTDM (the ted cluster), I needed to sequence the
genome of WAC04657. My initial attempts to isolate genomic DNA generated insufficient DNA
for whole genome sequencing. To increase my yields, I made modifications to the Qiagen
DNeasy protocol. The modified protocol focused on improving cell lysis. The following four
steps improved yields: 1) using a 12 h-old liquid culture for DNA extraction, 2) the addition of
glycine to the growth medium, 3) the use of a tissue grinder to break up cell clumps before
lysozyme treatment, and 4) an extended lysozyme treatment. The additional steps improved
DNA extraction from approximately 10 ng μL-1
to ≥100 ng μL-1
.
I submitted the resulting genomic DNA for sequencing by 454 pyrosequencing at the Farncombe
Institute at McMaster University. The genome was sequenced twice on 37.5% of a 454 plate.
Sequencing statistics can be found in Table 3.1. Technical issues caused shorter than expected
reads during the first sequencing run, which is why we sequenced the genome a second time. The
combined sequencing data were assembled using MIRA version 3.4.1.1 (May 2012) into 513
contigs (contig cutoff at ≥500 bp and ≥10× coverage). Putative open reading frames (ORFs)
were assigned using the program Prodigal (Hyatt et al., 2010). I searched the genome for Tmn16
homologs and found a single hit. I named this gene tedF1. Closer examination of this contig,
contig 68, revealed a short stretch of Type I PKS genes (tedS1) that were interrupted by the edge
of the contig (Figure 3.1). The contig contained an additional 15 genes. Four were likely to be
involved in 13-dTDM biosynthesis (each was given a ted name) while the remaining genes
40
appeared to be unrelated to the cluster or had unknown functions. Thus it appeared that contig 68
represented the left arm of the ted cluster with the remaining ted genes on the other side of tedS1.
At the time when I assembled this genome sequence, the genes required for forming the exo-
methylene unpublished and thus could not be used to identify the other arm of the cluster.
Therefore, I decided that the best way to identify the remaining genes would be to improve the
assembly, with a specific goal of closing the break in the PKS genes.
Table 3.1 Summary of high throughput sequencing runs.
WGS denotes whole genome sequence of WAC04657. aData reported for SMRT is a partial
dataset, only the reads in the “longest” file are shown.
Sample and
Technology
Number of
Reads
Read Length,
Range (bp)
Read Length,
Mode (bp)
Total Length
(Gbp)
WGS, 454 (first) 417,352 20 to 1040 350 0.135
WGS, 454 (second) 242,262 21 to 888 550 0.112
WGS, Illumina 3,483,228 251 251 0.874
PAC Clone, Illumina 183,874 251 251 0.046
WGS, SMRTa 331,452 50 to 30,328 1500 1.525
Figure 3.1 Contig 68 represents the partial ted gene cluster.
Contig 68 possesses a Tmn16 homolog (tedF1) and a partial PKS gene (tedS1). The contig also
has 15 additional genes. Only genes 68.01 to 68.06 have potential relevance to 13-dTDM
biosynthesis.
3.3.2 Illumina data did not improve the WAC04657 assembly
To improve the assembly, I took a two-pronged approach. The first approach was to re-sequence
the genome with the goal of increasing the depth of coverage. I reasoned that with more data, I
would be able to improve the assembly the PKS genes. I sequenced the whole genome again, but
this time with the Illumina technology (a.k.a. solexa) at the Farncombe Institute at McMaster
(sequencing statistics can be found in Table 3.1). Initial attempts to de novo assemble the hybrid
41
data with the MIRA assembler failed due to insufficient computer resources (memory
requirement exceeded my available 16 GB of RAM). Unfortunately, I was unfamiliar with cloud
computing at that time and thus did not attempt to use a ‘super computer’ to complete the
assembly. Instead, I resorted to using the Velvet assembler (which has a smaller memory
requirement) and performed a de novo assembly with the Illumina data only (Zerbino, 2002).
Despite the increased sequencing coverage, I was unable to assemble the data into fewer than
1000 nodes (n.b. a “node” is Velvet’s equivalent of a contig). I thus abandoned the Illumina data
and proceeded with my second strategy.
This second strategy involved isolating the complete ted cluster on a phage artificial
chromosome (PAC) and sequencing the resulting clone (Jones et al., 2013). I sent pelleted
WAC04657 cells to Bio S&T Inc. (Montreal, Canada) where they isolated DNA and cloned large
fragments into the artificial chromosome pESAC13. They built a library of 3,840 clones arrayed
into 384-well format. Bio S&T also screened the library by PCR to identify clones with the ted
cluster. As a marker for the left arm of the ted cluster, I designed primers which amplified a
region near the tedF1 gene. Unfortunately, I didn’t have any information about the right arm of
the cluster. At that time, I was under the belief that every biosynthetic gene cluster possessed a
short SARP regulator (an idea that myself and others [Liu et al., 2013] have since found to be
incorrect). A search of my current WAC04657 genome sequence revealed only one SARP on the
same contig as a PKS gene (contig 293) (Figure 3.2). I named this gene tedR. I reasoned that
contig 293 encoded the right arm of the ted cluster and provided Bio S&T with primers to
amplify tedR. Upon screening the library, Bio S&T identified two clones which possessed both
the tedF1 and the tedR amplicons. This confirmed that contig 293 was in close proximity to
contig 68. I selected one of the clones to work with (which I named PAC292) and sequenced it
with 1% of an Illumina flowcell at the Farncombe Institute (Table 3.1).
42
Figure 3.2 Contig 293 encodes the right arm of the ted cluster.
Contig 293 is the only contig from the 454 sequencing that possesses a SARP (tedR) on the same
contig as a PKS gene (tedS2). As a result, I hypothesized that this represents the right arm of the
ted cluster.
Using Velvet v1.2.10 with paired kmers of 89 bp, I assembled the sequence of PAC292 into six
nodes. Of these, two were discarded because they were very short (1 bp and 31 bp) and a third,
the E. coli phage ϕX174 genome, was discarded because it is a known sequencing spike-in
control. This left three nodes: node 1 (6.0 kb), node 4 (18.1 kb), and node 5 (94.7 kb). I manually
connected nodes 4 and 5 based on a 28 bp overlap on one side and, on the other side, by
sequence comparison to the genome of Streptomyces venezuelae ATCC 10712 (GenBank#
FR845719.1). This generated a closed circular plasmid of ~113 kb that lacked node 1 (Figure
3.3). My inability to scaffold node 1 suggested a misassembly. Analysis of the conserved
domains of node 1 indicated that it was part of a PKS system. A single cluster of PKS genes was
also found on the node 4/5 assembly suggesting that the misassembly was localized to this
region. Despite the misassembly, the PAC292 sequencing data confirmed that contigs 68 and
293 were neighbours separated by PKS genes. This supported the idea that contig 293 represents
the right arm of the ted cluster. Analysis of the modules on node 4/5 and node 1 showed that I
had assembled four full PKS modules (one loading and three extension modules) but, based on
the structure of 13-dTDM, I needed seven modules to build the full backbone. Thus, I concluded
that, due to the repetitive nature of the PKS genes, the assembly had “collapsed” in this region
and had combined the data from the seven PKS modules into a four modules.
43
Figure 3.3 Draft map of the PAC292 clone.
The PAC292 clone was sequenced by Illumina and assembled with Velvet. The resulting nodes
were built into scaffolds by hand. Not accounted for in the assembly is node 1. This suggests that
the sequence is misassembled, likely at the PKS genes. Thick arrowheads mark the point where
nodes 4 and 5 were joined. Lines with flat brackets denote the location of features of interest.
3.3.3 The full biosynthetic gene cluster for 13-deoxytetrodecamycin
After having made three attempts to sequence the ted cluster, I came to the realization that short
read technologies (i.e. 454 and Illumina) were inappropriate for sequencing the WAC04657
genome. I therefore decided to sequence WAC04657 by Pacific Bioscience’s SMRT sequencing
(colloquially called “PacBio”). The advantage of this technology is that read lengths are
significantly longer (Table 3.1). Moreover, the errors in the SMRT technology are independent
of GC-content thus making the sequencing of Streptomyces (70% GC) more reliable. I submitted
long-fragment DNA from WAC04657 to Genome Quebec (Montreal, Canada) for SMRT
sequencing on four SMRT cells. The sequencing generated 200,115 reads with a total length of
1.317 Gbp (Table 3.1). Using the HGAP method (Chin et al., 2013), the genome was assembled
by Genome Quebec into 7 contigs with an average of 169.7× coverage. I assigned putative ORFs
with the program Prodigal (Hyatt et al., 2010). Using the NCBI VecScreen tool
(http://www.ncbi.nlm.nih.gov/tools/vecscreen/), I screened the contigs for vector contamination.
Contigs 6 and 7 appeared to be clear vector contamination from pBR322 and were removed from
the genome record. Contig 5 also showed contamination, but a strong match to a vector could not
44
be found. Analysis of the putative ORFs on contig 5 showed that all the closest homologs were
from gammaproteobacteria, thus strongly suggesting that this contig was also a contamination.
Thus, contig 5 was removed from the genome record. Contig 0 showed vector contamination
from pGGC011 and pHSG664. The contamination was near the centre of contig 0 (at the 1.9 Mb
and 2.1 Mb positions) and fell within ORFs coding for a σ70
protein and a ribosomal protein. The
full-length plasmid sequences were BLASTed against contig 0 and found not to align to contig 0.
All this taken together suggested that the vector contamination in contig 0 was not true
contamination. As a result, the sequence of contig 0 was not modified.
Using the annotation program antiSMASH 2.0 (Blin et al., 2013), I identified 27 putative
biosynthetic gene clusters (Figure 3.4). These included three PKS clusters (including two Type I
and one Type II), eight non-ribosomal peptide synthase (NRPS) clusters, seven terpenoid
clusters, two siderophores, and several others. A preliminary comparison of each cluster to the
Gene Cluster Family (Doroghazi et al., 2014) database allowed me to predict the identity of
some of the clusters (Table 3.4). Within one of the Type I PKS clusters I was able to find both
tedF1 and tedR (red circle in Figure 3.4). Analysis of the cluster showed two fully assembled
PKS genes, tedS1 and tedS2, which encoded three and one PKS modules, respectively. Mirroring
the same problem experienced with the PAC292 assembly, the PKS genes did not possess the
expected seven modules required to synthesize the backbone of 13-dTDM. Again operating
under the assumption that the PKS genes were misassembled, I decided to manually assemble
the PKS genes from raw reads. I used a relaxed BLAST search to find raw SMRT reads which
started outside the PKS genes and read into them. While doing this, I found a single 19.5 kb read
that spanned the entire length of both PKS genes. The existence of this read suggested that the
PKS genes in the SMRT assembly were, in fact, properly assembled and accurate in length. This
inspired me to look more closely at a neighboring type I PKS cluster that was only 22.5 kb
downstream of tedS1 and tedS2 (blue circle in Figure 3.4). A closer analysis of the antiSMASH
data revealed that the clusters were in such close proximity that they overlapped (blue and red
bars in Figure 3.5). The adjacent cluster possessed two PKS genes (tedS3 and tedS4) which,
respectively, coded for two and one modules, suggesting that these genes could serve as the
missing three modules. Supporting this, the tedS3 and tedS4 genes lack the canonical
loading/first module expected for PKS enzymes and, therefore, cannot initiate polyketide
biosynthesis on their own. Additionally, tedS2 ends with a putative docking domain and tedS3
45
starts with a docking domain, suggesting that they could form a multiprotein complex (Dutta et
al., 2014; Gokhale et al., 1999). Thus, I hypothesized that these two clusters are not independent,
but instead together represent a single cluster — the ted cluster.
Figure 3.4 Location of the biosynthetic gene clusters in the genome of WAC04657.
The genome of WAC04657 has been assembled into five contigs (or unitigs) with a total length
of 7.761 Mb and possessing a predicted 27 biosynthetic gene clusters. Using antiSMASH, I
identified the location of the biosynthetic gene clusters found in the organism. Hollow circles
represent the location of putative biosynthetic gene clusters in the genome while the connected,
filled circles indicate the type of biosynthetic gene cluster at that location. The red and blue
circles mark two closely located Type I PKS clusters. The order of the contigs is unconfirmed;
“rev” denotes contigs whose nucleotide sequence was reversed-complemented compared to the
deposited sequence.
Figure 3.5 Position and overlap of PKS clusters, and comparison of the ted cluster as found
in WAC04657, S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202.
The blue and red bars denote the location and position of overlap of the two type I PKS clusters
that antiSMASH identified as distinct in WAC04657. For the purpose of visualization, the red
cluster is truncated at its 5’ end and the blue cluster is truncated at its 3’ end. The arrows
represent genes found in each of the organisms. Shaded regions linking the arrows denote that
the genes are syntenic. Hash marks represent breaks in contigs. Solid arrows represent genes
drawn to scale while dashed arrows (PKS genes) have been reduced in size and are not to scale.
46
Table 3.2 Proposed annotations for WAC04657's biosynthetic gene clusters.
Annotations are based on preliminary comparison to the Gene Cluster Family database (GFC db). T1-PKS, type 1 PKS; T2-PKS, type 2
PKS.
Unitig
Number
Cluster
Number
AntiSMASH
annotation
Query Protein Gene Cluster
Family
Frequency
in GFC db
Proposed
Identity
Other Notes
0 1 NRPS WP_062750444.1 NRPS_GCF.260 5
5 T2 PKS WP_062751471.1 PKS_II_GCF.12 5 cosmomycin
6 NRPS-
Butyrolactone
WP_062751572.1 NRPS_GCF.376 26
9 siderophore WP_062756830.1 NIS_GCF.60 346 desferoxamine
11 NRPS WP_062757016.1 NRPS_GCF.167 2
13 NRPS-T1
PKS
WP_062754669.1 NRPS_GCF.158 4
14 siderophore WP_062755433.1 NIS_GCF.15 275
1 1 NRPS WP_062757169.1 NRPS_GCF.483 6 same cluster as unitig 4, cluster 2
3 NRPS WP_062757392.1 n/a possible misannotation, likely flagged
by antiSMASH as a cluster because of
orphan adenylation domain
4a NRPS WP_062757544.1 NRPS_GCF.516 53
4b NRPS WP_062759194.1 NRPS_GCF.382 9
5 Terpene-T2
PKS
WP_062757680.1 PKS_II_GCF.6 186 spore pigment
6 T1 PKS KYG52514.1 Lant_GCF.91 2 ted cluster annotation based on work presented
in this thesis
7 T1 PKS KYG52530.1 n/a ted cluster no family identified, annotation based
on work presented in this thesis
2 1 NRPS WP_062759720.1 NRPS_GCF.47 8
3 NRPS WP_062759833.1 NRPS_GCF.6 4 all other representatives are found in
Rhodococcus spp.
