ENVIRONMENTAL REGULATION OF CYCLIC DI-GMP TURNOVER IN

VIBRIO CHOLERAE
By
Benjamin J. Koestler

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Microbiology and Molecular Genetics - Doctor of Philosophy
2014

ABSTRACT
ENVIRONMENTAL REGULATION OF CYCLIC DI-GMP TURNOVER IN

VIBRIO CHOLERAE
By
Benjamin J. Koestler

3’, 5’-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second
messenger that mediates the transition between a sessile, biofilm forming lifestyle
to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-diGMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the
second messenger. Although numerous bacterial behaviors are regulated by c-diGMP, the regulatory inputs of this system remain mostly undefined.
Here, I examine how the marine bacterium and human pathogen Vibrio

cholerae utilizes c-di-GMP signaling to interpret and respond to environmental
cues. The central hypothesis that underpins my research is that V. cholerae senses
environmental signals with DGCs and PDEs to modulate c-di-GMP concentrations
in different environments. As c-di-GMP is a widely conserved second messenger
utilized by many different bacteria, the mechanisms by which V. cholerae utilizes cdi-GMP in different environments can be applied to other bacterial systems to
further comprehend how they behave in and adapt to various surroundings.
To examine the influence of environmental factors on c-di-GMP synthesis and
hydrolysis, I have developed a novel method named The Ex vivo Lysate c-di-GMP
Assay (TELCA) that systematically measures total DGC and PDE cellular activity.

I have shown that V. cholerae grown in different environments exhibits distinct
intracellular levels of c-di-GMP, and using TELCA have determined that these
differences correspond to changes in both c-di-GMP synthesis and hydrolysis. These
findings highlight that modulation of both total DGC and PDE activity alters the
intracellular concentration c-di-GMP.
While searching for specific environmental cues that regulate c-di-GMP, I
have found that bile acids, a prevalent constituent of the human proximal small
intestine, increase intracellular c-di-GMP in V. cholerae. This bile-mediated
increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted
by intestinal epithelial cells. These findings lead me to propose that V. cholerae
senses distinct microenvironments within the small intestine, using bile and
bicarbonate as chemical cues, and responds by modulating intracellular c-di-GMP.
In addition to its function as a bacterial second messenger, c-di-GMP has
potent immunostimulatory properties in eukaryotes; these properties make c-diGMP a prime candidate for use as a vaccine adjuvant. Here, I present a novel
method of delivering c-di-GMP into eukaryotic cells using adenovirus. I have
demonstrated that c-di-GMP can be synthesized in vivo by delivering DGC DNA
into the cell, and that this c-di-GMP increases the secretion of numerous cytokines
and chemokines. This novel adenovirus c-di-GMP delivery system offers a more
efficient and cost-effective method to administer c-di-GMP as an adjuvant to
stimulate innate immunity.

ACKNOWLEDGEMENTS
I would like to acknowledge and thank C.M. Waters for providing mentorship
and guidance. I would also like to acknowledge T.M. Schmidt, R.A. Britton, and
S.H. Shiu for their guidance. I thank E.L. Reynolds and J.P. Massie for assistance
in data collection (Chapter 4). I also acknowledge A. Amalfitano for guidance and
S.S. Seregin, Y.A. Aldhamen, D.P.W. Rastall, S. Godbehere, J. Collins, and A.M.A.
Hunt for assistance in collecting data (Chapter 5). I am grateful for assistance from
L. Chen and A.D. Jones from the mass spectrometry facility. I would also like to
thank K. Whitehead for providing the mixture for SHB, B.R. Pursley for
constructing pBRP2, and C. Chan for constructing pANDA2. I thank S.M. Butler for
help proofreading and formatting this dissertation. And finally, I would like to
acknowledge the Rudolph Hugh Fellowship, the Russell B. DuVall Scholarship, and
the Marvin Hensley Fellowship.

iv

TABLE OF CONTENTS
LIST OF TABLES .................................................................................................... viii
LIST OF FIGURES.....................................................................................................ix
LIST OF ABBREVIATIONS .................................................................................... xii
CHAPTER 1 – Background .........................................................................................1
The bacterial second messenger cyclic di-GMP ...............................................1
Vibrio cholerae: marine bacterium and human pathogen...............................3
Innate Immune response to c-di-GMP .............................................................5
Summary ...........................................................................................................8
CHAPTER 2 - Exploring Environmental Control of Cyclic di-GMP Signaling in
Vibrio cholerae by Using the Ex Vivo Lysate Cyclic di-GMP Assay (TELCA)........10
Preface .............................................................................................................10
Bacterial strains and growth conditions ........................................................12
Synthesizing [13C]-c-di-GMP ..........................................................................13
Generation of lysates ......................................................................................14
Detection and quantification of c-di-GMP......................................................15
TELCA quantification of whole cell DGC activity .........................................17
TELCA quantification of whole cell PDE activity .........................................22
The local environment alters the intracellular concentration of c-di-GMP in
V. cholerae .......................................................................................................23
Growth environments modulate both c-di-GMP synthesis and
hydrolysis ........................................................................................................26
QS controls both total DGC and PDE activity in V. cholerae .......................29
CdgA contributes to the increased DGC activity at low cell density ............31
Discussion ........................................................................................................32
CHAPTER 3 – Bile Acids and Bicarbonate Inversely Regulate Intracellular
Cyclic di-GMP in Vibrio cholerae ..............................................................................37
Preface .............................................................................................................37
Growth conditions and molecular methods ...................................................38
Detection and quantification of intracellular c-di-GMP ................................40
Systematic screen of DGC and PDE activity .................................................41
Systematic screen of DGC and PDE expression ............................................41
Biofilm quantification .....................................................................................42
Bile increases the intracellular concentration of c-di-GMP in
V. cholerae .......................................................................................................43

v

The bile mediated increase of intracellular c-di-GMP is growth-phase
dependent ........................................................................................................44
Three DGCs are more active in the presence of bile .....................................45
The activity of PDEs in V. cholerae is not altered by bile .............................48
The three bile-inducible DGCs are inner membrane proteins ......................50
Three DGCs increase c-di-GMP synthesis in the presence of bile acids.......52
A toxR deletion mutant partially loses bile-mediated c-di-GMP increase ...53
Transcription of VC1295 is inhibited by bile acids ........................................55
Three DGCs and one HD-GYP account for bile-mediated c-di-GMP
induction ..........................................................................................................59
Deletion of the bile-responsive DGCs and PDE reduces bile induction of V.
cholerae biofilm formation ..............................................................................61
Bicarbonate decreases the bile-mediated increase of c-di-GMP in
V. cholerae .......................................................................................................62
Discussion ........................................................................................................65
Future directions .............................................................................................71
CHAPTER 4 – Intracellular c-di-GMP Negatively Correlates with the Growth Rate
and Ribosomal RNA of V. cholerae ...........................................................................76
Preface .............................................................................................................76
Measuring growth rate of V. cholerae ............................................................77
RNA extractions and quantification...............................................................78
Intracellular c-di-GMP and growth rate of V. cholerae are correlated .........78
Growth rate inhibition caused by ectopic DGC expression is
dose-dependent ................................................................................................79
Intracellular c-di-GMP concentration required for growth inhibition varies
for different DGCs ...........................................................................................83
Increased intracellular c-di-GMP reduces the expression of rRNA ..............86
Discussion ........................................................................................................88
Future directions .............................................................................................91
CHAPTER 5 - A novel Adenovirus Adjuvant Stimulates Innate Immunity by in vivo
cyclic di-GMP Synthesis ............................................................................................95
Preface .............................................................................................................95
DNA manipulation and virus construction ....................................................96
HeLa cell transformation ................................................................................97
Animal protocols .............................................................................................98
Quantitative PCR............................................................................................99
Cytokine and chemokine quantification ......................................................100
ELISPOT analysis.........................................................................................101
C. difficile challenge ......................................................................................101
Generating an adenovirus harboring a V. cholerae DGC ...........................102
Synthesis of c-di-GMP in a murine system ..................................................105

vi

C-di-GMP synthesized in vivo stimulates innate immunity in a mouse
model .............................................................................................................106
C-di-GMP synthesized in vivo lowers the effective antigen dose of
Ad5-TA...........................................................................................................110
Ad5-VCA0956 does not significantly enhance protection of Ad-TA against
C. difficile.......................................................................................................114
Discussion ......................................................................................................119
Future directions ...........................................................................................122
CHAPTER 6 – Conclusions .....................................................................................124
APPENDICES..........................................................................................................127
REFERENCES ........................................................................................................138

vii

LIST OF TABLES

Table 1

Description of DGC expression strain intracellular c-di-GMP levels
and growth inhibition ......................................................................... 82

Table 2

Cytokine and chemokine secretion of mice infected with Ad5 vectors
after 6 hours ...................................................................................... 111

Table 3

Cytokine and chemokine secretion of mice infected with Ad5 vectors
after 24h hours.................................................................................. 112

Table A1

Bacteria strain list ..................................................................................... 128

Table A2

Plasmid list ................................................................................................ 129

Table A3

Primer list .................................................................................................. 134

viii

LIST OF FIGURES

Figure 1

LC-MS/MS differentiation of [12C]-c-di-GMP and [13C]-c-di-GMP ....19

Figure 2

TELCA of [13C]-c-di-GMP synthesis of DGC and PDE ectopic
expression strains ...............................................................................21

Figure 3

TELCA of [13C]-c-di-GMP hydrolysis of DGC and PDE ectopic
expression strains ...............................................................................23

Figure 4

Growth curves of V. cholerae grown in different media....................25

Figure 5

Intracellular c-di-GMP concentrations of V. cholerae grown in
different media ....................................................................................26

Figure 6

TELCA of V. cholerae grown in different media ...............................28

Figure 7

TELCA of V. cholerae QS mutants ....................................................30

Figure 8

Intracellular c-di-GMP concentrations of V. cholerae grown with
different bile additives ........................................................................44

Figure 9

Intracellular c-di-GMP concentrations of V. cholerae during the course
of growth with bile ..............................................................................46

Figure 10

High-throughput screen of the in vivo activity of each of the 40 V.
cholerae DGCs with bile .....................................................................48

Figure 11

High-throughput screen of the in vivo activity of each of the 40 V.
cholerae PDEs with bile .....................................................................50

Figure 12

The amino acid sequence hydrophobicity and predicted signaling
domains of the bile-responsive DGCs ................................................51

Figure 13

Intracellular c-di-GMP concentrations of V. cholerae DGC expression
strains in the presence and absence of SHB .....................................54

ix

Figure 14

Intracellular c-di-GMP concentrations of V. cholerae ΔtoxR and ΔtoxT
deletion mutants in the presence of bile ............................................55

Figure 15

Relative transcription of DGCs and PDEs in the presence and absence
of bile ...................................................................................................57

Figure 16

Intracellular c-di-GMP concentrations of V. cholerae DGC and PDE
mutant strains in the presence and absence of bile ..........................60

Figure 17

Biofilm formation of V. cholerae DGC and PDE mutants in the
presence of bile ....................................................................................63

Figure 18

Intracellular c-di-GMP concentrations of V. cholerae grown in the
presence of bile and bicarbonate ........................................................64

Figure 19

Proposed model of V. cholerae c-di-GMP regulation in the human
small intestine ....................................................................................70

Figure 20

Linear regression of intracellular c-di-GMP concentrations on growth
rates of V. cholerae grown in different media ...................................79

Figure 21

Growth rates of V. cholerae DGC expression strains........................81

Figure 22

Linear regression of intracellular c-di-GMP concentrations on growth
rates of V. cholerae DGC expression strains .....................................84

Figure 23

Intracellular c-di-GMP concentrations of V. cholerae DGC expression
strains .................................................................................................86

Figure 24

Growth rates of V. cholerae DGC expression strains........................87

Figure 25

Relative rRNA abundance of V. cholerae DGC expression strains ..89

Figure 26

C-di-GMP synthesized in vivo by VCA0956 in HeLa cells ..............104

Figure 27

Infection of Ad5-VCA0956 in a murine model system ....................107

Figure 28

qRT-PCR of mouse liver gene transcripts 24 hours after infection with
Ad5 vectors ........................................................................................109

x

Figure 29

IFNγ ELISPOT analysis of mice vaccinated with Ad5-TA and Ad5
vectors ...............................................................................................115

Figure 30

C. difficile challenge of BALB/c mice ...............................................116

Figure 31

C. difficile challenge of BALB/c mice vaccinated with Ad5-TA and
Ad5-VCA0956....................................................................................118

xi

LIST OF ABBREVIATIONS

Ad5 ..................................................................................... Adenovirus type 5-derived
AI ....................................................................................Quorum sensing autoinducer

Bd ........................................................................................................ Below detection
BiC ................................................................................................. Sodium bicarbonate
BV ................................................................................................................ Bovine bile
CDAD .......................................................................... C. difficile associated diarrhea
c-di-AMP ............................................. Cyclic dimeric adenosine 3',5'-monophosphate
c-di-GMP ............................................ Cyclic dimeric guanosine 3',5'-monophosphate
cfu ................................................................................................. Colony forming unit
CHAPS .................... 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
CV ............................................................................................................ Crystal violet
CT .................................................................................................. Cholera enterotoxin
DGC ................................................................................. Diguanylate cyclase enzyme
GTP..................................................................................... Guanosine-5’ triphosphate
h.p.i ............................................................................................... Hours post injection
IM .......................................................................................................... Intramuscular
IPTG ................................................................. Isopropyl β-D1-thiogalactopyranoside
IV ............................................................................................................ Intravenously
LB ................................................................................................ Luria-Bertani media
LC-MS/MS ........... Liquid chromatography coupled with tandem mass spectrometry

xii

M.O.I........................................................................................ Multiplicity of infection

m/z ................................................................................................ Mass to charge ratio
OD......................................................................................................... Optical density
ORF .............................................................................................. Open reading frame
PBS ......................................................................................Phosphate buffered saline
PDE ................................................................................... Phosphodiesterase enzyme
ppGpp ..................................................................... Guanosine 3',5'-bispyrophosphate
pppGpp ..................................................... Guanosine 3'-diphosphate, 5'-triphosphate
QS ....................................................................................................... Quorum sensing
R.L.U ............................................................................... Relative luminescence units
rRNA ................................................................................................... Ribosomal RNA
SDS .......................................................................................... Sodium dodecyl sulfate
SFCs ................................................................................................. Spot forming cells
SHB ............................................................................................ Synthetic human bile
TELCA................................................................ The Ex vivo Lysate c-di-GMP Assay
TCP ......................................................................................... Toxin co-regulated pilus
TLR .................................................................................................... Toll-like receptor
Tris ......................................................... 2-Amino-2-hydroxymethyl-propane-1,3-diol
Vp ........................................................................................................... Viral particles
WT .................................................................................................................. Wild type

xiii

CHAPTER 1 – Background

The bacterial second messenger cyclic di-GMP
Nucleotide small molecules are utilized by organisms from across the tree of
life to relay signals from sensor proteins to target receptor proteins within the cell;
known bacterial second messengers include cyclic adenosine 3',5'- monophosphate
(cAMP), cyclic guanosine 3',5'-monophosphate (cGMP), guanosine 3',5'bispyrophosphate (ppGpp), guanosine 3'-diphosphate, 5'-triphosphate (pppGpp),
cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP), and cyclic dimeric
guanosine 3',5'-monophosphate (c-di-GMP). C-di-GMP was first discovered in 1985
by Benzimen et al. [1], and since has been predicted to be utilized in >75% of all
bacteria in representatives from every major bacterial phyla [2].
Diguanylate cyclase enzymes (DGCs) synthesize c-di-GMP from two GTP
molecules. These enzymes contain a conserved C-terminal GGDEF domain with a
GG[D/E]EF active site motif and a highly variable N-terminus that often encodes
conserved signal recognition domains such as GAF, PAS, or receiver domains [3-6].
Conversely, c-di-GMP specific phosphodiesterase enzymes (PDEs) hydrolyze c-diGMP and contain either a conserved C-terminal EAL or HD-GYP domain [7, 8].
Similar to DGCs, PDE domains are often linked to N-terminal signal recognition
domains.
C-di-GMP regulates numerous phenotypes in different bacteria (for a
comprehensive review of c-di-GMP controlled phenotypes, please refer to Romling et
1

al. [9]). Motility, the ability of a bacterial cell to move, has been shown to be
negatively regulated by c-di-GMP in many organisms [10-13]. Conversely, biofilm
formation, the bacterial behavior where cells excrete exopolysaccharides to form a
three-dimensional surface-associated community [14], is positively regulated by cdi-GMP. Biofilm-forming communities have been shown to play important roles in
environmental persistence and virulence [15]. C-di-GMP has been shown to
positively regulate biofilm formation in several pathogens including Pseudomonas

aeruginosa [11], Salmonella enterica [11], and Vibrio cholerae [16]. The differential
regulation of motility and biofilm formation by c-di-GMP indicates that c-di-GMP
mediates the transition between sessile and motile, virulent lifestyles.
A characteristic feature of c-di-GMP signaling networks is the relatively large
number of DGCs and PDEs encoded in individual bacterial genomes that
theoretically contribute to changes in the intracellular concentration of c-di-GMP.
For example, the bacteria V. cholerae encodes 61 distinct proteins predicted to
modulate c-di-GMP [17]. Because of this complexity, c-di-GMP signaling systems
are typically redundant, and it is challenging to understand on a systems level how
the concentration of c-di-GMP is controlled. Changes in c-di-GMP could be mediated
by modulation of total DGC activity, PDE activity, or both. It is not well understood
why some bacteria encode so many DGCs and PDEs, and the functions of the
variable N-terminal domains for most of these enzymes have not been
characterized. One hypothesis is that each DGC or PDE senses and responds to a

2

specific environmental signal by altering c-di-GMP synthesis or degradation activity
in the C-terminal domain.
A handful of environmental cues have been shown to influence intracellular
c-di-GMP in various bacteria. For example, specific environmental signals that
interact with DGCs and PDEs have been identified such as zinc in E. coli [18],
amino acids in P. aeruginosa [19], nitric oxide in Legionella pneumophila [20], and
norspermidine in V. cholerae [21]. Additionally, a DGC from Bordetella pertussis
and a DGC/PDE complex from E. coli bind oxygen to alter c-di-GMP turnover [22,
23]. There is also evidence that phototrophic bacteria utilize c-di-GMP to modulate
intracellular c-di-GMP in different light conditions [24, 25]. DGCs and PDEs have
also been identified in Xanthomonas campestris and Vibrio parahaemolyticus that
bind molecules associated with quorum sensing networks to modulate c-di-GMP
turnover [8, 26, 27]. However, these enzymes with known ligands represent only a
small fraction of the known DGCs and PDEs, and the environmental signals
recognized by the vast majority of DGCs and PDEs remain unidentified.

Vibrio cholerae: marine bacterium and human pathogen
One bacterium in which c-di-GMP signaling has been studied is V. cholerae.

V. cholerae, a gram-negative marine bacterium, is the causative agent of the human
diarrheal disease cholera. In aquatic environments, V. cholerae favors a sessile
lifestyle by preferentially forming biofilms on chitineous surfaces [28-30]. Upon
ingestion into a human host, V. cholerae expresses virulence and colonization

3

factors that lead to severe diarrhea, subsequently reseeding the bacteria back into
the environment. The pathogenesis of V. cholerae is dependent on two virulence
factors, cholera enterotoxin (CT) and the toxin c-regulated pilus (TCP) [31]. The
expression of both CT and TCP is controlled by the transcriptional regulators
ToxR/S, which coordinate with the transcriptional regulator ToxT to optimize
virulence expression in the human host [32, 33].
The transition of V. cholerae between aquatic environments and the human
host is mediated by c-di-GMP, which regulates biofilm formation, motility, and
virulence gene expression [16, 34-36]. It has been proposed that the intracellular cdi-GMP concentration in V. cholerae is relatively high in aquatic environments
leading to a sessile biofilm forming lifestyle, whereas in the human host, reduced cdi-GMP concentrations stimulate virulence factor expression [34]. Due to the
technical limitations of measuring intracellular c-di-GMP, this model has not been
directly tested, and the specific environmental cues that regulate the transition
between these two niches remain unknown.
Upon entering the human host, it is imperative that V. cholerae recognizes
environmental signals to mediate the transition from an aquatic bacterium to a
human pathogen. One signal prevalent in the small intestine is bile. Bile is an
antimicrobial substance secreted from the liver into the proximal small intestine
that aids in digestion by emulsifying lipids. The composition of secreted bile is
heterogeneous and includes inorganic salts, cholesterol, phospholipids, pigments,
and bile acids [37]. Bile acids are derived from cholesterol in the liver and are
4

processed by hepatic cells to produce a mixture dominated by taurine and glycine
conjugates [37, 38]. Bile acids have detergent properties that enable the interaction
between bile and digestive lipids. For a complete review on the properties of bile,
refer to Begley et al. [39].
The interplay between enteric bacteria and bile is extensive. In bacteria,
there is evidence that bile causes oxidative stress, DNA damage, and perturbs the
cell membrane [40-42]. To counteract this stress, V. cholerae upregulates the porin
protein ompU in a toxR-dependent manner to increase bile resistance [43]. V.

cholerae also employs six resistance-nodulation-division family efflux pumps that
have been implicated in bile resistance [44-46]. Additionally, V. cholerae increases
biofilm formation in a vpsR dependent manner in the presence of bile acids,
presumably to increase resistance to the deleterious effects of bile [47]. It is clear
that the sensing of bile by V. cholerae leads to distinct physiological changes, but a
connection between bile and c-di-GMP has not been described in V. cholerae or any
other bacterium.

Innate Immune response to c-di-GMP
In addition to its role regulatory role as a bacterial second messenger, c-diGMP is a potent stimulator of innate immunity in eukaryotic organisms.
Presumably, as c-di-GMP is an intracellular second messenger unique to bacteria, it
is recognized by eukaryotic organisms as a signal of bacterial invasion. This
stimulation of innate immunity occurs at least in part through the eukaryotic

5

protein STING, which senses pathogen derived nucleic acids in the cytoplasm and
subsequently activates a signaling cascade to stimulate a type-I interferon response
[48, 49]. Studies show that the presence of c-di-GMP can trigger the production of
IL-2, IL-4, IL-5, IL-6, IL-8, IL-12p40, IL-17, IP-10, TNF-α, KC, MIP-1α, MIP-1β,
MIP-2, MCP-1, RANTES, IFN-β, IFN-γ, stimulate the NLRP3 inflammasome
pathway, and promote the recruitment and activation of macrophages, NK-cells, αβ
conventional T-cells, and enhance DC maturation [49-57]. Furthermore, in vivo
studies have shown that co-administration of purified c-di-GMP with an antigen
confers increased protection of animals in several different murine challenge
models, including those utilizing Staphylococcus aureus, Klebsiella pneumoniae,
and Streptococcus pneumoniae [53-55, 58].
Because c-di-GMP activates a robust immune response, there has been
ongoing focus on using c-di-GMP as an adjuvant to improve vaccine efficacy [59].
Adjuvants are compounds administered alongside vaccine antigens for the purpose
of enhancing the longevity, potency, or reducing the effective dose of the antigen
without introducing toxic side effects. This is accomplished by stimulating the
innate arm of the immune system, resulting in increased cytokine and chemokine
production and upregulation of proinflammatory genes [60], which then enhances
antigen recognition and response [61]. The development of novel adjuvants may be
critical to the success of vaccines targeting diseases for which vaccinations have
previously failed such as Clostridium difficile, human immunodeficiency virus,
malaria, and cancer. Despite the demand, currently there are few adjuvants
6

approved for human use. The most commonly used adjuvants are aluminum-salt
(Alum)-based; however, these adjuvants suffer drawbacks including local reactions
to administration, inadequate T-cell responses, allergic IgE-type responses, and are
ineffective with specific types of antigens [62]. Other less-commonly utilized
adjuvants include oil and water emulsions, lipopolysacharide derivatives, selfassembling viral nanoparticles, and cholera toxin B subunit [62]. While each
adjuvant offers different advantages and disadvantages, there is a large demand for
novel adjuvants that can be paired with and improve vaccine antigens. Because cdi-GMP has effective immunostimulatory properties, it makes an ideal candidate
for a novel adjuvant [59].
Despite the properties that make c-di-GMP a promising adjuvant, there are
currently practical limitations to using c-di-GMP as a vaccine adjuvant. The studies
examining the adjuvant properties of c-di-GMP utilized mucosal, intramuscular
(IM), or systemic administration of chemically synthesized c-di-GMP [53-55, 58].
While this method has been shown to effectively activate innate immunity, it lacks
efficiency and the mechanism by which c-di-GMP enters the cytoplasm of cells to
stimulate STING is unknown. Furthermore, chemical synthesis of pharmaceutical
grade c-di-GMP in large quantities is currently cost prohibitive. There is a need for
an efficient and cost effective method of delivering c-di-GMP in vivo for use as an
adjuvant.

