DOSR a response regulator essential for hypoxic dormancy in mycobacterium bovis BCG 1

DOSR – A RESPONSE REGULATOR ESSENTIAL FOR
HYPOXIC DORMANCY IN Mycobacterium bovis BCG





BOON KA KHIU CALVIN
(B.Sc. Hons.)
IMPERIAL COLLEGE OF LONDON







A THESIS SUBMITTED FOR THE DEGREE OF
DOCTORATE OF PHILOSOPHY




INSTITUTE OF MOLECULAR AND CELL BIOLOGY
NATIONAL UNIVERSITY OF SINGAPORE

2003

ii
Acknowledgement


I would like to express my heartfelt gratitude to my supervisor A/P Thomas Dick for
his constant guidance, stimulating discussions and encouragements in the tough times
encountered during the course of this project. His contributions go beyond scientific
thinking. He had had a major influence on the further development of my strengths
and realization of my weaknesses. For this, I am very grateful.

My sincere thanks to the members of my PhD Supervisory Committee A/P Wang
Yue, Dr. Anthony Ting and Dr. Michael Sprengart for their constructive suggestions
and guidance.

I am very grateful to Dr. Robert Qi and Li Rong for performing the mass
spectrometric analysis that lead to the identification of DosR, which had been the
central feature of this thesis. I am also thankful to Dr. Alice Tay for providing
excellent sequencing services.

Special thanks to Bernadette Oei-Murugasu for her help and advice during the
learning stages, to Indra for his critical comments on the thesis, to Bee Huat, Boon
Heng, Michael, Pam, Raymond and other past and present members of the
mycobacterium lab for their friendship, advice and stimulating discussions.

Last but not least, I would like to express my deepest gratitude to my parents for their
support and encouragement throughout the years of my education. Without them, I
would not be writing this thesis. Finally, I am very grateful to my beautiful wife Boon
Tin for her enduring love and confidence in me that had been my constant source of

strength throughout these years.




iii
Table of Contents

Acknowledgement ii
Table of contents iii
List of Figures vii
List of Tables x
Abbreviations xi
Summary xiv


Chapter 1
1. Introduction 1

1.1 Bacilli Persist in vivo 2
1.2 Hypoxia could be a Factor in Persistence 3
1.3 Discovery of the Dormancy Response: Wayne’76 Culture Model 5
1.4 Temporal Analysis of Dormancy: Wayne’96 Culture Model 9
1.5 Other ‘Hypoxia’ Culture System 13
1.6 The Hypoxia Induced in vitro Dormancy Response is Poorly Understood 15

Chapter 2
2. Materials and Methods 20

2.1 Materials

2.1.1 Vectors 20
2.1.1 Bacterial Strains 21
2.1.2 Chemicals and Reagents 21
2.1.3 Growth Media 22

2.2 Mycobacterial Culture
2.2.1 The Wayne Dormancy Culture Model 23
2.2.2 The Aerated Stationary Phase Culture Model 23
2.2.3 Determination of C.F.U 24

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2.3 DNA and RNA Methods 24
2.3.1 Restriction Digest of DNA 25
2.3.2 Blunt Ending of DNA Fragments 25
2.3.3 Dephosphorylation of DNA Fragments 26
2.3.4 Ligation of DNA Fragments 26
2.3.5 Agarose Gel Electrophoresis 26
2.3.6 Elution of DNA from Agarose Gels 27
2.3.7 Precipitation of DNA 28
2.3.8 Preparation of E.coli Electrocompetent cells 28
2.3.9 Transformation of E.coli by Electroporation 29
2.3.10 Preparation of BCG Electrocompetent cells 29
2.3.11 Transformation of BCG by Electroporation 29
2.3.12 Mini Preparation of Plasmid DNA 30
2.3.13 Maxi Preparation of Plasmid DNA 31
2.3.14 Mini Preparation of Genomic DNA 32
2.3.15 PCR 33
2.3.16 Sequencing of DNA 34
2.3.17 Two Step RT-PCR 35

2.3.18 Southern Blotting and Hybridization 36
2.3.19 Preparation of BCG Total RNA 38
2.3.20 Northern Blotting and Hybridization 38
2.3.21 Screening of M.bovis BCG Genomic Library 40

2.4 Protein Methods
2.4.1 Preparation of BCG Protein Lysate 42
2.4.2 Determination of Protein Concentration 42
2.4.3 Two-dimensional Gel Electrophoresis 43