4 2 NRPS WP_062761293.1 NRPS_GCF.483 6 same cluster as unitig 1, cluster 1
47
3.3.4 The edges of the ted cluster were identified by comparative genomics
One of the challenges with studying biosynthetic gene clusters is identifying the edges of the
cluster. That is, which genes are actually part of the cluster and which are just neighbours. The
annotation program antiSMASH designates edges by including all genes within a fixed distance
from the synthase genes and does not consider gene function (Blin et al., 2013). Thus, these
edges are, by design, inaccurate and liberal. The experimental method for identifying edges
involves gene annotation followed by knock outs (e.g. tetronomycin (Demydchuk et al., 2008)).
With the growing amount of genomic data available, it is now possible to annotate cluster edges
informatically. To do this, the same gene cluster is identified in other organisms and the clusters
are compared. Genes which are conserved are considered to be part of the cluster. This strategy
has already been used to automatically identify the edges of biosynthetic gene clusters
(Doroghazi and Metcalf, 2013; Doroghazi et al., 2014).
To find the ted cluster in other organisms, I searched the NCBI’s refseq database using TedF1 as
a query. I found three hits which shared ≥85% identity and 100% query coverage with TedF1.
These hits originated from the genomes of Streptomyces atroolivaceus ATCC 19725,
Streptomyces globisporus NRRL B-2293, and Streptomyces sp. LaPpAH-202. The next closest
hit was found in Streptomyces sp. CNH099 and shared only 64% identity across 96% of the
query (results are current as of May 12th
, 2016). While the majority of hits were found in
Streptomyces, other genera identified included Micromonospora, Actinoplanes, Saccharothrix,
Frankia, Herbidospora, Dactylosporangium, and Verrucosispora suggesting that these
organisms also produce tetronate molecules. Using the ted cluster as a guide, I searched the
genomes of S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202 and identified a ted-like
cluster in each organism. I aligned the ted cluster in each organism (notice grey bars in Figure
3.5) and took note of percent identity (Figure 3.6). In this way, I was able to clearly identify a
core set of conserved genes which constitute the ted cluster (Figure 3.7, Table 3.3, Table 3.4).
This core cluster is bounded by the genes tedF1 on the left-hand side and tedT on the right-hand
side of the cluster. Found in this core cluster are:
all the genes predicted to be required for the synthesis of the tetronate ring (tedF1
to tedF5)
48
four PKS proteins (tedS1 to S4)
three transcriptional regulatory proteins (tedL, R, and T)
a major facilitator superfamily (MFS) transporter (tedM) and ATP-binding
cassette (ABC) transporter (tedA1 to A4)
seven tailoring enzymes (tedC, D, E, G, H, I, and J)
In addition to these genes, there are a set of six genes which are only partially conserved across
all of the clusters. These are named tedX1 to X6. Genes like tedX1 (a putative 4’-PPT transferase)
and tedX6 (a putative thioesterase) have clear relevance to small molecule biosynthesis, but their
lack of conservation suggests they are non-essential or that their function can be supplied in
trans by homologs in other clusters. Other genes like tedX2 (a putative 3’,5’-cyclic AMP
phosphodiesterase), and tedX3 to X5 (possible lantibiotic cluster) are unlikely to be required for
biosynthesis of 13-dTDM. Instead, these genes, if associated with 13-dTDM, are more likely to
be involved in signaling (cAMP pathway) or, in the case of the lantibiotic genes, may produce a
molecule whose activity could synergizes with 13-dTDM. The last gene found in the cluster is
tedU. This gene is found only in WAC04657. Bioinformatic analysis was unable to identify a
putative function for this gene, though it does possess a conserved domain of unknown function,
DUF4157.
Figure 3.6 The ted biosynthetic gene cluster is well conserved in WAC04657 compared to S.
atroolivaceus, S. globisporus, and S. sp. LaPpAH-202.
The percent identity of each protein in the ted cluster was compared to the ted cluster found in
WAC04657. Nearly all the proteins fall above 70% identity. The two proteins which deviate
from this trend are TedG in S. atroolivaceus and TedX1 in S. globisporus. Additional details can
be found in Table 3.4.
49
Figure 3.7 Proposed biosynthetic gene cluster for 13-dTDM as found in WAC04657.
The genes tedX3, tedX4, and tedX5 are found only S. atroolivaceus and S. globisporus, but their
position relatively to the WAC04657 ted cluster is marked with a brace. Genes are drawn to
scale.
50
Table 3.3 Predicted function of ted cluster genes.
Function was annotated by searching the protein sequence for conserved domains in the NCBI
Conserved Domain Database or, in the case of the PKS genes, with antiSMASH.
Name Notes
tedX1 4’-PPT transferase
tedX2 3’,5’-cyclic AMP phosphodiesterase; CpdA-like protein
tedX3 lantibiotic cyclase; lanC-like gene
tedX4 lantibiotic dehydratase; thiopeptide-type bacteriocin biosynthesis domain
tedX5 small peptide
tedF1 tetronate ring tailoring enzyme; fkbH-like gene
tedF2 tetronate ring tailoring enzyme; fkbH-associated ACP
tedB thioesterase
tedS1 polyketide synthase: [AT(Ac), ACP], [KS, AT(mal), DH, KR, ACP], [KS, AT(mal),
DH, KR, ACP]
tedS2 polyketide synthase: docking domain, [KS, AT(mal), DH, ER, KR, ACP], docking
domain
tedM major facilitator superfamily (MFS) efflux pump
tedL large ATP-binding LuxR (LAL)-type regulator
tedC flavin reductase, potentially uses the coenzyme F420
tedR Streptomyces antibiotic regulatory protein (SARP) type regulator
tedD FMN-linked alkanal monooxygenase; potentially uses F420
tedE FMN-linked alkanal monooxygenase; potentially uses F420
tedA4 ABC transporter: ATP-binding protein
tedA3 ABC transporter: permease
tedA2 ABC transporter: permease
tedA1 ABC transporter: substrate-binding protein
tedG ferredoxin
tedH cytochrome P450
tedF3 tetronate ring tailoring enzyme; acyl-transferase
tedF4 tetronate ring tailoring enzyme; hydrolase
tedI F420-dependent oxidoreductase
tedJ short chain dehydrogenase/reductase; possible Diels-Alderase for decalin ring
formation
tedF5 tetronate ring tailoring enzyme; fabH-like gene
tedU predicted protein; conserved domain DUF4157
tedS3 polyketide synthase: docking domain, [KS, AT(mal), DH, ER, KR, ACP], [KS,
AT(mmal), DH, KR, ACP]
tedS4 polyketide synthase: docking domain, [KS, AT(mal), ACP]
tedT tetR-family regulator
tedX6 thioesterase
51
Table 3.4 Comparison of the ted cluster proteins found in S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202 against those
found in S. str. WAC04657.
In several instances there were proteins that were not reported on the NCBI databases but could be identified by annotating the open
reading frames with Prodigal from the whole genome nucleotide sequence. Asterisk (*) denotes a homolog that was manually
identified by Prodigal, but is not recorded in the NCBI databases. The pound symbol (#) denotes that this organism lacked a syntenic
homolog. N/A denotes that a comparison to WAC04657 was not possible. The ‘pseudogene’ flag denotes that the gene was recorded
on the NCBI as incomplete. Typically these genes are missing their start or stop codon for various reasons. The ampersand (&) is used
here to mark a group of genes in S. sp. LaPpAH-202 that appear in place of the tedS2 genes (WP_018471144.1, WP_018471145.1,
WP_018471146.1, WP_018471147.1). These are likely to represent a misassembly rather than legitimate proteins. Gene
Name
WAC04657 Gene Length
(aa)
S. atroolivaceus
Gene
Length
(aa)
% ID to
WAC04657
S. globisporus
Gene
Length
(aa)
% ID to
WAC04657
S. sp. LaPpAH-202
Gene
Length
(aa)
% ID to
WAC04657
TedX1 KYG52512.1 228 # N/A * 53 # N/A
TedX2 KYG52513.1 289 # N/A WP_030692210.1 286 92 # N/A
TedX3 # WP_051709958.1 450 N/A WP_037675149.1 515 N/A # N/A
TedX4 # WP_033304711.1 1098 N/A WP_037675146.1 1065 N/A # N/A
TedX5 # * N/A * N/A # N/A
TedF1 KYG52514.1 631 WP_033304710.1 627 86 WP_030692209.1 630 95 WP_018471140.1 327 85
TedF2 KYG52515.1 80 WP_033304709.1 78 79 WP_030692208.1 80 88 WP_018471141.1 78 82
TedB KYG52516.1 277 WP_051709955.1 282 76 WP_051845343.1 287 87 WP_018471142.1 281 71
TedS1 KYG52517.1 4178 pseudogene N/A pseudogene N/A pseudogene N/A
TedS2 KYG52518.1 2219 pseudogene N/A pseudogene N/A & N/A
TedM KYG53018.1 486 WP_033305898.1 484 88 WP_037676056.1 486 93 WP_018471148.1 483 87
TedL KYG52519.1 905 WP_033305892.1 968 80 WP_030694121.1 969 85 WP_018471149.1 935 80
TedC KYG53019.1 157 WP_051710287.1 157 83 WP_030694122.1 162 93 WP_037817114.1 166 81
TedR KYG52520.1 256 WP_033305891.1 255 88 WP_037676053.1 256 95 WP_018471151.1 255 86
TedD KYG52521.1 353 WP_033305890.1 351 84 WP_030694124.1 350 84 WP_018471152.1 351 82
TedE KYG52522.1 356 WP_033305889.1 354 84 WP_030694125.1 354 87 * N/A
TedA4 KYG53020.1 536 WP_033305888.1 572 81 WP_030694126.1 547 94 * N/A
TedA3 KYG52523.1 310 WP_033305887.1 308 78 WP_051845691.1 265 96 WP_018471155.1 309 77
TedA2 KYG52524.1 310 WP_033305886.1 310 87 WP_030694128.1 310 96 WP_018471156.1 310 85
TedA1 KYG52525.1 542 WP_033305885.1 542 84 WP_030694129.1 542 93 WP_018471157.1 542 85
TedG KYG52526.1 75 WP_051710284.1 81 63 WP_037676060.1 75 88 WP_037817137.1 76 76
TedH KYG52527.1 411 WP_051710281.1 411 85 WP_051845688.1 411 90 WP_020571985.1 410 85
TedF3 KYG52528.1 255 WP_033305884.1 255 89 WP_030694132.1 255 97 WP_018471160.1 255 89
TedF4 KYG53064.1 392 WP_051710279.1 393 81 WP_051845689.1 380 90 WP_018471161.1 395 82
TedI KYG52529.1 292 WP_033305883.1 292 78 WP_051845690.1 292 75 WP_018471162.1 292 74
TedJ KYG52530.1 491 WP_033305882.1 489 79 WP_030694135.1 488 91 WP_018471163.1 476 82
TedF5 KYG52531.1 344 WP_033305881.1 344 90 WP_030694136.1 344 95 WP_018471164.1 344 89
TedU KYG53062.1 424 # N/A # N/A # N/A
TedS3 KYG53063.1 4005 pseudogene N/A pseudogene N/A pseudogene N/A
52
TedS4 KYG53021.1 1099 WP_033305381.1 1067 79 WP_051844737.1 1088 89 WP_018469694.1 1065 80
TedT KYG52532.1 181 WP_033305380.1 181 92 WP_030689881.1 181 97 WP_018469693.1 181 91
TedX6 KYG52533.1 264 # N/A WP_030689880.1 252 91 WP_018469692.1 251 77
53
3.4 Discussion
The sequencing of Streptomyces genomes began in 2002 with Streptomyces coelicolor. In the
field of drug discovery, this was significant because the genome sequence revealed that
S. coelicolor encoded as many as 30 biosynthetic gene clusters, yet had been characterized to
produce only half as many molecules (Bentley et al., 2002; Nett et al., 2009). Given that S.
coelicolor is the most studied member of the genus, it suggested that the biosynthetic potential of
less well-studied Streptomyces was staggering. Since then, the genomes of 486 Streptomyces
species (648 Streptomyces strains total) have been deposited into the NCBI’s RefSeq database
(search done in July 2016). This makes Streptomyces the most sequenced geneus in the RefSeq
database (Table 3.5). Yet the interesting question is ‘why’ the scientific community has spent so
much effort sequencing Streptomyces. At a cursory glance, the answer is exactly what one would
expect — Streptomyces produce antibiotics and other drugs. But more specifically, Streptomyces
genome sequences are informative. Since the genome sequence can be used to partially predict
the structure of an antibiotic, there is tangible value in the sequencing a streptomycete. This has
enabled some exceptional bioinformatic analyses to be applied to biosynthetic gene clusters (e.g.
Doroghazi et al., 2014), yet one of the rate limiting factors in these analyses is contiguity of the
assemblies. While genome assembly is becoming more accessible to the average scientist, the
process still presents numerous challenges. Adding to the difficulty is the fact that Streptomyces
have a high-GC content and that PKS clusters are highly repetitive. These features make
Streptomyces genomes and their biosynthetic gene clusters harder to sequence and more difficult
to assemble.
54
Table 3.5 The top 20 most sequenced genera based on unique species name from the
NCBI’s RefSeq database.
Data was collected in July 2016 by downloading the RefSeq “assembly_file.txt” (found on the
ftp site) and removing strains which had duplicate “species_taxid”. The remaining
organism_name data was then broken by white space, the genus extracted (first element of the
list), and the frequency of each genus was counted and reported as the “Sequenced Species”.
Gram Stain denotes whether members of the genus are, based on 16S phylogeny, classified as
gram positive or gram negative. Pathogenic is an anthropomorphic descriptor which
approximately attempts to classify the genus as being a pathogen of humans.
Rank Genus Sequenced
Species
Gram
Stain
Pathogenic
1 Streptomyces 486 +
2 Pseudomonas 288 - +
3 Bacillus 254 +
4 Lactobacillus 171 +
5 Mycobacterium 145 + +
6 Paenibacillus 128 +
7 Acinetobacter 119 - +
8 Streptococcus 117 + +
9 Burkholderia 110 - +
10 Vibrio 106 - +
11 Rhizobium 104 -
12 Enterobacter 102 + +
13 Candidatus 100 n/a n/a
14 Corynebacterium 95 +
15 Clostridium 93 + +
16 Mesorhizobium 85 -
17 Sphingomonas 85 -
18 Prevotella 75 - +
19 Pseudoalteromonas 74 -
20 Mycoplasma 74 + +
In this chapter I reported sequencing the genome of WAC04657 with three distinct technologies.
With Illumina I generated an assembly of >1000 contigs, with the 454 technology I assembled
the data into 513 contigs, and with the PacBio data I assembled the genome into 5 contigs. A
comparison against all of the assemblies in the Streptomyces RefSeq database suggests that other
researchers are having a similar experience with sequencing Streptomyces genomes (Figure 3.8).