7

Summary
The broad goal of this study is to understand how the model bacterium V.

cholerae uses c-di-GMP signaling to interpret and respond to changing
environmental signals. The central hypothesis that underlies my research is that V.

cholerae senses environmental signals with different DGC and PDE proteins to
modulate c-di-GMP concentrations in different environments. The rationale for this
study is that c-di-GMP is a conserved second messenger utilized by a broad
spectrum of bacteria, and the mechanisms by which V. cholerae utilizes c-di-GMP
signaling in different environments can be applied to other bacterial systems to
further comprehend how they similarly respond and adapt to their environments.
Furthermore, as c-di-GMP has potent immunostimulatory properties, knowledge of
this signaling system can be appropriated to develop new applications for clinical
settings.
To show that a changing environment impacts c-di-GMP signaling globally, I
have demonstrated that intracellular c-di-GMP concentrations of V. cholerae vary
when V. cholerae is grown in different environments. I developed The Ex Vivo
Lysate Cyclic di-GMP Assay (TELCA) to examine total cellular DGC and PDE
activity, and used TELCA to show that these variations of c-di-GMP concentrations
in different environments are due to changes in both c-di-GMP synthesis and
hydrolysis. Using TELCA, I was also able to show that quorum sensing regulation
of c-di-GMP levels in V. cholerae is due primarily to decreased DGC activity during
transitions from low- to high-cell-density.
8

I have also begun to explore specific environmental cues that modulate c-diGMP in V. cholerae, and I have shown that bile acids increase intracellular c-diGMP in V. cholerae. I determined that this increase is due to multiple factors
including altered c-di-GMP synthesis activity of three DGCs and reduced expression
of one PDE. Additionally, this bile-mediated increase in intracellular c-di-GMP is
quenched by bicarbonate, another common component of the human digestive tract.
Finally, I have applied the knowledge I have gained regarding active DGCs of

V. cholerae to develop a novel adenovirus adjuvant that delivers DGC DNA into
eukaryotic cells, resulting in c-di-GMP synthesis in vivo and a robust innate
immune response. Additionally, this adenovirus construct harboring a DGC gene is
able to reduce the effective dose of a C. difficile vaccine antigen to stimulate IFNγ
production of splenocytes. This novel method of in vivo c-di-GMP synthesis offers a
more efficient and cost-effective alternative to using c-di-GMP as a vaccine
adjuvant.

9

CHAPTER 2 - Exploring Environmental Control of Cyclic didi-GMP Signaling in

Vibrio cholerae by Using the Ex Vivo Lysate Cyclic didi-GMP Assay (TELCA)
Preface
Quorum sensing (QS), the process of bacterial cell-cell communication
mediated by AIs that are constitutively secreted and accumulate with cell density,
is a fundamental chemical signaling system in bacteria. Sensing of AIs by bacteria
provides information on the local population density and composition to
appropriately regulate bacterial behaviors such as competence, biofilm formation,
and virulence [63-68]. In V. cholerae, QS is mediated by two AI molecules (CAI-1
and AI-2) that are produced in tandem [67]. In the low-cell-density state, AI
concentrations are low and their cognate receptors function as kinases ultimately
leading to phosphorylation of the response regulator LuxO. Phosphorylated LuxO
upregulates the transcription of four small regulatory RNAs, which subsequently
suppress translation of the master QS regulator HapR [69, 70]. When the
concentration of AIs increase, they bind the AI receptors switching them to
phosphatases, leading to dephosphorylation of LuxO, deactivation of the sRNAs,
and induction of HapR expression [71]. In V. cholerae, biofilm formation is
repressed in the high-cell-density QS state via direct repression of extracellular
polysaccharide synthesis genes by HapR [66, 72].
Both the QS and c-di-GMP signaling systems are inextricably linked in V.
cholerae [73]. At low cell density, the intracellular c-di-GMP concentration of V.

10

cholerae is relatively high, promoting biofilm formation and inhibiting motility.
Upon transition to the high-cell-density state the intracellular c-di-GMP
concentration is suppressed, decreasing biofilm formation [66, 72]. In V. cholerae
the transcription of 18 of the 61 predicted c-di-GMP turnover enzymes are
differentially regulated at low and high cell density [66, 72, 74]. However, because
of the complexity of this signaling system it remains unclear if the QS-mediated
changes in c-di-GMP levels in V. cholerae are being driven by changes in c-di-GMP
synthesis, hydrolysis or both.
To further the exploration of the environmental modulation of c-di-GMP and
the regulatory connections between QS, c-di-GMP, and biofilm formation in V.

cholerae, I have developed a novel approach, hereby named The Ex vivo Lysate c-diGMP Assay (TELCA), which enables the quantification of c-di-GMP synthesis and
hydrolysis of a whole-cell lysate. I have shown that growing V. cholerae in divergent
environments can lead to a 20-fold difference in the intracellular concentration of cdi-GMP, and these distinct c-di-GMP concentrations can be attributed to both
changes in c-di-GMP synthesis and hydrolysis. I also used TELCA to show that
alteration of both c-di-GMP synthesis and hydrolysis between the low- and high-cell
density QS states contributes to the QS-mediated change of intracellular c-di-GMP.
I further show that deletion of the DGC CdgA, which is transcriptionally regulated
by HapR and has been predicted to be a key DGC in the induction of biofilm
formation [72, 74-76], significantly reduces the elevated DGC activity associated
with low cell density, highlighting the sensitivity of TELCA. TELCA provides a new
11

method to study complex c-di-GMP signaling networks from a systems perspective.
The findings of this chapter have been published in the Journal of Applied and
Environmental Microbiology [77].

Bacterial strains and growth conditions
The V. cholerae El Tor biotype strain C6706str2 was used for all experiments
[78]. All strains, plasmids, and primers used are listed in Tables A1-3. The
construction of the expression plasmids for VCA0956 and VC1086 are described
elsewhere [72, 79]. Unless otherwise specified, cultures were grown in LuriaBertani media (LB, 1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, Accumedia) at
35°C with shaking at 220 rpm. MOPS media was purchased from Teknova and
prepared as per manufacturer’s instruction. The composition of AKI, AB, ASW, and
M9 media are described elsewhere [80-83]. MOPS, AB, and M9 were supplemented
with glucose as a carbon source (0.2% for MOPS and AB, 0.4% for M9). For
experiments involving the expression of DGCs or PDEs from plasmids, media was
supplemented with kanamycin (Sigma) at 100 µg/mL and the inducer isopropyl βD1-thiogalactopyranoside (IPTG) at 0.1 mM.
The construction of the ∆hapR and ∆luxO strains has been described
previously [67, 68]. To generate the ∆cdgA strain, natural transformation and
homologous recombination were used. Briefly, Phusion DNA polymerase (NEB) was
used to amplify approximately 500 bp upstream and downstream of cdgA
(VCA0074) using the primers VCA0047KO-1 (upstream) and VCA0047KO-2

12

(downstream), and VCA0047KO-3 (upstream) and VCA0047KO-4 (downstream)
(Table A3). Fusion PCR was then used to join these products upstream and
downstream of a chloramphenicol resistance cassette (cat) bordered by FLP
recombination target (FRT) sites from the plasmid pKD3 [84]. This DNA was
introduced to V. cholerae by inducing natural transformation using a modified
protocol from Marvig and Blokesch [85]. V. cholerae was grown on chitin flakes in
modified M9 media [82]. This media (M9-T) contained the standard concentration of
M9 salts supplemented with 5 mM CaCl2, 3.6 mM MgSO4, and 4.5 mM N-acetyl-Dglucosamine as a carbon source and was balanced to a pH of 6.0. Concentrated
DNA was introduced to the culture, and homologous recombination events were
selected by growing the culture on LB supplemented with 1 µg/mL chloramphenicol.
The cat gene was excised by ectopically expressing the FLP recombinase from the
vector pTL17 [86].

Synthesizing [13C]-c-di-GMP
[13C]-c-di-GMP was synthesized from guanosine-[13C]-5’ triphosphate ([13C]GTP, Sigma) using the purified DGC WspR-R242A as previously described [87]. The
reaction consisted of 56 µM WspR-R424A, 20 µM [13C]-GTP, 24 mM Tris-Cl, 5 mM
MgCl2, 45 mM NaCl, and EnzCheck Phosphate Assay Kit reaction buffer mix
(Invitrogen) at a total volume of 100 µL, hypothetically yielding 10 µM [13C]-c-diGMP (two GTP molecules per molecule c-di-GMP). The reaction was run overnight
followed by treatment for 30 minutes at 37°C with Antarctic phosphatase (NEB) in
its corresponding reaction buffer to remove any excess [13C]-GTP. The reaction was
13

heated at 100°C for 10 minutes to inactivate the Antarctic phosphatase and
precipitate protein. After cooling to room temperature, the reaction was centrifuged
at max speed for 10 minutes to remove the precipitated protein, and the
supernatants were removed and stored at -20°C until use. [13C]-c-di-GMP synthesis
was confirmed using liquid chromatography with mass spectrometry as described
below.

Generation of lysates
Cell lysates for TELCA were generated by inoculating overnight cultures of

V. cholerae 1:1000 into 30 mL of the specified media with the appropriate
antibiotics or inducer. Cultures grown to the desired OD600 as indicated were
centrifuged at 7800 rpm to pellet the cells. The pellets were then resuspended in 15
mL TELCA reaction buffer, which consisted of 25 mM Tris, 5 mM MgCl2, and 5 mM
NaCl balanced to a pH of 7.5. The cultures were then processed three times using a
M110-P processor (Microfluidics) at 20,000 psi producing a clear lysate. A protein
assay (Bio-Rad) was performed according to the manufacturer’s instructions to
quantify the total protein content, and each lysate was diluted to 0.1 mg/mL in
TELCA reaction buffer. To measure c-di-GMP synthesis, 100 mM [13C]-GTP stock
was added to a final concentration of 1 mM (10 µL [13C]-GTP stock in 990 µL lysate
diluent for timecourse experiments, 1 µL [13C]-GTP stock in 99 µL lysate diluent for
endpoint experiments). To measure c-di-GMP hydrolysis, 10 µM [13C]-c-di-GMP
stock was added to a final concentration of 100 nM (10 µL [13C]-c-di-GMP stock in
990 µL lysate diluent for timecourse experiments, 1 µL [13C]-c-di-GMP stock in 99
14

µL lysate diluent for endpoint experiments). The concentrations are within the
physiological ranges of the natural concentrations of GTP and c-di-GMP found in a
bacterial cell [79, 88]. The reactions were then mixed and incubated at 37°C until
the experimental endpoint, and then the reaction was stopped by mixing 100 µL
reaction to 100 µL of phenol:chloroform:isoamyl alcohol (25:24:1). The mixture was
centrifuged at max speed for 5 minutes, and then the aqueous phase was removed
and stored at -80°C until analysis.

Detection and quantification of c-di-GMP
For the quantification of intracellular c-di-GMP, overnight cultures grown in
the corresponding media were used to inoculate 2 mL cultures (1:1000). Cultures
were grown to the appropriate OD600 (OD600, AKI: 0.5-1.0; LB: 0.5-0.8; ASW: 0.2-0.4;
MOPS: 0.4-0.8; AB: 0.3-0.7; M9: 0.2-0.4), and a 1.5 mL aliquot of culture was
centrifuged at maximum speed for 30 seconds. The supernatant was removed, and
the pellet was resuspended in 100 µL of cold extraction buffer (40% acetonitrile/40%
methanol/0.1 N formic acid). The slurry was incubated at -20° C for 20 minutes, and
then the insoluble fraction was pelleted by centrifugation for 10 minutes at
maximum speed. The supernatant was collected and stored at -80°C. Prior to
analysis, the extraction buffer was evaporated using a vacuum manifold, and the
pellet was resuspended in 100 µL water. [12C]-c-di-GMP and [13C]-c-di-GMP was
quantified using an Acquity Ultra Performance liquid chromatography system
(Waters) coupled with a Quattro Premier XE mass spectrometer (Waters) as
previously described [79]. Chromatographic separation of 10 µL sample was
15

accomplished using reverse phase separation on a Waters BEH C18 2.1 x 50 mm
column and a flow rate of 0.3 mL/minute, using the following gradient: solvent A (10
mM tributylamine and 15 mM acetic acid in 97:3 water:methanol) to solvent B
(methanol), 0 minutes; A=99%:B=1%, 2.5 minutes; A=80%:B=20%, 7.0 minutes;
A=35:B=65%, 7.5 minutes; A=5%:B=95%, 9.01 minutes; 99%:B=1%, 10 minutes;
gradient ends.
After separation, the detection of c-di-GMP was carried out using
electrospray ionization with multiple reaction monitoring in negative ion mode
where [13C]-c-di-GMP was detected with a m/z of 709.16 -> 354.31 and [12C]-c-diGMP with a m/z of 689.16 -> 344.31. The MS detection was optimized using the
following parameters: capillary voltage, 3.5 KV; cone voltage, 50 V; collision energy,
34 V; source temperature, 120°C; desolvation temperature, 350°C; cone gas flow, 50
L/h (nitrogen), 800 L/h; collision gas flow (nitrogen), 0.15 mL/min; and multiplier
voltage, 650 V. The concentration of c-di-GMP was determined by generating an 8point standard curve (1:2 dilutions) of chemically synthesized c-di-GMP (Biolog)
ranging from 1.9 nM to 250 nM. The intracellular c-di-GMP concentration was
calculated by dividing the c-di-GMP concentration of the sample by the total
intracellular volume of bacteria extracted. This was estimated by multiplying the
average cell volume of the bacterial cell, determined by measuring individual cell
dimensions using differential image contrast microscopy, and the number of
bacterial cells in the extract.

16

TELCA quantification of whole cell DGC activity
I sought to develop a systems approach to examine if changes in the levels of
c-di-GMP in V. cholerae could be attributed to the modulation of net DGC and/or
PDE activity. The rationale for developing this method is that current approaches to
directly measure c-di-GMP primarily utilize liquid chromatography coupled with
tandem mass spectrometry (LC-MS/MS) of cell extracts [89-91] or an in vivo FRET
biosensor to quantify c-di-GMP at the single cell level [92]. While these methods
allow accurate quantification of the average intracellular c-di-GMP in the cell, they
cannot distinguish if differences in the intracellular c-di-GMP are being driven by
changes in c-di-GMP synthesis or hydrolysis. Consider a case where the levels of cdi-GMP are elevated by growth in a different environment. This increase in c-diGMP can be due to increased DGC activity, decreased PDE activity, or modulation
of both. One cannot predict a priori solely based on the concentration of c-di-GMP
how this change is mediated. My method, named TELCA, quantifies total DGC or
PDE activity of cell lysates using [13C]-containing substrates to allow the
differentiation of newly synthesized or degraded c-di-GMP from natural occurring cdi-GMP by mass spectrometry. The substrate for the DGC assay measures
synthesis of [13C]-c-di-GMP from exogenous [13C]-GTP whereas the PDE assay
monitors the degradation of exogenous [13C]-c-di-GMP.
To assess how effectively TELCA measures cellular DGC activity, extracts
were generated from cultures of wild type (WT) V. cholerae ectopically expressing
an active DGC (VCA0956), an active PDE (VC1086), or the vector control. Note that
17

WT V. cholerae has a relatively low intracellular concentration of c-di-GMP at the
growth state used for these experiments (mid-exponential growth, OD600 of 0.5-0.8)
due to QS mediated reduction in c-di-GMP levels at high cell density. I hypothesized
that lysates produced from cells expressing VCA0956 would more rapidly
synthesize [13C]-c-di-GMP from [13C]-GTP than lysates with the empty vector
control or overproducing the PDE VC1086. The cells from all three cultures were
harvested and lysed using a Microfluidics M110-P Processor, which exposes the
cells to high shear and impact force resulting in a uniform lysate. This process
causes minimum disruption of protein conformation and thus is well suited to
generate lysates for this assay. [13C]-GTP was added to 0.1 mg/mL total protein
from each extract to a final concentration of 1.0 mM, and aliquots of the reaction
were removed at specified timepoints followed by quenching with a phenolchloroform extraction. The concentration of newly synthesized [13C]-c-di-GMP was
quantified using LC-MS/MS. To differentiate [13C]-c-di-GMP from naturally
occurring [12C]-c-di-GMP, I measured a mass to charge ratio (m/z) of 709.16 >
354.31 to detect [13C]-c-di-GMP and 689.16 > 344.31 for [12C]-c-di-GMP as
previously described (Fig. 1A) [79]. The TELCA reaction generated a signal at the
same retention time of chemically synthesized [12C]-c-di-GMP standards at a m/z of
709.16 > 354.31 with no signal at 689.16 > 344.31, indicating that this detection
method is sufficient to discriminate between these two carbon isotope forms of c-diGMP (Fig. 1B).

18

Figure 1. LCLC-MS/MS differentiation
differentiation of [12C]C]-c-didi-GMP and [13C]C]-c-didi-GMP. (A)
Molecular structure of c-di-GMP with the m/z used for detection and quantification.
The top m/z describes the half of the molecule observed after fragmentation (as
indicated by the line) while the bottom m/z represent the entire molecule. The
values for both [12C]- and [13C]-c-di-GMP are indicated. (B) The spectrum of [13C]-cdi-GMP. The top contains a peak at 5.4 minutes with a m/z of 709.16 > 354.31,
indicating the presence of [13C]-c-di-GMP. The bottom contains no peak at 5.4
minutes with a m/z of 689.16 > 354.31, indicating that no [12C]-c-di-GMP is present.

19

A non-linear regression with a one-phase decay model (Graphpad Prism) was
used to analyze the increase in [13C]-c-di-GMP concentrations over time. I
constrained the maximum yield of the model to 0.5 mM [13C]-c-di-GMP as this is the
maximum theoretical yield of the reaction. The lysate from V. cholerae expressing
the DGC produced significantly more (two tailed Student’s T-Test, p < 0.05) [13C]-cdi-GMP over time, yielding 10.2 fold and 17.1 fold more c-di-GMP than the vector
control and PDE lysates, respectively, after 120 minutes (Fig. 2). Furthermore, the
rate constant for the DGC lysate, which represents the increase of c-di-GMP
synthesis per unit time expressed as inverse minutes (6.9±1.1x10-6 min-1, R2=0.76)
was significantly different (sum-of-squares F-test, p < 0.05) than those of the vector
control lysate (5.9±0.5x10-7 min-1, R2=0.90) and the PDE lysate (4.1±0.4x10-7 min-1,
R2=0.87). This result indicates that TELCA is able to differentiate the cellular DGC
activity of lysates containing a higher concentration of active DGCs. Furthermore, I
found that lysates generated from V. cholerae expressing the PDE produced 1.7 fold
less [13C]-c-di-GMP than the vector control lysate after 120 minutes and exhibited a
lower rate constant, indicating that additional PDEs in the lysate can reduce the
net c-di-GMP synthesis by hydrolyzing [13C]-c-di-GMP post synthesis.
Of note, [13C]-c-di-GMP accumulated in all three lysates including the PDE
overexpression strain suggesting that given excess GTP, DGC activity supersedes
PDE activity in these conditions over the time course examined. A subset of DGCs
exhibit feedback inhibition by an allosteric RXXD inhibition site in close proximity
to the DGC active site [10]. VCA0956 does not have a predicted inhibition site;
20

600
[ 13 C]-c-di- GMP
(nM)

Vector
PDE
400

DGC

200

0
1

10

100

Time
(minutes)
C]--c-di
di--GMP synthesis of DGC and PDE ectopic expression
Figure 2. TELCA of [13C]
strains. TELCA was applied to lysates of V. cholerae strains ectopically expressing
an active DGC (VCA956, solid squares), an active PDE (VC1086, empty squares),
and an empty vector control (empty circles). [13C]-GTP was added to each lysate and
[13C]-c-di-GMP synthesis was tracked over time using LC-MS/MS. A non-linear
regression using a two-phase exponential decay model was applied to each lysate.

however, it is possible that the c-di-GMP synthesis of another DGC subject to
feedback inhibition would have an altered synthesis profile. Additionally, it should
be noted that I was not able to detect any hybrid c-di-GMP composed of one [12C]GTP and one [13C]-c-di-GMP (detection m/z of 709.16 > 344.31), indicating that the
native [12C]-GTP is depleted upon addition of [13C]-GTP. Furthermore, there was no
[12C]-c-di-GMP detected in the vector control lysate or the PDE lysate at any
timepoint. However, in the DGC lysate, I did detect [12C]-c-di-GMP in low
abundance alongside synthesized [13C]-c-di-GMP suggesting that if [12C]-GTP is to

21

be used in lieu of [13C]-GTP, additional measures would be required to compensate
for the existence of native c-di-GMP in certain strains.

TELCA quantification of whole cell PDE activity
I performed a similar experiment to determine if TELCA is effective at
differentiating the PDE activity of different bacterial strains; however, instead of
using [13C]-GTP as the initial substrate, [13C]-c-di-GMP was added. To generate
[13C]-c-di-GMP, a c-di-GMP synthesis reaction was performed using [13C]-GTP and
the DGC WspR that contained a mutation in the RXXD allosteric inhibition site
(R242A, [87]). The remaining labeled GTP was removed by treating the reaction
with Antarctic phosphatase, although quantification of enzymatically synthesized
[13C]-c-di-GMP using LC-MS/MS indicated that the reaction went to completion,
generating the expected hypothetical yield. I observed that the PDE lysate more
rapidly degraded [13C]-c-di-GMP than both the vector control lysate and the DGC
lysate (Fig. 3), as the PDE lysate retained an average of 20.5% [13C]-c-di-GMP after
120 minutes whereas the vector control lysate and the DGC lysate retained
averages of 69.6% and 73.9% of the [13C]-c-di-GMP, respectively. The difference
between the PDE lysate and both the vector control and DGC lysates were
significantly different (two tailed Student’s T-Test, p < 0.05). Likewise, a non-linear
regression with a one-phase decay model with a plateau constraint of zero was
applied to the data, and I found that the rate constant of the PDE lysate
(1.9±0.6x10-2 min-1, R2=0.66) was significantly higher (sum-of-squares F-test, p <
0.05) than both the vector control lysate (2.5±1.0x10-3 min-1, R2=0.35) and the DGC
22

lysate (2.0±0.9x10-3 min-1, R2=0.27). As expected, there were no notable differences
between the DGC-expressing lysate and the vector control lysate suggesting that a
difference in DGC activity does not impact the PDE activity. From these data, I
conclude that TELCA is sufficient to differentiate both the c-di-GMP synthesis and
hydrolysis potential of whole-cell lysates.