2.5 Computational Analysis 45

Chapter 3
3. Identification of Dormancy Induced Proteins 47

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3.1 Analysis of the Dormancy Response in BCG using 2-D Gel 48
Electrophoresis
3.2 Protein Identification via Mass Spectrometry 53
3.3 Transcript Levels of Dormancy Induced Proteins 57
3.4 Analysis of Dormancy Induced Proteins in Aerated Stationary
Phase Cultures 60
3.5 Computational Analysis of Dormancy Induced Proteins 64
3.5.1 HspX 64
3.5.2 23kD Putative Response Regulator 65
3.5.3 32kD Conserved Hypothetical Protein 67
3.5.4 16kD Conserved Hypothetical Protein 67

3.6 Conclusion 70

Chapter 4
4. Functional Chracterization of the Dormancy Specific Response
Regulator Rv3133c 71

4.1 Molecular Characterization of the dosR Locus; Generation of Gene
Replacement and Rescue Constructs. 72
4.1.1 Genomic Organization of the BCG Rv3133c locus 72
4.1.2 Generation of BCG ∆dosR::km Replacement Construct 75
4.1.3 Isolation of Single Recombinants Clones for the Generation of
∆dosR::km 78
4.1.4 Isolation of ∆dosR::km from Single Recombinants 83
4.1.5 Generation of BCG ∆Rv3132c::km Replacement Construct 87
4.1.6 Isolation of Single Recombinants Clones for the Generation of
∆Rv3132c::km 89
4.1.7 Isolation of ∆Rv3132c::km from Single Recombinants 92
4.1.8 Construction of Rescue Construct pCB4 for ∆dosR::km and
∆Rv3132c:: km 98


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4.2 Phenotypic Analysis of ∆dosR::km and ∆Rv3132c::km 99
4.2.1 Reduction in Viability of BCG ∆dosR::km 99
4.2.2 Moderate Survival Phenotype of BCG ∆Rv3132c:: km 103
4.2.3 Regulation of Dormancy Induced Proteins by DosR 106
4.2.4 Minor Role of Rv3132c in the Regulation of the Dormancy
Induced Proteins 108
4.2.5 Wild type-like Survival of BCG ∆dosR::km and ∆Rv3132c:: km
in Aerated Stationary Phase Cultures 108

4.3 Conclusion 112

Chapter 5
5. Discussion 113

5.1 Proteins Induced in the Wayne’96 Dormancy Culture System 114
5.1.1 HspX 115
5.1.2 Rv2626c 116
5.1.3 Rv2623 117
5.1.4 DosR 118

5.2 Transcript Levels of the Four Dormancy Induced Proteins are
Elevated in Dormant Bacilli 119
5.3 DosR is the Master Regulator of Dormancy 121
5.4 Rv3132c is Required but not Essential for Dormancy 122
5.5 DosR but not Rv3132c is Essential for the Regulation of the
Dormancy Induced Proteins 124
5.6 DosR Function is Conserved in M. smegmatis 127
5.7 Hypoxic Dormant Bacilli and Persistence in vivo 128

References
134

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List of Figures

Chapter 1

Figure 1.3.1 The Wayne’76 standing culture model 9

Figure 1.4.1 The Wayne’96 in vitro dormancy culture model
12
Figure 1.6.1 Hallmarks of bacilli grown in the Wayne’76 and’96 in vitro
dormancy culture model
18

Chapter 3

Figure 3.1.1 Growth of BCG in the Wayne dormancy culture system 50
Figure 3.1.2 Temporal proteome of BCG grown in the Wayne dormancy
culture system using pH 3 to 10 isoelectric focusing strips 51
Figure 3.1.3 Temporal proteome of BCG grown in the Wayne dormancy
culture system using pH 4 to 7 isoelectric focusing strips 52
Figure 3.1.4 Under Loading Experiments using pH 4 to 7 isoelectric
focusing strips 54
Figure 3.2.1 Protein identification by mass peptide fingerprinting and
sequence tag analysis 55
Figure 3.3.1 Steady state levels of mRNAs of dormancy-induced proteins
in growing and hypoxic stationary phase cultures 59
Figure 3.4.1 Growth of BCG cultures in aerated roller bottles 62
Figure 3.4.2 Temporal proteome of BCG grown in aerated roller bottle
cultures using pH 4 to 7 isoelectric focusing strips 63
Figure 3.5.2.1 Multiple alignment of Rv3133c with other response regulators
with known phosphorylation sites 66
Figure 3.5.1 Domain structure of the four dormancy induced proteins 69
Figure 4.1.1.1 The genomic organization of the Rv3133c locus in
Mycobacterium tuberculosis and Mycobacterium bovis BCG 73