That is, the assembly of Illumina and 454 sequencing result in more contigs (i.e. less complete
assemblies) than PacBio sequencing (note that the number of assembled contigs is not a product
of the sequencing technology per se, but a product of the read length [recall read lengths reported
in Table 3.1]). My data suggests that for anyone who is going to sequence a Streptomyces
55
genome, the best single-pass technology is PacBio sequencing. The drawback of PacBio is the
added cost. To sequence the WAC04657 genome by PacBio cost $2000 CAD while Illumina
cost only $500 CAD. The 454 technology, which is now discontinued, cost about $3000 CAD.
When sequencing just a single genome, the added cost of PacBio is trivial compared to the
quality of data generated but, when large numbers of genomes need to be sequenced, Illumina is
the most cost-effective strategy. Lastly, it should be noted that there is a common belief that
PacBio sequencing suffers from a high error rate. While this is certainly true of the raw reads, the
HGAP assembly method (used to assemble the WAC04657 genome) has been reported to have
99.999% accuracy (Chin et al., 2013).
Figure 3.8 Number of contigs in each Streptomyces genome assembly divided by sequencing
technology.
For the sake of illustration, only the major methods of sequencing have been shown. Everything
else, including assemblies without reported sequencing methods, has been pooled into the
“other” category. Notably, this includes all of the complete genome assemblies which used
numerous methods to finish the genome (e.g. S. coelicolor). Data was taken from the “
*_assembly_statistics.txt” file found for each Streptomyces assembly reported on the RefSeq
Database. Data was collected in July 2016.
With the genome of WAC04657 sufficiently assembled, I was able to easily identify the ted
cluster. Many antibiotics have unique features which can be used to accurately identify their
56
gene cluster. In the case of 13-dTDM, that feature was the tetronate ring. The same strategy has
been used for other tetronate molecules and for glycosylated compounds, non-ribosomal
peptides, enediynes, phosphonates, and halogenated compounds (Daduang et al., 2015; Ju et al.,
2015; Kersten et al., 2013; Liao et al., 2016; Mohimani et al., 2014; Rudolf et al., 2016). Once
the ted cluster had been identified in WAC04657, it was then possible to identify the ted cluster
in other organisms. While I used TedF1 as a query to search the NCBI BLAST databases, the
high sequence identity between all the ted-homologs suggests that most of the proteins in the
cluster would have been sufficient as a query (Figure 3.6, Table 3.4). By comparing the clusters
from each organism, I was able to map the edges of the ted cluster. This edge-mapping strategy
is powerful, but suffers from a limitation: how do you know if an unconserved gene should be
excluded from a cluster? That is to say, what if these unconserved genes contribute to minor
modifications in the molecule’s structure? Certainly, the most reliable method of identifying the
edges of the cluster would be to knock out all the neighbouring genes, but this may not always be
possible with wild-isolate organisms. Thus, future users should keep in mind that the method of
edge-mapping presented here provides an excellent prediction for the core cluster, but may miss
genes unique to just one of the clusters.
In closing, the identification of the ted cluster is the first time that a gene cluster for a TDM-
group molecule has been reported. Given that TDM has been classified as a “miscellaneous”
tetronate molecule, this genome sequence provides valuable information about the molecules on
the periphery of this large family. The same cluster is also found in several other Streptomyces
which suggests that they are also producers of TDM-family molecules. Based on the work
presented here, we can make two broad predictions which will be explored in the next chapter.
Those predictions are: 1) that manipulating the ted cluster in WAC04657 will cause
perturbations in the production of 13-dTDM, and 2) that S. atroolivaceus, S. globisporus, and S.
sp. LaPpAH-202 also produce 13-dTDM.
57
58
Chapter 4. The ted Cluster Produces the Tetrodecamycins
Contributions by others to this work. All work was independently performed.
59
4.1 Abstract
In Chapter 2, I identified a novel antibiotic, 13-dTDM, with activity against MRSA. This
molecule was produced by S. sp. str. WAC04657. To facilitate future work on this molecule, I
decided that I wanted to identify the biosynthetic gene cluster in WAC04657 which is
responsible for producing this molecule. Thus, in Chapter 3, I reported sequencing the genome of
WAC04657 and my identification of the ted cluster. The goal of this current chapter was to
provide experimental evidence to support the hypothesis that the ted cluster is responsible for
producing 13-dTDM. To this end, I built a 13-dTDM-null strain of WAC04657 and a 13-dTDM
overexpression strain of WAC04657 by, respectively, disrupting tedF1 gene and overexpressing
tedR gene. By analyzing extracts of these strains I observed altered 13-dTDM expression as well
as the altered expression of a four additional molecules. I purified one of these molecules and
discovered that it was a novel molecule related to the TDM-group. I named it W5.9. As reported
in Chapter 3, the ted cluster was also identified in S. atroolivaceus, S. globisporus, and S. sp.
LaPpAH-202. While S. sp. LaPpAH-202 was refractory to genetic manipulation, I found that
overexpression of tedR in S. atroolivaceus and S. globisporus induced the expression of two
molecules. By purifying both these molecules, I identified them as TDM and dhTDM. The
experiments reported in this chapter support the conclusion that the ted cluster is responsible for
biosynthesizing not only 13-dTDM but the entire TDM-group of molecules.
60
4.2 Introduction
In the proceeding chapters, I reported on the discovery of 13-deoxytetrodecamycin (13-
dTDM), an antibiotic with anti-MRSA activity that is produced by S. sp. strain WAC04657. This
molecule is the most recently discovered member of a group of molecules known as the
tetrodecamycin-group (TDM-group). The two other members of the group, derived from S.
nashvillensis MJ885-mF8, are tetrodecamycin (TDM) and dihydrotetrodecamycin (dhTDM)
(Figure 4.1) (Tsuchida et al., 1994, 1995a, 1995b). One of the key structural features of the
TDM-group molecules is a five-membered lactone ring called a tetronate ring. Biosynthesis of
this ring requires the action of five dedicated enzymes (Kanchanabanca et al., 2013; Sun et al.,
2008).
Figure 4.1 Structures of the TDM-group molecules.
By searching the genome sequence of WAC04657 for the tetronate-ring enzymes, I was able to
identify a putative 13-dTDM biosynthetic gene cluster— the ted cluster. Using this cluster as a
query, I was also able to identify ted-like clusters in three other bacteria: S. atroolivaceus, S.
globisporus, and S. sp. LaPpAH-202. The ted cluster contains a variety of biosynthetic genes,
including PKS genes (tedS1 to tedS4), tailoring genes, and tetronate biosynthesis genes (tedF1 to
tedF5). Of particular relevance in this chapter is the Tmn16-homolog TedF1. This protein is
responsible for di-dephosphorylating 1,3-bisphosphoglycerate and loading the resulting glyceryl
moiety onto TedF2 (Sun et al., 2008). The glyceryl is subsequently used to form the tetronate
ring. Other genes found in the ted cluster are three transcriptional regulators: tedR, tedL, and
tedT. Together, these proteins are predicted to coordinate the expression of the biosynthetic
genes. Of particular relevance in this chapter is the small SARP-family regulator tedR. Small
SARPs are generally characterized as master ON-OFF switches for the cluster in which they
reside. Small SARPs are almost exclusively positive regulators and are not subject to allosteric
61
regulation (Liu et al., 2013). As a result, their expression is directly correlated with production of
their cognate antibiotic.
The goal of this chapter is to provide experimental evidence that the ted cluster is responsible for
producing 13-dTDM. To do this, I genetically manipulate the ted cluster to generate a 13-dTDM
overexpression and 13-dTDM-null strain. In the process, I observe that the ted cluster is
responsible for producing a series of related molecules. I further confirm the involvement of the
ted cluster in producing TDM-group molecules by studying the secondary metabolism of S.
atroolivaceus, S. globisporus, and S. sp. LaPpAH-202. By the end of this chapter, I will have
shown that the ted genes produce 13-dTDM, TDM, dhTDM, and the novel molecule W5.9.
4.3 Results
4.3.1 The ted cluster is responsible for producing 13-deoxytetrodecamycin
Based on the work in the previous chapter, I predicted that the ted cluster was responsible for
biosynthesizing 13-dTDM. To test this hypothesis, I initially planned to introduce the full ted
cluster into a chassis strain and heterologously express the molecule. Unfortunately, the PAC
library I ordered did not capture the full ted cluster. Thus, I designed an alternate set of
experiments which involved genetically manipulating the ted cluster directly in WAC04657. I
wanted to generate two phenotypes. The first was a 13-dTDM-null phenotype and the second
was a 13-dTDM overexpression phenotype. To generate the 13-dTDM-null phenotype, I aimed
to inactive an enzyme essential for 13-dTDM biosynthesis. The most obvious candidates were
tedF1 or the tedS genes. Due to the repetitive nature of PKS genes I was concerned that I would
be unable to specifically amplify the tedS1 gene, so I focused on making a clean knock-out of
tedF1. In my initial attempt to make a ΔtedF1 strain, I designed a vector that would replace
tedF1 gene with a mini-gene composed of six codons. Despite building the knockout plasmid, I
was unable to introduce/recombine the plasmid into WAC04657.
Given that Streptomyces generally undergo low rates of homologous recombination and coupled
with the fact that WAC04657 is experimentally uncharacterized, I opted next to make a
disruption mutant (i.e. insertional inactivation via a single crossover) (Kieser et al., 2000). Again,
I focused on tedF1 because of its essentiality as well as its large size (1896 bp). In this case, size
was important because I wanted to include a large stretch of homologous DNA to facilitate the
62
homologous recombination event while also breaking the gene up into two non-functional
pieces. I cloned a 798 bp fragment of the tedF1 gene into the suicide vector pOJ260 and
introduced this into WAC04657 by conjugation. The conjugation efficiency was extremely low,
but resulted in four exconjugants. Of these, three were confirmed by PCR to have a tedF1
disruption. Analysis of the fermentations of the tedF1-mutants revealed that the mutants did not
produce 13-dTDM (the alternate name for 13-dTDM is W8.0; see Figure 4.2). This confirmed
that tedF1 (and by proxy, the ted cluster) is essential for 13-dTDM biosynthesis. Interestingly,
several other peaks were also lost in the tedF1 disruption mutant. These molecules were named
W4.9, W5.9, W6.3, and W6.4. The naming system used here denotes the producing organism
(“W” for WAC04657) and the elution time from the HPLC column. These molecules are
discussed further in section 4.3.2.
Figure 4.2 Genetic manipulation of the ted cluster in WAC04657 results in changes in 13-
dTDM (W8.0) production as well as altered expression of several other molecules.
Differences in production of the TDM-family molecules can be seen after both, A) 24 h growth,
and B) 48 h growth. The line labelled “+ tedR” marks the ermE*p-tedR expressing strain; EV
denotes a strain of WAC04657 possessing the pSET152-ermE*p empty vector control plasmid;
“tedF1 dis.” denotes a strain of WAC04657 which has a disruption mutation in the tedF1 gene.
Dotted lines mark molecules which are responsive to genetic manipulation of the ted gene
cluster.
63
The next phenotype I wanted to generate was an overexpression of 13-dTDM. In the ted cluster
there are three regulatory proteins: tedR, tedL, and tedT. Examination of tedL and tedT showed
that they are, respectively, an LAL-family and a TetR-family regulator. These regulators are
subject to allosteric control and, based on sequence, their role as an activator or repressor cannot
be predicted. This makes genetic manipulation of these regulators unpredictable. By contrast,
tedR is a member of the small SARP-family of regulators. These regulators are, as a general rule,
transcriptional activators (Liu et al., 2013). Moreover, overexpression of small SARPs in other
systems has been shown to drive expression of their cognate molecule (Narva and Feitelson,
1990). Thus, I predicted that by overexpressing tedR in WAC04657, I should be able to
overproduce 13-dTDM.
I built a plasmid in which the expression of tedR was under the control of a strong, constitutive
promoter (ermE*p) in the integrative plasmid pSET152. While integration can have side effects,
this plasmid is used ubiquitously in the field because of its stability (Gverzdys, 2011). I
introduced the tedR plasmid and the empty vector control into WAC04657 by conjugation. On
the conjugation plate I observed two different colony morphologies: those overproducing a dark
brown pigment (“brown”) and those that did not (“non-brown”). I selected three exconjugants
from each morphotype for further study. The basis of these differences was not investigated,
though I predict that the difference is due to integration of the plasmid into a secondary attB sites
(Combes et al., 2002). Both morphotypes overproduced 13-dTDM compared to the control
strain. For the sake of clarity, only the results from the “non-brown” morphotype are shown
(Figure 4.2). This result confirmed that the tedR was able to control production of 13-dTDM.
Notably, I also observed increased production of the same molecules that were lost in the tedF1-
disruption: W4.9, W5.9, W6.3, and W6.4. Taking the tedF1 and tedR data together, the results
strongly support the hypothesis that the ted cluster is responsible for producing 13-dTDM.
4.3.2 The ted cluster in WAC0657 produces a novel TDM-group molecule
Comparison of the tedR overexpression strain and the tedF1 disruption strain revealed four
additional molecules (not counting 13-dTDM) whose expression was also responsive to genetic
manipulation of the ted cluster. These molecules were named W4.9, W5.9, W6.3, and W6.4.
Since these molecules were responsive to the ted cluster manipulations, I reasoned that they were
64
relatives of 13-dTDM. To examine this possibility, I purified small quantities of each molecule
and measured their masses by high-resolution ESI-MS. The molecules had masses ranging from
318 Da to 352 Da, thus putting them in the same mass range as 13-dTDM (Table 4.1). To study
these molecules further, I purified the molecule produced in the largest quantity, W5.9. I tested
this compound for antibacterial activity against B. subtilis, S. aureus, M. luteus, MRSA, S.
epidermidis, E. faecalis, E. coli, B. cepacia, A. baumannii, and K. pneumoniae. While 13-dTDM
had MICs against all the tested Gram positive bacteria in the range of 1 to 8 μg mL-1
, W5.9 had
no activity against any of these strains even at concentrations as high as 64 μg mL-1
. None-the-
less, I decided to elucidated W5.9’s structure.
65
Table 4.1 Observed properties of the TDM-group molecule identified in this study.
Note that the UV-Vis Absorbance Maximum (Abs. Max.) was recorded on an HPLC in a solution of 40% aqueous acetonitrile + 0.1%
formic acid.