[ 13 C]-c-di-GMP
(nM)

100

Vector
PDE
DGC

50

0
1

10

100

Time
(minutes)
Figure 3. TELCA of [13C]C]-c-didi-GMP hydrolysis of DGC and PDE ectopic expression
strains. TELCA was applied to lysates of V. cholerae strains ectopically expressing
an active DGC (VCA956, solid squares), an active PDE (VC1086, empty squares),
and an empty vector control (empty circles). [13C]-c-di-GMP was added to each
lysate and the hydrolysis was tracked over time using LC-MS/MS. A non-linear
regression using a two-phase exponential decay model was applied to each lysate.

The local environment alters the intracellular concentration of c-di-GMP in
V. cholerae
How the extracellular environment impacts the synthesis and degradation of
intracellular c-di-GMP is poorly understood. It has been proposed that extracellular
23

stimuli, such as oxygen, light, amino acids, and AIs, regulate intracellular c-di-GMP
by altering the activity of DGCs and PDEs [8, 19-24, 26, 27]. Also, many DGCs and
PDEs have domains predicted to be involved in sensing extracellular ligands [93]. I
hypothesized that V. cholerae growing in different environments would exhibit
distinct concentrations of c-di-GMP due to alterations in the environmental inputs
controlling DGC and PDE activity. To test this hypothesis, I quantified the
intracellular c-di-GMP concentration of V. cholerae in six different growth media
using LC-MS/MS. For the purposes of this study, I consider AKI [80], LB, and ASW
complex, carbon rich media, while MOPS, AB [81] and M9 [82] are considered
defined, carbon poor media. I sampled from each media during the latter half of
steady-state growth, which I define as 1-2 doublings from stationary phase. This
time point was determined by generating growth curves for V. cholerae in each
different media (Fig. 4).
I found that V. cholerae demonstrated differences of up to 20-fold in the
intracellular concentration of c-di-GMP when grown in these different media (Fig.
5). Consistent with prior studies, I measured the concentration of c-di-GMP in LB to
be 101.2±52.7 nM [79]. The complex, carbon rich media AKI also produced relatively
low intracellular c-di-GMP that was statistically indistinguishable from LB,
measuring as 113.9±43.3 nM. In contrast, the carbon poor media AB and M9
exhibited significantly higher levels of intracellular c-di-GMP at 529.4±307.2 nM
and 2379.9±745.8 nM, respectively, and M9 media had significantly higher
intracellular c-di-GMP than AB media. Interestingly, the intracellular c-di-GMP of
24

10

LB
AKI

OD 600

1

ASW
0.1

MOPS
AB

0.01

M9

0.001
0

200

400
Time
(minute s)

600

800

Figure 4. Growth curves of V. cholerae grown in different media. Cultures of V.
cholerae were inoculated 1/1000 into 50 mL of the designated media, and OD600
readings were taken at regular time intervals. Each point represents the mean of
three replicates, and the error bars indicate standard error of the mean.

V. cholerae grown in MOPS more resembled LB and AKI, measuring at 126.2±41.5
nM; in contrast, intracellular c-di-GMP of V. cholerae grown in ASW was modestly
higher, measuring at 251.6±75.5 nM. Thus, these results support the idea that c-diGMP signaling systems primarily function as environmental sensors and modulate
c-di-GMP accordingly. Moreover, the high concentration of c-di-GMP observed in M9
media suggests the range of physiologically relevant concentrations of c-di-GMP
experienced by V. cholerae is greater than currently appreciated.

25

4000

Intrace llular c-di-GMP
(nM)

3000
2000
1000
800
600
400
200
0

AKI
α

LB
β

ASW
χ

MOPS
δ

AB
ε

M9
φ

χεφ

χεφ

αβδφ

χεφ

αβδφ

αβχδε

Figu
Figure 5. Intracellular cc-didi-GMP concentrations
concentrations of V. cholerae grown in different
media.
media. V. cholerae was grown in 6 different media, and cells were harvested during
late-exponential growth. The intracellular concentration was quantified using LCMS/MS and calculated using cell counts, determined by OD600 and plating, and cell
size, determined by microscopy. Error bars represent standard deviation and
corresponding symbols indicate statistical significance, which was determined using
a two-tailed Student’s T-Test (p < 0.05).

Growth environments modulate both c-di-GMP synthesis and hydrolysis
While it is clear that the different growth environments alter intracellular cdi-GMP concentrations of V. cholerae, it is unclear if these changes are being driven
by modulation of DGC and/or PDE activity. I addressed this question using TELCA
on cultures that were grown in four of the six different growth media examined in
Fig. 5. I hypothesized that [13C]-c-di-GMP synthesis would be greater when cells

26

were grown in both AB and M9 media, as the intracellular c-di-GMP of cells grown
in these media is elevated. [13C]-GTP was added to lysates generated from V.

cholerae grown in each environmental condition, and [13C]-c-di-GMP synthesis was
quantified after 60 minutes using LC-MS/MS (Fig. 6A). Analogous to the
intracellular c-di-GMP measurements, V. cholerae grown in AKI and LB exhibited
similar c-di-GMP synthesis. Consistent with my hypothesis, growth of V. cholerae in
AB media exhibited significantly elevated c-di-GMP synthesis, corresponding to its
elevated intracellular c-di-GMP concentration. Interestingly, however, cells grown
in M9 media showed a lower amount of c-di-GMP synthesis than those grown in AB,
even though M9 produced the highest intracellular concentration of c-di-GMP that I
observed.
Based on my analysis of DGC activity I hypothesized that cells grown in M9
must exhibit lower PDE activity relative to the other media to account for the high
concentration of c-di-GMP. To test this hypothesis, TELCA was used to quantify
[13C]-c-di-GMP hydrolysis of cell lysates (Fig. 6B). Consistent with the intracellular
c-di-GMP measurements, cells grown in AKI and LB had similar PDE activity; cells
grown in AB media also had similar c-di-GMP hydrolysis to AKI and AB. However,
in support of my hypothesis, cells grown in M9 media had significantly lower PDE
activity compared to cells grown in the other media conditions. TELCA analysis of

V. cholerae grown in these four different media suggests that suppressed PDE
activity increases intracellular c-di-GMP in M9 media, while elevated DGC activity
increases intracellular c-di-GMP in AB media.
27

A

13
[ C]-c-di-GMP
S ynthesized (nM )

100
80
60
40
20
0

AKI

LB

AB

M9

AKI

LB

AB

M9

B

[

13

C]-c-di-GMP
Percent Los s

100
80
60
40
20
0

Figure 6. TELCA of V. cholerae grown in different media. (A) TELCA was applied to
lysates of WT V. cholerae grown in different media. [13C]-GTP was added to each
lysate and the amount of [13C]-c-di-GMP synthesis was quantified using LC-MS/MS.
(B) TELCA was applied to lysates of WT and QS mutants of V. cholerae. [13C]-c-diGMP was added to each lysate, and the amount of [13C]-c-di-GMP hydrolysis was
quantified using LC-MS/MS. The percent loss was calculated by comparing each
lysate to a no-protein control reaction. All cultures were grown in triplicate, and the
error bars indicate the standard deviation from the mean. Brackets indicate
statistical significance using a one-tailed Student’s T-Test (p<0.05).

28

QS controls both total DGC and PDE activity in V. cholerae
As mentioned above, QS modulates the intracellular concentration of c-diGMP in V. cholerae to regulate biofilm formation, but the specific DGCs and PDEs
involved in this regulation are not fully understood. This question is challenging to
address as QS controls the transcription of 18 proteins that potentially alter c-diGMP levels [66, 72]. Moreover, DGCs and PDEs could have differential enzymatic
activities at low versus high cell density. To address this question, I used TELCA to
determine if QS modulates the levels of c-di-GMP in V. cholerae by altering DGC
and/or PDE activity. To analyze DGC and PDE activity of V. cholerae in different
QS states, the WT, ∆hapR, and ∆luxO mutant strains were analyzed. The ∆hapR
strain is a QS mutant that is locked in low cell density as HapR is the master high
cell density transcriptional regulator, whereas the ∆luxO strain is a QS mutant
locked in high cell density as the Qrr sRNAs are never expressed. Importantly,
cultures were grown and harvested at an OD600 of 0.2-0.3, corresponding to the lowcell-density state. Therefore, I expected the WT strain to be in low cell density and
mimic the ∆hapR mutant. Indeed, TELCA analysis indicated that the WT and the
∆hapR lysates exhibited significantly elevated c-di-GMP synthesis compared to the
∆luxO mutant (Fig. 7A), consistent with these strains having a higher concentration
of c-di-GMP [66, 72]. Analysis of the DGC activity of WT cells grown to high cell
density (compare Fig. 6A, LB to Fig. 7A, WT) indicates that the DGC activity
decreases as cells reach a quorum. In fact, the DGC activity of the ∆luxO mutant at

29

A
13
[ C]-c-di-GM P
S ynthesized (nM)

100
80
60
40
20
0
WT

∆ hapR

∆ luxO

∆ cdgA

WT

∆ hapR

∆ luxO

∆ cdgA

B

[

13

C]-c-di-GMP
Percent Los s

100
80
60
40
20
0

Figure 7. TELCA of V. cholerae QS mutants. (A) [13C]-GTP was added to each lysate
and the amount of [13C]-c-di-GMP synthesis was quantified using LC-MS/MS. (B)
[13C]-c-di-GMP was added to each lysate, and the amount of [13C]-c-di-GMP
hydrolysis was quantified using LC-MS/MS. The percent loss was calculated by
comparing each lysate to a no-protein control reaction. All cultures were grown in
triplicate, and the error bars indicate the standard deviation from the mean.
Brackets indicate statistical significance using a one-tailed Student’s T-Test
(p<0.05).

30

low cell density (Fig. 7A) is quite similar to that of WT V. cholerae grown to high
cell density (Fig. 6A, LB).
I performed an analogous experiment to quantify the impact of QS on net
PDE activity. The relative PDE activity was determined for each of the WT, ∆hapR,
and ∆luxO lysates by measuring the percent loss of [13C]-c-di-GMP. I found that the
percent loss of [13C]-c-di-GMP was significantly higher in the ∆luxO strain
compared to the ∆hapR mutant and WT strain (Fig. 7B). The PDE activity of WT
cells harvested at low cell density was indistinguishable from the ∆hapR mutant.
This result would suggest that PDE activity is increased as cells transition from low
to high cell density. However, a comparison the WT strain grown to high cell
density (40.6 ± 8.3 nM; Fig. 6B, LB) and the WT strain grown at low cell density
(46.6 ± 8.7 nM; Fig. 7B) reveals a similar PDE activity. Indeed, the PDE activity of
WT grown to high cell density (40.6 ± 8.3 nM; Fig. 6B, LB) was lower than the
∆luxO mutant (72.2 ± 8.0 nM; Fig. 7B). This difference in PDE activity between the
WT strain and ∆luxO mutant was evident even if the WT strain was grown to
stationary phase (data not shown). Thus, in regards to PDE activity the ∆luxO
strain has a more dramatic phenotype than WT V. cholerae growing in LB.

CdgA contributes to the increased DGC activity at low cell density
The above experiments suggest that increased DGC activity at low cell
density contributes to the higher concentration of c-di-GMP. CdgA (VCA0074) is a
well-studied DGC that is repressed in the high-cell-density state and is important

31

for biofilm formation [72, 75, 94-97]. Based on a large body of evidence that
indicates CdgA plays an important role in bridging c-di-GMP signaling and QS (see
discussion), I hypothesized that CdgA significantly contributed to the increased
DGC activity I observed at low cell density. To test this hypothesis and determine if
TELCA can detect the contribution of a single DGC to total cellular activity, lysates
from the ∆cdgA mutant grown to low cell density were analyzed. I observed a 36.4%
decrease in DGC activity in the ∆cdgA strain compared to the WT strain, although
the differences did not quite reach statistical significance (p=0.06) (Fig. 7A). As
expected, there was no significant difference in the net PDE activity of the ∆cdgA
strain and the WT strain (Fig. 7B). These results confirm previous genetic studies
that implicate CdgA as a major contributor to the increased DGC activity observed
in the low-cell-density state in V. cholerae, and further illustrate the applicability of
TELCA to analyze complex c-di-GMP signaling pathways.

Discussion
The bacterial second messenger c-di-GMP is a central regulator of biofilm
formation and motility in bacteria. Indeed, bioinformatic studies indicate that DGCs
and PDEs are encoded by more than 80 percent of all sequenced bacteria [2]. C-diGMP controls many important phenotypes in bacteria including biofilm formation,
motility, production of virulence factors, and lifecycle progression, and this list
continues to grow [11, 13, 16, 98-100].

32

Net changes in DGC or PDE activity leading to different intracellular c-diGMP concentrations can be difficult to parse as bacteria often encode multiple
DGCs and PDEs. Furthermore, it is often challenging to resolve differences in c-diGMP signaling pathways in single DGC or PDE mutants due to this redundancy
and the inherently low concentration of c-di-GMP in many bacterial species (for
example, see [101]). To circumvent these challenges, I developed TELCA to quantify
total cellular c-di-GMP synthesis and hydrolysis activities. The [13C]-labeled
substrates TELCA utilizes are stable, safe to use, and can be distinguished from
naturally occurring [12C]-c-di-GMP using mass spectrometry. To my knowledge, this
is the first description of an in vitro assay to measure cellular DGC or PDE activity.
To test TELCA, I examined strains of V. cholerae ectopically expressing
either a GGDEF or an EAL enzyme and observed enhanced DGC and PDE activity,
respectively. These experiments validated the approach and demonstrated that I
can differentiate lysates with increased synthesis and hydrolysis activities. As an in

vitro assay, disruption of the cell might alter the activity of localized or membrane
bound proteins in the lysate. However, it should be noted that CdgA is a predicted
integral inner membrane protein yet I was able to quantify a loss in cellular DGC
activity of a ∆cdgA mutant using TELCA, showing that CdgA remained active
during the assay.
Regulatory inputs of the c-di-GMP signaling network remain undercharacterized. The consensus of the field is that c-di-GMP turnover enzymes bind
and respond to environmental ligands, host derived metabolites, or are directly
33

modified by post-translational regulatory mechanisms to regulate c-di-GMP
synthesis or hydrolysis, thus enabling the bacterium to fine tune intracellular c-diGMP to its environment [6]. Indeed, I showed that when V. cholerae is cultured in
different media, the intracellular c-di-GMP varies significantly (Fig. 5).
Surprisingly, the changes in the c-di-GMP levels were dramatic with cells grown in
M9 exhibiting 20 times more c-di-GMP than cells grown in LB. This result suggests
that V. cholerae, and potentially other bacteria, experience a much wider range of cdi-GMP concentrations than previously appreciated. Moreover, cells grown in
defined, carbon poor media demonstrated slower growth rates and higher levels of cdi-GMP compared with complex, carbon rich media.
The increased c-di-GMP observed in AB and M9 could be generated by three
distinct processes: 1) increased DGC activity, 2) decreased PDE activity, or 3)
modulation of both DGC and PDE activity. TELCA analysis revealed that c-di-GMP
in AB media was driven by increased DGC activity while the c-di-GMP increase in
M9 was driven by reduced PDE activity. This result highlights that bacteria can
and do control intracellular c-di-GMP by regulating both c-di-GMP synthesis and
hydrolysis. Moreover, one cannot distinguish the relative DGC and PDE inputs
based solely on measuring the intracellular concentration of c-di-GMP.
I applied TELCA to further understand how QS controls intracellular c-diGMP in V. cholerae. By analyzing mutants that lock the bacteria in the low- or
high-cell-density state, I determined that DGC activity is increased at low cell
density and decreases as the cells enter a quorum. Alternatively, I observed
34

increased PDE activity of the ∆luxO high-cell-density mutant, compared to a locked
low-cell-density mutant and the WT strain. However, I did not observe changes in
PDE activity of the WT strain at different cell densities; thus, the ∆luxO mutant
exhibits a more pronounced modulation of PDE activity compared to the WT strain.
A discrepancy in phenotypic expression of the WT and ∆luxO mutant is not
unprecedented. For example, the ∆luxO mutant is deficient for colonization of the
infant mouse whereas the WT is proficient [68]. Therefore, these strains should not
be considered equivalent. The analysis of the ∆luxO mutant does indicate that QS
in V. cholerae has the potential to modulate cellular PDE activity. I speculate that
in other conditions not examined here, in which the Qrr sRNAs are more fully
repressed at high cell density, the WT strain would exhibit density dependent PDE
activity. My results clearly show that in LB V. cholerae QS control of c-di-GMP
occurs through modulation of c-di-GMP synthesis but not c-di-GMP degradation.
These results experimentally validate previous transcriptional studies that have
shown that the expression of both DGCs and PDEs are differentially regulated by
QS [66, 72, 74].
One protein that has been implicated in QS-mediated control of c-di-GMP is
the DGC CdgA. It has been previously shown that CdgA is capable of synthesizing
c-di-GMP in V. cholerae [79]. The regulation of cdgA is controlled by QS, as cdgA is
repressed at high cell density via direct binding of HapR to the promoter region [72,
74, 75]. Furthermore, CdgA actively regulates biofilm formation as a ∆cdgA mutant
strain demonstrates decreased colony rugosity [76] and ectopic expression of cdgA
35

leads to increased biofilm formation [79]. Also of note, a ∆hapR∆cdgA double mutant
strain has reduced biofilm formation compared to the ∆hapR mutant, indicating
that CdgA is involved in the HapR mediated increase in biofilm formation [102].
Taken together, this evidence suggested that repression of cdgA by HapR is one
mechanism by which QS controls the intracellular concentration of c-di-GMP and
biofilm formation. To confirm these previous genetic studies and quantify the
contribution of CdgA to total DGC activity, I created a deletion mutant of cdgA and
measured the relative c-di-GMP synthesis and hydrolysis potential using TELCA.
My results are consistent with previous findings as the ∆cdgA strain had reduced cdi-GMP synthesis compared to the ∆hapR and WT strains while having similar c-diGMP synthesis as the ∆luxO mutant (Fig. 7). Importantly, this finding
demonstrates that TELCA is able to distinguish the input of one DGC to total
cellular DGC activity.
TELCA is a straightforward technique that can be utilized to quantify total
DGC or PDE activity for any bacterium. This method allows a systems level
analysis of total c-di-GMP synthesis or degradation activity and can serve as a
complementary approach to the current methods of c-di-GMP detection to further
unravel complex c-di-GMP signaling networks.

36

CHAPTER 3 – Bile Acids and Bicarbonate Inversely Regulate Intracellular Cyclic
didi-GMP in Vibrio cholerae

Preface
I hypothesized that bile is an environmental signal sensed by V. cholerae to
recognize and adapt to growth in the human environment by modulating c-di-GMP
signaling pathways. Based on current models postulating that V. cholerae reduces
intracellular c-di-GMP in the human host, I predicted that bile acids would reduce
global c-di-GMP concentrations. Surprisingly, I discovered that bile acids increase
intracellular c-di-GMP. A screen of the activity of all 61 V. cholerae c-di-GMP
turnover enzymes in the presence and absence of bile acids identified three DGCs
that showed increased c-di-GMP synthesis in the presence of bile acids.
Furthermore, a screen on the expression of all 61 V. cholerae c-di-GMP turnover
enzymes revealed that bile acids inhibited the expression of one PDE. Deletion of
these four enzymes abolished the induction of c-di-GMP by bile and negated the
ability of V. cholerae to form biofilms in the presence of bile. Bicarbonate, a
biological pH buffer secreted by intestinal epithelial cells in the small intestine,
suppressed the bile-induced increases in intracellular c-di-GMP. I propose that bile
and bicarbonate inversely control c-di-GMP levels in V. cholerae, allowing this
bacterium to sense and adapt to local environmental niches within the small
intestine.

37

Growth conditions and molecular methods
All strains, plasmids, and primers used are listed in Tables A1-3. The V.

cholerae El Tor biotype strain C6706str2 was used for all experiments [78] and
Escherichia coli strains DH10B (Invitrogen) and S17-λpir [103] were used to harbor
and conjugate plasmid DNA into V. cholerae. The construction of the ∆vpsL, ∆luxO,
and ∆hapR strains has been described elsewhere [67, 68, 72]. For all experiments,
unless otherwise specified, cultures were grown in LB media at 35°C with shaking
at 220 rpm. When necessary, media was supplemented with kanamycin (Sigma) at
100 µg/mL or chloramphenicol (Sigma) at 10 µg/mL. The inducer isopropyl β-D1thiogalactopyranoside (IPTG) was added at 0.1 mM when required.
Synthetic human bile (SHB) is a mixture of six purified conjugated bile acids
added to LB media at physiologically relevant concentrations to mimic the human
small intestine [37, 38, 104, 105]. All bile acids were purchased from Sigma. The
conjugated bile acids added were taurocholate (0.46 mM), glycocholate (0.93 mM),
taurochenodeoxycholate (0.46 mM), glycochenodeoxycholate (0.93 mM),
taurodeoxycholate (0.32 mM), and glycodeoxycholate (0.64 mM). Bovine bile (BV,
Sigma) was supplemented at 0.4% w/v. 3-[(3-Cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS) was supplemented at 0.4% w/v, and sodium dodecyl
sulfate (SDS) was supplemented at 0.01 % w/v due to its potent bactericidal activity.
Taurine and glycine were each supplemented at 4 mM. Sodium bicarbonate (BiC)
was supplemented when indicated at 0.3% w/v (49.2 mM), consistent with CT

38

inducing conditions [80], while 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris)
was supplemented when indicated at 0.6% w/v (49.5 mM).
All of the protocols used in this study for DNA manipulation and plasmid
construction were performed as previously described [106]. The DNA polymerase
Phusion (Thermo Scientific) was used for all PCR reactions. The expression
plasmids for the DGCs and PDEs were constructed as described elsewhere [79].
Briefly, these plasmids allow controlled expression of each DGC encoded by V.

cholerae via induction of the Ptac promoter with IPTG. To construct the HD-GYP
expression plasmids, each HD-GYP gene was amplified from the V. cholerae
chromosome, then inserted into the pEVS143 vector using the EcoRI and BamHI
insertion sites, as previously described [72]. The identification of the c-di-GMP
reporter plasmid 6:C9-lux is described elsewhere [107]. The DGC mutant allele
plasmids were generated using the Lightning Site-Directed Mutagenesis kit
(Agilent) with the DGC expression plasmid as template using the primers listed in
Table A3. The VC2497-lux reporter was constructed by amplifying the promoter of
VC2497 from the V. cholerae chromosome by PCR, then inserting it into the
pBBRlux vector using the SacI and BamHI insertion sites.
To generate the DGC and toxR deletion strains, natural transformation and
homologous recombination were used. A PCR product was generated that contained
a chloramphenicol resistance cassette (cat) bordered by FLP recombination target
(FRT) sites from the plasmid pKD3 [84], flanked by 500 bp upstream and
downstream of the targeted gene using the primers KO1/KO2 and KO3/KO4. The
39

PCR products generated with these promoters were fused to the cat gene using
zipper PCR. Natural competence was induced by ectopically expressing tfoX
(VC1153) [63] from the Ptac promoter using the plasmid pANDA2. pANDA2 was
constructed by amplification of VC1153 with the primers CMW464 and CMW465
and insertion of this product into the EcoR1/BamH1 sites of the plasmid pEVS143.
Homologous recombination events were selected by growing the culture on LB
supplemented with chloramphenicol at 1 µg/mL. The cat gene was then removed by
ectopically expressing a FLP recombinase on the vector pTL17 [86]. The toxT
deletion mutant was constructed using allelic exchange as previously described via
the plasmid pBK51, a derivative of the plasmid pKAS32 [108].

