CELL ENVELOPE STRESS RESPONSE AND MECHANISMS OF ANTIBIOTIC RESISTANCE IN

196 Pages·2010·8.01 MB·English

CELL ENVELOPE STRESS RESPONSE AND
MECHANISMS OF ANTIBIOTIC RESISTANCE
IN BACILLUS SUBTILIS

A Dissertation
Presented to the Faculty of the Graduate School
of Cornell University
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy

by
Anna-Barbara Geertruida Hachmann
May 2010
© 2010 Anna-Barbara Geertruida Hachmann
CELL ENVELOPE STRESS RESPONSE AND
MECHANISMS OF ANTIBIOTIC RESISTANCE
IN BACILLUS SUBTILIS
Anna-Barbara Geertruida Hachmann, Ph. D.
Cornell University 2010

The bacterial cell envelope, consisting primarily of the cell membrane and the
cell wall, is the most important physical and structural barrier. The cell wall provides
the cell with structural strength and protects it from lysis due to the high turgor. The
cytoplasmic membrane functions as a molecular sieve, controlling the transport of
specific proteins, and nutrients. Because many aspects of the cell envelope are
specific to bacteria, it is also a prime target for antibiotics. To date, at least 17 classes
of antibiotics are available for treatment; however, to each class bacteria have
developed resistance. This selective pressure within bacteria not only originates from
the widespread use of antibiotics, it is also inherent in the natural environment of
many soil dwelling prokaryotes which evolved to produce antibiotics as signaling
molecules or for nutrient competition. Here, we have investigated the response of the
Gram-positive model bacterium Bacillus subtilis to commonly used cell envelope
active antibiotics. By combining global analytical techniques, including microarray
analyses, proteomic studies, transposon mutagenesis, whole genome sequencing, and
fluorescence and electron microscopy, we obtained a clearer picture of the response of
B. subtilis to daptomycin, moenomycin, ramoplanin, fosfomycin, and duramycin. In
addition, we discuss mechanisms of resistance to these antibiotics.
BIOGRAPHICAL SKETCH
Anna-Barbara Geertruida Hachmann, née Kleijn, was born on December 5th,
1978 in Bielefeld, Germany and grew up in the town of Tecklenburg.
After graduating from the Graf-Adolf-Gymnasium Tecklenburg in 1998, she
worked for one year as laboratory assistant at Wiewelhove GmbH, a pharmaceutical
company that specializes in contract manufacturing of solid forms in Ibbenbüren,
Germany. She was responsible for analyzing raw materials and proprietary medicinal
products according to the Pharmacopoea Europaea in the Department of Quality
Control. It was during this time that she decided to persue a research career with a
pharmaceutical background.
In October 1999 Anna began her studies of pharmacy at the Friedrich-Schiller
University in Jena, Germany. From October 2003 until May 2004, she joined Dr.
Hendrik van Veen in the Department of Pharmacology at the University of
Cambridge, UK, where she explored structure activity relationships of the ATP-
binding cassette multidrug transporter LmrA in Lactococcus lactis. She returned to
Jena, to complete her pharmacy degree. In December 2004 she received her European
pharmacist license.
Anna began her graduate studies at Cornell University in January 2005 where
she joined Professor John D. Helmann’s group in the Department of Microbiology.
She was fascinated about studying mechanisms of antibiotic resistance at the level of
transcriptional regulation in the Gram-positive model bacterium Bacillus subtilis. This
dissertation summarizes her major findings.
Following the completion of her PhD, she will begin post-doctoral research
with Professor Jon Clardy and Dr. David Rudner at Harvard Medical School, Boston.
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ACKNOWLEDGMENTS
I am sincerely grateful to my advisor and mentor, Professor John D. Helmann,
who has supported me throughout my graduate studies with his patience and
knowledge while allowing me room to investigate different aspects of antibiotic
resistance. This dissertation would not have been possible without his guidance.
It is a pleasure to thank my committee members, Associate Professor Ruth
Collins, Professor Tadhg Begley, and Assistant Professor Hening Lin for their support
and helpful advice. In addition, I would like to thank Professor Esther Angert,
Professor Joseph Peters, Dr. Qiaojuan Shi, and Professor Anthony Hay for their
assistance with various techniques.
During my graduate studies I was fortunate to work with a friendly and
cheerful group of fellow students who encouraged me along the way and shared the
excitement of “re”search. It was a privilege to work with Ahmed Gaballa, who is a
wonderful mentor and friend, and has the ability to always put a smile on your face. I
would like to thank the members of the Helmann lab and Winans lab, in particular
Shawn MacLellan, Yun Luo, Veronica Guariglia-Oropeza, Tina Wecke, as well as
Showey Yazdanian, Alex Fishman, Dominika Zgid, Paulina Gonzalez-Morelos, and
my class mates for their friendship and many fond memories. I owe Cornell Outdoor
Education and my climbing partners my love for climbing and the outdoors.
All this would not have been possible without the constant support from my
husband and my family. With this I would also like to thank Nancy and Robert Nead
(including the Zoo) for making us feel home away from home.
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TABLE OF CONTENTS