Name Alternate
Name(s)
Producing
Strain(s)
Retention
Time
(min)
UV-Vis
Abs Max
(nm)
Molecular
Formula
Mol Wt
(g mol-1
)
ESI-MS
(m/z), Found
ESI-MS
(m/z),
Calculated
Mass
Accuracy
(ppm)
W4.9 WAC04657 4.9 260 C18H24O7 352 353.1598 353.1600 -0.6
dihydro-
tetrodecamycin
(dhTDM)
A5.4,
G5.4
S. atroolivaceus,
S. globisporus
5.4 251 C18H24O6 336 337.1651 337.1651 0.0
W5.9 WAC04657 5.9 271 C18H24O6 336 337.1664 337.1651 3.9
W6.3 WAC04657 6.3 243 C18H22O5 318 341.1364 341.1365 -0.3
tetrodecamycin
(TDM)
A6.4,
G6.4,
W6.4
WAC04657,
S. atroolivaceus,
S. globisporus
6.4 271 C18H22O6 334 335.1496 335.1495 0.3
13-deoxy-
tetrodecamycin
(13-dTDM)
W8.0 WAC04657 8.0 271 C18H22O5 318 319.1548 319.1545 0.9
66
Compound W5.9 exhibited a pseudomolecular ion that corresponded to the molecular formula
C18H24O6 based on high resolution UPLC-ESI-MS (m/z 337.1664 [M+H]+; calculated m/z
337.1651 [M+H]+) indicating seven degrees of unsaturation. Given that W5.9 is a product of the
ted gene cluster, I began my structural elucidation with the assumption that the molecule would
be structurally related to 13-dTDM. Indeed, extensive analysis of the 1D (1H, 13C CRAPT) and
2D (1H-13C HSQC, 1H-1H COSY, 1H-13C HMBC) NMR spectra confirmed that W5.9
possesses a decalin ring system (C-7 to C-18) very similar to the one found in 13-dTDM but
lacks the characteristic tetronate ring (Figure 4.3B, Table 4.2). All NMR spectra can be found in
the appendix. The positions of C-6 and C-5 were extrapolated relative to the decalin ring through
HMBC correlations: δH 1.16 (H-17), δH 1.76 (H-8), δH 2.31 (H-16) to δC 199.35 (C-6); and δH
4.31(H-15) to δC 162.79 (C-5) (Figure 4.3B). The downfield chemical shift of C-6 (δC 199.35)
suggested an enone ether while C-5 (δC 162.79) indicated the presence of a carbonyl ester next
to C-15 (δC 86.27). HMBC correlations were observed between C-1 (a methyl group, δC 21.84)
to C-2 and to C-3: δH 1.50 (H-1) to δC 106.21 (C-2) and to δC 199.77 (C-3). The chemical shift
of C-2 (δC 106.21) indicated a hemiacetal while the chemical shift of C-3 (δC 199.77) would be
representative of a ketone. Due to the lack of HMBC correlations for position C-4, I employed
computer-assisted structure elucidation (ACD/Structure Elucidator, version 14.00, Advanced
Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2015) to generate all
possible structures within my constraints (Moser et al., 2012). To select the best structure, I
evaluated the computer generated structures based on two general criteria: 1) that the chemical
shifts of the proposed structure made sense with the observed chemical shifts, and 2) that the
structure possessed a carbon backbone which could be synthesized by the PKS machinery
encoded in the cluster. In the best generated structure (the current structure for W5.9), C-4 (δC
104.53) was assigned to the α carbon of the enone. C-4 was predicted to neighbour C-6 (the β
carbon) as well as carbonyls at C-3 and C-5. Indeed, the structure has a continuous carbon chain
extending through C-7, C-6, C-4 and C-5 with oxygens attached at C-6 and C-5. This is
congruent with formation of this carbon chain by the same PKS machinery responsible for
producing 13-dTDM. There are a few examples of molecules that share structural similarity in
terms of the five-membered ring moiety which I have used as a point of comparison for the
NMR shifts of W5.9. Enalin A (Figure 4.3C) is a natural product produced by Verruculina enalia
No. 2606 which has a similar five-membered furan as W5.9 (Lin et al., 2002). In particular, the
chemical shifts of C-1, C-2, and C-3 in W5.9 are in agreement with the chemical shifts of the
67
equivalent atoms in Enalin A (namely the carbons in the methyl, hemiacetal, and ketone
functional groups). The synthetic molecule 5-t-butyl-4-methoxycarbonyl-2,3-dihydrofuran-2,3-
dione (Figure 4.3C) was used as a point of comparison for the alkene in molecule W5.9 (Stadler
et al., 2001). Indeed, C-4 and C-6 in W5.9 are in agreement with the chemical shifts of the
equivalent alkene in the synthetic molecule. Thus, taking all the above information together, I
propose the structure for W5.9 is the structure found in Figure 4.3A.
Figure 4.3 Supporting data for the structural elucidation of molecule W5.9.
A) The structure of W5.9 with the carbon atoms numbered. B) 1H-
1H COSY (bold lines) and
1H-
13C HMBC (arrows) draw onto the structure of W5.9. HMBC data is not shown where COSY
correlations were observed. C) Comparison of observed chemical shifts of enalin A and the
synthetic molecule 5-t-butyl-4-methoxycarbonyl-2,3-dihydrofuran-2,3-dione against the
chemical shifts of W5.9. Numbers represent the chemical shift of the associated carbon atom.
68
Table 4.2 13
C and 1H NMR data for W5.9 in CDCl3.
Position δC δH Mult (JH-H)
1 21.84 (CH3) 1.50 s
2 106.21 (C)
3 199.77 (C)
4 104.53 (C)
5 162.79 (C)
6 199.35 (C)
7 45.42 (C)
8 47.41 (CH) 1.76 m
9 28.30 (CH2) 1.11 m
1.95 d (10.50)
10 27.34 (CH2) 1.38 m
1.87 d (11.67)
11 25.61 (CH2) 1.17 m
1.76 m
12 28.30 (CH2) 1.04 m
2.09 d (11.19)
13 43.03 (CH) 1.22 m
14 72.81 (CH) 3.94 dd (6.31)
15 86.27 (CH) 4.31 t (5.65)
16 34.70 (CH) 2.31 m
17 18.19 (CH3) 1.16 s
18 11.69 (CH3) 0.99 d (6.93)
4.3.3 S. atroolivaceus and S. globisporus are producers of TDM and
dhTDM
Given the presence of the ted cluster in S. atroolivaceus, S. globisporus and S. sp. LaPpAH-202,
I predicted that these organisms also produced 13-dTDM. I acquired S. atroolivaceus, S.
globisporus, and S. sp. LaPpAH-202 and performed extractions of each organism grown on
MYM media. Unexpectedly, none of the organisms produced 13-dTDM or W5.9 (Figure 4.4).
Note that S. sp. LaPpAH-202 produced a peak which eluted at the same time as 8.0 min but,
based on the UV-Vis profile, this molecule was distinct from 13-dTDM. I hypothesized that the
reason I was not observing 13-dTDM or W5.9 was because the clusters were unexpressed under
the current laboratory conditions. To circumvent any endogenous regulation, I decided to
heterologously express tedR from WAC04657 (tedRW) in each of these organisms. I successfully
introduced the ermE*p-tedRW plasmid (the same as used in Section 4.3.1) into S. atroolivaceus
and S. globisporus. I was unable to introduce the plasmid into S. sp. LaPpAH-202 despite being
69
able to introduce the control vector. As a result, I proceeded with only S. atroolivaceus and S.
globisporus. I purified three isolates from each conjugation and tested the resulting strains for the
production of 13-dTDM on MYM agar. Again, none of the +tedR strains produced 13-dTDM or
W5.9. Instead, I observed a de novo induction of A5.4 and A6.4 in S. atroolivaceus and I
observed increased expression of G6.4 in S. globisporus (Figure 4.5). With prolonged
incubation, I also found that S. globisporus produced G5.4 (denoted by small arrow in Figure
4.4). Notably, WAC04657 also produces a ted-dependent molecule which elutes at 6.4 min
(W6.4, recall Figure 4.2) but does produce a molecule eluting at 5.4 min. The data for these
molecules is summarized in Table 4.1.
70
Figure 4.4 Wild-type S. atroolivaceus, S. globisporus, and S. sp. LaPpAH-202 do not
produce 13-dTDM or W5.9.
S. sp. LaPpAH-202 appears to produce a molecule which elutes at the same time as 13-dTDM,
but analysis of the UV-Vis profile of this molecule revealed that it is not 13-dTDM. Each
organism was grown on MYM agar for either four or seven days before performing extractions.
Absorbance on the HPLC was monitored at 271 nm. Dotted lines indicate the elution time of
W5.9 and 13-dTDM. Small arrow in S. globisporus traces indicate the elution of molecule G5.4.
71
Figure 4.5 Heterologous overexpression of tedRW in S. atroolivaceus and S. globisporus
stimulates production of TDM-group molecules.
A) The stimulation of the molecules A5.4 and A6.4 is particularly clear in S. atroolivaceus,
shown here after 5 days of growth. B) In S. globisporus, there is a significant increase in the
amount of G6.4 produced after 24 h growth, though stimulation of G5.4 is considerably less
obvious. EV denotes the pSET152-ermE*p empty vector containing organism, while “+ tedR”
marks the ermE*p-tedR expressing strain.
Comparison of the UV-Vis absorbance spectrum for the 6.4 min molecules revealed that they
shared an absorption maximum at 271 nm. This suggested that they may be the same compound.
I purified small quantities of W6.4, A6.4 and G6.4 and found that they had an observed mass of
335.1496 [M+H]+. This corresponds to the molecular formula C18H22O6, the same as that
reported for TDM (Tsuchida et al., 1995b). Tandem MS/MS showed that all of the 6.4 minute
molecules fragment to the same daughter ions with the same relative intensities, thus confirming
that these are the same molecule (Figure 4.6). I purified large quantities of G6.4 and analyzed the
molecule by NMR. Analysis of the 1D and 2D spectra (Table 4.3) and comparison to the
published NMR spectra for TDM revealed that the G6.4 (and A6.4 and W6.4 by association) is
TDM (Tsuchida et al., 1995b). All NMR spectra can be found in the appendix. The same strategy
was taken for the 5.4 min molecules. The UV-Vis maximum for these molecules was 251 nm.
MS revealed the mass of the molecules to be 337.1651 [M+H]+ which corresponds to the
molecular formula C18H24O6. Tandem MS/MS confirmed that the molecules fragmented to the
same daughter ions with the same relative intensity (Figure 4.6). Based on the UV-Vis
absorbance profile and the molecular formula, I predicted that the molecules were dhTDM. I
purified large amounts of A5.4 and analyzed the molecule by NMR. Analysis of the 1D and 2D
spectra (Table 4.3) and comparison to the published NMR spectra for dhTDM confirmed that
A6.4 (and G5.4 by association) is dhTDM (Tsuchida et al., 1995b). All NMR spectra can be
72
found in the appendix. Note that additional peaks found in the A5.4 NMR spectra suggest an
impurity or instability.
In summary, the data presented in this chapter confirmed that the ted cluster in WAC04657 is
responsible for producing not just 13-dTDM, but four other TDM-group molecules as well.
These include the novel molecule W5.9 and TDM. Fermentations of S. atroolivaceus and S.
globisporus revealed that they did not produce 13-dTDM, but instead produce TDM and
dhTDM. When all the data are taken together, they strongly supports the conclusion that the ted
cluster is responsible for producing the TDM-group molecules.
73
Figure 4.6 Tandem MS-MS data of the molecules eluting at 6.4 and 5.4 minutes suggests
that they are the same molecule.
Molecules W6.4, A6.4 and G6.4 fractionate to the same daughter ion pattern with the same
relative intensitities. This strongly supports the conclusion that these molecules are identical.
The fractionation pattern of molecules A5.4 and G5.4 also leads to the same conclusion.
74
Table 4.3 13
C and 1H NMR data for TDM and dhTDM.
Tetrodecamycin (TDM)
Dihydrotetrodecamycin (dhTDM)
(CDCl3)
(CD3OD)
Position δC δH mult (JH-H)
δC δH mult (JH-H)
1 163.93 (C)
172.22 (C)
2 100.94 (C)
100.65 (C)
3 165.29 (C)
184.89 (C)
4 148.07 (C)
75.62 (CH) 4.99 m
5 95.90 (CH2) 5.31 d (2.81)
18.19 (CH3) 1.49 d (6.81)
5.38 d (2.81)
6 193.62 (C)
199.77 (C)
7 53.21 (C)
54.39 (C)
8 41.87 (CH) 1.51 m
44.59 (CH) 1.4 m
9 23.84 (CH2) 1.45 m
25.87 (CH2) 1.27 m
1.64 m
1.66 m
10 25.71 (CH2) 1.16 qt (13.18, 3.57)
27.87 (CH2) 1.14 m
1.79 m
1.7 m
11 20.97 (CH2) 1.59 m
22.60 (CH2) 1.43 m
1.63 m
1.66 m
12 41.81 (CH2) 1.35 td (13.56, 4.39)
39.94 (CH2) 1.09 m
1.99 m
2.13 m
13 78.88 (C)
73.84 (C)
14 81.63 (CH) 4.26 d (1.28)
79.99 (CH) 3.52 s
15 86.49 (CH) 4.72 t (1.66)
95.09 (CH) 4.83 m
16 32.06 (CH) 2.15 qd (7.39, 1.23)
33.80 (CH) 2.79 m
17 17.32 (CH3) 1.23 s
17.83 (CH3) 1.25 s
18 14.20 (CH3) 1.05 d (7.41)
14.58 (CH3) 0.93 d (7.41)
4.4 Discussion
One of the major issues in Streptomyces-based antibiotic discovery is the Dereplication Problem
(Baltz, 2006). This problem describes the re-discovery of known compounds produced by
different strains of Streptomyces. With increasing amounts of genomic information, it is
becoming clear that the basis for this problem is the horizontal transfer of gene clusters (Cheng et
al., 2016). The ted cluster is no different. This cluster is found in four distinct Streptomyces
species. In this chapter I confirm that the ted cluster is responsible for producing 13-dTDM, but
also discover that this same cluster is responsible for producing W5.9, TDM, and dhTDM. What
is surprising about this is that these nearly identical clusters give rise to some common molecules
(e.g. TDM) but also to some unique molecules (e.g. 13-dTDM and W5.9).
75
My initial thought was that the production of the different TDM-group molecules would be a
product of differences in gene content. While there certainly are differences in the
presence/absence of some ted genes (namely tedX1-X2, tedX3-X5, and tedU), there are few
correlations which explain the production of the end-products in a satisfactory manner. The only
two good correlations are: 1) tedU being correlated with 13-dTDM and W5.9 production; and 2)
tedX3-X5 being correlated with dhTDM production. While tedU may indeed be responsible for
some interesting chemistry, tedX3-X5 are annotated as lantibiotic synthesis enzymes. While it
cannot be currently ruled out, I think tedX3-X5 are unlikely candidates for tailoring dhTDM. This
suggests that the diversity of molecules generated is caused by something else. Instead of a “one
gene one function” model, accumulation of certain molecules may occur because of mutations to
the tailoring enzymes. Indeed, given that 13-dTDM, TDM, and dhTDM are incrementally
different, it is logical to suggest that they each represent a different step in the biosynthetic
pathway (Figure 4.7).