Detection and quantification of intracellular c-di-GMP
All c-di-GMP quantifications were analyzed using liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) as described in Chapter 2.
Unless otherwise specified, cultures were grown to an OD600 between 0.6 and 1, and
a 1.5 mL aliquot of this culture was removed and centrifuged for 30 seconds at
maximum speed. The supernatant was immediately removed and the pellet was
resuspended in 100 µL of cold extraction buffer (40% acetonitrile/40% methanol/0.1
N formic acid) and incubated for 20 minutes at -20°C. The insoluble fraction was
pelleted in a bench top centrifuge at 4°C for 5 minutes and the supernatant was
collected and stored at -80°C. Prior to mass spectrometry, the extraction buffer was
evaporated using a vacuum manifold. The pellet was then resuspended in 100 µL
water.
40

Systematic screen of DGC and PDE activity
To determine the in vivo activity of each V. cholerae DGC, each of the 40
DGC expression vectors was conjugated into a ∆vpsL strain of V. cholerae harboring
a separate reporter vector encoding a luciferase-transcriptional fusion of a c-di-GMP
inducible promoter located within the open reading frame (ORF) of VC1673 (6:C9-

lux, [107]). These strains were then grown in solid white 96-well clear-bottom plates
(Costar 3903) in 150 µL of LB or LB with SHB in the presence of IPTG inoculated
as a 1/100 from an overnight culture. Luminescence and OD600 values were recorded
in a Spectramax M5 Platereader (Molecular Devices) after 8 hours of growth, and
were reported as relative luminescence units (R.L.U.). The screen for PDE activity
was performed in the same manner as the DGC assay with the addition of a third
vector, pBRP02, which carry the allele of the DGC qrgB under pTac control.
pBRP02 has the Vibrio harveyi qrgB allele cloned into the vector pMMB67eh [109],
allowing it to coexist with the PDE expression vectors and the 6:C9-lux reporter
plasmid.

Systematic screen of DGC and PDE expression
DGC-gfp (GGDEF domain proteins) and PDE-gfp (GGDEF + EAL and EAL
domain proteins) transcriptional fusion plasmids were constructed as previously
described [72]. Strains containing the plasmids were grown in triplicate overnight
and then inoculated 1/100 in 150 µL LB or LB with SHB in a Costar black, clearbottom 96 well plate (cat. # 3904). Cultures were grown for 8 hours, and then

41

fluorescence was quantified (excitation 475, emission 510) using a M5 Spectramax
plate reader (Molecular Devices). Relative fluorescence was quantified by dividing
the fluorescence by the OD600 reading of the culture. PDE-lux transcriptional fusion
plasmids were constructed as previously described [66]. Strains containing the
plasmids were grown in triplicate overnight and then inoculated 1/100 in 150 µL LB
or LB with SHB in a Costar white, clear-bottom 96 well plate (cat. # 3903). Cultures
were grown for 6 hours, and then luminescence was quantified using an Envision
plate reader (PerkinElmer). Relative luminescence was quantified by dividing the
luminescence by the OD600 reading of the culture.

Biofilm quantification
Static biofilm formation was determined using a modified protocol from [47].
Overnight planktonic cultures of V. cholerae and V. cholerae DGC mutants were
grown in LB media, and cultures were diluted 1:1000 into 1 mL LB or LB with BV
as specified in 17 x 100 mm polystyrene test tubes (BD Falcon). These tubes were
incubated at 35°C for 24 hours without shaking. The supernatant was then
removed, an absorbance reading of the supernatant was taken (OD600), and the
biofilm was gently washed with approximately 2 mL of phosphate buffer saline
(PBS). The biofilm was then stained with 0.41% crystal violet (CV) in 12% ethanol
for 3 minutes followed by three washes with PBS, elution of the CV in 10 mL 95%
ethanol, and OD570 measurement for each biofilm. Each OD570 biofilm measurement
was normalized to the OD600 measurement of the planktonic culture to account for
differences in growth.
42

Bile increases the intracellular concentration of c-di-GMP in V. cholerae
It has been proposed that V. cholerae has a high intracellular c-di-GMP
concentration in environmental reservoirs where it predominantly exists as surface
associated biofilm communities and reduces c-di-GMP upon entrance into the
human host, leading to the activation of virulence factor expression [35]. As bile is a
prevalent constituent of the human small intestine, I hypothesized that V. cholerae
would reduce c-di-GMP levels in response to bile. To test this hypothesis, I grew
wild type V. cholerae in the presence of a crude bovine bile extract (BV) or synthetic
human bile (SHB) and quantified the intracellular c-di-GMP concentration using
LC-MS/MS. BV was supplemented to the media at 0.4% w/v, a concentration which
is physiologically relevant to that found in the small intestine [39, 110]. SHB is a
mixture of six purified bile acids that replicate the physiological concentrations of
bile acids found in the human small intestine [37, 38, 104, 105]. To my surprise,
growth in both BV and SHB-containing media significantly increased the
intracellular concentration of c-di-GMP 4.4 and 3.6-fold, respectively (Fig. 8).
To determine if the increase in intracellular c-di-GMP was due to the
detergent activity of the bile acids, I grew V. cholerae in the presence of either the
anionic detergent SDS (0.01% w/v) or the zwitterionic detergent CHAPS (0.4% w/v).
I observed no significant difference in intracellular c-di-GMP upon addition of either
detergent, suggesting that this increase in intracellular c-di-GMP is specific to bile
acids. As the SHB mixture of bile acids contains bile acids conjugated to either
taurine or a glycine, it was possible that these moieties were causing the increase in
43

intracellular c-di-GMP. However, there was no significant difference in the
intracellular c-di-GMP concentration of V. cholerae grown with the addition of
either taurine or glycine at equivalent concentrations (Fig. 8). Therefore, I conclude

1000

*
*

800
600
400
200

+

C
H
A
LB
PS
+
Ta
ur
in
LB
e
+
G
ly
ci
ne

SD
S
+
LB

LB

+
LB

LB

+

B
V

SH
B

0
LB

Intracellular c-di-GMP
(nM)

that bile acids increase the intracellular concentration of c-di-GMP in V. cholerae.

Figure 8. Intracellular cc-didi-GMP concentrations of V. cholerae grown with different
bile additives. V. cholerae was grown in LB media and LB media with bovine bile
(BV), synthetic human bile (SHB), the detergents SDS and CHAPS, and the amino
acids taurine and glycine. Intracellular c-di-GMP was measured using LC-MS/MS.
The reported values indicate the mean. Error bars indicate standard deviation, and
* indicates statistical significance compared to LB as determined by a Student’s
two-tailed T-Test (n=3, p < 0.05).

The bile mediated increase of intracellular c-di-GMP is growth-phase dependent
The experiments described thus far analyzed cultures grown to late
exponential growth. I therefore examined the temporal dynamics of c-di-GMP
induction by bile at various points during bacterial growth. When grown in
44

standard culture conditions with shaking, there is no significant difference in the
doubling time of V. cholerae in the presence or absence of SHB (LB: 22.3 ± 0.3
minutes, LB + SHB: 21.6 ± 0.6 minutes). I measured intracellular c-di-GMP in three
growth phases in the presence and absence of SHB: early exponential growth (OD600
0.2-0.3), late exponential growth (OD600 0.6-0.8), and stationary phase (OD600 1.01.2). When grown in LB alone, I observed elevated c-di-GMP in early exponential
growth, and the concentrations of c-di-GMP decreased as the cell density increased.
These observations are consistent with previous studies showing that c-di-GMP is
elevated in the low-cell-density QS state [66, 72]. I observed significant increases of
the intracellular c-di-GMP concentration in the presence of bile in early exponential
and late exponential growth phases (Fig. 9). Alternatively, no significant induction
of c-di-GMP was observed in stationary phase. These results indicate that bile
induction of c-di-GMP is growth phase dependent. I speculated that QS might be
responsible for density-dependent bile induction of c-di-GMP. However, bile
induction of c-di-GMP was unchanged in V. cholerae mutants locked in either the
low- or high-cell density QS state showing this process is independent of QS (data
not shown).

Three DGCs are more active in the presence of bile
As SHB increases intracellular c-di-GMP, I hypothesized that specific DGCs
in V. cholerae would exhibit increased activity in the presence of SHB. To identify
these DGCs, I developed a high-throughput screen where the in vivo activity of each
of the 40 V. cholerae DGCs could be determined in the presence and absence of
45

Intracellular c-di-GMP
(nM)

4000
3000
2000
1000
0
Early
Exponential
Growth

Late
Exponential
Growth

Stationary
Phase

Figure 9. Intracellular cc-didi-GMP concentrations
concentrations of V. cholerae during the course of
growth with bile.
bile. All intracellular c-di-GMP concentrations were determined using
LC-MS/MS. Black bars indicate strains grown in LB and grey bars indicate strains
grown in LB + SHB. The reported values indicate the mean and the error bars
indicate the standard deviation from the mean. Brackets indicate statistical
significance as determined by a one-tailed Student’s T-Test (n=3, p > 0.05).

SHB. This screen utilized a series of expression vectors carrying every V. cholerae
DGC under the control of the IPTG-inducible Ptac promoter and a common
ribosome binding site [79]. Therefore, I infer in this system that differences in
activity are not due to changes in gene expression. In addition to the DGC
expression vector, each strain contained a separate reporter plasmid that carried a
transcriptional fusion of a c-di-GMP inducible promoter located within the ORF of
VC1673 with the luciferase operon (named 6:C9-lux, [107]). These vectors were
introduced into a ∆vpsL strain of V. cholerae which abrogates biofilm formation and
eliminates interference of aggregate formation on the analysis of reporter gene
expression. In the absence of additional c-di-GMP generated by exogenous
46

expression of DGCs, the reporter plasmid does not produce significant luminescence
in the presence or absence of SHB (Fig. 10, vector control). C-di-GMP production by
active DGCs leads to increased luciferase production, as can be seen by induction of
luminescence by expression of the constitutively active DGC qrgB from V. harveyi
(Fig. 10, qrgB). Induction of this transcriptional reporter is dependent on c-di-GMP
synthesis as expression of qrgB*, an allele of qrgB harboring a mutation in the
active site of the protein, does not induce luminescence (Fig. 10, qrgB*).
6:C9-lux expression was determined upon induction of each DGC in the
presence of LB and LB with SHB. Seven DGCs showed reduced luminescence
expression in the presence of SHB, although the extent of this reduction was not
large (< 3.5-fold). It is not currently known if this reduction of expression is related
to bile inhibition of the DGC activity of these proteins, unrelated transcriptional
regulation of 6:C9-lux, or a non-specific effect of SHB on luminescence. Due to the
large number of DGCs that are negatively affected (7, p <0.01), the relatively low
reduction, and that bile increases the total intracellular c-di-GMP concentration, I
favor the latter two possibilities. Nevertheless, three DGCs demonstrated more
than a five-fold fold increase luminescence in the presence of SHB (p<0.01, Fig. 10);
these DGCs are VC1067 (7.0 fold increase), VC1372 (47.6 fold increase), and
VC1376 (6.7 fold increase).

47

1.5
1.5×
×10 7
1.0
1.0×
×10 7
5.0
5.0×
×10 6

*

*
QrgB
QrgB*
Vector Control
VC0900
VC1029
VC1067
VC1104
VC1185
VC1216
VC1353
VC1367
VC1370
VC1372
VC1376
VC1593
VC1599
VC2224
VC2285
VC2370
VC2454
VC2697
VCA0049
VCA0074
VCA0165
VCA0217
VCA0557
VCA0560
VCA0697
VCA0848
VCA0939
VCA0956
VCA0960
VCA0965
VCA1082
VC0072
VCA0785
VC0398
VC0653
VC0658
VC0703
VC1934
VC2750
VCA0080

0

Relative Lumine scence Units

*

GGDEF

GGDEF + EAL

Figure 10. HighHigh-throughput screen of the in vivo activity of each of the 40 V.
cholerae DGCs with bile.
bile. V. cholerae strains containing a DGC expression plasmid
and the 6:C9-lux reporter plasmid were measured for luminescence in LB (black)
and LB with SHB (grey). Expression plasmids encoding qrgB and its mutant allele
counterpart qrgB* were included as positive and negative controls, respectively. The
vector control indicates expression of 6:C9-lux in the absence of protein induction.
The error bars indicate standard deviation. Statistical significance (*) was
determined for cultures exhibiting a positive fold change (LB + SHB / LB) greater
than two as determined by a Student’s one tailed T-Test (n=3, p < 0.01).

The activity of PDEs in V. cholerae is not altered by bile
The hydrolysis of c-di-GMP is driven by c-di-GMP-specific PDE enzymes,
which contain either a C-terminal EAL or HD-GYP domain. Like DGC enzymes,
these proteins are also modular and the variable N-terminal domain is thought to

48

respond to environmental stimuli. I hypothesized that an increase in intracellular cdi-GMP in response to bile acids could be caused by a repression of PDE activity. To
examine this possibility, I developed a second high-throughput screen to analyze the
enzymatic activity of the 29 predicted PDEs of V. cholerae. The sequence of one
EAL protein, VC0515, was highly divergent from the sequenced N16961 strain upon
amplification from the genome, and was thus excluded from analysis; this is
consistent with other studies reporting sequence variance [72]. Similar to the screen
above, each V. cholerae PDE is expressed from an inducible expression vector that
has been introduced into a ∆vpsL strain of V. cholerae containing the 6:C9-lux
reporter vector. A third vector (pBRP02) was introduced containing the
constitutively active DGC qrgB, effectively increasing the baseline c-di-GMP
concentration and subsequently the baseline luminescence values. Robust reporter
gene expression can be seen in a strain expressing qrgB with a vector control that
has no exogenous PDE expression (Fig. 11, vector control).
Strains of V. cholerae expressing each PDE alongside the DGC qrgB were
grown in LB and LB with SHB, and the expression of 6:C9-lux was quantified. 22 of
the 29 PDEs showed appreciable decreases (> 2-fold, p <0.01) in luminescence when
grown in LB compared to the vector control (Fig. 11), indicating that these PDEs
are actively hydrolyzing c-di-GMP. However, none of the PDEs demonstrated any
appreciable difference when grown in LB with SHB compared to LB alone (Fig. 11).
Thus, I conclude that SHB does not affect the c-di-GMP hydrolysis of PDEs in the
conditions I examined here.
49

6.0 ×10 7
4.0 ×10 7
2.0 ×10 7
Vector Control
VC0072
VCA0785
VC0398
VC0653
VC0658
VC0703
VC1934
VC2750
VCA0080
VC1087
VC1295
VC1348
VC2340
VC2497
VCA0210
VCA0681
VCA0895
VCA0931
VC0137
VC1086
VC1211
VC1592
VC1641
VC1652
VC1710
VC1851
VCA0101
VCA0536
VCA1083

0

Re lative Lumine sce nc e Units

GGDEF + EAL

HD-GYP

EAL

Figure 11. HighHigh-throughput screen of the in vivo activity of each of the 40 V.
cholerae PDEs with bile.
bile. V. cholerae strains containing a PDE expression plasmid,
the 6:C9-lux reporter plasmid, and the qrgB expression plasmid were measured for
luminescence in LB (black bars) and LB with SHB (grey bars). The vector control
indicates expression of 6:C9-lux with qrgB induction and without PDE expression.
The error bars indicate standard deviation (n=3).

The three bile-inducible DGCs are inner membrane proteins
I analyzed the predicted domain structure and subcellular localization of the
three bile inducible DGCs to search for commonalities. An analysis of the relative
hydrophobicity of the amino acid sequence of these three DGCs using the program
toppred predicts that all contain transmembrane spanning domains (Fig. 12, [111113]). The DGC VC1067 (GI: 15641080), which has also been referred to as cdgH

50

B

2
0
-2
-4

0

4
2
0
-2
-4

0

200
400
600
Amino Ac id

C

Hydrophobicity
Value

4

Hydrophobicity
Value

Hydro pho bic ity
Value

A

100 200 300
Amino Acid

4
2
0
-2
-4

0

200
400
Amino Acid

Figure 12. The amino acid sequence hydrophobicity and predicted signaling
domains of the bilebile-responsive DGCs. The amino acid sequence of (A) VC1067, (B)
VC1372, and (C) VC1376 was used to predict transmembrane domains using the
toppred program. The dotted line indicates the cutoff value for a potential
transmembrane domain. A depiction of the potential N-terminal signaling domains
and transmembrane domains (grey rectangles) and the C-terminal GGDEF domain
of each DGC are shown below each respective hydrophobicity plot. The images
were adapted from the SMART database [113].

51

[94], is predicted to contain two sequential periplasmic substrate-binding domains
(PBPb) in the N-terminus while VC1376 (GI: 15641388), which has also been
referred to as cdgM [97], is predicted to contain a conserved CHASE domain.
VC1372 (GI: 15641384) does not contain any conserved protein domains in the Nterminus; however, analysis of the relative hydrophobicity of the amino acid
sequence using toppred predicts 6 sequential intra-membrane spanning domains.

Three DGCs increase c-di-GMP synthesis in the presence of bile acids
To directly examine if SHB increases the DGC activity of these three
enzymes, I quantified the intracellular c-di-GMP concentration of strains ectopically
expressing VC1067, VC1372, or VC1376 grown in the presence and absence of SHB.
I similarly analyzed expression of active site mutant alleles of these genes
(GG[D/E]EF -> AA[D/E]EF) that render them incapable of c-di-GMP synthesis. As
addition of SHB increases the intracellular c-di-GMP concentration of V. cholerae
(Fig. 8), I hypothesized that the strains expressing each AA[D/E]EF mutant would
show increased intracellular c-di-GMP in the presence of bile similar to that of the
WT strain. Moreover, induction of the bile-activated DGCs would lead to more c-diGMP synthesis in the presence of bile versus LB alone.
When grown in LB media, there was no notable difference between strains
expressing any of the AA[D/E]EF alleles and the vector control in LB media. As
expected growth of the vector control or DGC mutants in bile showed similar
increases in c-di-GMP. Induction of the DGCs VC1067 and VC1376 produced

52

significantly increased amounts of c-di-GMP in LB alone, leading to intracellular cdi-GMP concentrations of 48.4±7.0 µM and 19.3±10.0 µM, respectively (Fig. 13).
Alternatively, the intracellular c-di-GMP of the VC1372 expression strain in the
absence of bile (116.9±17.4 nM) was not significantly altered compared to the vector
control (263.7±119.7 nM), indicating that this DGC does not produce c-di-GMP in
LB media alone. Importantly, the intracellular concentration c-di-GMP was
increased significantly upon ectopic expression of VC1067, VC1372, and VC1376 in
the presence of LB with SHB versus LB alone resulting in intracellular
concentrations of 89.2±31.5 µM, 4.3±0.4 µM, and 67.4±20.1 µM c-di-GMP,
respectively (Fig. 13). These data together indicate that VC1067, VC1372, and
VC1376 synthesize more c-di-GMP in the presence of bile acids.

A toxR deletion mutant partially loses bile-mediated c-di-GMP increase
One system that has been implicated in the bile response of V. cholerae is the
ToxR virulence regulatory network. The transcriptional regulator ToxR controls the
expression of the virulence regulator ToxT, as well as the expression of CT and
TCP[114, 115]. Furthermore, ToxR mediates bile resistance in V. cholerae by
altering the expression of ompU and ompT [43, 116]. It has also been shown that
bile acids alter the protein activity of ToxR [117]. I hypothesized that bile acids act
through ToxR or ToxT to alter the transcription of c-di-GMP turnover enzymes
resulting in increased global c-di-GMP concentrations. To determine if bile
mediated changes in ToxR or ToxT transcription regulation effect intracellular c-diGMP, I generated ∆toxR and ∆toxT deletion strains and measured intracellular c-di53

Intr acellular c -di-GMP
(nM)

150000
100000
50000
6000
4000
2000

V
ec
to
r

C
on
tr
ol
V
V
C1
C1
06
06
7
7
(A
AD
E
F)
V
V
C
C
13
13
72
72
(A
A
E
E
F)
V
V
C
C1
13
37
76
6
(A
AD
E
F)

0

Figure 13.
13. Intracellular c-didi-GMP concentrations of V. cholerae DGC expression
strains in the presence and absence of SHB. The c-di-GMP concentrations of the
empty vector strain or the VC1067, VC1372, or VC1376 expression strains were
quantified with LC-MS/MS. The intracellular c-di-GMP of strains expressing alleles
containing mutations in the active site motif of each DGC were also quantified. The
black bars indicate strains grown in LB, while the grey bars indicate strains grown
in LB with SHB. Error bars indicate standard deviation. Brackets indicate
statistical significance, which was determined using a Student’s one tailed T-Test
(n=3, p < 0.05).

GMP in the presence and absence of SHB. I found that while the ∆toxR strain
showed no significant difference to the WT strain in LB alone, intracellular c-diGMP was 44.9% lower in the ∆toxR strain compared to WT in LB with SHB (Fig.
14). Similar to the ∆toxR strain, the intracellular c-di-GMP of the ∆toxT strain is

54

not discernibly different in LB. In contrast, the intracellular c-di-GMP of the ∆toxT
strain is modestly reduced in LB with SHB compared to WT, however this
difference is not statistically relevant. These results indicate that the
transcriptional regulator ToxR plays a role in the bile-mediated increase in

Intrace llular c-di-GMP
(nM)

intracellular c-di-GMP.

600

400

200

0
WT

∆ toxR

∆ toxT

Figure 14. Intracellular cc-didi-GMP concentrations of V. cholerae ∆toxR and ∆toxT
deletion mutants in the presence of bile.
bile. LC-MS/MS was used to quantify
intracellular c-di-GMP. Black bars indicate strains grown in LB and grey bars
indicate strains grown in LB with SHB. The reported values indicate the mean and
the error bars indicate the standard deviation from the mean. Brackets indicate
statistical significance as determined by a one-tailed Student’s T-Test (n=6, WT and
∆toxR, n=3, ∆toxT, p > 0.05).