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Figure 4.1.2.1 Summary of the cloning strategy for the construction of the

dosR gene replacement construct 76
Figure 4.1.2.2 dosR locus and gene replacement constructs 77

Figure 4.1.3.1 Streak plates of wild type strains, dosR single and double
recombinants mutants 79
Figure 4.1.3.2 PCR strategy for screening of ∆dosR::km legitimate single
recombination events 81
Figure 4.1.3.3 PCR analysis using genomic DNA extracted from single
recombinants and double recombinants isolated during the
generation of ∆dosR::km 82
Figure 4.1.4.1 PCR strategy for screening of ∆dosR::km 84
Figure 4.1.4.2 Southern blot analysis of dosR gene replacement mutants 86
Figure 4.1.5.1 Summary of the cloning strategy for the construction of the
Rv3132c gene replacement construct 88
Figure 4.1.5.2 dosR locus, Rv3132c gene replacement construct 89
Figure 4.1.6.1 Streak plates of wild type strains, Rv3132c single and double
recombinants mutants 90
Figure 4.1.6.2 PCR strategy for screening of ∆Rv3132c::km legitimate single
recombination events 91
Figure 4.1.7.1 PCR strategy for screening of ∆Rv3132c::km 93
Figure 4.1.7.2 PCR analysis using genomic DNA extracted from single
recombinants and double recombinants isolated during the
generation of ∆Rv3132c::km 94
Figure 4.1.7.3 Southern blot analysis of Rv3132c gene replacement mutants 96
Figure 4.1.7.4 Summary of dosR locus, gene replacement constructs and
rescue plasmid 97
Figure 4.2.1.1 Growth of wild type BCG, ∆dosR::km1 and ∆dosR::km1 (pCB4)
strains in the Wayne dormancy culture system 101
Figure 4.2.1.2 Survival of wild type BCG and ∆dosR::km1 and ∆dosR::km1
(pCB4) strains in the Wayne dormancy culture system 102

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Figure 4.2.2.1Growth of wild type BCG, ∆Rv3132c::km1 ,∆Rv3132c::km1
(pCB4) and ∆dosR::km1 strains in the Wayne dormancy culture
system 104
Figure 4.2.2.2Survival of wild type BCG, ∆Rv3132c::km1 and ∆Rv3132c::km1
(pCB4) strains in the Wayne dormancy culture system 105

Figure 4.2.3.1Two-dimensional gel electrophoresis analyses of protein extracts
from wild type BCG and ∆dosR::km1, ∆dosR::km1 (pCB4) and
∆Rv3132c::km1 strains grown in the Wayne dormancy culture
system 107
Figure 4.2.5.1Growth of wild type BCG, ∆dosR::km1 and ∆Rv3132c1::km
strains in the aerated stationary phase culture system 110
Figure 4.2.5.2Survival of wild type BCG, ∆dosR::km1 and ∆Rv3132c1::km
strains in the aerated stationary phase culture system 111

Chapter 5

Figure 5.5.1 A working model for the molecular mechanisms of the
dormancy response. 126




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List of Tables

Chapter 3

Table 3.2.1 Dormancy induced proteins identified by nanoelectrospray
tandem mass spectrometry 56

Table 3.3.1 Primer sequences used for RT-PCR and isolation of probes
for Northern hybridization 58

Chapter 4

Table 4.1.1.1 Sequences of primers employed in the sequencing of the
genomic fragment containing the BCG Rv3133c locus 74

Table 4.1.3.1 Primer sequences used for PCR screening of clones isolated
during the generation of ∆dosR::km and ∆Rv3132c:: km 80


xi
Abbreviations

A
600
Absorbance at λ600nm
Amp ampicilin
Amp
R
ampicillin resistant
ATP adenosine 5’-triphosphate

BCG Mycobacterium bovis BCG
BSA bovine serum albumin

o
C degrees Celsius
cDNA complementary deoxyribonucleic acid
c.f.u colony forming units
CHAPS 3-3-cholamidopropyl-dimethylammonio-1-1-propane sulfonate
Ci Curie
µCi microCurie

DEPC diethylenepyrocarbonate
DNA deoxyribonucleic acid
DNase deoxyribonuclease
DTT 1,4-dithiothreitol