In my proposed biosynthesis, the linear carbon backbone is formed by TedS1-S4. The backbone
is then cyclized with a glyceryl moiety (via the activities of TedF1, F2, and F3) to give rise to the
immature tetronate ring while simultaneously releasing the linear chain from the final module of
the PKS. The tetronate ring is dehydrated through an acetylation-elimination reaction (via TedF4
and F5) which gives rise to the exocyclic methylene. The decalin ring is then formed, possibly
through the action of the putative Diels-Alderase TedJ (homology to GenBank#AFV71312.1)
(Tian et al., 2015). To form the heterocycle, I propose that an epoxide is formed on the alkene of
the decalin ring which then undergoes a nucleophilic attack by the hydroxyl of the tetronate ring.
This forms the seven-membered heterocycle and simultaneously generates a hydroxyl on C-14.
This mirrors a similar reaction proposed for the formation of the heterocycle of abyssomicin
(recall Figure 1.1) (Gottardi et al., 2011). The resulting product is 13-dTDM. The addition of a
hydroxyl at C-13 would give rise to TDM and subsequent reduction of the exo-methylene would
give rise to dhTDM. This pathway is elegant, but it brings about two questions. First, why
doesn’t WAC04657 produce dhTDM? Given that dhTDM appears to be produced in smaller
quantities than TDM (e.g. Figure 4.5) and, in WAC04657, TDM is already produced in small
quantities (Figure 4.2), dhTDM is likely present but below the limit of detection. The second
question regarding the pathway is, how does this pathway generate W5.9? At this time, it is not
obvious how this molecule is produced, though I would like to suggest a working hypothesis.
76
Perhaps W5.9 results from a failure to form the tetronate ring. So, instead of reacting with the
glyceryl moiety, the backbone can self-cyclize resulting in simultaneous release from the PKS
and formation of the carbonyl ether linkage between C-5 and C-15. The furan ring can then be
made by reacting this molecule with a three carbon molecule like methylglyoxal thus generating
C-1, C-2, and C-3. Indeed, it is appealing to think that tedU may participate in this reaction
though there is currently no evidence to support this idea.
Figure 4.7 Proposed biosynthesis for 13-dTDM, TDM, and dhTDM.
Details for the biosynthesis can be found in the text.
One final observation worth commenting on is the lack of TDM-group molecules produced by S.
sp. LaPpAH-202. It is probably worth clarifying that this strain doesn’t produce any “obvious”
TDM-group molecules. Many of the TDM-group molecules absorb light at 271 nm, thus making
them particularly easy to identify by HPLC. As a result, it is possible that LaPpAH-202 is
producing molecules from the ted cluster, but they may have a unique UV-Vis pattern.
77
Alternatively, I think it is more likely that the cluster is unexpressed. This was my thought when
I attempted to introduce the tedRW gene into LaPpAH-202. The fact that I was able to introduce a
control vector but not the ermE*p-tedRW plasmid suggests that the tedRW gene is somehow
causing toxicity. Perhaps there is a loss-of-function mutation in the resistance genes. Thus, when
the cluster is expressed, the cells cannot protect themselves.
In closing, the data in this chapter confirmed that the ted cluster is responsible for producing 13-
dTDM as well as several other TDM-group molecules. Additionally, I describe the creation of an
overproducer strain of 13-dTDM which can be used in future studies of this molecule. The gene
tedRW was also shown to be effective at driving overexpression of TDM-group molecules in S.
atroolivaceus and S. globisporus, suggesting that these organisms could be used as a source of
TDM and dhTDM.
78
79
Chapter 5. Summary, Future Directions, and Conclusions
5.1 Thesis Summary
WAC04657 is a wild-isolate Streptomyces that produces an antibiotic activity against MRSA.
Using bioactivity-guided assays, I purified and characterize the antibiotic responsible for this
activity. This molecule, 13-dTDM, is a novel antibiotic that belongs to the TDM-group of
molecules. Testing 13-dTDM in pure form confirmed that this molecule has anti-MRSA activity.
MICs against Gram positive organisms were found to be in the range of 1 to 8 μg mL-1
.
Since the 13-dTDM is produced in small quantities, I decided to rationally engineer an
overproducer strain of this molecule to facilitate future work. To do so, I sequenced the genome
of WAC04657 with several different technologies. Ultimately, I found PacBio to be the most
effective and assembled the genome into five contigs. By searching the genome for proteins
involved in tetronate ring biosynthesis, I was able to find the putative biosynthetic gene cluster. I
named this the ted cluster. By using TedF1 as a query in a BLAST search of the NCBI databses,
I found the same cluster in three other organisms: S. atroolivaceus, S. globisporus, and S. sp.
LaPpAH-202. By comparing the ted cluster found in each organism, I was able to map the
conserved “core” of the cluster.
To confirm that the ted genes were responsible for producing 13-dTDM, I disrupted one of the
biosynthetic enzymes (tedF1) and overexpressed a transcriptional regulator (tedR). Consistent
with my hypothesis, the strain with the tedF1 disruption failed to produce 13-dTDM while the
tedR overexpression strain overproduced 13-dTDM. This confirmed the ted cluster’s
involvement in 13-dTDM production. During these experiments, I observed that the expression
of four other molecules was altered by the mutants in the same was a 13-dTDM. I purified the
most abundant of these, W5.9, and found that it is related to 13-dTDM but lacks the
characteristic tetronate ring. Next, I overexpressed the tedRW gene in S. atroolivaceus and S.
globisporus. I discovered that they did not produce 13-dTDM or W5.9, but were producers of
TDM and dhTDM. These experiments also revealed that WAC04657 is a producer of TDM.
5.2 Future Directions
80
5.2.1 Overproducer strain of TDM
From very early in my thesis it has been my goal to build an overproducer strain of 13-dTDM.
Yet my experience over the course of this thesis has led me to believe future efforts should focus
on TDM, not 13-dTDM. In the fourth chapter I showed that yields of the TDM-group molecules
can be increased by overexpressing the tedR gene. While this strategy certainly works, I have
preliminary data which suggests that the degree of overproduction is relatively moderate. Given
that the tedR gene is being driven by a very strong promoter yet the increase in 13-dTDM is
limited, this suggests that 13-dTDM is already being produced at near maximum velocity. Thus,
its production must be limited by some other factor (likely metabolic). Further, the data in this
thesis clearly illustrates that the amount of TDM produced by S. atroolivaceus and S. globisporus
is much greater than the amount of 13-dTDM being produced by WAC04657 (compare Figure
4.2, Figure 4.4, and Figure 4.5). Thus, taking all this information together, I propose that the best
way to further study these molecules is to study TDM, not 13-dTDM.
To someone taking over this project, I would initially recommend finding a set of ideal
conditions for extracting the molecule. To do this, I recommend performing a series of
extractions, both on different media types as well as at different time points. I would perform this
work with the +tedR strains of S. atroolivaceus and S. globisporus. The use of the +tedR strain is
recommended because it both increases yields of TDM and circumvents any endogenous
regulation of the gene cluster. Additionally, our group has also started working with liquid
cultures extracted with resins and seen improved yields; this may be worth considering. Lastly,
consideration should be given to “ease of use” of the strain. I will immediately mention that S.
globisporus grows quickly, sporulates prodigiously, and is easy to conjugate. In contrast, S.
atroolivaceus is slower growing and does not sporulate well on MYM, though conjugations
proceed well.
If the yields provided by this are insufficient for larger experiments (e.g. mouse studies), then I
cautiously advise returning to the PAC library and screening for the half of the ted cluster which
PAC292 did not capture. It may be possible to combine the two halves of the cluster into the full
cluster. Introduction of the tedW cluster into a chassis strain is expected to increase 13-dTDM
yields. Moreover, making mutations in the cluster would be facilitated by having the cluster
81
available in an E. coli host strain. Extending upon this idea, it would be of value to
overexpress/delete the tedL and tedT genes to see if yields can be further increased.
5.2.2 TDM-group molecules as drugs
One of the largest unanswered questions in my thesis is whether TDM could be used as a
clinically-useful antibiotic. The ability of TDM to inhibit the growth of MRSA is valuable.
Encouragingly, in one of the original papers, TDM is briefly described as having no toxicity to
mice when injected intraperitoneally up to 100 mg kg-1
(Tsuchida et al., 1994). By contrast,
certain synthetic precursors of TDM have shown cytotoxicity against human cell lines (Tsuchida
et al., 1995c). These two results are in conflict. A clear answer is needed about the toxicity of the
TDMs so that they can be considered as antibacterials or as alternate drugs (anti-cancer, anti-
helmenthic, immunological, anti-fungal, etc.). Moving forward, suggested experiments would be
to: 1) determine cell culture cytotoxicity; 2) determine toxicity in a whole animal; and 3)
determine if the compound can be used as an in vivo antibiotic (possibly through the use of a S.
aureus abscess model in mice).
5.2.3 Mechanism of Action of the TDM-group molecules
One of the most interesting features of 13-dTDM and TDM is the Michael acceptor in their
structure. This suggests that they act by covalently binding to their target (an idea explored in
Gverzdys et al., 2016). If this is true, then TDM could be used as a probe for molecular biology.
By attaching a fluorophore or some other moiety to the 14-OH position (e.g. Tsuchida et al.,
1995c), TDM could be used to specifically label its molecular target. To do this, I initially
envisioned labelling the 14-OH with biotin and performing a pulldown experiment. Early
attempts to synthesize the molecule resulted in loss of antibiotic activity against whole cells
(Gverzdys & Kramer, unpublished results). The reason for the loss of activity was not
investigated though it is possible that the biotin label altered the permeability of the molecule or
interfered with the ability of the molecule to interact with its target. Regardless, there are several
other related proteomic strategies that could be used to find the target. For example, instead of
performing this experiment with whole cells, the biotinylated TDM can be mixed with a protein
extract, thus removing problems associated with cell permeability. Alternatively, the use of
biotin could be abandoned for a different functional group (e.g. an alkyne handle for use with
click chemistry) (a discussion on affinity tags can be found in Ziegler et al., 2013). Another
82
viable method would be a whole-cell proteomics approach. Again assuming that TDM binds
covalently to its target, comparative mass spectrometry could be used to assay for proteins which
have been “labelled” by TDM relative to an untreated control (either by a shift in their mass or
by altered susceptibility to protease treatment). Even if the interaction is not covalent, a method
like TICC (“target identification by chromographic coelution,” briefly discussed in Section 1.2)
also provides a powerful method for target identification (Chan et al., 2012). While I am
confident that the molecular target of TDM can be identified, there are two primary
considerations that should be made when performing these experiments. First, it is possible that
the target may be a membrane protein. If this is the case, the above proposed experiments may
have to be redesigned to ensure efficient extraction of membrane proteins. Second, one of the
challenges of working with TDM is its poor solubility in aqueous solutions. Thus, I would
recommend performing these experiments on solid agar impregnated with TDM. Cells can then
be plated on the agar, incubated, and then collected by washing the plate with saline.
5.2.4 Curious features of the ted cluster
Outside of the above major directions, there are four smaller questions that I think also merit
study: 1) is TedU responsible for producing W5.9; 2) why don’t S. atroolivaceus and S.
globisporus produce 13-dTDM; 3) are tedX3-X5 responsible for producing another molecule that
acts synergistically with the TDMs; and 4) why is there an ABC-importer in the ted cluster.
Is TedU responsible for producing W5.9? W5.9 is of particular interest because my proposed
biosynthesis cannot account for it. As I suggested above, it is enticing to think that W5.9 is a
consequence of TedU’s activity. TedU is further made interesting by its almost complete lack of
homologs. A BLAST search of the RefSeq database finds numerous hits from other
actinomycetes but only a single homolog with >90% query coverage. Most of these other hits
align to the domain of unknown function (DUF4157) on the N-terminus but not the rest of the C-
terminus. Is this protein a jumping gene? Could it be of viral origin? Studying this protein could
assign the first known function to DUF4157 and shed light on the biosynthesis of W5.9. The
most logical way to start studying this protein would be to overexpress tedU in S. globisporus
and look for disruption of normal TDM biosynthesis.
Why don’t S. atroolivaceus and S. globisporus produce 13-dTDM? This question digs into the
enzymatic details about the biosynthesis of the TDM-group molecules. My biosynthetic pathway
83
requires that 13-dTDM be produced by any strain which produces TDM, yet it was never
observed in S. atroolivaceus or S. globisporus. A simple explanation for this is that conversion of
13-dTDM to TDM occurs very rapidly in S. atroolivaceus and S. globisporus. Then why does
13-dTDM accumulate in WAC04657? Perhaps WAC04657 possesses a mutation in (or in the
expression of) the tailoring enzyme which catalyzes this step. I remind the reader that TDM is
still produced by WAC04657, just in very small amounts. This suggests that, if there is a
mutation, this mutation is reducing the efficiency of the reaction, not completely inactivating it.
Assuming this idea is correct, then a way to test this would be to make mutations in S.
globisporus and see if these mutations result in loss of TDM and dhTDM and accumulation of
13-dTDM.
Are the genes tedX3-X5 responsible for producing another molecule that acts synergistically
with TDM? The genes tedX3 to X5 are present in only S. atroolivaceus and S. globisporus, and
they appear to code for a lantibiotic gene cluster. Since the lantibiotic cluster and the ted cluster
are oriented divergently from each other (recall Figure 3.7), it is enticing to assume that they are
co-expressed. Further, it would be exciting to know if they act synergistically. The first step to
studying the lantibiotic would be to express it in a chassis strain. Given the small size of these
genes (~5 kb), they are a reasonable target for PCR. When expressed in the chassis, the produced
molecule could be purified and subjected to synergy studies with TDM and dhTDM.
Why is there an ABC-importer in the ted cluster? This may be the one question that I have
thought about the most in the past year. The genes tedA1 to A4 encode an ABC-transporter. At
first glance, I assumed this transporter was an exporter and thus a resistance factor. Yet the gene
tedA1 is a putative substrate-binding protein, strongly suggesting that this transporter is an
importer (Figure 5.1A). This brings up a very interesting question: why would this cluster need
an importer? The protein tedG (which is immediately upstream of tedA1) is a ferredoxin. Perhaps
the cluster imports its own iron. Alternatively, perhaps the transporter imports a signal molecule
that interacts with TedL or TedT. The signaling molecule idea is alluring because it suggests that
the cluster could regulate its own expression as opposed relying on a transcription factor from
the core genome (Figure 5.1B). The advantage to such regulation is that, after being horizontally
transferred to a new host (or a host receiving a new gene cluster), the expression of the new gene
cluster could be easily controlled by the host (Figure 5.1C). I refer to this model as my
Plug’n’Play model. This would be in contrast to having to “hardwire” the cluster into the genetic
84
framework by serendipitously mutating the promoter of the cluster’s master regulator so that a
pleiotropic regulator can activate expression of the antibiotic at the appropriate time. Indeed, I
have some preliminary co-culture data which suggests that WAC04657 dis. tedF1 can induce
TDM production in S. atroolivaceus on MYM agar (recall that wild-type S. atroolivaceus does
not produce TDM on MYM). This supports the idea of a diffusible signal, though it is hard to
rule out if this is a primary metabolite or another molecule. Thus, the best approach may be to
purify TedA1, bind it to a column, and flow cell lysate over the column. The bound signal can
then be eluted and identified by MS, or the protein can be eluted and crystallized with the bound
signal molecule.