Transcription of VC1295 is inhibited by bile acids
I hypothesized that the presence of bile could also regulate the transcription
of DGCs or PDEs to control intracellular c-di-GMP concentration. Specifically, bile
could increase the expression of DGCs or decrease the expression of PDEs to

55

increase intracellular c-di-GMP. To examine this possibility, I measured the relative
transcription of 51 GGDEF, GGDEF+EAL, and EAL proteins in LB and LB with
SHB by growing strains of ∆vpsL V. cholerae containing transcriptional fusions of
approximately 500 bp DNA upstream of each gene to gfp. After 8 hours of growth, I
found that there was less than a 2-fold difference in fluorescence between all
GGDEF, GGDEF + EAL, and EAL strains grown in the presence and absence of bile
(Fig. 15 A and B) suggesting that in the conditions examined here, regulation of
GGDEF and EAL gene transcription by bile does not play a significant role in the
bile mediated increase of intracellular c-di-GMP.
I also examined the relative transcription of the 8 HD-GYP proteins in LB
and LB with SHB using transcriptional fusions of each promoter, defined as
approximately 500 bp upstream of each HD-GYP gene, to the lux operon. The HDGYP gene VC1087 is encoded in a putative operon with the EAL gene VC1086, and
thus the expression is presumed to be synonymous with VC1086 (Fig. 15 B). The
luminescence of each reporter strain was determined after 6 hours of growth in
either LB or LB with SHB. The expression of six of the genes encoding HD-GYP
proteins was not significantly changed in LB with SHB compared to LB (> 2-fold),
whereas the expression of VC2497 was modestly increased in the presence of SHB
(2.2-fold, p < 0.05). Importantly, the expression of VC1295 (GI: 15641308) was
decreased 2.8 fold when grown in the presence of SHB (Fig. 15C, p < 0.05). VC1295
appears to be an active PDE when ectopically expressed in V. cholerae (Fig. 11).

56

3000
2000
1000
VC0900
VC1029
VC1067
VC1104
VC1185
VC1216
VC1353
VC1367
VC1370
VC1372
VC1376
VC1593
VC1599
VC2224
VC2285
VC2454
VC2697
VCA0049
VCA0074
VCA0165
VCA0217
VCA0557
VCA0560
VCA0697
VCA0848
VCA0939
VCA0956
VCA0960
VCA0965
VC2370

0

Relative Fluorescence Units

A

GGDEF

Figure 15. Relative transcription of DGCs and PDEs in the presence and absence of
bile. (A) V. cholerae strains containing a transcriptional fusion of each GGDEF
protein promoter to gfp was grown in LB (black) or LB with SHB (grey). The
fluorescence in each environmental condition was quantified after 8 hours of growth
(n=3). (B) V. cholerae strains containing a transcriptional fusion of each GGDEF +
EAL or EAL protein promoter to gfp was grown in LB (black) or LB with SHB
(grey). The fluorescence in each environmental condition was quantified after 8
hours of growth (n=3). (C) V. cholerae strains containing a transcriptional fusion of
each promoter driving expression of an HD-GYP to luciferase were grown in LB
(black) or LB with SHB (grey). Luminescence was quantified after 6 hours of growth
(n=4) in each environmental condition. All cultures were normalized to an OD600
reading. Error bars indicate standard deviation. * indicates statistical significance
from the LB condition, determined by a one-tailed Students T-Test (p < 0.05).

57

C

58

HD-GYP
VCA0931

7.5 ×10

1.5 ×10

10 7
GGDEF + EAL

VCA0895

VCA0681

VCA0210

VC2497

VC2340

VC1348

VC1295

0

10 6

Relative Luminescence Units

B

EAL

*
VCA0536

VCA0101

VC1851

VC1710

VC1652

VC1641

VC1592

VC1211

VC1086

VC0515

VC0137

VCA0080

VC2750

VC1934

VC0703

VC0658

VC0653

VC0398

VCA0785

VC0072

VCA1082

0

12500

25000

Relative Fluorescence Units

Figure 15 (cont’d)

This result indicates that bile acids decrease the expression of VC1295, possibly
resulting in decreased c-di-GMP hydrolysis contributing to increased intracellular cdi-GMP.

Three DGCs and one HD-GYP account for bile-mediated c-di-GMP induction
To determine if the DGCs VC1067, VC1372, and VC1376 contribute to the
bile-mediated increase of intracellular c-di-GMP in V. cholerae, I constructed
unmarked V. cholerae DGC deletion mutants and examined SHB mediated
induction of c-di-GMP (Fig. 16). Similar to my previous findings, the intracellular cdi-GMP concentration of V. cholerae increased 4.1 fold in the presence of SHB in the
WT strain. Both the ∆VC1067 and ∆VC1376 single mutants showed a modest but
significant reduction of c-di-GMP when grown in bile compared to the WT strain,
having 29.6% and 24.9% less c-di-GMP, respectively. The ∆VC1372 single mutant
and ∆VC1372∆1376 double mutant were not statistically different from the WT
strain. Importantly, the ∆VC1067∆VC1372∆VC1376 triple mutant strain exhibited
the greatest reduction of intracellular c-di-GMP in the presence of bile, losing 46.8%
of the intracellular c-di-GMP compared to the WT strain in LB with SHB (p< 0.05).
These results suggest that VC1067, VC1372, and VC1376 function redundantly in
the bile-mediated c-di-GMP induction.
As the expression of the HD-GYP VC1295 was inhibited by bile acids, I
constructed an unmarked V. cholerae VC1295 deletion mutant and quantified
intracellular c-di-GMP in the presence and absence of SHB. In LB alone, the

59

Intrac ellular c-di-GMP
(nM)

1500

1000

500

∆ VC1295
∆ VC1067
∆ VC1372
∆ VC1376

∆ VC1295

∆ VC1067
∆ VC1372
∆ VC1376

∆ VC1372
∆ VC1376

∆ VC1376

∆ VC1372

∆ VC1067

WT

0

Figure 16. Intracellular cc-didi-GMP concentrations of V. cholerae DGC and PDE
mutant strains in the presence and absence of bile. LC-MS/MS was used to quantify
c-di-GMP after growth in LB (black) or LB with SHB (grey). The reported values
indicate the mean and the error bars indicate standard deviation. Brackets indicate
statistical significance as determined by a one-tailed Students T-Test (n=9-10, p <
0.05).

intracellular c-di-GMP was modestly increased 1.8 fold in the ∆VC1295 mutant
compared to WT (Fig. 16, p < 0.05). This result is expected as deletion of an active
PDE will increase intracellular c-di-GMP. However, the intracellular c-di-GMP
concentrations of the ∆VC1295 strain grown in the presence of SHB were
indistinguishable from WT. I hypothesized that both activation of DGC activity and
transcriptional regulation of VC1295 contribute to bile induction of c-di-GMP. To
test this, I created a quadruple ∆VC1295∆VC1067∆VC1372∆VC1376 mutant and
60

measured intracellular c-di-GMP in the presence and absence of SHB. Similar to
the ∆VC1295 single mutant, when grown in LB alone the quadruple mutant had
elevated intracellular c-di-GMP compared to the WT strain (2.4 fold, p < 0.05).
Importantly, this strain showed no change in intracellular c-di-GMP in the presence
of SHB. This indicates that these four proteins are responsible for the bile-mediated
changes in intracellular c-di-GMP.

Deletion of the bile-responsive DGCs and PDE reduces bile induction of V. cholerae
biofilm formation
It has been previously reported that BV (i.e., bovine bile) increases biofilm
formation of V. cholerae, and this induction is dependent on the transcriptional
regulator vpsR [47]. VpsR binds c-di-GMP to regulate the transcription of biofilm
genes [107]. I wondered if the levels of c-di-GMP measured in the various DGC and
PDE mutants strains with and without bile would correlate with biofilm formation.
To test this, I performed a static biofilm assay where the wild type, ∆vpsL mutant,
and the DGC and PDE V. cholerae mutant strains were grown in polystyrene test
tubes containing LB or LB with BV without shaking followed by crystal violet (CV)
staining of the resulting attached biofilm. The ∆vpsL mutant cannot produce
exopolysaccharide and thus does not form biofilms. BV was used to induce biofilm
formation to remain consistent with prior studies [47, 117] and because it induced
more robust biofilm formation in this assay than SHB (data not shown).

61

I observed that all cultures of V. cholerae grew to a significantly lower optical
density, as measured by OD600, after static growth in the presence of BV. To account
for these growth differences, the biofilm formation of each culture was normalized
to the OD600 of the planktonic culture. Consistent with previous reports [47], BV
increased biofilm formation in the WT strain 2.7-fold, and this response was
eliminated in the ∆vpsL strain (Fig. 17). While the ∆VC1067, ∆VC1376, and
∆VC1372∆1376 mutants all showed modest to no loss of bile induced c-di-GMP (Fig.
16), these mutants no longer exhibited significant bile-induced biofilm formation.
Only the ∆VC1372 mutant induced biofilm formation in response to bile, indicating
that this DGC contributes less to biofilm formation. As expected, the triple DGC
mutant showed the lowest level of biofilm formation and was not responsive to bile
addition. Although the ∆VC1295 strain had modestly elevated c-di-GMP in LB,
deletion of VC1295 had no noticeable impact on biofilm formation. Like the triple
mutant, the DGC/PDE quadruple mutant exhibited low biofilm formation that was
unresponsive to bile addition. These results demonstrate that the bile responsive
DGCs are required for c-di-GMP dependent biofilm formation in response to bile
acids. However, the HD-GYP VC1295 did not contribute to biofilm formation in this
assay.

Bicarbonate decreases the bile-mediated increase of c-di-GMP in V. cholerae
As c-di-GMP has been shown to repress virulence gene expression in V.

cholerae, it remained puzzling as to why bile would increase c-di-GMP [35]. I
hypothesized that additional signals in the small intestine could override this
62

0.8
0.6
0.4

∆ VC1295
∆ VC1067
∆ VC1372
∆ VC1376

∆ VC1295

∆ VC1067
∆ VC1372
∆ VC1376

∆ VC1372
∆ VC1376

∆ VC1376

∆ vpsL

WT

0.0

∆ VC1372

0.2

∆ VC1067

Biofilm Fo rmatio n
(OD 570 CV / OD 600 p lanktonic)

1.0

Figure 17.
17. Biofilm formation of V. cholerae DGC and PDE mutants in the presence
of BV.
BV. Biofilm formation was quantified in test tubes using CV. A strain containing
a mutation in the vpsL gene was included as a negative control. The CV value was
normalized by the OD600 value of the planktonic culture to account for differences in
growth. The reported values indicate the mean, and error bars indicate standard
deviation from the mean. Brackets indicate statistical significance as determined by
a Student’s one tailed T-Test (n=3, p <0.05).

induction. Another major component of the human small intestine is bicarbonate,
which is secreted by the pancreas as well as the small intestinal epithelial cells.
Bicarbonate acts as a pH buffer to neutralize acids secreted by the stomach [118,
119]. Bicarbonate has also been implicated in virulence gene regulation in V.

cholerae [120]. As bicarbonate is abundant in the small intestine, I hypothesized
that it may contribute to the regulation of intracellular c-di-GMP in V. cholerae. To
examine if bicarbonate impacted bile induction of c-di-GMP, I measured
63

intracellular c-di-GMP of V. cholerae when grown in the presence and absence of
SHB and bicarbonate. I observed no significant difference in intracellular c-di-GMP
of V. cholerae in the presence of bicarbonate alone (Fig. 18). However, growth of V.

cholerae in the presence of both SHB and bicarbonate completely abolished bile

500

*

400
300
200
100

Tr
is

B
iC
SH
B
+
LB

LB

+

SH
B

+

+

+
LB

LB

+

SH
B

B
iC

0
LB

Intracellular c-di-GMP
( µM)

induction of c-di-GMP (Fig. 18).

Figure 18. Intracellular
Intracellular cc-didi-GMP concentrations of V. cholerae grown in the
presence of bile and bicarbonate. LC-MS/MS was used to quantify c-di-GMP ofV.
cholerae grown in LB, LB with bicarbonate (BiC), or LB with SHB and different
supplements. The reported values indicate the mean and the error bars indicate the
standard deviation. * indicates statistical significance compared to LB as
determined by a one-tailed Student’s T-Test (n=3, p > 0.05).

As bicarbonate acts as a buffer in the small intestine, I hypothesized that
bicarbonate suppression of the bile induction of c-di-GMP was due to pH changes. I
tested this idea by growing V. cholerae in the presence of SHB with the pH buffer

64

Tris. The pH of LB media and LB with SHB media was approximately the same and
did not change significantly over the course of growth (pH LB, pre growth: 7.3, post
growth: 7.0; pH LB with SHB, pre growth: 7.3, post growth: 7.1). Upon addition of
bicarbonate, the pH increased substantially (pH LB with SHB and BiC, pre growth:
8.1, post growth: 8.7). The addition of Tris caused a similar increase in pH (pH LB
with SHB and Tris, pre growth: 8.7, post growth: 8.6). Analogous to bicarbonate, the
addition of Tris to LB with SHB media inhibited the normal induction of c-di-GMP
by bile (Fig. 18), indicating that this inhibition of bile induced c-di-GMP is pH
dependent.

Discussion
Bile is an abundant component of the human small intestine, and thus a
probable physiological cue for V. cholerae to recognize upon entry into this
environment. A number of lines of evidence suggest that bile is an important signal
in the transition of V. cholerae between environmental and infectious lifestyles. Bile
acids increase the expression of ompU in a toxR-dependent manner to increase bile
resistance, indicating that the classical biotype of V. cholerae is capable of sensing
the presence of bile [116]. There are conflicting reports regarding bile control of
virulence factor expression. Bile acids were reported to negatively regulate the
expression of the TCP and CT in a toxT-dependent manner in a classical biotype,
demonstrating that there is a link between bile and virulence [121]. Contrary to this
finding, bile acids were reported to induce CT and TCP in a toxR-dependent manner
in a classical V. cholerae biotype and a tcpP-dependent manner in the El Tor
65

biotype used in this study [117, 122]. From these studies, it is clear that V. cholerae
responds to bile to induce a number of physiological changes. In this work, I explore
the connections between bile and c-di-GMP.
Based on the prevailing V. cholerae disease model hypothesizing that c-diGMP levels are decreased upon infection, I predicted that bile acids would decrease
intracellular c-di-GMP concentrations. Contrary to this prediction, I found that bile
acids increase the intracellular c-di-GMP concentration of V. cholerae (Fig. 8). This
finding suggests that the dynamics of the c-di-GMP signaling system in the human
host are more complex than previously appreciated. Furthermore, I have found the
difference in the bile mediated change of c-di-GMP is largest during exponential
growth and that at stationary phase this difference was negated. Consistent with
these findings, it has been shown that intracellular c-di-GMP is depleted at high
cell density, and that these changes are due in part to quorum sensing [66, 72, 77].
My results indicate that this regulation is dominant over the increase in
intracellular c-di-GMP caused by bile.
To begin to understand how V. cholerae modulates its intracellular c-di-GMP
in response to bile, I determined if the activity of any DGCs or PDEs were affected
by bile using a novel high-throughput in vivo assay. This assay is easily adaptable
to examine the response of DGCs and PDEs to any environmental cue, and it can be
modified to explore other bacteria if a suitable in vivo reporter of c-di-GMP levels is
available. Three DGCs (VC1067, VC1372, and VC1376) exhibited increased c-diGMP synthesis activity in the presence of SHB (Fig. 10, 13). The DGC activity of
66

VC1372 appeared to be absolutely dependent on bile whereas bile modulated the
basal activities of VC1067 and VC1376. All of these DGCs are important for the
bile-induced increase of intracellular c-di-GMP and biofilm formation of V. cholerae
(Fig. 16, 17).
The mechanisms by which these three DGCs respond to bile acids are
currently unknown. All three DGCs are predicted to be associated with the inner
membrane. VC1067 has been implicated in biofilm formation as it induces rugosity
associated phenotypes in V. cholerae [94]. Furthermore, it has been shown that both
VC1067 and VC1376 actively produce c-di-GMP and stimulate biofilm formation
[79, 94], and a VC1376 mutant strain also demonstrates lower vpsL expression [97].
VC1376 has also been implicated in the indole-induced increase in biofilm formation
[96]. Furthermore, all three DGCs also have been shown to repress motility when
ectopically expressed in V. cholerae; this is particularly interesting as the levels of
motility repression of the VC1372 and VC1376 expression strains do not seem to
correlate with the intracellular c-di-GMP levels reported here (Fig. 13) [123].
Analysis by BLAST revealed that homologous sequences to VC1372 are only found
in a few Vibrio species dominated by strains of V. cholerae. A previous study has
also noted that VC1372 is unique to V. cholerae amongst the genus Vibrio [124].
The phylogenetic link of VC1372 with an enteric human pathogen, the
domain structure of VC1372, and my observation that the activity of VC1372 is
absolutely dependent on the presence of bile suggest the physiological cue which
controls VC1372 is bile. Bile acids are known to interact with the cell membrane
67

due to their detergent activity [41]. Moreover, I observed bile-mediated activation of
VC1372 in E. coli (data not shown), an orthologous system with no clear homolog to
VC1372. Alternatively, as both VC1067 and VC1376 maintain robust activity upon
exogenous expression even in the absence of bile, I postulate these DGCs might be
controlled indirectly by bile through sensing perturbations in the membrane.
Additionally, the expression of the PDE VC1295 was inhibited by bile acids
(Fig. 15C). This HD-GYP actively hydrolyzes c-di-GMP in both LB and LB with
SHB, but bile acids do not affect this activity (Fig. 11). VC1295 is predicted to be
composed of 492 amino acids and contains 6 predicted N-terminal transmembrane
domains preceding a HAMP domain linked to the HD-GYP domain [111-113]. The
mechanism governing this transcriptional regulation of VC1295 by bile remains
unknown. Analysis of the VC1295 promoter region reveals motifs resembling the
ToxR binding site (-127 bp, TCAAA-N11-TTAAA [114]). While this gene is not listed
amongst the known genes regulated by ToxR [33] there is evidence that the activity
of ToxR is altered by bile [117], suggesting that the ToxR regulon could be altered
when bile acids are present. As a ∆toxR strain of V. cholerae partially loses the bileinduced increase in c-di-GMP (Fig. 14), I hypothesize that in the presence of bile,
ToxR inhibits the expression of VC1295. Although VC1295 contributed to bileinduced c-di-GMP, I did not observe any effect of VC1295 on biofilm formation. I
speculate that this result is due to the distinct experimental differences in which cdi-GMP and biofilm formation were measured.

68

Another important host derived cue is bicarbonate, a biological pH buffer that
is highly abundant in the human small intestine [119]. Bicarbonate has been shown
to be important for virulence, as bicarbonate is critical for in vitro production of CT
[80]. Bicarbonate is capable of activating V. cholerae virulence gene expression via
the transcriptional regulator toxT [120]. I have shown that bicarbonate is able to
suppress the bile-mediated induction of c-di-GMP in V. cholerae. Furthermore, this
regulation is driven by changes in pH, as the bile-mediated induction is similarly
repressed by Tris (Fig. 18). It is possible that the change in pH alters the structure
of bile so that it no longer triggers the c-di-GMP synthesis activity of the DGCs.
Alternatively, the bicarbonate could directly interact with DGCs or PDEs to
competitively alter intracellular c-di-GMP.
As bile has strong antimicrobial properties [125], it may be physiologically
advantageous for V. cholerae to increase c-di-GMP to grant elevated tolerance to
bile acids and other stresses associated with the intestinal environment. Other
studies have indicated that biofilm formation is important for increased acid shock
tolerance and protection from bile acids [47, 75], and biofilms increase infectivity
and intestinal colonization in a mouse infection model [126]. Indeed, I confirmed
that BV induces biofilm formation in V. cholera and showed that all three bileresponsive DGCs were required for bile-induction of biofilm formation.
I propose that bile stimulates a high intracellular c-di-GMP concentration in
the intestinal lumen (Fig. 19). Upon penetration of the mucosal layer where the
bicarbonate concentration and thus the pH is elevated [127], the response to bile is
69

abrogated leading to a corresponding decrease in intracellular c-di-GMP. The
physiological consequences of spatial alteration of c-di-GMP within the small
intestine remain to be determined although I speculate c-di-GMP could be
modulating biofilm formation, motility, and virulence gene expression. My model
predicting high c-di-GMP concentrations in the lumen and decreased c-di-GMP
proximal to the intestinal epithelial cells is consistent with previously described
virulence gene regulatory models for V. cholerae and S. enterica [120, 128, 129].

Intestinal
Lumen

Bile ↑
Bicarbonate (pH) ↓
c-di-GMP ↑

Mucus
Lining

Bile ↓
Bicarbonate (pH) ↑
c-di-GMP ↓

Intestinal
Epithelial
Cells
Intestinal Epithelial Cells

Figure 19
19. Proposed model of V. cholerae c-didi-GMP regulation in the human small
intestine. C-di-GMP is elevated in the lumen, where the concentration of bile is
elevated and the concentration of bicarbonate is low. Upon entry into the mucosal
layer where bile is low and the bicarbonate concentration is elevated, c-di-GMP is
repressed..

70

My findings indicate that both bile and bicarbonate are environmental cues
that modulate c-di-GMP signaling in V. cholerae and facilitate the transition from
aquatic environments to the human host. They suggest that modulation of c-diGMP levels by V. cholerae upon entry into the human host is more complex than
previously appreciated, and that both bile and bicarbonate act together to inversely
regulate the intracellular concentration of c-di-GMP to presumably enable the
bacteria to adapt and thrive in the diverse intestinal environment.