BIOGRAPHICAL SKETCH iii
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS v
LIST OF FIGURES vi
LIST OF TABLES viii

CHAPTER 1
Introduction 1

CHAPTER 2
Genetic Analysis of Factors Affecting Susceptibility of Bacillus subtilis
to Daptomycin 31

CHAPTER 3
Pgsa depletion leads to High Daptomycin Resistance in Bacillus subtilis 65

CHAPTER 4
Tn7SX Transposon Mutagenesis: a Tool to Study Antibiotic Resistance
Mechanisms in Bacillus subtilis 99

CHAPTER 5
The Transglycosylation Inhibitors Ramoplanin and Moenomycin Induce
Distinct Transcriptional Responses 147

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LIST OF FIGURES

Figure 1.1. Electron micrograph of Bacillus subtilis. 1
Figure 1.2. Peptidoglycan crosslinking by bi-functional penicillin binding proteins. 2
Figure 1.3. Peptidoglycan biosynthesis in B. subtilis. 3
Figure 1.4. Structure of teichoic acids in B. subtilis. 4
Figure 1.5. Essentiality of teichoic acids. 5
Figure 1.6. Membrane lipid biosynthesis in B. subtilis. 7
Figure 1.7. Cell envelope stress response systems in B. subtilis. 8
Figure 1.8. Induction of cell envelope stress response systems in B. subtilis. 9
Figure 1.9. Targets of antibiotic action during cell wall synthesis. 11
Figure 1.10. Structure of the cyclic lipopeptide daptomycin. 13
Figure 1.11. Proposed mechanism of action of daptomycin. 14
Figure 1.12. Structure of the glycolipid moenomycin. 16
Figure 1.13. Moenomycin interaction with transglycosylase. 17
Figure 1.14. Imaging of peptidoglycan synthesis with fluorescent ramoplanin. 18
Figure 1.15. Structure of the ramoplanin dimer. 19
Figure 1.16. Antibiotic resistance profile of soil inhabiting bacteria. 20
Figure 1.17. Resistance levels against tested antibiotics. 21
Figure 1.18. Increase of antibiotic resistance versus decrease of new antibiotics. 21
Figure 1.19. Part of the bacitracin stress response in B. subtilis. 22
Figure 1.20. Mechanism of vancomycin resistance in enterococci. 23
Figure 1.21. Inactivation of β-lactams by β-lactamase. 24
Figure 1.22. MprF mediated resistance to cationic antimicrobial peptides. 24

Figure 2.1. Daptomycin stimulon in B. subtilis. 41
Figure 2.2. Daptomycin-BDP inserts preferentially at new division septa and in
a spiral pattern. 51
Figure 2.3. Daptomycin-BDP staining of stationary phase cells. 52
Figure 2.4. Correlation between daptomycin-BDP staining and anionic
phospholipid content and distribution. 54
Figure 2.5. Cluster analysis of B. subtilis microarray studies with 40 different
antimicrobial agents. 57

Figure 3.1. Daptomycin-BDP inserts in a spotted pattern and at cell poles and
division septa in DapR1. 76
Figure 3.2. Transmission electron micrographs of W168. 78
Figure 3.3. TEM of DapR1 reveal a thicker cell wall at the poles and irregular
septum placement compared to W168. 79
Figure 3.4. Comparison of DapR1 and W168 transcriptome. 81
Figure 3.5. DapR1 and W168 gene expression ratios after daptomycin treatment. 81
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Figure 3.6. Cytoplasmic proteome. 84
Figure 3.7. Extracellular proteome. 85
Figure 3.8. Cell wall proteome. 86
Figure 3.9. Muramic acid quantification in DapR1 and W168. 94

Figure 4.1. The Tn7SX transposon. 101
Figure 4.2. Synthesis of phosphatidylethanolamine. 106
Figure 4.3. Structure and target of moenomycin. 107
Figure 4.4. Growth inhibition of W168 and a sigM deletion by moenomycin. 108
Figure 4.5. Distribution of moenomycin resistant Tn7SX insertions. 111
Figure 4.6. Regular and alternative cell wall synthesis pathways in E. faecium. 120
Figure 4.7. Putative interaction network of ybcI. 122
Figure 4.8. Expression of the dinG ypmA ypmB aspB operon. 124
Figure 4.9. Putative interaction network of ypmB. 125
Figure 4.10. Tn7SX transposon insertions in ybfM and psd. 128
Figure 4.11. Affinity of moenomycin to truncated HMW PBPs. 129
Figure 4.12. aprE regulatory network. 131
Figure 4.13. Structure of the NRPS produced lipopeptide plipastatin. 134
Figure 4.14. Synthesis of the low-molecular-weight thiol bacillithiol in B. subtilis. 136