85
Figure 5.1 Plug n’ Play model for control of a biosynthetic gene cluster with a conserved
peptide signal.
A) The ABC transporter in the ted cluster is composed of four genes. TedA1 is annotated as a
putative oligopeptide-binding substrate-binding protein. TedA2 is a permease. TedA3 is a
permease. TedA4 is an ATP-binding protein. B) In my proposed model, a conserved peptide
signal is encoded in the core of the Streptomyces genome. The signal is released and re-imported
back into the cell by the ABC importer encoded within the biosynthetic gene cluster. The peptide
86
signal then interacts with an appropriate transcriptional regulator (perhaps a TetR-family
regulator or an LAL-family regulator) which ultimately stimulates production of the cognate
antibiotic. C) The advantage of this form of regulation is that a bacterium which receives a new
biosynthetic gene cluster via horizontal gene transfer can immediately control the new cluster.
As long as the core genome encodes the appropriate peptide signal and that the cluster is able to
respond to that signal, than the host organism needs only to control expression of the signal in
order to control expression of a new cluster. This model is inspired by CSH from B. subtilis
(Lazazzera and Grossman, 1998).
5.3 Conclusion
I am proud to say that the work reported in this thesis will significantly advanced the field’s
knowledge about the TDM-group of molecules, and also contributes to the larger field of
tetronate molecules. During my investigations I found two new TDM-group molecules and also
identified the biosynthetic gene cluster responsible for producing all the TDM-group molecules.
Each of these discoveries has brought with it many mysteries and questions. Many of these
questions are bigger than the TDM-group of molecules. Indeed, the work described here has
started to tease at truly interesting questions in the field. For example, how do molecules evolve
and how do you generate diversity of structures? Hopefully the work I presented here and the
work I complete in the future will somehow contribute to answering these exciting questions.
87
88
Chapter 6. Materials and Methods
6.1 General Experimental Procedures
6.1.1 Equipment information
HPLC was performed on a Waters Alliance e2695 equipped with an inline Waters 2998
photodiode array and a Waters Fraction Collector III. High-resolution mass spectra were
measured using a Waters Acquity UPLC-Xevo G2-S QTof. UV/visible spectra were recorded
with a Beckman DU-530 Spectrophotometer. Optical rotation was measured on a Rudolph
Research Analytical Autopol IV automatic polarimeter. 1D and 2D NMR data for 13-dTDM
were acquired on a Bruker Avance III 700 MHz NMR spectrometer equipped with a 5 mm QNP
cryoprobe operating at 700.17 MHz for 1H NMR and 176.08 MHz for
13C NMR. For W5.9,
TDM, and dhTDM, 1H NMR and 2D NMR spectra were acquired on an Agilent DD2-700 MHz
NMR spectrometer with a 1H-19F {13C/15N} 5 mm Triple Resonance Cold Probe; 13
C NMR
spectra were acquired on an Agilent DD2-500 MHz NMR with an XSens Cold Probe. DNA
oligonucleotides were ordered from Integrated DNA Technologies (IDT, www.idtdna.com).
Sanger sequencing was performed by either MOBIX Labs at McMaster University or TCAG at
the University of Toronto.
6.1.2 Primers used
Table 6.1 Primers used in this thesis.
# Name Sequence Notes
1 PSET FWD GCT GGC GAA AGG GGG
ATG T
Forward primer for sequencing inserts
in the pSET152 multiple cloning site
2 PSET REV TAG CTC ACT CAT TAG
GCA CC
Reverse primer for sequencing inserts
in the pSET152 multiple cloning site
52 SCI68.04upFw TAT TCT AGA TCC TCG
TGG GTT CGC GGA CG
For building tedF1 KO plasmid,
amplicon is 1.2 kb, partner is primer
53
53 SCI68.04upRv TAT GGA TCC CTC GAC
CAT TCC CAG CTC TC
For building tedF1 KO plasmid,
amplicon is 1.2 kb, partner is primer
52
54 SCI68.04dnFw TAT GGA TCC CTG CAC
CAG TGA AGA ACC TC
For building tedF1 KO plasmid,
amplicon is 1.2 kb, partner is primer
55
89
55 SCI68.04dnRv TAT GAA TTC CTA CCG
TCT CCG AGT TTC GG
For building tedF1 KO plasmid,
amplicon is 1.2 kb, partner is primer
54
61 68.04F CGC CGA GGT TCT TCA
CTG GTG CAG
For building tedF1 KO plasmid,
amplifies the full tedF1 gene, amplicon
is 2kb
62 68.04R AAG GGC GGC TTT AGA
ACG CTC GCC AA
For building tedF1 KO plasmid,
amplifies the full tedF1 gene, amplicon
is 2kb
63 68.04disF AAC ACG GTG TGG GGC
GGC GT
For building tedF1 KO plasmid, For
amplifying the tedF1 fragment from
WAC04657, partner is primer 64,
amplicon is 0.8 kb
64 68.04disR GCT GAG CAG GAA GTT
GTC GAT GAC
For building tedF1 KO plasmid, For
amplifying the tedF1 fragment from
WAC04657, partner is primer 63,
amplicon is 0.8 kb
65 M13F GTA AAA CGA CGG CCA
GT
common sequencing primer
66 M13R CAG GAA ACA GCT ATG
AC
common sequencing primer
67 293.05F AGG AGG TCA CAC AAG
ACA TGC GAT TCG AAA
TC
For amplifying tedR from WAC04657,
amplicon is 0.8 kb, partner is primer
68, introduces a consensus RBS
(AGGAGG) (please refer to the work
of Pak (2010) )
68 293.05R ACC GCT CGT TTC ACG
CCG CT
For amplifying 293.05 from
WAC04657, amplicon is 0.8 kb,
partner is primer 67
69 PAC.68.04f CCG AGC ATC CGG ACA
CAC
for screening PAC library for contig
68, amplicon is 448 bp, partner is
primer 70
70 PAC.68.04r AGC AGA CGC GCG TTG
GC
for screening PAC library for contig
68, amplicon is 448 bp, partner is
primer 69
71 PAC.293.05f CTG AGC GCT GCC AGT
CTC A
amplicon is 605 bp, partner is primer
72
72 PAC.293.05r AGC GCG TAG AGC GGA
CC
amplicon is 605 bp, partner is primer
71
84 pESAC13 fwd CTT GAC ATT GTA GGA
CTA TAT TGC TCT
Use of this primer was not reported in
this thesis, for sequencing in out of
pESAC13 from the P1 rep side into the
genomic DNA
85 pESAC13 rev CTT CTG TAT GTA CTG
TTT TTT GCG ATC T
Use of this primer was not reported in
this thesis, for sequencing out of
pESAC13 from the sacB side into the
chromosomal DNA
90
81 16S rRNA for AGA GTT TGA TCC TGG
CTC AG
For amplifying 16S rRNA for the
purpose of sequencing and genotyping,
use a 2 min extension time
82 16S rRNA rev ACG GCT ACC TTG TTA
CGA CTT
For amplifying 16S rRNA for the
purpose of sequencing and genotyping,
use a 2 min extension time
6.1.3 Strains used
Table 6.2 Personal strains used in this thesis.
F# Name Notes
31 B. subtilis 168 indicator strain
43 E. coli ATCC 25922 indicator strain
61 E. coli ET12567/pUZ8002 +
pSET152-ermE*p-null
for conjugating pSET152-ermE*p-null into
Streptomyces
82 E. coli ET12567/pUZ8002 Conjugation donor strain (Flett et al., 1997)
91 S. sp. Ja2b + pSET152-ermE*p-
null
apramycin resistance, F#176 with F#61 integrated
into the genome
93 E. coli XL1 Blue basic cloning E. coli from Stratagene
176 S. sp. strain WAC04657 sequenced, previously known as S. sp. Ja2b or S.
cinereus, received from Michael Hart who received
it from the Wright Lab at McMaster, TDM-group
producer
201 E. coli DH5a + pOJ260 suicide vector, apra resistance, oriT (Bierman et al.,
1992)
205 E. coli + pHP45omega spectinomycin resistance, ampicillin resistance,
Justin Nodwell Collection #728 (Prentki and Krisch,
1984)
206 E. coli XL1 Blue + pSET152-
ermE*p-null (version LK)
apramycin resistance, received from Leslie
Cutherbertson (LC#230) who received it from
Lindsay Kalan in Wright Lab at McMaster , differs
from the typical pSET152 plasmid because it already
has the ermE*p promoter built in (Bibb et al., 1985,
1985)
224 E. coli XL1 Blue + pOJ260-Dn
fragment (in BamHI/EcoRI site)
apramycin resistance
229 E. coli XL1 Blue + pOJ260-
Up+Dn
apramycin resistance
231 E. coli XL1 Blue + pOJ260-
Up+Omega+Dn
apramycin resistance, spectinomycin resistance
237 E. coli XL1 Blue + pOJ260-Up
(in XbaI/BamHI site)
apramycin resistance
263 E. coli XL1 blue + pOJ260-
68.04 fragment (cand. 2)
apramycin resistance; published as pOJ260-tedF1
(frag.) (Gverzdys and Nodwell, 2016)
267 Micrococcus luteus indicator strain
91
268 E. coli XL1 blue + pSET152-
ermE*p-293.05
apramycin resistance, backbone was F#206,
published as pSET152-ermE*p-tedR (Gverzdys and
Nodwell, 2016)
270 WAC04657 68.04 disruption
clone 1
apramycin resistance, F#263 integrated into genome,
TDM-group-null phenotype
271 WAC04657 68.04 disruption
clone 2
apramycin resistance, F#263 integrated into genome,
TDM-group-null phenotype
272 WAC04657 68.04 disruption
clone 4
apramycin resistance, F#263 integrated into genome,
TDM-group-null phenotype
273 WAC04657 + pSET152-
ermE*p-293.05 Brown 1
apramycin resistance, F#268 integrated into genome,
produced a brown pigment when picked on
conjugation plate, TDM-group overexpression
phenotype
274 WAC04657 + pSET152-
ermE*p-293.05 Brown 2
apramycin resistance, F#268 integrated into genome,
produced a brown pigment when picked on
conjugation plate, TDM-group overexpression
phenotype
275 WAC04657 + pSET152-
ermE*p-293.05 Brown 3
apramycin resistance, F#268 integrated into genome,
produced a brown pigment when picked on
conjugation plate, TDM-group overexpression
phenotype
276 WAC04657 + pSET152-
ermE*p-293.05 No Brown 1
apramycin resistance, F#268 integrated into genome,
did not produce a brown pigment when picked on
conjugation plate, TDM-group overexpression
phenotype
277 WAC04657 + pSET152-
ermE*p-293.05 No Brown 2
apramycin resistance, F#268 integrated into genome,
did not produce a brown pigment when picked on
conjugation plate, TDM-group overexpression
phenotype
278 WAC04657 + pSET152-
ermE*p-293.05 No Brown 3
apramycin resistance, F#268 integrated into genome,
did not produce a brown pigment when picked on
conjugation plate, TDM-group overexpression
phenotype
351 E. coli + 4504-1-3J PAC library
clone (white cap), 5' ted cluster
contains DNA from unitig 1 from 702 to 802 kb (5'
side of the ted cluster)
352 E. coli + 4504-1-16M PAC
library clone (blue cap), not ted
cluster
contains DNA from unitig 1 from 280 to 356 kb
(does not contain ted cluster)
356 Streptomyces atroolivaceus
ATCC 19725
From Cederlane (ATCC)
357 Streptomyces globisporus ssp.
globisporus NRRL B-2293
From USDA Agriculture Research Services
412 Streptomyces LaPpAH-202 From Caitlin Carlson in Cameron Currie's Lab (U. of
Wisconsin-Madison)
419 S. atroolivaceus + pSET152-
ermE*p-null 1
apramycin resistance, F#356 with F#61 integrated
into the genome
420 S. atroolivaceus + pSET152-
ermE*p-null 2
apramycin resistance, F#356 with F#61 integrated
into the genome
92
421 S. atroolivaceus + pSET152-
ermE*p-null 3
apramycin resistance, F#356 with F#61 integrated
into the genome
422 S. atroolivaceus + pSET152-
ermE*p-tedR(WAC04657) 1
apramycin resistance, F#356 with F#268 integrated
into the genome, TDM-group overexpression
phenotype
423 S. atroolivaceus + pSET152-
ermE*p-tedR(WAC04657) 2
apramycin resistance, F#356 with F#268 integrated
into the genome, TDM-group overexpression
phenotype
424 S. atroolivaceus + pSET152-
ermE*p-tedR(WAC04657) 3
apramycin resistance, F#356 with F#268 integrated
into the genome, TDM-group overexpression
phenotype
425 S. globisporus + pSET152-
ermE*p-null 1
apramycin resistance, F#357 with F#61 integrated
into the genome
426 S. globisporus + pSET152-
ermE*p-null 2
apramycin resistance, F#357 with F#61 integrated
into the genome
427 S. globisporus + pSET152-
ermE*p-null 3
apramycin resistance, F#357 with F#61 integrated
into the genome
428 S. globisporus + pSET152-
ermE*p-tedR(WAC04657) 1
apramycin resistance, F#357 with F#268 integrated
into the genome, TDM-group overexpression
phenotype
429 S. globisporus + pSET152-
ermE*p-tedR(WAC04657) 2
apramycin resistance, F#357 with F#268 integrated
into the genome, TDM-group overexpression
phenotype
430 S. globisporus + pSET152-
ermE*p-tedR(WAC04657) 3
apramycin resistance, F#357 with F#268 integrated
into the genome, TDM-group overexpression
phenotype
Table 6.3 Nodwell shared BSL2 strains used in this thesis.
BSL2# Name Notes
JN44 S. aureus ATCC 29213
JN45 P. aeruginosa PAO1
JN47 S. epidermidis ATCC 12228
JN48 S. aureus ATCC BAA-41 methicillin-resistant
JN49 S. aureus ATCC BAA-44 multidrug-resistant
JN50 E. faecalis ATCC 29212
JN51 B. cepacia ATCC 25416
JN52 A. baumannii ATCC 19606
JN53 K. pneumoniae ATCC 13883
6.1.4 General culture methods
All E. coli strains and indicator strains were grown at 37 °C on LB agar in a stationary incubator
or in LB broth with shaking at 200 rpm. If a strain was labeled as a BSL2 strain (Table 6.3) then
liquid cultures of these strains were grown in 15 mL conical tubes with the cap on to prevent the
release of aerosols from the culture. Streptomyces were almost exclusively grown on MYM agar.