Future directions
In the search to identify specific environmental signals that affect c-di-GMP
levels, I found that bile acids increase intracellular c-di-GMP in V. cholerae.
Furthermore, I showed that bile acids increase the c-di-GMP synthesis of three
DGCs. However, the mechanisms that govern the behavior of these three enzymes
are unknown. One common attribute that these proteins share is that they all have
predicted trans-membrane domains. It is possible that changes in the cell
membrane caused by bile acids could serve as a signal to alter DGC activity. In
support of this, it has been shown that temperature mediated changes in membrane
properties can alter protein behavior and initiate phenotypic changes [130, 131]. I
hypothesize that bile acids are acting through DGC transmembrane domains to
influence c-di-GMP synthesis by altering the physical properties of the lipid
membrane. To test this hypothesis, I propose to examine the effect of temperature
on the bile-induced increase c-di-GMP. V. cholerae cultures can be grown at either
35 ºC or 20 ºC in the presence and absence of bile acids. Membrane fluidity can be
71

assessed using fluorescence anisotropy as previously described [132] and
intracellular c-di-GMP can be measured using LC-MS/MS. In addition, the VC1067,
VC1372, and VC1376 ectopic expression strains and their mutant allele
counterparts also should be examined at the two temperatures in the presence and
absence of bile. If membrane fluidity is affecting the c-di-GMP synthesis of these
proteins, I expect reduced c-di-GMP synthesis in the presence of bile at the lower
temperatures.
Alternatively, it is possible that bile induces composition changes of the lipid
membrane of V. cholerae, which then alters the c-di-GMP synthesis of the three
bile-responsive DGCs. This phenomenon has been observed in Lactobacillus reuteri,
where bile acids alter the lipid membrane profile [133]. To test the effect of
membrane composition on c-di-GMP synthesis, I propose ectopically express fatty
acid biosynthesis enzymes to artificially modify the fatty acid composition of the cell
membrane. Increased expression of the fatty acid biosynthesis gene fabF in B.

subtilis promotes the proportion of long-chain fatty acids leading to decreased
membrane fluidity, while expression of the gene fabH promotes branched-chain
fatty acids and increases membrane fluidity [132, 134]. Either the fabF gene
homolog (VC2019) or the fabH gene homologs (VC2023, VCA0751) can be ectopically
expressed alongside each of the three bile-responsive DGCs, and intracellular c-diGMP can be measured using LC-MS/MS. Fluorescence anisotropy can confirm
changes in membrane fluidity. I hypothesize that the DGCs expressed alongside

fabF (reduced membrane fluidity) will have reduced c-di-GMP synthesis, whereas
72

DGCs expressed alongside fabH (increased membrane fluidity) will have increased
c-di-GMP synthesis.
In addition to the three DGCs that increased c-di-GMP synthesis in the
presence of bile, I demonstrated that bile acids decrease the expression of one PDE,
VC1295. However, the regulators driving bile-mediated expression changes of
VC1295 are undefined. As I have identified putative ToxR binding sites in the
promoter region of VC1295, and I have demonstrated that a ∆toxR mutant reduces
induction of c-di-GMP in the presence of bile acids, I hypothesize that ToxR binds to
the promoter region in the presence of bile acids to inhibit VC1295 expression. In
support if this, it has been shown that the transcriptional regulation activity of
ToxR is altered in the presence of bile acids, and that this regulation is dependent
on the transmembrane domain of ToxR [117]. To test the hypothesis that bile and
ToxR drive VC1295 expression, I propose introducing the VC1295-lux vector in the
∆toxR mutant strain and examining the expression of VC1295 in the presence and
absence of bile. I expect that the luminescence will be unchanged in the presence of
bile acids in the ∆toxR mutant strain. Electromobility gel-shift assays (EMSA) can
be performed as previously described [107] using purified ToxR protein and DNA
from the promoter region of VC1295 in the presence of increasing amounts of
purified bile acids [135]. If ToxR is interacting with the VC1295 promoter in the
presence of bile, I expect to see increased binding of DNA in the presence of bile
acids. Finally, I propose creating mutations in the predicted ToxR binding sites on
the VC1295-lux promoter vector, and examining luminescence in the WT strain in
73

the presence and absence of bile acids. These experiments will elucidate the role
ToxR plays in altering the expression of VC1295 and increasing intracellular c-diGMP concentrations in the presence of bile acids.
While bile acts to increase intracellular c-di-GMP in V. cholerae, I have
shown that bicarbonate can quench this response; however, it remains unclear how
this occurs. To explore this question, I propose to comprehensively examine the
expression of genes in V. cholerae cultures grown in LB, LB with bicarbonate, LB
with SHB, and LB with SHB and bicarbonate. To do this, RNA can be extracted
from each culture and whole transcriptome shotgun sequencing (RNAseq) can be
used to quantify gene expression. I expect that differences in gene expression will be
observed in cultures grown in the presence of SHB versus SHB with bicarbonate.
After genes have been identified that are differentially regulated in the presence of
bicarbonate, genomic knockout mutant strains can be made of these target genes
and the intracellular c-di-GMP of these strains can be determined when grown in
the presence of SHB and SHB with bicarbonate.
I demonstrated that the deletion of the enzymes responsible for the bilemediated c-di-GMP induction reduces bile-induced biofilm formation. However, the
full physiological implications of increased c-di-GMP in the presence of bile and
subsequent consequences on the pathogenesis of V. cholerae have yet to be
determined. To ascertain if increased c-di-GMP is beneficial for bile tolerance, I
propose competing the WT V. cholerae strain and the V. cholerae quadruple
DGC/PDE mutant strain in the presence and absence of bile and measuring relative
74

fitness. Neutral markers can be inserted into the genomes of each strain, such as
antibiotic resistance genes or fluorescent proteins, to differentiate the strains. I
hypothesize that the quadruple mutant will have lower competitive fitness in the
presence of bile acids compared to the WT strain. As controls, the ∆vpsL strain
should be competed against the WT strain to determine the role of biofilm formation
plays in competitive fitness. Likewise, as positive controls, ∆toxR and ∆ompU
mutants should be competed against WT in the presence and absence of bile, as
these genes are important for bile resistance and fitness [43, 116, 136]. To
determine the role bile-induced c-di-GMP plays in vivo, I propose determining the
competitive index of the quadruple DGC/PDE mutant to the WT strain of V.

cholerae in an infant mouse model as previously described [35, 116]. While the bile
composition of a mouse is different from that of a human [137], the infant mouse
model has been used to study the effects of proteins related to bile resistance in
pathogenesis [45, 46, 116]; thus I expect that this model will be sufficient to
examine the role of c-di-GMP in bile-related fitness. I expect that the quadruple
mutant has significantly reduced competitive index in a mouse model. These
experiments will clarify the role c-di-GMP plays in vivo during V. cholerae
pathogenesis.

75

CHAPTER 4 – Intracellular cc-didi-GMP Negatively Correlates with the Growth Rate
and Ribosomal RNA of V. cholerae

Preface
It has been observed that ectopic DGC expression can cause a growth
deficiency in both V. cholerae and E. coli [4, 79]. It remains unknown as to why
ectopic DGC expression would affect growth rate. Many factors influence bacterial
growth rate; the growth rate of bacteria is positively correlated with the ribosomal
RNA (rRNA) gene copy number, and this phenotype is associated with how bacteria
respond to resource availability [138]. Furthermore, ribosome abundance in the cell
correlates with growth rate, although it remains unclear if ribosome depletion
inhibits growth rate or if growth rate inhibition causes ribosome depletion [139146]. In support of the former, it has been shown that (p)ppGpp accumulates in the
cell upon nutrient deprivation, altering GTP homeostasis and inhibiting RNA
synthesis in the cell resulting in reduced growth rate [143, 147-151].
There are numerous phenotypes controlled by c-di-GMP including motility,
biofilm formation, and virulence [11, 13, 16, 98]. Here, I examine the hypothesis
that c-di-GMP negatively regulates the growth rate of V. cholerae. I have shown
that there is a correlation between intracellular c-di-GMP and growth rate of V.

cholerae grown in different media. Furthermore, I have shown that ectopic DGC
expression represses the growth rate in V. cholerae in a dose-dependent manner,
and that the efficiency of this repression varies between different DGCs. Finally, I

76

have shown that ectopic expression of the DGC VC1067 reduces rRNA in the cell.
Together, I propose that c-di-GMP is negatively regulating growth rate in V.

cholerae by reducing intracellular ribosomes via an unidentified molecular
mechanism.

Measuring growth rate of V. cholerae
Growth curves were determined by taking OD600 readings of liquid cultures
at regular intervals. For cultures grown in 150 µL volumes in clear 96-well plates
(Greiner), OD600 readings were taken with a SpectraMax M5 plate reader
(Molecular devices) every 10 minutes for a period of 15 hours. For cultures grown in
2 mL volumes in 14 mL round-bottom polystyrene tubes (Falcon), OD600 readings
were taken with a DU 700 series spectrophotometer (Beckman Coulter)
approximately every 30 minutes for a period of 6 hours. All cultures were grown at
35 ºC, with shaking at 220 rpm, and DGC expression strains were supplemented
with kanamycin at 100 µg/mL. Relative growth rates were determined by plotting
each curve in Graphpad Prism and calculating area under the curve. The area
under the curve values for each strain at each inducer concentration were
normalized to the area under the curve of a no-inducer control. For calculating
specific growth rates, growth curves were analyzed with the grofit package in R
[152, 153]. C-di-GMP was quantified using LC-MS/MS as described in Chapter 2.

77

RNA extractions and quantification
For RNA quantification, 5mL of LB media supplemented with 100 µg/mL
kanamycin and 0.1 mM IPTG in 25 mL Erlenmeyer flasks were inoculated with 5
µL overnight V. cholerae cultures and grown to an OD600 of approximately 0.6-0.8
(late exponential growth). Cultures were then diluted to an OD600 of 0.5, and RNA
extractions were performed using Trizol reagent (Ambion, Life Technologies) as per
the manufacturer’s instructions. Total RNA was quantified using a NanoDrop 1000
(Thermo Scientific). rRNA was then quantified in each sample using a 2100
Bioanalyzer (Agilent).

Intracellular c-di-GMP and growth rate of V. cholerae are correlated
In Chapter 2, I showed that intracellular c-di-GMP concentrations of V.

cholerae vary when the bacteria are grown in different growth media (Fig. 5) and I
observed that these concentrations tended to be higher in minimal media, where the
growth rate was slower (Fig. 4). I hypothesized that intracellular c-di-GMP may be
negatively correlated with bacterial growth rate. To examine this hypothesis that cdi-GMP and growth rate are connected, I determined the doubling times (in
minutes) during exponential growth of V. cholerae grown in different media (Fig. 4)
by selecting two timepoints during exponential growth in each environment and logtransforming the fold-change of OD600-2 and then multiplying this value by the time
elapsed. The doubling time of V. cholerae grown in LB determined here is consistent
with previous studies [154]. Growth rate (divisions per hour) was determined by

78

dividing 1 hour by the doubling time; these growth rates were then regressed on
intracellular c-di-GMP concentrations (Fig. 20). I found that there was a negative
trend of intracellular c-di-GMP and growth rate, although the slope bordered
outside of statistical significance from zero (p=0.056). Furthermore, the goodness of
fit (R2=0.163) indicates that only a modest portion of this relationship can be
attributed to intracellular c-di-GMP. This analysis provides preliminary evidence

Intr acellular c -di-GMP
(nM)

that there is a relationship between intracellular c-di-GMP and growth rate.

3000
2000
1000
0

1

2

3

4

Growth Rate
(Doublings / Hour)
Figure 20
20. Linear regression of intracellular cc-didi-GMP concentrations on growth
rates of V. cholerae grown in different media. Dotted lines indicate 95% confidence
intervals. Goodness of fit (R2) was 0.163, and non-zero significance (p) was 0.056.

Growth rate inhibition caused by ectopic DGC expression is dose-dependent
Previous studies have indicated that ectopic expression of a DGC can inhibit
bacterial growth [4, 79], corroborating my hypothesis that c-di-GMP negatively
affects growth rate. I wanted to further explore the dynamics of the relationship
79

between growth rate repression and ectopic DGC expression; specifically, I sought
to confirm these previous observations and determine if this inhibition of growth is
DGC dose-dependent. I utilized two DGC inducible expression vectors encoding
VC1067 and VC2285 to alter the intracellular concentration of c-di-GMP. These two
DGCs have been shown to synthesize high levels of intracellular c-di-GMP upon
IPTG induction (Chapter 3, [79]). I measured the growth of V. cholerae strains
harboring these DGC expression vectors at IPTG concentrations ranging from 0.002
mM to 0.1 mM by growing the strains in a 96-well plate and taking OD600 readings
every 10 minutes for 15 hours. The growth rate of each strain at each IPTG
concentration was determined by fitting each growth curve to an exponential
growth model using the program grofit [152, 153], and determining the maximum
slope of each curve (Fig. 21). In an empty vector negative control, I observed no
change in growth rate across the inducer gradient. In contrast, both the VC1067
and VC2285 expression strains showed a dose-dependent reduction in growth rate.
The VC2285 strain showed a greater reduction in growth at all timepoints
compared to the VC1067 strain. This is consistent with observations that the
VC2285 strain produces more c-di-GMP that the VC1067 strain [79]. I conclude
from these data that ectopic DGC expression inhibits growth rate in a dosedependent manner.
I next sought to determine what concentrations of c-di-GMP lead to growth
inhibition in V. cholerae. I selected nine DGC expression strains and grew them in 8
different IPTG concentrations ranging from 0.0005 mM to 1.0 mM. The relative
80

Max Gro wth Rate
(mu)

0.003
Vector Control
VC1067
VC2285

0.002

0.001

0.000
0.001

0.01

0.1

IPTG Conce ntratio n (mM)
Figure 21
21. Growth rates of V. cholerae DGC expression strains.
strains. V. cholerae DGC
expression cultures were grown in a 96-well plate and growth rate was determined
at a range of IPTG concentrations. The max growth rate (mu) indicates the
maximum slope during log-phase growth of OD600 curves fit to exponential growth
models. Each point represents the mean of three replicates, and error bars indicate
standard deviation.

growth rate of each strain was determined by measuring OD600 at regular intervals,
then determining the area under each curve. Intracellular c-di-GMP of each strain
was determined at each inducer condition during late-stationary growth using LCMS/MS as previously described [79]. Then, the growth rate of each strain at each
inducer concentration was regressed on the intracellular c-di-GMP concentrations. I
found that 4 of the 9 regressed strains had slopes significantly different from zero,
indicating that these DGCs are capable of inhibiting growth of V. cholerae upon
ectopic expression (Table 1).

81

Expression
strain

Max
intracellular
c-di-GMP
(nM)

Min
Area under
the curve

Slope

Nonzero
significance
(p < 0.01)

r2

-1.0e-5
*
0.88
± 8.1e-7
-8.5e-5
VC1104
3476.9
0.64 ± 0.05
*
0.65
± 1.3e5
-2.8e-5
VC1216
5840.2
0.86 ± 0.08
0.12
± 2.0e-5
-7.6e-5
VC1353
1596.2
0.94 ± 0.07
0.14
± 4.1e-5
1.5e-4
VC1599
888.7
0.91 ± 0.04
0.07
± 1.1e-4
-2.4e-5
VC2224
22249.3
0.5 ± 0.05
*
0.83
± 2.3e-6
-1.3e-4
VC2454
809.5
0.82 ± 0.03
0.03
± 1.5e-4
-1.1e-4
VCA0074
3515.7
0.51 ± 0.09
*
0.59
± 2.0e-5
-2.6e-4
VCA0165
541.0
0.88 ± 0.07
0.20
± 1.1e-4
Table 1. Description of DGC expression strain intracellular cc-didi-GMP
levels and growth inhibition. Max intracellular c-di-GMP indicates the
highest level of intracellular c-di-GMP over the IPTG gradient. Min area
under the curve indicates the lowest area under the curve value of each
strain grown in the corresponding IPTG concentration. Linear regression
analysis of c-di-GMP and growth rate was performed, and the resulting
slopes are displayed here. The nonzero statistical significance and the r2
values are also shown.
VC1067

98111.4

0.11 ± 0.03

To compare the concentrations of c-di-GMP required to inhibit growth rate
across different DGCs, the maximum c-di-GMP produced by each DGC expression
strain capable of inhibiting growth was regressed on the minimum growth rate
observed for each strain. I separately analyzed the DGC expression strains that
were unable to significantly alter growth rate (Fig. 22A). I found that strains
expressing DGCs capable of inhibiting growth rate did so in a c-di-GMP dose82

dependent manner, where the slope demonstrated a significant negative deviation
(p < 0.05). Interestingly, two DGCs (VC1104 and VCA0074) inhibited growth rate at
c-di-GMP concentrations less than that of the DGC VC1216, which did not show any
significant growth rate inhibition.
I went on to analyze the dynamics of growth inhibition of the four DGCs that
inhibited growth rate of V. cholerae. Two of the DGC expression strains, VC1104
and VCA0074, produced similar levels of c-di-GMP upon expression resulting in
similar levels of growth rate inhibition (Fig. 22B). In contrast, the VC1067 and
VC2224 strains required significantly more intracellular c-di-GMP to achieve the
same level of growth rate inhibition as either the VC1104 or VCA0074 strains.
From this, I conclude that certain DGCs are able to inhibit growth rate at lower
concentrations of c-di-GMP than other DGCs, suggesting there is signaling
specificity within this pathway.

Intracellular c-di-GMP concentration required for growth inhibition varies for
different DGCs
The growth rate inhibition of ectopic DGC expression has been attributed to
c-di-GMP toxicity [4]. However, my data indicate that DGCs inhibit growth rate at
different concentrations of c-di-GMP, suggesting that this inhibition is occurring
independent of generalized c-di-GMP toxicity. I hypothesize that c-di-GMP is
regulating growth rate in V. cholerae through a specific, unknown mechanism. I
selected two DGCs that inhibit growth rate at different c-di-GMP concentrations;

83

Re lative Gro wth Rate
(Are a under the curve )

A

1.2

text
0.8

0.4

0.0

10 3

10 4

10 5

B

Relative Growth Rat e
(Area under the curve)

Intrace llular c-di-GMP
(nM)

VC1067
VC1104
VC2224
VCA0074

1.0

0.5

0.0
10 2

10 3
10 4
Intrace llular c-di-GMP
(nM)

10 5

Figure 22
22. Linear regression of intracellular cc-didi-GMP concentrations on growth
rates of V. cholerae DGC expression strains. (A) Regression analysis of maximum
intracellular c-di-GMP and maximum growth rate of different DGCs of V. cholerae.
Dotted lines indicate linear regression of DGCs that significantly inhibit growth
(black circles) or DGCs that do not significantly inhibit growth (grey squares). (B)
The intracellular c-di-GMP concentrations of individual DGCs was regressed on
relative growth rates over a range of IPTG concentrations. Each point represents
the mean of three replicates. Error bars indicate standard deviation.

84

these DGCs are VC1067 and VCA0074. Intracellular c-di-GMP was measured using
LC-MS/MS at an inducer concentration that was predicted to produce equal levels of
growth rate inhibition at different c-di-GMP concentrations, which was 0.1 mM
IPTG (Fig. 22B). I found that VC1067 produced 4.0-fold more intracellular c-diGMP at this inducer concentration than VCA0074 (p < 0.05, Fig. 23). In contrast,
intracellular c-di-GMP concentrations of strains containing mutant alleles
(GG[D/E]EF -> AA[D/E]EF) of either VC1067 or VCA0074 were indistinguishable
from those of the vector control.
I then measured growth of the VC1067 and VCA0074 strains grown at an
IPTG concentration of 0.1 mM to confirm that ectopc expression of each of these
DGCs inhibits growth rate at equivalent levels (Fig. 24A). These curves were used
to calculate the doubling time of each strain by fitting each curve to an exponential
growth equation using grofit [152, 153] and deriving the maximum slope. The
doubling time was determined from the slope by dividing log10(2) by each slope
value. I found that the doubling time of both the VC1067 and VCA0074 expression
strains was significantly increased over the vector control and their respective
active site mutant alleles (Fig. 24B). To account for growth rate deficiency caused by
the burden of ectopically expressing each DGC, the doubling time of the VC1067
and VCA0074 expression strains was divided by the doubling time of their
respective mutant allele counterpart expression strains. The VC1067 strain
demonstrated a 1.47-fold increase in doubling time over its mutant allele, whereas
the VCA0074 strain demonstrated a 1.45–fold increase in doubling time. I thus
85

50000

30000

*
VC
A

00
74

00
74
VC
A

VC
10

67
VC
10

C
on
tr
ct
or
Ve

67
*

10000

ol

Intrac ellular c-di-GMP
(nM)

70000

DGC Expression Strain

Figure 23
23. Intracellular cc-didi-GMP concentrations of V. cholerae DGC expression
strains.
strains. Cultures were grown in the presence of 0.1 mM IPTG. Bars indicate the
mean of three replicates, and error bars indicate standard deviation. Brackets
indicate statistical significance, which was determined using a two-tailed Students
T-test (p < 0.05).

conclude that while the VC1067 strain synthesizes significantly more c-di-GMP at
this inducer concentration compared to the VCA0074 strain, the growth rate is
virtually indistinguishable.

Increased intracellular c-di-GMP reduces the expression of rRNA
As my evidence indicates that intracellular c-di-GMP is negatively impacting
growth rate, I sought the mechanism governing this phenomenon. Numerous
studies have demonstrated that growth rate and ribosome abundance are
inextricably linked [139-151]. I hypothesized that c-di-GMP negatively affects the
transcription of rRNA in V. cholerae. This would lead to decreased ribosomes in the
86

A

OD 600

1

Vector Control
VC1067
VC1067*
VCA0074
VCA0074*

0.1

0.01

0

100

100
Doubling Time
(min)

B

200
Time
(min)

1.47-fold

80

300

1.45-fold

*
*

60
40

VC
10
67
V
C
10
67
*
V
C
A
00
74
V
C
A
00
74
*

V
ec
to
r

C
on
tr
ol

20

DGC Expre ssio n Strain
Figure 24.
24. Growth rates of V. cholerae DGC expression strains. (A) Growth curves
of DGC expression strains grown in the presence of 0.1 mM IPTG. OD600 readings
were taken at regular intervals (B) Doubling times were determined from growth
curves. Each bar represents the mean of three replicates, and error bars indicate
standard deviation. * indicates statistical significance from their respective mutant
alleles, as determined by a Students two-tailed T-test (p < 0.05).

87

cell, resulting in slower protein synthesis and subsequently slower cell division. To
examine this hypothesis, RNA extractions were performed on the VC1067
expression strain and its mutant allele on cultures normalized to equal cell density.
16S and 23S rRNA was quantified using a 2100 Bioanalyzer (Agilent). Relative
RNA abundance was determined by normalizing the respective RNA abundance by
the initial RNA concentration of each sample. I found that strains ectopically
expressing VC1067 strain had 39.6% and 32.2% less 16S and 23S rRNA,
respectively, compared to the VC1067* mutant allele (p < 0.05, Fig. 25). These data
support the hypothesis that c-di-GMP regulates growth rate by altering ribosome
abundance in the cell.

Discussion
The regulation of bacterial growth rate has been the subject of study for over
50 years [146]. Indeed, the rate at which bacteria are able to reproduce has large
implications for pathogenesis and epidemiology of disease, and involves core
biological functions that are widely conserved amongst bacteria. The concept of a
second messenger controlling bacterial growth rate is not novel. It has been shown
that the small nucleotide signal (p)ppGpp regulates growth in response to metabolic
stresses [143, 147-151]. Here, I explore the connection between growth rate
regulation and c-di-GMP. I have shown that there is a correlation between
intracellular c-di-GMP and growth rate in V. cholerae grown in different media, as
well as V. cholerae ectopically expressing different DGCs (Fig. 20, 21, 22). It has

88

80000
60000
40000

16S

VC1067

VC1067*

0

VC1067*

20000

VC1067

Re lative RNA Abundance

100000

23S

Figure 25.
25. Relative rRNA abundance of V. cholerae DGC expression strains. The
rRNA of RNA extractions from V. cholerae strains ectopically expressing VC1067
(black) or the active-site mutant allele VC1067* (grey) were quantified using a 2100
Bioanalyzer. Each bar represents the mean of five replicates, and error bars
indicate standard deviation. Brackets indicate statistical significance, as
determined using a Students two-tailed T-test (p < 0.05).

been noted in previous studies that ectopic DGC expression leads to growth rate
inhibition in E. coli and V. cholerae [4, 79].
I have shown that growth rate inhibition is dependent on the c-di-GMP
synthesis of DGCs, as expression of DGC mutant alleles had no significant impact
on growth rate (Fig. 24). This inhibition has been previously attributed to a c-diGMP toxicity associated with unnaturally high intracellular c-di-GMP
concentrations in the cell. However, my data indicate that this is not necessarily the
case, as growth rate inhibition was observed upon the expression of VC1104, while
89

expression of VC1216 and VC1353 produced equal or greater concentrations of c-diGMP while failing to inhibit growth (Fig. 22). Furthermore, I also observed that the
DGCs VC1067 and VCA0074 produce different levels of intracellular c-di-GMP yet
produce the same growth rate inhibition. These findings suggest that growth rate
inhibition is subject to DGC signaling specificity, a phenomenon where c-di-GMP
produced by certain DGCs effects downstream phenotypes at concentrations
different from other DGCs. Other studies have validated the concept of high
specificity c-di-GMP signaling for phenotypes such as biofilm formation [79, 91, 98,
155, 156].
Although the mechanisms by which c-di-GMP inhibits growth have not yet
been fully characterized, I have shown that ectopic expression of a DGC can lead to
decreased 16S and 23S rRNA in the cell (Fig. 25). This change in intracellular
ribosomes could account for the decreased growth rate observed in cells containing
elevated c-di-GMP (Fig. 24). Growth rate has been correlated with rRNA gene copy
number in bacteria, and it is well established that ribosome abundance in the cell is
correlated with growth rate [139-151]. Furthermore, it has been shown that
disrupting the rRNA copy number in E. coli has negative implications for fitness
[157]. Transcriptional regulation of rRNA has been associated with nutrient
abundance; specifically, during periods of starvation, the stringent response is
activated, resulting in increased (p)ppGpp and simultaneous repression of rRNA
transcription [143, 147-151]. Thus it makes sense that c-di-GMP, a second

90

messenger affected by environmental factors, would regulate growth rate by
altering ribosome abundance.
From these studies, I postulate two hypotheses to explain the phenomenon of
c-di-GMP-dependent growth rate inhibition I observe. The first hypothesis is that cdi-GMP is inhibiting rRNA transcription through some yet unidentified effector
protein. I propose that a subset of DGCs, such as VC1104 and VCA0074, specifically
regulate growth rate through this unknown factor. It is possible c-di-GMP acts
through a similar mechanism as (p)ppGpp, which binds RNA polymerase to
regulate transcription [158]. The second hypothesis is that DGC expression is
leading to a depletion of GTP, which subsequently alters growth rate. Previous
studies have shown that GTP depletion can lead to growth rate inhibition in B.

subtilis [159]. Furthermore, the growth rate regulator (p)ppGpp has also been
shown to be a critical regulator of GTP homeostasis [151]. I propose that DGCs that
produce very high amounts of c-di-GMP, such as VC1067, inhibit growth by
disrupting GTP homeostasis. These hypotheses have not yet been tested.