E.coli Escherichia coli
EDTA ethylenediaminetetraacetic acid

g gram
µg microgram
Get gentamycin
Get
R
gentamycin resistant
Get
S
gentamycin sensitive

Hyg hygromycin
Hyg
R
hygromycin resistant

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Kan kanamycin
Kan
R
kanamycin resistant
Kan
S
kanamycin sensitive
kb kilobases
kD kilodalton
Klenow large fragment of E.coli DNA polymerase I
kV kilo volts

L litre
µl microlitre

M moles per litre
mA miliamperes
µA microamperes
µl microlitre
µM micromolar
mg miligrams
ml millilitre
MOPs 3-N-morpholinopropmesulfonic acid
mRNA messenger RNA

nm nanometer

OD optical density

PBS phosphate-buffered saline
PDA piperazine diacrylamide
p.f.u plaque forming unit

RNA ribonucleic acid
RNase ribonuclease
rpm revolutions per minute


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SDS sodium dodecyl sulfate
Suc sucrose
Suc
R
sucrose resistant
Suc
S
sucrose sensitive

TCEP Tris-carboxyethyl-phosphine
TEMED NNN’N’-tetra-methylethylenediamide
Tris tris (hydroxymethyl) aminomethane

UV Ultra-violet

V Volts

X-gal 5-bromo-4-chloro-3-indolyl-β-galactopyranoside

xiv
Summary


The persistence of Mycobacterium tuberculosis (MTB) despite long chemotherapy is a
major obstacle in the effective treatment and eradication of the disease. As such,
understanding persistence is vital to the global control of tuberculosis. Several lines of
evidence indicate that hypoxia could be a factor in persistence. The discovery that
MTB has the ability to adapt and survive hypoxia in vitro by shifting down to non-
replicative drug resistant dormant form raises the question whether the bacilli in vivo
are in a similar physiological state and whether they play a role in the observed
persistence of the disease. However, the molecular mechanisms of the hypoxia-
induced dormancy response are poorly understood. The lack of molecular dormancy
markers and dormancy mutants hamper investigators from providing direct evidence
that hypoxic dormant bacilli exist in vivo and contribute to the persistence of the
disease.

The first part of this study aims to further define the hypoxic dormancy response by
identifying dormancy dependent proteins via two-dimensional electrophoresis. Using
the Wayne’96 in vitro dormancy culture system and the attenuated BCG strain of the
tubercle bacilli as a model organism, the temporal proteome profile during the
dormancy response was defined. Four proteins were found to be induced upon entry
into dormancy. They are the alpha-crystallin homologue HspX, a response regulator
Rv3133c, and the conserved hypothetical proteins Rv2623 and Rv2626c. Induction of
Rv3133c and Rv2623 appears to be dormancy specific. Hence these proteins are
useful markers for the demonstration of hypoxic dormant bacilli in vivo.

Response regulators are phosphorylation dependent transcription factors known to be
involved in adaptation of bacteria to diverse conditions. Therefore, the hypoxic
dormancy specific up regulation of Rv3133c response regulator indicated that this

protein could play a role in the adaptation to dormancy survival and the induction of
the other 3 dormancy-induced proteins.

xv
In the second part of this work, a functional characterization of Rv3133c was carried
out. Inspection of the Rv3133c locus revealed that the Rv3132c gene, which encodes a
histidine protein kinase, overlaps the Rv3133c gene by 1 base pair thereby indicating
that the two proteins could form a ‘dormancy’ two-component signalling system. To
define the function of Rv3133c and its candidate cognate sensor kinase Rv3132c, gene
replacement mutants were constructed and analysed in the Wayne’96 in vitro culture
system.

The Rv3133c mutant showed a drastic loss of viability during hypoxic dormancy.
Thus, the loss of this dormancy specific response regulator resulted in the loss of the
ability of the bacilli to adapt and to survive hypoxic dormancy. In addition, the loss of
induction of the other three dormancy-induced proteins was observed in the Rv3133c
mutant background. Hence, the induction of these dormancy proteins is dependent on
Rv3133c. Based on these two functions, dormancy survival and regulation, the
Rv3133c gene was named dosR for
d
ormancy survival regulator. In contrast, the
Rv3132c mutant displayed a moderate dormancy phenotype. This suggests that other
‘dormancy’ kinases may be involved in the regulation of DosR.

Taken together, this work provides conclusive evidence that dosR is the master
regulator of the dormancy response. The dosR mutant is the first dormancy specific
mutant. It represents a useful tool to investigate the relevance of hypoxic dormant
bacilli in persistence in vivo.