93
Growth temperature for Streptomyces was 30 °C. Spore stocks were made on MYM agar
following common methods, with the added note that S. atroolivaceus was grown on R2 agar
without sucrose (R2-S agar) (Kieser et al., 2000). Where needed, media was supplemented with
apramycin (50 μg mL-1
), chloramphenicol (25 μg mL-1
), kanamycin (50 μg mL-1
), or nalidixic
acid (25 μg mL-1
).
Additional notes about each of the Streptomyces I used are as follows. Note that the sporulation
data is based on 6 days of growth at 30 °C, though sporulation often occurs sooner.
WAC04657 sporulated well on MS, R2YE, MYM, ISP4, and R2-S. It sporulated poorly on YPD.
It made nice, flat colonies which adhered well to ISP4 (3 days at 30 °C) which were amenable
for velvet cloth replicating. It sporulated slowly on ISP2 (3 days at 30 °C). It made tiny colonies
which adhered well to MS agar (3 days at 30 °C), again useful for replicating. WAC04657
colonies didn’t adhere well to MYM, but they grew and sporulated very well on this media. As a
result, MYM was my general culture media for WAC04657, but I had to be extremely careful
when performing soft agar overlays of colonies on this media.
For S. atroolivaceus, it sporulated well on RS-2 and ISP4, but sporulated poorly on MS, YPD,
R2YE, and MYM. While I observed that this strain was the strongest producer of TDM (result
needs to be confirmed), it was an inconsistent sporulator (i.e. it was difficult to work with). I had
had some preliminary luck getting prodigious sporulation by plating on R2-S and incubating at
room temperature for an extended period of time (note that I wrapped the plate with parafilm to
prevent it from drying out).
For S. globisporus, it sporulated well on MYM agar, but sporulated very poorly on MS, YPD,
R2YE, ISP4, and R2-S.
For S. sp. LaPpAH-202, it sporulated well on MS, R2YE, MYM, ISP4, R2-S, and IWL4 agar,
but sporulated poorly on YPD, LB, and ISP2.
6.1.5 Media recipes
LB media was made by dissolving 10 g of peptone from casein, 5 grams of yeast extract, and 10
g of NaCl either with or without 20 g of agar in 1 L of filter purified water.
94
MYM media was made by dissolving 4 g D-(+)-maltose monohydrate, 4 g yeast extract, and 10 g
malt extract either with or without 20 g Bacto brand agar (specifically) in 1 L of 1:1 tap
water:filter purified water. 2 mL per L of trace elements (Kieser et al., 2000) were added to the
molten agar immediately before use. If I was using the agar while still molten from the
autoclave, then I added the trace elements prior to autoclaving.
R2-S (“R2 minus S”) was made by first making a base and then adding additional ingredients to
it before pouring the plates. The base was made with 0.313 g of K2SO4, 12.65 g of MgCl2⋅6H2O,
0.125 g of Difco casamino acids, 12.6 g of dextrose and water to 1 L. The base was then divided
into 250 mL portions in 500 mL flasks and 6.32 g of Bacto brand agar was added to each flask. If
liquid media was required, then the agar was omitted and the RS-2 base put into 100 mL bottles.
After autoclaving, additional ingredients were added (note: I will provide two numbers for each
ingredient, the first number will be for a 250 flask and the second, bracketed number will be for a
100 mL bottle). To the base, I added 3.125 mL (1.25 mL) of 0.5% KH2PO4, 25 mL (10 mL) of
3.68% CaCl2⋅2H2O, 4.7 mL (1.88 mL) of 20% proline, 31.25 mL (12.5 mL) of 5.73% TES at
pH7.2, 0.625 mL (0.250 mL) of trace elements (Kieser et al., 2000), and 1.56 mL (0.624 mL) of
1 N NaOH.
R2YE was made in almost the same fashion as R2-S. The first difference is that I added 128.75 g
of sucrose to the base. After autoclaving, the same ingredients as for R2-S were added to the
base, except that after the addition of TES and before the addition of trace elements, I added
15.63 mL (6.25 mL) of 10% yeast extract (note: when making the 10% yeast extract solution, it
needs to be gently heated to dissolve).
MS agar was made of a mixture of two solutions. Solution A was made by mixing 60 g of soy
flour with 1.8 L of tap water in a 2 L flask. The soy flour was added slowly to the flask while
simultaneously heating and stirring. Solution B was made by adding 60 g of mannitol to 1.2 L of
tap water in a 2 L flask and swirling to dissolve. Then, 200 mL of solution B and 300 mL of
warm solution A were added to a fresh 2 L flask. 8 g of Bacto brand agar was added to each flask
and the solution was autoclaved for two 15 min cycles. If the MS plates were intended for
conjugations, then 1 mL of 1M MgCl2 was added to each 100 mL of MS agar after reheating the
agar and before pouring plates.
95
ISP2, also called yeast malt extract (YM) agar, was made by dissolving 4 g of yeast extract, 10 g
of malt extract, and 4 g of dextrose either with or without 20 g of Bacto brand agar in 1 L of
filter purified water.
ISP4 is purchased as a powder from BD (product number 277210). It was prepared as described
by the manufacturer.
YPD was made by dissolving 10 g of yeast extract, 20 g of peptone, and 20 g of dextrose with or
without 20 g of agar in 1 L of filter purified water.
IWL4 was made by dissolving 37 g of ISP4 powder, 0.5 g of yeast extract, 1.0 g of tryptone in 1
L of water (note that the ISL4 already has agar in it). After autoclaving, 20 mL per L of 1 M
MgCl2 were added to the media.
YEME is a media which is used for isolating DNA from Streptomyces. In particular, it has
sucrose to act as an osmoprotectant, glycine to weaken the peptidoglycan, and MgCl2 to assist
with the activity of the lysozyme. It was made by dissolving 3 g of yeast extract, 5 g of peptone,
3 g of malt extract, 10 g of dextrose, 110 g of sucrose in 1 L of water. It was aliquoted into 100
mL portions in bottles. After autoclaving, add 0.5 mL of 1 M MgCl2 and 3.3 mL of 15% glycine
were added to each 100 mL of YEME base.
6.1.6 General PCR Protocol
Table 6.4 Standard PCR reagents.
Reagent Volume (μL)
Primer 1 (10 μM) 2.5
Primer 2 (10 μM) 2.5
Template 1.0
dNTPs (25 mM) 1.0
Thermopol Buffer (10x) 5.0
Vent (NEB) 1.0
DMSO 0 to 5 μL
H2O to 50 μL
The thermocycler is programed as follows:
96
95 °C for 10 minutes
30 cycles of:
30 s at 95 °C
30s at Ta
text at 72 °C
2 min at 72 °C
Please note that while the above protocol is the one that I used throughout this thesis, I strongly
recommend switching to a more modern polymerase. Recently, I have had excellent results with
the KOD Xtreme Hot Start DNA Polymerase (EMD Millipore) using the protocols provided with
the kit.
6.2 DNA purification methods
6.2.1 Plasmids and PAC clone isolation
Plasmids were isolated using a variety of commercially available kits. PAC clones were purified
using the QIAGEN Midi Kit while following the protocol for very low-copy plasmids and using
the QIAGEN-tip 100.
6.2.2 DNA isolation for phylogenetic tree
To isolate WAC04657 chromosomal DNA, a 250-mL baffled flask with 25 mL of MYM broth
and 2.5 mL of 10% glycine was inoculated with WAC04657 spores and incubated for 12 h in a
shaking incubator. Note that the WAC04657 failed to produce brown pigment and resembled a
culture of E. coli. The culture was not very dense and did not produce the typical Streptomyces
smell. After 12 hours, I was able to collect about 40 mg of cells (50 μL worth). The cells were
collected by centrifugation at 3000 × g for 10 min and all but 3 mL of the supernatant was
removed. The loose pellet was suspended in the remaining fluid and aliquots were iteratively
pelleted in a microcentrifuge at 16,000 × g for 1 min until all supernatant had been removed.
DNA was then extracted from the cells with the DNeasy Blood and Tissue kit (Qiagen) using the
“Pretreatment for Gram positive Bacteria” protocol provided by the manufacturer.
6.2.3 DNA isolation for WGS 454 and Illumina Sequencing
DNA was extracted in the same manner as reported in section 6.2.2.
6.2.4 DNA isolation for PacBio Sequencing
97
WAC04657 was cultured and pelleted in the same fashion as described in section 6.2.2. The
DNA was then extracted from the cells using the QIAGEN Genomic Tip 20/G kit and buffers. I
followed a modified version of the manufacturer’s protocol. In the early steps of the protocol I
used the the lysis protocol from the 100/G tip (instead of the 20/G tip protocol). That is, I
suspended the entire culture in 3.5 mL of lysis buffer using a Teflon or ground-glass tissue
disruptor and incubated, as described in the manual, to lyse the cells. I was careful to ensure that
the lysis went to completion (approx. 3 hrs on a rocker in a stationary incubator set to 37 °C).
The presence of particulates floating in the lysate was an indicator of incomplete lysis. Any
particulates were removed by centrifuging. The entire lysate was then applied to the 20/G tip
(using slight pressure applied with a 5 mL syringe). The 20/G protocol was then continued as
described by the manufacturer.
Moving forward, I would recommend studying the growth conditions of WAC04657 grown in
liquid YEME as a source of cells for DNA extraction.
6.3 Bioassays
6.3.1 Colony diffusion assays
Petri dishes (9 cm diameter) with 25 mL of MYM agar were spotted with 105 CFU of
WAC04657 spores suspended in 5 μL of 0.85% saline. The droplets were dried under laminar air
flow. Plates were incubated for 48 h at 30 °C and overlaid with 5 mL molten, extra-soft LB agar
(2.5 g agar in 1L LB broth) containing 0.5% (v/v) of indicator organism from overnight culture.
Plates were incubated at 37 °C overnight and zones of clearance were measured from the edge of
the Streptomyces colony to the edge of the zone of clearance.
6.3.2 Solid Agar MICs
LB agar was melted and cooled to 55 °C in a water bath. Two-fold dilutions of molecule from
64 μg mL-1
to 1 μg mL-1
(final concentration in agar) were added to the molten agar and poured
into petri dishes. Overnight cultures of each indicator organisms were diluted to OD600 0.001 in
LB broth and 5 μL of this was spotted onto the antibiotic plates and allowed to dry. Plates were
then incubated for 16 h at 37 °C. The MIC was defined as minimum concentration of molecule at
which no growth of the indictor organism could be visually detected.
98
6.4 Molecule purification and HPLC methods
6.4.1 HPLC Methods
Table 6.5 HPLC Method 1
Method Name: “WAC04657 crude C18 ana col"
A = H2O + 0.1% Formic Acid, B = HPLC Acetonitrile-190 + 0.1% Formic Acid
flow = 1 mL min-1
column temp. = 35℃
column = XSELECT CSH C18 5 μm 4.6×150 mm
injection volume: 20 μL
time (min) %B
0 20
3 40
8 40
8.5 95
10.5 95
11 20
15 20
Table 6.6 HPLC Method 2
Method Name: “5_9 PFP ana col”
A = H2O + 0.1% Formic Acid, B = HPLC Acetonitrile-190 + 0.1% Formic Acid
flow = 1 mL min-1
column temp. = 35℃
column = Phenomenex 250 x 4.60 mm Luna 5u PFP(2) 100A
time (min) % B
isocratic 25
Table 6.7 HPLC Method 3
Method Name: "6_3 and 6_4 PFP ana col"
A = H2O + 0.1% Formic Acid, B = HPLC Acetonitrile-190 + 0.1% Formic Acid
flow = 1 mL min-1
column temp = 35℃
column = Phenomenex 250 x 4.60 mm Luna 5u PFP(2) 100A
time (min) %B
0 28
10 28
15 32
15.5 80
17.5 80
18 28
23 28
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Table 6.8 HPLC Method 4
Method name: “8_0 PFP ana col”
A = H2O + 0.1% Formic Acid, B = HPLC Acetonitrile-190 + 0.1% Formic Acid
flow = 1 mL min-1
column temp. = 35℃
column = Phenomenex 250 x 4.60 mm Luna 5u PFP(2) 100A
Note: when I ran this method with numerous injections, it was important to wash frequently with
100% MeCN to reduce the column backpressure
time (min) % B
0 20
3 40
12 40
12.5 20
16.5 20
6.4.2 Small-scale crude extract analysis of Streptomyces
105 colony forming units of Streptomyces spores were suspended in 50 μL of 0.85% saline and
then were spread as a lawn onto small petri dishes containing 10 mL MYM agar. The plates were
then incubated at 30 °C. After the desired growth time (growth time varied, see individual
figures for length of time incubated), the agar and cell mat were cut into 1 cm2 chunks and
extracted with 10 mL of ethyl acetate overnight. The extract was separated from the agar chunks
by filtration through Whatman paper, collected in test tubes, and dried in a centrifugal evaporator
set to a maximum temperature of 30 °C. The dried extract was suspended in 200 μL of 50%
aqueous acetonitrile + 0.1% formic acid by sonication, washed with an equal volume of hexane,
and centrifuged at 21,000 × g for 30 min at 4 °C to remove particulates. The samples were then
loaded into robovials and analyzed on the HPLC using HPLC method 1 (Table 6.5). Analysis of
the traces was typically performed on the computer attached to the HPLC using the softward
provided by the manufacturer. For the purpose of publication, HPLC traces were exported as a
table, graphed in R, and then manipulated with Adobe Illustrator.
6.4.3 TLC, bioautography, and initial identification of 13-dTDM
A crude extract of WAC04657 was dissolved in chloroform to 4 mg mL-1
and 10 μL was spotted
on a silica TLC plate. The TLC plate was developed in 1:9 methanol:chloroform and visualized
by UV at 254 nm or by bioautography. Bioautography was performed by overlaying the TLC
plates with extra-soft LB agar mixed with a 0.5% (v/v) culture of B. subtilis 168. Plates were
100
incubated overnight at 37 °C, sprayed with thiazolyl blue tetrazolium bromide dye (MTT) (5 mg
mL-1
in H2O), and incubated for 1 h at 37 °C before visualizing. From a TLC plate run in
parallel, the band of silica gel corresponding to the zone of activity was scraped off the
aluminum backing and extracted with chloroform. The resulting extract was dried under vacuum,
suspended in DMSO, and subjected to HPLC using HPLC method 1 (Table 6.5).