Future directions
C-di-GMP has been shown to regulate numerous bacterial phenotypes, and
more have yet to be identified [73]. I have shown that growth rate and c-di-GMP are
correlated and that ectopic expression of select DGCs results in c-di-GMP dependent
growth rate inhibition. This is significant, as the growth rate has important
implications in bacterial ecology and pathogenesis [138, 143, 157, 160]. The

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mechanism governing the c-di-GMP mediated inhibition of growth rate is unknown.
I have shown that the DGCs VC1067 and VCA0074 produce significantly different
levels of c-di-GMP in the cell yet result in the same level of growth rate inhibition.
In order to explore this relationship and determine how this growth rate inhibition
is occurring, I propose extracting RNA from both the VC1067 and VCA0074
expression strains. RNAseq can then be performed on extracts from both cultures to
identify differentially regulated transcripts. As controls, the vector control and the
active site mutant allele strains should be included. Since these two DGCs inhibit
growth at different concentrations of c-di-GMP, they may be acting through two
distinct pathways. I propose focusing on differentially regulated genes targeting
known growth rate factors, metabolic enzymes, and genes of unknown function. V.

cholerae deletion mutants of promising targets will be made, and subsequently
analyzed for growth in the presence and absence of elevated intracellular c-di-GMP.
An alternative method for determining the mechanism of c-di-GMP mediated
growth rate inhibition is to utilize experimental evolution to generate V. cholerae
strains that do not exhibit growth rate inhibition at high c-di-GMP concentrations.
To do this, I propose creating a c-di-GMP strain with a neutral-site chromosomal
insertion of the VC2285 gene under the control of the pTac promoter. VC2285 will
be used due to its ability to synthesize very high levels of c-di-GMP and its robust
inhibition of growth rate (Fig. 21, [79]). As I have shown that expression of VC2285
inhibits growth, I postulate that if VC2285 is grown and subsequently passaged
repeatedly, spontaneous suppressor mutants will arise in the population that will
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be able to grow faster in the presence of high levels of c-di-GMP. These mutants will
be able to out-compete their ancestors and overtake the population over the course
of the experiment. This strain can be passaged in the presence of the inducer IPTG
once a day; when cultures arise that exhibit faster growth rates than their
ancestors, members of the culture population will be isolated and whole-genome
sequencing will be used to identify where on the chromosomes the mutations
occurred.
Alternatively, it is possible that while c-di-GMP and growth rate are
correlated, c-di-GMP is not directly regulating growth rate. An alternative
hypothesis is that growth rate is regulating intracellular c-di-GMP. This could be
occurring through protein dilution [161], where protein synthesis cannot keep up
with cell division during rapid growth, resulting in different DGC and PDE
abundance in the cell and subsequently altering intracellular c-di-GMP
concentrations. To examine this hypothesis, I propose artificially altering the
growth rate of V. cholerae using a continuous culture chemostat, and measuring
intracellular c-di-GMP at different growth rates. I hypothesize that c-di-GMP would
be lower at faster dilution rates where the culture is growing faster, and higher c-diGMP in the slower dilution rates where growth rate is lower.
Another mechanism by which DGC expression could be altering growth rate
is by interacting with the (p)ppGpp signaling network. It has been shown that
(p)ppGpp negatively regulates growth rate in response to nutrient deprivation [143,
147-151] . I postulate that c-di-GMP regulates (p)ppGpp synthetases and hydrolases
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to alter intracellular (p)ppGpp concentrations and subsequently regulates growth
rate. To examine this hypothesis, I propose to use LC-MS/MS to quantify (p)ppGpp
of the VC1067 DGC expression strain and its mutant allele counterpart over a
range if inducer concentrations. I expect that the VC1067 strain would have
elevated (p)ppGpp concentrations compared to the VC1067* mutant strain, and that
this relationship would be dose dependent. One important regulatory target of
(p)ppGpp is GTP homeostasis [151]; this is particular relevance, as c-di-GMP is
synthesized from GTP. To determine if there is any link between intracellular c-diGMP, (p)ppGpp, and GTP, I propose to also measure the intracellular concentration
of GTP and GTP precursors of cells expressing either VC1067 or the VC1067*
mutant allele. Concentrations of GTP are approximately 5 mM in E. coli [88]; in
contrast, intracellular c-di-GMP of the VC1067 expression strain in the presence of
0.1 mM IPTG (where growth rate is inhibited) is approximately 50 µM. Thus, as one
c-di-GMP molecule is synthesized from two GTP molecules and assuming V.

cholerae has similar intracellular GTP homeostasis, I would expect that ectopic
expression of VC1067 could detract approximately 2% of the normal intracellular
GTP pool. Whether the extent of these changes on intracellular GTP or fluxes of
GTP precursors would be enough to effect growth rate is yet to be determined.

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CHAPTER 5 - A novel Adenovirus Adjuvant Stimulates Innate Immunity by in vivo
cyclic didi-GMP Synthesis

Preface
Thus far, I have described the interaction between environmental factors and
intracellular c-di-GMP, and its impact on the physiology of V. cholerae. As a
byproduct of this research, I have determined which of the V. cholerae DGCs
actively produce c-di-GMP (Fig. 2, 10, 13, 22, 23). I sought to use this information to
develop a clinical application. Other studies have demonstrated that c-di-GMP has
potent immunostimulatory properties [49-57]. I postulated that a DGC from V.

cholerae could be introduced into a eukaryotic cytoplasm to synthesize c-di-GMP in
vivo and subsequently activate the innate arm of the immune system.
One vehicle that has been used for antigen and adjuvant delivery is
replication deficient adenovirus based vectors [162]. Adenovirus vectors transduce
large fragments of DNA into a wide range of cells in order to synthesize proteins in

vivo, and gene expression can be modulated and even localized to specific cell types.
Unlike other types of viral delivery systems, DNA delivered by adenovirus vectors
does not integrate into the genome and thus circumvents the danger of insertional
mutagenesis [163]. Additionally, adenovirus vectors can be produced cost-efficiently
in high abundance. Importantly, adenovirus vectors are currently being used in
human clinical trials world-wide [164].

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Here I have investigated whether an adenovirus vector could be used to
deliver DGC DNA into eukaryotic cells to synthesize c-di-GMP in vivo,
manipulating a host-pathogen interaction to activate the innate arm of the immune
system and improve the immune response against co-administered antigen. I have
constructed an adenovirus containing an allele encoding the V. cholerae DGC
VCA0956. I have shown that this DGC is capable of synthesizing c-di-GMP when
virally transduced into cell culture lines. Furthermore, I have demonstrated that
use of the VCA0956 adenovirus construct in a murine system results in c-di-GMP
synthesis and subsequent upregulation of genes associated with innate immunity as
well as increased secretion of numerous cytokines and chemokines. Finally, I have
shown that c-di-GMP produced by VCA0956 in vivo functions as an adjuvant by
reducing the effective dose of a C. difficile toxin A antigen necessary to generate a
potent immune response. I propose that this novel adenovirus c-di-GMP delivery
system offers a more efficient and cost-effective method for use of c-di-GMP as an
adjuvant to stimulate innate immunity.

DNA manipulation and virus construction
All strains, plasmids, and primers used are listed in Tables A1-3. All of the
DNA manipulation and plasmid construction was performed as previously described
[82]. The DGC alleles were amplified from V. cholerae El tor strain C6706 or the
plasmids pCMW75 and pCMW98 using the DNA polymerase Phusion (New
England Biolabs) and the oligonucleotides listed in Table A3 (IDT). This product
was then inserted into the plasmid pShuttle-CMV [165] by digesting with Kpn1 and
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XhoI (Fermentas), and then ligated with a T4 DNA ligase (Invitrogen). E. coli strain
DH10B (Invitrogen) was used for harboring plasmid DNA, and sequence fidelity
was confirmed by sequencing (Genewiz). The active site mutant alleles were
generated using the QuickChange Lightning site-directed mutagenesis kit (Agilent).
A first-generation, human Adenovirus type 5-derived (Ad5) replication
deficient vector (deleted for the E1 and E3 genes) was used in this study [166].
Recombination, viral propagation of the Ad5 vectors, and subsequent virus
characterization was performed as previously described [166, 167]. Viral particle
number and infectious units titer (TCID/ml) was determined by optical density
measurement at 260 nm and validated as previously described [168].Construction of
the Ad5-Null and Ad5-TA is described elsewhere [169, 170]. All virus constructs
were confirmed to be RCA negative using RCA PCR and direct sequencing methods
[166] and the bacterial endotoxin content was found to be < 0.15 EU per mL [166].
All procedures with recombinant adenovirus constructs were performed under BSL2 conditions.

HeLa cell transformation
All transfections of plasmid DNA into HeLa cells was performed with the
TransIT-HeLaMONSTER transfection kit (Mirus) in 6-well plates with 2.5 µg
plasmid DNA. For HeLa cell infections with adenovirus vectors, cells were infected
with 2.0*109 viral particles (multiplicity of infection, M.O.I. of 500). Cell cultures
were all checked for confluence and morphology before and after transfection and

97

infection using microscopy. After 24 hours of growth at 37 °C in 5% CO2, the cells
were dissociated using 300 µL 0.25% trypsin, and then cells were resuspended in 4
mL PBS and then pelleted by centrifugation at 1600 RPM at 4 °C. Afterwards the
cells were resuspended in 100 µL extraction buffer (40% acetonitrile, 40% methanol,
and 0.1 N formic acid). The cell lysate was incubated at -20 °C for 30 minutes, and
then centrifuged at max speed for 10 minutes. The extraction buffer was removed
from the pelleted debris and stored at -80 °C until analysis. Immediately prior to
analysis, the extraction buffer was evaporated using a vacuum manifold, and the
samples were rehydrated in 100 µL water. C-di-GMP was quantified as described
in Chapter 2.

Animal protocols
Adult BALB/c WT male mice (6-8 weeks old) were used for all animal
experiments (Jackson Laboratory). For c-di-GMP quantification and innate studies,
mice were anesthetized using isofluorane, and 2x1011 adenovirus viral particles (vp)
per mouse (200 µL total volume, suspended in PBS) were administered
intravenously (IV) via retro-orbital injection. After administration, mice were
monitored every 6 hours by lab personnel for mortality and other health parameters
in accordance with Michigan State EHS and IACUC. After 24 hours the mice were
sacrificed, and the spleen and liver were isolated from each animal. Each tissue was
placed in 500 µL PBS, and then the tissue suspension was homogenized using an
Omni Tissue Homogenizer (Omni International). 300 µL of homogenate was added
to an equal volume of equilibrated Phenol Solution (Sigma). The homogenate-phenol
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solution was then vortexed and then centrifuged at 15,000 rpm for 10 minutes. The
aqueous phase was removed and added to 500 µL chloroform. The mixture was
vortexed and then centrifuged at 15,000 rpm for 10 minutes. The aqueous phase
was then removed and stored at -80 °C until analysis.

Quantitative PCR
Quantitative PCR was used to determine adenovirus abundance from DNA
extracted from liver tissue as previously described [171]. Ad5 genome copy numbers
were quantified using an ABI 7900HT Fast Real-Time PCR system and the SYBR
Green PCR Mastermix (Applied Biosystems) in a 15 µL reaction using a primer set
for the Ad5 Hexon gene that has been previously described [172]. All PCRs were
subjected to the following procedure: 95.0 °C for 10 minutes, followed by 40 cycles of
95.0 °C for 15 seconds and 60.0 °C for 1 minute. Standard curves to determine the
number of viral genomes per liver cell were run in duplicate and consisted of 6 halflog dilutions using DNA extracted from purified Ad5 virus [166]. As an internal
control, liver DNA was quantified using primers spanning the GAPDH gene [171]
and standard curves were generated from total genomic DNA. Melting curve
analysis was performed to confirm the quality and specificity of the PCR (data not
shown).
To determine relative abundance of specific liver-derived RNA transcript,
reverse transcription was performed on RNA derived from the liver tissue using
SuperScript III (Invitrogen) and random hexamers (Applied Biosystems) as per the

99

manufacturer’s instruction. RT reactions were diluted to a total volume of 60 µL,
and 2 µL from each sample was used as template for subsequent PCR. Quantitative
PCR was subsequently performed as described above using an ABI 7900HT Fast
Real-Time PCR system and SYBR Green PCR Mastermix (Applied Biosystems)
using primer sets that have been previously described [166]. The comparative Ct
method was used to determine relative gene expression using GAPDH to
standardize expression levels across all samples. Relative expression changes were
calculated by comparing experimental levels of liver transcript to levels of liver
transcript derived from mock-treated animals.

Cytokine and chemokine quantification
Cytokine and chemokine concentrations were quantified from plasma
samples using a Bio-Plex multiplex bead array system (Bio-Rad). At 6 and 24 hours,
blood samples were taken from mice using heparinized capillary tubes and EDTAcoated microvettes (Sarstedt). The samples were centrifuged at 3,400 rpm for 10
minutes to isolate plasma. Samples were assayed for 23 independent cytokines and
chemokines (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL12-p40, IL-13,
IL-17, IFN-γ, G-CSF, GM-CSF, Eotaxin, KC, MCP-1, MIP-1α, MIP-1β, RANTES,
and TNF-α) as per the manufacturer’s instructions (Bio-Rad) via Luminex 100
technology (Luminex).

100

ELISPOT analysis
For adaptive immunity studies, mice were administered adenovirus ranging
from 1x106 to 5x109 vp per mouse suspended in 25 µL PBS via IM injection into the
tibialis anterior of the right hindlimb. To measure antigen specific recall responses,
mice were sacrificed and the spleen was harvested after 14 days. Splenocytes were
isolated and ex vivo stimulated with immunogenic peptides from C. difficile TA
library as previously described [170]. ELISpot analysis for was performed as
previously described [170] using 96-well multiscreen high-protein binding
Immobilon-P membrane plates (Millipore) and the Ready-Set Go IFN-γ mouse
ELISpot kit (eBioscience). Spots were photographed and counted using an
automated ELISpot reader system (Cellular Technology).

C. difficile challenge
C. difficile mouse challenges were performed as previously described [173,
174]. Mice were administered Ad5 vectors; after 10 weeks, mice were administered
cefoperazone in their drinking water at 0.5 mg / mL for 10 days, followed by 2 days
with untreated drinking water. Immediately prior to the challenge, weights of each
mouse were recorded. Mice were then administered 100 µL C. difficile CD2015
spores by oral gavage. Mice were monitored twice a day, and the endpoint was
determined when mice lost ≥20% of their initial body weight.
All animal procedures were reviewed and approved by the Michigan State
University EHS and IACUC. Care for the mice was provided in accordance with
101

PHS and AAALAC standards. Plasma and tissue samples were collected and
handled in accordance with the Michigan State University Institutional Animal
Care and Use Committee.

Generating an adenovirus harboring a V. cholerae DGC
There is an abundance of literature demonstrating that c-di-GMP is able to
act as a vaccine adjuvant by stimulating the innate immune system [59]. However,
these studies utilized chemically synthesized c-di-GMP delivered intramuscularly or
intravenously, which is cost prohibitive and inefficient in delivery. Because c-diGMP is synthesized from GTP and GTP is abundant in the cytoplasm of eukaryotic
organisms, I postulated that a DGC expressed under the control of a strong
eukaryotic promoter/enhancer element would lead to c-di-GMP synthesis within the
eukaryotic cell and subsequent enhancement of downstream innate immune
responses. To identify a DGC that would produce c-di-GMP in the cytoplasm of a
eukaryotic cell, I examined the two DGCs VCA0848 and VCA0956 from V. cholerae,
as V. cholerae is a well-studied model system for c-di-GMP signaling and many V.

cholerae DGCs have been shown to synthesize c-di-GMP in high concentrations [79].
Additionally I examined the DGC qrgB from V. harveyi, which has been shown to
synthesize c-di-GMP in V. cholerae [72]. I selected these three DGCs since they had
no predicted N-terminal regulatory or trans-membrane domains and canonical
GGDEF domains and active site motifs. Ectopic expression of VCA0956 has been
shown to increase biofilm formation in both V. cholerae and Vibrio vulnificus [79,

102

175], repress motility in V. cholerae [123], and increase intracellular c-d-GMP in V.

cholerae and Shewanella oneidensis [35, 77, 176].
To determine if any of these DGCs are able to synthesize c-di-GMP in a
eukaryotic cytoplasm, I constructed plasmids containing each DGC under the
control of the constitutive CMV promoter/enhancer in the plasmid pShuttleCMV. I
also constructed corresponding vectors containing the same DGC alleles with a
mutation in the active site of the GGDEF domain (GG[D/E]EF -> AA[D/E]EF).
These plasmids were transfected into HeLa cells, and c-di-GMP levels were
measured in cell lysates after 24 hours using LC-MS/MS. I found that eukaryotic
cells transfected with both the VCA0848 and VCA0956 alleles produced detectable
levels of c-di-GMP; in contrast, no detectable c-di-GMP was observed in cells
transfected with the qrgB allele, or the active site mutant alleles and mock
treatment controls (Fig. 26A). The VCA0848 vector produced 31.6-fold more c-diGMP than the VCA0956 vector. Cell cultures were checked by microscopy and I
observed no discernible morphological differences between expression of VCA0848,
VCA0956, and the controls. Furthermore, trypan blue staining indicated that
treatment with VCA0848 or VCA0956 did not appear to impact overall cell viability.
Additionally, HeLa cells transfected with the VCA0956 plasmid and measured 48
hours after had less intracellular c-di-GMP, suggesting that c-di-GMP synthesized
intracellularly is transient (data not shown). These results indicate that VCA0848
and VCA0956 are capable of synthesizing c-di-GMP in the cytoplasm of a eukaryotic
cell.
103

A
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Figure 26. CC-didi-GMP synthesized in vivo by VCA0956 in HeLa cells. LC-MS/MS was
used to quantify c-di-GMP in HeLa cells. (A) HeLa cells were transfected with
plasmid vectors containing the VCA0956 allele or the active site mutant allele,
VCA0956*. (B) HeLa cells were infected with Ad5 vectors. Bars represent the mean
of 3 replicates; error bars indicate standard deviation. bd indicates below detection.

104

I selected VCA0956 for further analysis, as this DGC has been the most
extensively characterized. The pShuttleCMV-VCA0956 plasmid and its mutant
allele counterpart were used to construct and purify to high concentration the
respective recombinant Ad5-based vectors. To confirm that the VCA0956 Ad5
construct, herein referred to as Ad5-VCA0956, was able to produce c-di-GMP in a
eukaryotic cytoplasm, I infected HeLa cells (500 M.O.I.) with the Ad5-VCA0956 and
Ad5-VCA0956 mutant allele (Ad5-VCA0956*) adenovirus vectors and measured cdi-GMP using LC-MS/MS after 24 hours. The Ad5-Null vector, an adenovirus
construct carrying no transgene, was also included as a negative control. I found
that cells infected with the Ad5-VCA0956 produced high concentrations of c-diGMP, whereas cells infected with the Ad5-VCA0956* or the Ad5-Null produced no
detectable c-di-GMP (Fig. 26B). Importantly, similar to VCA0956 plasmid
transfections, infection with Ad5-VCA0956 had no noticeable impact on cell
morphology or viability. These results demonstrate that an adenovirus vector can be
used to deliver a V. cholerae DGC into a eukaryotic cell to synthesize c-di-GMP.

Synthesis of c-di-GMP in a murine system
As the Ad5-VCA0956 vector is capable of producing c-di-GMP in HeLa cells in

vitro, I next determined if my vector produces c-di-GMP in vivo in a murine model
system. Therefore, BALB/c mice (n=3) were IV injected with the Ad5-Null, Ad5VCA0956, or the Ad5-VCA0956* vectors and quantitative PCR was utilized to
measure adenovirus genomes in the spleen and liver of injected mice at 24 hours
post injection (h.p.i.). I observed comparable Ad5 genome counts for each treatment
105

in both the liver and spleen (Fig. 27A). Consistent with previous reports that the
predominant tropism of adenovirus is in the liver [172, 177, 178], there were
significantly more Ad5 genomes in the liver cells than in the spleen cells. I then
measured c-di-GMP in both the liver and spleen using LC-MS/MS, and found that
the Ad5-VCA0956 vector produced detectable c-di-GMP in both tissues, whereas the
Ad5-Null and Ad5-VCA0956* vectors produced no detectable c-di-GMP (Fig. 27B).
The concentration of c-di-GMP was consistent with the abundance of Ad5-VCA0956
genomes per cell, as the amount of c-di-GMP was significantly higher in the liver
tissue than the spleen. These data indicate that the Ad5-VCA0956 vector is capable
of initiating c-di-GMP synthesis in a murine model system.