Figure 5.1. Structure of the lipoglycodepsipeptide ramoplanin. 149
Figure 5.2. Inhibition of transglycosylation by ramoplanin. 150
Figure 5.3. Structure of a ramoplanin dimer at the membrane interface. 150
Figure 5.4. Crystal structure of moenomycin. 151
Figure 5.5. Ramoplanin stimulon in B. subtilis. 158
Figure 5.6. Ramoplanin stimulon in B. subtilis liaR deletion. 159
Figure 5.7. Ramoplanin stimulon of a liaR deletion versus wild-type CU1065. 160
Figure 5.8. Cluster analysis of B. subtilis with 40 antibiotics. 160
Figure 5.9. Teichoic acid and peptidoglycan synthesis. 162
Figure 5.10. Moenomycin stimulon in B. subtilis. 164
Figure 5.11. Overexpression of sigV in B. subtilis W168. 167
Figure 5.12. Overexpression of sigV in B. subtilis Δ7 ECF deletion strain. 168
Figure 5.13. Antibiotic sensitivity profile of ECF σ mutants. 169
Figure 5.14. Promoter consensus sequence of σW, σX, and σM. 170
Figure 5.15. Promoter consensus sequence of σM and σV. 172
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LIST OF TABLES

Table 1.1. Cell envelope active antibiotics with their respective targets. 10

Table 2.1. Strains used in this study. 38
Table 2.2. Oligonucleotides used in this study. 39
Table 2.3. Daptomycin stimulon. 42
Table 2.4. Minimum inhibitory concentration of B. subtilis mutants with
altered membrane composition, or deletion of transcriptional regulators. 46

Table 3.1. Strains and oligonucleotides used in this study. 72
Table 3.2. Minimum inhibitory concentrations of B. subtilis wild-type and DapR1. 74
Table 3.3. Minimum inhibitory concentrations of B. subtilis mutants. 74
Table 3.4. Comparison of cell wall thickness of W168 and DapR1. 77
Table 3.5. Fold change of cytosolic protein expression of DapR1 and W168. 87
Table 3.6. Fold change of extracellular protein expression of DapR1 and W168. 88
Table 3.7. Single nucleotide polymorphisms in genes or intergenic regions. 91
Table 3.8. Fatty acid methyl esther analysis of B. subtilis wild-type and DapR1. 93

Table 4.1. Strains and oligonucleotides used in this study. 102
Table 4.2. Tn7 proteins and their roles during transposition. 104
Table 4.3. High molecular weight penicillin binding proteins in B. subtilis. 107
Table 4.4. Moenomycin resistant Tn7SX insertions in W168, sigM, and pbpDFG. 112
Table 4.5. Moenomycin resistant Tn7SX insertions summary. 116
Table 4.6. Regulon members of the YdfHI two-component regulatory system. 118
Table 4.7. Fosfomycin sensitive Tn7SX insertions in W168. 137

Table 5.1. Strains and oligonucleotides used in this study. 154
Table 5.2. Ramoplanin stimulon. 157
Table 5.3. Moenomycin stimulon and σV regulon. 165
Table 5.4. σV and σM specific regulon members. 172

Table S3.1. Gene expression of DapR1 and W168 without daptomycin treatment. 177
Table S3.2. Gene expression of DapR1 and W168 with daptomycin treatment. 186

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CHAPTER 1
INTRODUCTION

1.1 The cell envelope of Gram-positive bacteria
In Gram-positive bacteria, the cell is enclosed by the cytoplasmic membrane
and the cell wall, which consists of peptidoglycan (PG) as well as wall- and lipo-
teichoic acids (Fig. 1.1, Fig. 1.2). As a three-dimensional lattice of about 50 nm
thickness, the cell wall functions as the most important physical barrier, providing the
cell with structural strength and protecting it from lysis due to the high turgor (2, 65).
These properties and its specificity to bacteria also render the cell wall a prime target
for antibiotics (61) (Fig. 1.3).

Figure 1.1. Electron micrograph of Bacillus subtilis.
The enlarged frame highlights the cytoplasmic membrane in dark gray (CM) and the
cell wall in lighter gray (CW). Scale bar represents 0.2 µm.
1