6.4.4 Purification of 13-dTDM
Four hundred mL of MYM agar was poured into 24 cm × 32 cm dishes (i.e. Betty Crocker metal
baking trays), inoculated with approximately 6×107 colony forming units of WAC04657 spores,
and incubated for 5 days. The agar and cell mat was macerated and extracted with an equal
volume of ethyl acetate overnight. The extract was gravity filtered, dried in a rotary evaporator
under vacuum, dissolved in 2 mL of 50% aqueous acetonitrile, and loaded onto a 5 g Sep-Pak
C18 column (Waters). The column was washed first with 10 mL of H2O and then 10 mL of 30%
aqueous acetonitrile. 13-dTDM was eluted with 70% aqueous acetonitrile and dried by
lyophilization. This was then suspended in 50% aqueous acetonitrile, washed with hexane, and
subjected to an initial round of HPLC purification using HPLC method 1 (Table 6.5) while
monitoring at 271 nm. 13-dTDM eluted at approx. 8.0 min. The molecule was collected and
dried by lyophilization. After dissolving in DMSO, another round of HPLC using HPLC method
4 (Table 6.7) was used to purify the molecule further. 13-dTDM eluted at 6.75 min. The
molecule was collected and dried by lyophilization, extracted with chloroform, centrifuge to
remove the precipitate, and dried under vacuum to yield pure 13-dTDM.
6.4.5 Purification of W5.9
6.4 L of solid MYM agar culture of WAC04657 + pSET152-ermE*p-tedR was incubated for
48 h, macerated, and extracted with an equal volume of ethyl acetate overnight. The resulting
extract was filtered through cotton followed by a coffee filter followed by Whatman #1 filter
paper and dried under vacuum. The resulting oily, brown extract (481.7 mg) was suspended in
2 mL of 50% aqueous acetonitrile and loaded onto a Sep-Pak C18 column (Waters). The column
was washed with 10 mL each of 100% H2O and 30% aqueous acetonitrile. Molecule W5.9 min
was eluted with 70% aqueous acetonitrile, concentrated in a centrifugal evaporator (max. temp of
30 ℃) and brought to dryness by lyophilization (60.3 mg). This was dissolved in 500 μL of 50%
aqueous acetonitrile + 0.1% formic acid and, after a hexane wash and filtration through a 0.45
101
μm filter, the extract was purified by using HPLC method 1 (Table 6.5). The peak eluting at 5.9
minutes was collected and lyophilized to yield a white powder (5.7 mg). This was suspended in
50% aqueous acetonitrile + 0.1% formic acid, further purified using HPLC method 2 (Table 6.6).
The peak which eluted at 12.4 minutes was collected. After lyophilization, this yielded pure
W5.9 (2.6 mg).
6.4.6 Purification of TDM
Either S. atroolivaceus + pSET152-ermE*p-tedR or S. globisporus + pSET152-ermE*p-tedR
were grown on MYM agar for 4 days. The agar and cell mat was macerated, extracted with an
equal volume of ethyl acetate then concentrated to dryness. The extract was then suspended in
pure acetonitrile + 0.1% formic acid, washed with hexane and filtered through a 0.45 μm filter,
and separated by HPLC using HPLC method 1 (Table 6.5). The peak eluting at 6.4 minutes was
collected and lyophilized. The resulting molecule was suspended in 100% acetonitrile + 0.1%
formic acid and separated by HPLC using HPLC method 3 (Table 6.7) while monitoring at 271
nm. The peak eluting at 14.4 min was collected and lyophilized to yield pure TDM.
6.4.7 Purification of dhTDM
Either S. atroolivaceus + pSET152-ermE*p-tedR or S. globisporus + pSET152-ermE*p-tedR
were grown on MYM agar for 4 days. The agar and cell mat was macerated, extracted with an
equal volume of ethyl acetate then concentrated to dryness. The extract was then suspended in
pure acetonitrile + 0.1% formic acid, washed with hexane and filtered through a 0.45 μm filter,
and separated by HPLC using HPLC method 1 (Table 6.5). The peak eluting at 5.4 minutes was
collected and lyophilized. The resulting molecule was suspended in 100% acetonitrile + 0.1%
formic acid and separated by HPLC using HPLC method 3 (Table 6.7) while monitoring at 250
nm. The peak eluting at 11.2 min was collected and lyophilized to yield dhTDM.
6.5 Plasmid assembly and manipulation of Streptomyces
6.5.1 tedF1 knock out plasmid
102
Figure 6.1 Plasmid 231 map.
The plasmid was built as shown in Figure 6.1.To build this plasmid, I first cloned fragments of
genomic DNA from upstream and downstream of the tedF1 gene. To amplify the “up frag”, I
used the general PCR protocol (section 6.1.6) with the following details: primer 52 and 53,
template was WAC04657 chromosomal DNA (F#176), 5% v/v DMSO, Ta of 55 °C, text of 1 min
30 sec. The same method was followed to amplify the “dn frag”, except with primers 54 and 55.
The resulting amplicons were 1.2 kb. The amplicons were ligated into pOJ260 (F#201). The up
frag was inserted into the XbaI/BamHI sites resulting in plasmid F#237. The dn frag was ligated
into the EcoRI/BamHI sites resulting in plasmid F#224. Sequences of the up and dn frag were
confirmed with the M13F and M13R primers (primers 65 and 66). Once sequences were
confirmed, I excised the up frag from F#237 by digesting the plasmid with XbaI and BamHI. I
ligated the up fragment into the XbaI/BamHI site of F#224. The resulting plasmid was F#229. I
excised the omega fragment (spectinomycin resistance cassette) from pHP45Ω (F#205) by
digesting with BamHI and PstI (PstI was used to remove a co-eluting band). I gel extracted the 2
kb fragment and ligated this into the BamHI sit of F#229. The resulting plasmid was F#231.
Diagnostic digests were used to confirm proper assembly of the plasmid.
6.5.2 tedF1 disruption plasmid
The full length version of tedF1 was first amplified for use as a template for subsequent rounds
of PCR. I used the general PCR protocol (section 6.1.6) with the following details: primer 61 and
62, template was WAC04657 chromosomal DNA (F#176), 5% v/v DMSO, Ta of 55 °C, text 2
min 3 sec. The generated amplicon was 2.0 kb. The amplicon was purified using the QIAquick
PCR purification system as described by the manufacturer. To generate the tedF1 fragment, I
used the general PCR protocol (section 6.1.6) with the following details: primers 63 and 64,
103
template was the primer 61/62 amplicon, no DMSO, Ta 55 °C, text 53 sec. The generated
amplicon was 0.8 kb. This was purified using the QIAquick PCR purification system as
described by the manufacturer. Amplicon 63/64 was blunt-end cloned into the EcoRV site of
pOJ260 (F#201) to yield plasmid F#263.
6.5.3 tedR overexpression plasmid
tedR was amplified using the general PCR protocol (section 6.1.6) with the following details:
primer 67 and 68, template was WAC04657 chromosomal DNA (F#176), Ta of 55 °C, text of 53
sec. The generated amplicon was 0.8 kb. This was purified using the QIAquick PCR purification
system and blunt-end cloned into the EcoRV site of pOJ260 (F#201) to yield plasmid F#268.
Orientation was checked by diagnostic digest and sequence fidelity was checked by sequencing.
6.5.4 Streptomyces strain construction
Plasmids were introduced into E. coli ET12567/pUZ8002 by the heat shock protocol. The
resulting strains were used as donors in the standard E. coli-Streptomyces conjugation protocol
(Gust et al., 2006) (http://strepdb.streptomyces.org.uk/redirect/protocol_V1_4.pdf). Slight
modifications to the protocol were made. Instead of washing the donor E. coli in 10 mL of fresh
LB several times, cells were pelleted and transferred to microfuge tubes. Washing was then
performed at 16,000 × g using 1 mL volumes with four washing steps. Streptomyces were often
not heat shocked prior to conjugation. For WAC04657, due to the rapid speed that it grows, I
sometimes left the plates at room temperature to incubate instead of at 30 °C. Additionally, a
glass spreader was sometimes used to spread the antibiotic solution. If conjugations were not
working well on MS agar, then conjugations were also attempted on R2-S agar.
6.6 Genome analysis
6.6.1 Phylogenetic tree
16S rDNA was sequenced using the primers 81 and 82 resulting in a sequence of 1335 bp after
trimming. 16S rDNA sequences from other organisms were obtained by performing a BLASTn
search of the NCBI databases with the WAC04657 rDNA sequence as the query. The resulting
sequences were aligned with MUSCLE (Edgar, 2004), trimmed, and used in MEGA6 (Tamura et
104
al., 2013) to build the phylogenetic tree (parameters: model T92+G+I, ML tree, 500 bootstrap
replicates).
6.6.2 WAC04657 454 sequencing
The genome was sequenced on a 454 GS FLX+ instrument at McMaster University using 37.5%
of a sequencing plate. The genome was assembled using MIRA version 3.4.1.1 (Chevreux et al.,
2004) into 513 contigs. Open reading frames were assigned using Prodigal (Hyatt et al., 2010).
6.6.3 WAC04657 Illumina sequencing
The genome was sequenced on 15% of a MiSeq Illumina flowcell (2×250 bp) at the Farncombe
Institute at McMaster University. The data was assembled using velvet v1.2.10 (Zerbino, 2002).
6.6.4 PAC clone screening and sequencing
To screen the PAC clone, primers 69 and 70 were designed to be specific to contig 68 with an text
of 30 sec which generated an amplicon of 448 bp; primers 71 and 72 were specific to contig 293
with an text of 40 sec which generated an amplicon of 605 bp. Trial amplifications were
performed using the general PCR protocol (section 6.1.6) with the following details: primers as
mentioned above, template was WAC04657 chromosomal DNA (F#176), 0% v/v DMSO, Ta of
55 °C, text as mentioned above.
PAC292 (formerly F#292, but re-entered in my strain collection as F#351) was purified and
sequenced on 1% of a MiSeq Illumina plate (2×250 bp) at the Farncombe Institute at McMaster
University. The data was assembled using velvet v1.2.10 (Zerbino, 2002) with paired kmers of
89 bp.
6.6.5 WAC04657 PacBio sequencing
Genome sequencing was performed at Genome Quebec (Montreal, Canada) using the SMRT bell
library prep and four SMRT sequencing cells on a PacBio RSII sequencer. The genome was
assembled by Genome Quebec using the HGAP method (Chin et al., 2013) which resulted in
eight contigs (numbered 0 to 7, also called unitigs). These contigs were screened for vector
contamination using the NCBI’s Vecscreen tool (http://www.ncbi.nlm.nih.gov/tools/vecscreen/).
Open reading frames were assigned using Prodigal (Hyatt et al., 2010). Protein annotations were
105
made by manually searching the NCBI Conserved Domain Database (CDD;
http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Gene clusters were annotated using
AntiSMASH 2.0 (Blin et al., 2013). The genomic data was searched using the standalone version
of the BLAST suite (Camacho et al., 2009).
6.6.6 Accession number for WAC04657
The genome sequence for Streptomyces str. WAC04657 was deposited at
DDBJ/EMBL/GenBank under the accession LQYF00000000.
6.6.7 Identification of the ted genes in other organisms
To find the ted cluster in other organisms, the TedF1 protein sequence was used as a query to
search the NCBI’s RefSeq databases (http://BLAST.ncbi.nlm.nih.gov/). The genome sequences
and protein sequences of these organisms were downloaded and searched for all the genes in
WAC04657’s antiSMASH designated ted cluster. Genes which showed good homology (>50%
percent identity as identified by BLASTp search) and synteny were marked as common between
the clusters.
106
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Chapter 7. Appendix: NMR Spectra
7.1 13-dTDM NMR spectra
Figure 7.1 1H NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
118
Figure 7.2 13
C DEPTq NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
119
Figure 7.3 1H-
13C HSQC NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
120
Figure 7.4 1H-
13C HSQC-TOCSY NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
121
Figure 7.5 1H-
1H COSY NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
122
Figure 7.6 1H-
13C HMBC NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
123
Figure 7.7 1H-
1H NOESY NMR spectra for 13-dTDM.
The molecule was dissolved in CDCl3.
124
7.2 W5.9 NMR spectra
Figure 7.8 1H NMR spectra of molecule W5.9.
The molecule was dissolved in CDCl3.
125
Figure 7.9 CRAPT NMR spectra of molecule W5.9.
The molecule was dissolved in CDCl3.
126
Figure 7.10 1H-13
C HSQC spectra of molecule W5.9.
The molecule was dissolved in CDCl3.
127
Figure 7.11 1H-1H COSY spectra of molecule W5.9.
The molecule was dissolved in CDCl3.
128
Figure 7.12 1H-13C HMBC NMR spectra of molecule W5.9.
The molecule was dissolved in CDCl3.
129
7.3 TDM NMR traces
Figure 7.13 1H NMR spectra of molecule G6.4 (TDM).
The molecule was dissolved in CDCl3.
130
Figure 7.14 CRAPT NMR spectra of molecule G6.4 (TDM).
The molecule was dissolved in CDCl3.
131
Figure 7.15 1H-13C HSQC NMR spectra of molecule G6.4 (TDM).
The molecule was dissolved in CDCl3.
132
Figure 7.16 1H-1H COSY spectra of molecule G6.4 (TDM).
The molecule was dissolved in CDCl3.
133
5
Figure 7.17 1H-13C HMBC NMR spectra of molecule G6.4 (TDM).
The molecule was dissolved in CDCl3.
134
7.4 dhTDM NMR spectra
Figure 7.18 1H NMR spectra of molecule A5.4 (dhTDM).
The molecule was dissolved in CD3OD.
135
Figure 7.19 CRAPT NMR spectra of molecule A5.4 (dhTDM).
The molecule was dissolved in CD3OD.
136
Figure 7.20 1H-13C HSQC NMR spectra of molecule A5.4 (dhTDM).
The molecule was dissolved in CD3OD.
137
Figure 7.21 1H-1H COSY spectra of molecule A5.4 (dhTDM).
The molecule was dissolved in CD3OD.
138
Figure 7.22 1H-13C HMBC NMR spectra of molecule A5.4 (dhTDM).
The molecule was dissolved in CD3OD.
139
Chapter 8. Appendix: Sequencing Receipts
140
141
142
143
Copyright Acknowledgements
Explicit copyright permission is not required for reproduction of the following articles in this
thesis:
Gverzdys, T., Hart, M.K., Pimentel-Elardo, S., Tranmer, G. & Nodwell, J.R. (2015). “13-Deoxytetrodecamycin, a new tetronate ring-containing antibiotic that is
active against multi-drug-resistant Staphylococcus aureus” J. Antibiot. 68:698-
702. doi: 10.1038/ja.2015.60
Gverzdys, T., & Nodwell, J.R. (2016). “Biosynthetic Genes for the Tetrodecamycin
Family of Antibiotics” J. Bacteriol. 198(14):1965-1973. doi:
10.1128/JB.00140-16
Gverzdys, T., Kramer, G. & Nodwell, J.R. (2016). “Tetrodecamycin: An Unusual and
Interesting Tetronate Antibiotic” Bioorg. Med. Chem. doi:
10.1016/j.bmc.2016.05.028