C-di-GMP synthesized in vivo stimulates innate immunity in a mouse model
It has been previously shown that adenovirus vectors stimulate several proinflammatory innate immune response genes [162, 166, 171]. To examine if the
Ad5-VCA0956 alters the profile of innate immune gene expression compared to the
Ad5 vector alone, Balb/c mice (n=3) were IV injected with Ad5-Null, Ad5-VCA0956,
and Ad5-VCA0956* and qRT-PCR was utilized to quantify the expression levels of
several liver gene transcripts at 24 h.p.i. Infection with Ad5-VCA0956 had no
observable effect on the health of the mice. I found that the Ad5-Null treatment was
able to stimulate 6 of the 12 markers examined (> 2-fold; ADAR, MCP-1, TLR2,
IP10, Oas1a, RIG1) (Fig. 28). These results are consistent with previous studies
demonstrating that the adenovirus vector alone is capable of altering gene
expression in the liver [165, 166]. The expression of four genes was significantly
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Spleen

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Figure 27
27. Infection of Ad5Ad5-VCA0956 in a murine
murine model system. Mice were infected
with Ad5 vectors intraveneously. (A) After 24 hours qPCR was used to quantify Ad5
genomes in liver cells (black) or spleen cells (hashed). Data were normalized to
internal GADPH control. (B) LC-MS/MS was used to quantify c-di-GMP extracted
from the liver (black) or spleen (checkered). Bars represent the mean of 3 replicates;
error bars indicate standard deviation. bd indicates below detection.
107

(p < 0.05) higher in the Ad5-VCA0956 treatment compared to the Ad5-VCA0956*
treatment (Fig. 28A); these include the IFN-responsive gene ADAR, the monocyte
and basophil chemotractant MCP-1, the toll-like receptor (TLR) signaling pathway
gene MyD88, and the pattern recognition receptor TLR2. It is worth noting that cdi-GMP sensing in the cytoplasm is thought to be independent of TLRs [53].
Additionally, the expression of three genes was significantly (p < 0.05) repressed in
the Ad5-VCA0956 treatment compared to the Ad5-VCA0956* treatment (Fig. 28B):
the pro-inflammatory interleukin genes IL18 and IL1β, and the interferon
transcription factor IRF3. Interestingly, IRF3 has been shown to interact with
STING to initiate a c-di-GMP-mediated host type I interferon response [48, 179,
180].
In the cytoplasm, c-di-GMP interacts with STING to activate IRF3, NF-κβ,
and the p38/JNK/ERK MAP kinase signaling pathways, resulting in increased
production of numerous cytokines and chemokines characteristic of an innate
immune response [48]. To determine if the Ad5-VCA0956 vector is capable of
increasing cytokine and chemokine secretion, I directly quantified the abundance of
cytokines and chemokines in the plasma of mice treated with Ad5-VCA0956 using a
multiplexed assay system at 6 (Table 2) and 24 (Table 3) h.p.i. Consistent with prior
studies showing that the adenovirus vector stimulates the secretion of proinflammatory cytokines and chemokines [166, 167], I observed 9 cytokines and

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Fold c hange
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(Ad5 treated / Mock)

A

Figure 28
28. qRTqRT-PCR of mouse liver gene transcripts 24 hours after infection with
Ad5 vectors. Ad5-Null (striped), Ad5-VCA0956 (black), or Ad5-VCA0956* (grey)
were normalized to internal GADPH control. Fold change indicates each value
normalized to values measured from mock treated mice. Results are separated into
liver gene expression increased by Ad5-VCA0956 (A), decreased by Ad5-VCA0956
(B), or unaffected by Ad5-VCA0956 (C). Bars represent the mean of 3 replicates;
error bars indicate standard deviation. Brackets indicate statistical significance,

109

which was determined using a two-tailed Student’s t-test (P < 0.05).
chemokines that were modestly induced in the Ad5-Null treated mice compared to
the naïve mice (IFN-γ, MCP-1, G-CSF, MIP-1α, IL-6, MIP-1β, IL-12p40, KC,
RANTES; > 3-fold), and these differences were greatest at the 6-hour time point. I
found that 15 cytokines and chemokines were significantly increased in the plasma
of the Ad5-VCA0956 treated mice compared to the control Ad5-VCA0956* treated
mice at one or both of the two timepoints. Furthermore, for the majority of cytokines
and chemokines examined, the largest differences observed were at the 24 hour
time point, indicating that the effect of Ad5-VCA0956 is both more potent and
longer lasting than that of the adenovirus vector alone. The induction of most of
these cytokines and chemokines are consistent with other studies examining the
immunostimulatory effects of c-di-GMP [50, 52-56]. Interestingly, I observed
increases in IL-1α, G-CSF, and Eotaxin levels in the Ad5-VCA0956 injected mice,
which have not been previously reported to be induced by c-di-GMP. These data
together indicate that the Ad5-VCA0956 vector is capable of inducing a robust
innate response beyond that of the adenovirus vector alone in a murine model
system.

C-di-GMP synthesized in vivo lowers the effective antigen dose of Ad5-TA
The purpose of an adjuvant is to enhance the efficacy of a paired antigen by
increasing the longevity, potency, or reducing the effective dose. To determine how
well the Ad5-VCA0956 construct functions as a vaccine adjuvant, I tested if Ad5VCA0956 could enhance the adaptive response to a C. difficile antigen. C. difficile, a

110

6 h.p.i
Mock
Ad5-Null
Ad5-VCA0956
Ad5-VCA0956*
26.93
33.96
39.19
35.67
IL-1α
± 5.33
± 3.90
± 11.51
± 18.20
1266.47
597.54
601.11
792.85
IL-1β
± 976.22
± 54.53
± 135.01
± 177.15
2.70
4.26
4.11
3.51
IL-4
± 0.39
± 0.47
± 0.33
± 0.33
27.89
137.62
2836.67
135.95
ILIL-6
± 31.57
± 25.88
± 313.53**
± 72.95
26.65
45.46
32.71
IL-10
bd
± 11.62
± 3.5
± 9.65
511.64
3351.06
3847.84
4165.28
IL-12(p40)
± 210.70
± 368.94
± 733.63
± 601.5
164.30
171.12
231.81
156.88
IL-13
± 205.70
± 85.67
± 44.87
± 13.23
1033.46
1578.95
2582.84
2181.27
Eotaxin
± 286.53
± 204.69
± 189.22
± 413.21
93.43
1265.03
3722.93
1173.55
G-CSF
± 100.88
± 771.43
± 577.59**
± 235.67
84.27
142.15
85.35
GM-CSF
bd
± 6.55
± 14.05
± 41.35
3.66
28.14
452.73
37.45
IFNIFN-γ
± 4.81
± 4.64
± 90.62*
± 4.85
5.16
257.61
346.74
174.94
KC
± 5.48
± 96.01
± 39.01
± 136.14
4309.67
7146.20
4816.93
MCPbd
MCP-1
± 213.69
± 1166.34**
± 2357.41
7.98
47.50
61.10
48.89
MIP-1α
± 5.56
± 10.61
± 5.88
± 20.07
73.61
582.11
1022.01
754.20
MIP-1β
± 92.93
± 108.91
± 105.93
± 354.18
22.10
71.37
109.03
67.26
RANTES
± 11.30
± 4.45
± 10.51
± 16.77
15.17
345.81
1091.77
463.09
ILIL-12(p70)
± 2.33
± 81.94
± 331.08**
± 106.69
Table 2. Cytokine and chemokine secretion of mice infected with Ad5 vectors after
6 hours.
hours. Mice were infected IV with either Ad5-Null (n=2), Ad5-VCA0956 (n=3), or
Ad5-VCA0956 (n=3). Values indicate the mean pg / mL ± standard deviation.
Asterisks (*) indicate a statistically significant difference between Ad5-VCA0956
and Ad5-VCA0956*, which was determined using a two-way ANOVA test
combined with a Bonferroni posttest (* p <0.05; ** p < 0.01).
111

24 h.p.i.
Mock
Ad5-Null
Ad5-VCA0956
Ad5-VCA0956*
9.99
15.96
115.58
18.78
ILIL-1α
± 2.16
± 4.12
± 30.86**
± 5.57
1056.48
441.68
723.60
642.02
IL-1β
± 950.52
± 6.21
± 63.81
± 259.42
2.88
2.79
6.08
3.25
ILIL-4
± 0.13
± 0.25
± 0.87**
± 0.47
640.05
IL-6
4.18
bd
27.03
± 291.76
6.22
212.84
11.91
ILIL-10
bd
± 4.09
± 145.12*
± 5.82
309.78
989.84
11602.17
1069.21
ILIL-12(p40)
± 63.47
± 20.73
± 3585.45**
± 335.21
55.80
19.00
287.97
13.08
ILIL-13
± 68.56
± 16.51
± 7.54*
± 8.15
836.39
3974.88
743.67
Eotaxin
539.42
± 229.64
± 894.88**
± 187.39
94.72
302.85
156212.26
300.03
G-CSF
± 46.39
± 56.98
± 59031.70**
± 38.42
216.35
GM-CSF
15.06
bd
bd
± 16.78
17.10
IFN-γ
bd
bd
bd
± 4.60
5.61
43.02
1159.46
61.28
KC
± 6.84
± 11.94
± 523.33*
± 57.88
169.58
55001.74
327.11
MCPbd
MCP-1
± 12.19
± 19457.26**
± 38.26
12.85
303.94
14.24
MIPMIP-1α
2.45
± 8.24
± 27.35**
± 9.31
39.55
40.73
1291.42
92.01
MIPMIP-1β
± 50.19
± 0.69
± 360.00**
± 73.37
11.5
53.69
3657.80
68.55
RANTES
± 4.33
± 3.49
± 1455.71**
± 14.17
7.19
6.45
90.28
8.95
IL-12(p70)
± 4.39
± 5.43
± 6.22
± 9.74
Table 3. Cytokine and chemokine secretion of mice infected with Ad5 vectors after
24 hours. Mice were infected IV with either Ad5-Null (n=2), Ad5-VCA0956 (n=3),
or Ad5-VCA0956 (n=3). Values indicate the mean pg / mL ± standard deviation.
Asterisks (*) indicate a statistically significant difference between Ad5-VCA0956
and Ad5-VCA0956*, which was determined using a two-way ANOVA test combined
with a Bonferroni posttest (* p <0.05; ** p < 0.01).

112

Gram-positive spore-forming anaerobic bacteria, is the leading causative agent of
noscomial infections leading to diarrheal disease in the developed world. C. difficile
associated diarrhea (CDAD) represents nearly 1% of all hospital stays in the United
States and can lead to septicemia, renal failure, and toxic megacolon [181].
Incidents and mortality of C. difficile infections are rising in the U.S., and the
economic burden on the health care system is reported to be in the billions of dollars
[181-185]. Furthermore, to date there are no approved effective vaccine treatments
available for CDAD treatment or prevention [186].
A previously developed adenovirus vector that expresses the immunogenic
portion of the C. difficile toxin A (Ad5-TA) protects mice from a toxin challenge by
generating a humoral and T-cell response specific to toxin A in a murine model
system [170]. I hypothesized that supplementing this vaccine with the Ad5VCA0956 adjuvant would enhance this humoral and T-cell response due to the
strong pro-inflammatory innate response it generates. I therefore vaccinated mice
by IM injection with varying concentrations of the Ad5-TA vector in combination
with the Ad5-VCA0956 vector in equal ratio ranging from 1*106 to 5x109 viral
particles (vp). After two weeks, I assessed TA specific T-cell responses in the spleens
of the naive and vaccinated animals using an IFNγ ELISpot assay, utilizing the 15mer peptide (VNGSRYYFDTDTAIA) that has been previously shown to elicit the
secretion of IFNγ in splenocytes of mice immunized with the Ad5-TA vector [170]. I
found that co-injection of equal amounts of the Ad5-TA and the mutant DGC allele
vector Ad5-VCA0956* produced no induction of IFNγ secreting T-cells over that of
113

naïve splenocytes at viral doses of 1x106 and 1x107, but did generate significant
IFNγ producing T-cells at 1x108 and 5x109 (Fig. 29, white squares). Although coinjection of 1x106 Ad5-TA with Ad5-VCA0956 did not produce increased IFNγ
levels, I observed significantly increased (p < 0.05) IFNγ producing T-cells at a dose
of 1x107, as compared to cells derived from the DGC mutant treated control (Fig. 29,
black squares). No c-di-GMP was detected in the liver of mice infected with Ad5VCA0956 at the 5x109 dose after 14 days, suggesting that even at high doses, IM
administration of Ad5-VCA0956 does not lead to long-lasting c-di-GMP production
at distal sites (data not shown). Thus, I conclude that c-di-GMP synthesized by Ad5VCA0956 lowers the effective dose to generate an adaptive response to Ad5-TA.

Ad5-VCA0956 does not significantly enhance protection of Ad-TA against C. difficile
To determine if the Ad5-VCA0956 construct increases the efficacy of the Ad5TA vaccine in vivo, I decided to test the adjuvant-vaccine combination in a
cefoperazone-treated mouse C. difficile challenge model [173, 174]. I first sought to
determine the optimum dose of C. difficile to administer to effectively challenge the
mice. Naïve mice were administered cefoperazone (0.5 mg/mL in drinking water) for
10 days, followed by a two day rest period. Mice were then administered varying
doses ranging from 5*103 to 5*106 of hyperinfectious C. difficile CD2015 (NAP-1,
027 ribotype [187]) spores by oral gavage. The strain of C. difficile used is a
clinically isolated strain from a hospital in Southeast Michigan (unpublished).
Weights of the mice were monitored for a four day period. Mice were sacrificed upon

114

1000
800

SFCs pe r
6
10 sp lenocyt es

600
*

400
150
100
50

6

7

8

9

1x
10

1x
10

1x
10

5x
10

N
ai
ve

0

Ad5-TA +
Ad5-VCA0956
Ad5-VCA0956*
Figure 29
29. IFNγ
IFNγ ELISPOT analysis of mice vaccinated with Ad5Ad5-TA and Ad5
vectors.
vectors. Mice were administered IM varying doses of both Ad-TA and either AdVCA0956 (black) or Ad-VCA0956* (grey). After 14 days, splenocytes were ex vivo
stimulated with a C. difficile specific peptide and the number of IFNγ secreting
splenocytes was determined using ELISPOT. Each point represents an individual
animal. Lines indicate the mean of the replicates, and error bars indicate standard
error. * indicates statistical significance using a two-way ANOVA test combined
with a Bonferroni posttest (P < 0.05).

losing >20% of their initial body weight. I found that none of the mice administered
the lowest dose lost >20% of their initial body weight (Fig. 30 A and B). However,
when the dose was increased, I observed significantly more weight loss. The

115

Perce nt survival

A

100
80
60

5*
5*
5*
5*

106
105
104
103

cfu
cfu
cfu
cfu

5*
5*
5*
5*

106
105
104
103

cfu
cfu
cfu
cfu

40
20
0

B

Percent Body Weight

0

1

2
3
Time (Days)

4

110
100
90
80

0

1

2
3
Time (Days)

4

Figure 30.
30. C. difficile challenge of BALB/c mice. Mice were challenged with varying
doses of C. difficile CD2015 spores. (A) Survival of mice challenged with varying
doses of C. difficile. Mice were sacrificed after losing > 20% of their initial body
weight. (B) Weight loss of mice challenged with C. difficile. Each point represents
the mean of 5 replicates. Error bars indicate standard error.

116

greatest weight loss was observed in the 5*105 dose of C. difficile, resulting in 80%
mortality by day 4 of the challenge.
To determine how effective my Ad5-VCA0956 adjuvant was at increasing the
protective properties of Ad5-TA, I vaccinated mice with 1*107 vp of both the Ad5-TA
vaccine antigen and the Ad5-VCA0956 adjuvant vector; this dose was selected as it
produced a significant difference in the ELISPOT analysis (Fig. 29). As controls, I
also vaccinated mice with equal doses of the Ad5-TA vaccine with the Ad5VCA0956* and Ad5-Null vectors. After 10 weeks, mice were administered
cefoperazone for 10 days, followed by a 2-day rest. Mice were then challenged with
5*105 C. difficile spores. I found that both the mortality and weight loss of all mice
treated with the Ad5-TA was less than the naïve mice (Fig. 31). However, I did not
observe any significant differences between the mice that received the Ad5VCA0956 treatment compared to either the Ad5-VCA0956* or the Ad5-Null
treatments. And while the average weight loss at the end of the challenge was
lowest in the Ad5-VCA0956 treated mice, this difference was not statistically
significant from that of the Ad5-VCA0956* or Ad5-Null treated mice. Thus, I
conclude from this experiment that in the conditions examined here, the Ad5VCA0956 does not contribute to the effectiveness of the Ad5-TA vaccine.

117

Percent survival

A

100

Naive
Ad-VCA0956
Ad-VCA0956*
Ad-Null

80
60
40
20
0

B

Percent Body Weight

0

1

2
3
Time (Days)

4

110
Naive
Ad-VCA0956
Ad-VCA0956*
Ad-Null

100
90
80
70
0

1

2
3
Time (Days)

4

Figure 31. C. difficile challenge of BALB/c mice vaccinated with Ad5Ad5-TA and Ad5Ad5VCA0956. Mice were treated with equal doses of Ad5-TA and either Ad5-VCA0956
(squares), Ad5-VCA0956* (up-triangles), or Ad5-Null (down-triangles). (A) Survival
of mice challenged with C. difficile. Mice were sacrificed after losing > 20% of their
initial body weight. (B) Weight loss of mice challenged with C. difficile. Naïve: N=5,
Ad5-VCA0956: N=9, Ad5-VCA09568 and Ad5-Null: N=15. Error bars indicate
standard error.

118

Discussion
With a current demand for novel vaccines that target difficult-to-treat
diseases, it is crucial to have adjuvants to pair with these vaccines to optimize
efficacy. Currently, there are a limited number of adjuvants available for clinical
use, and there is a need for new adjuvants which can enhance the efficacy of
vaccines to improve immunological protection [61, 188]. Numerous studies have
implicated c-di-GMP as a promising novel adjuvant. Indeed, this second messenger
molecule has been shown to stimulate a robust type I interferon response and
increase the secretion of numerous cytokines and chemokines to initiate a balanced
Th1/Th2 response, as well as stimulate the inflammasome pathway and immune
cell activation/recruitment [49-57]. My approach to utilize c-di-GMP as an adjuvant
is novel in that it utilizes an adenovirus vector to deliver c-di-GMP producing
enzyme DNA into cells, thereby synthesizing the adjuvant in vivo and
circumventing the inefficient delivery of IM or IV administration of chemically
synthesized c-di-GMP. Adenovirus vectors are promising in that they are costefficient to produce and can efficiently deliver specific antigens or adjuvants into
cells for in vivo production, greatly reducing the cost of using c-di-GMP as an
adjuvant.
I have demonstrated that an adenovirus vector carrying a bacterial DGC is
capable of synthesizing c-di-GMP in both human and mouse model systems.
Consistent with other reports utilizing c-di-GMP, synthesis of c-di-GMP by virally
mediated transduction of the DGC gene in vivo increases the secretion of numerous
119

cytokines and chemokines [50, 52-57]. These induced cytokines and chemokines
include signals characteristic of both Th1 (Table 2 and 3, IFNγ, IL-12) and Th2
(Table 2 and 3, IL-4, IL-6) type responses. Additionally, c-di-GMP production from
Ad5-VCA0956 enhances activation of the innate immune system by activating TLR
signaling (Fig. 28, TLR2, MyD88). It appears however that c-di-GMP synthesized in

vivo negatively regulates the expression of inflammasome-dependent pathways in
hepatocytes (Fig. 28, IL-1β, IL-18). The significance of this finding is unclear,
especially as it has been reported that c-di-GMP activates the NLRP3
inflammasome pathway [51]. Importantly, I observed no signs of poor cell
physiology or health in my cell cultures and animal models. Furthermore, my data
indicate that the c-di-GMP synthesized by the Ad5-VCA0956 vector is transient,
and thus should enhance antigen recognition and response while minimizing any
potentially unwanted long term effects associated with administration, such as
autoimmune activation [189].
I have also shown that c-di-GMP synthesized in vivo reduces the effective
antigen dose of Ad5-TA, a vaccine antigen which targets the toxin of the human
pathogen C. difficile. Reducing the dose required to initiate an adaptive immune
response is of particular significance as high viral particle doses can lead to global
toxicities, endothelial cell activation, and liver damage [171, 177, 190-192]. C-diGMP has been shown to enhance protection against other pathogens including S.

aureus, K. pneumoniae, and S. pneumoniae [53-55, 58], indicating that c-di-GMP
has broad antigen-adjuvant synergy. Although I have only demonstrated an
120

enhanced adaptive response to C. difficile in vitro, Ad5-VCA0956 can be coupled
with other vaccines to enhance antigen recognition efficacy. While c-di-GMP is a
bacteria-specific second messenger, the Ad5-VCA0956 stimulated c-di-GMP innate
immune response could enhance protection in viral or cancer vaccine systems as
well.
I believe that the approach of synthesizing c-di-GMP in vivo offers numerous
advantages over directly administering c-di-GMP as a mucosal, intramuscular, or
intravenous adjuvant; these include lower cost, more efficient adjuvant delivery,
and a more robust innate immune response. Furthermore, adjustments can be made
to alter and optimize the Ad5-VCA0956 for clinical use. V. cholerae contains 40
predicted DGC alleles within its genome, and it has been shown that ectopic
expression of these different DGCs results in different intracellular c-di-GMP
concentrations [79]. Hence intracellular expression of other DGCs could produce
different amounts of c-di-GMP in eukaryotic cells, increasing the efficiency of this
adjuvant. Alternatively, other types of second messengers could be used to
stimulate innate immunity. One example would be to express a diadenylate cyclase
to synthesize the related bacterial second messenger cyclic di-AMP in vivo. Cyclic
di-AMP has similarly been shown to induce a robust innate immune response
through STING mediated recognition [193, 194]. Additionally, different promoters
could be used in lieu of the CMV promoter to produce localized or temporally
controlled c-di-GMP production in the body.

121

Future directions
With a current demand for development of vaccines for otherwise difficult to
treat infectious diseases, vaccine adjuvant development has been proposed as a
promising research avenue. I have shown that an adenovirus vector harboring a
DGC gene results in c-di-GMP synthesis in eukaryotic cells, leading to robust
stimulation of the innate immune system. In this study, I identified two DGCs that
produced c-di-GMP upon transfection into HeLa cells (Fig. 26A). The DGC VCA0848
produced significantly more c-di-GMP upon transfection than the VCA0956 strain.
As my data indicate that this DGC is more efficient at synthesizing c-di-GMP in the
eukaryotic cell environment, I hypothesize that an Ad5 construct harboring this
DGC will produce more intracellular c-di-GMP than the Ad5-VCA0956 vector in

vivo. This is significant, as less adenovirus would be required to stimulate innate
immunity and thus would increase the effect of c-di-GMP specific innate immune
activation compared to Ad5-stimulated innate immunity. Furthermore, reducing
the dosage of Ad5 required to initiate an innate immune response would reduce the
adverse effects associated with Ad5 treatment [171, 177, 190-192]. I propose
creating an Ad5-VCA0848 construct and determining its effect on innate and
adaptive immunity as described in Chapter 4.
Previous studies demonstrated that administration of purified c-di-GMP
leads to an innate immune response that conveys protection against S. pneumonia,

K. pneumonia, and S. aureus [53, 54, 58]. As the Ad5-VCA0956 is able to synthesize
c-di-GMP in vivo, I hypothesize that the Ad5-VCA0956 produces a more efficient
122

stimulation of innate immunity and thus leads to a more robust response. I propose
to test the Ad5-VCA0956 in a S. pneumonia challenge model to see if the vector is
capable of protecting animals against acute bacterial infection [58]. As a control, the
Ad5-VCA0956* and the Ad5-Null vectors should be included to account for any
protection granted by the vector or expressing a bacterial protein in an animal.
Additionally, purified c-di-GMP should be administered intranasally as a control to
determine if the Ad5-VCA0956 is more effective than c-di-GMP.
The Ad5-VCA0956 construct is able to increase the adaptive response of Ad5TA at lower doses (Table 2 and 3, Fig. 29). However, treatment of mice with Ad5VCA0956 in combination with Ad5-TA did not increase protection in a C. difficile
mouse challenge model compared with Ad5-VCA0956* or the empty Ad5-Null
vector. The dose I utilized in this challenge was the dose that increased IFNγ
production of splenocytes compared to the negative controls (Fig. 29). It is possible
that this dose is not sufficient to produce other cytokines or chemokines important
for enhancing an adaptive immune response. I postulate that the Ad5-VCA0956
vector could increase the protection of Ad5-TA at higher doses. I propose repeating
this challenge with different doses of the Ad5-VCA0956 while maintaining the Ad5TA dose. Additionally, this experiment should be repeated with the Ad5-VCA0848
to determine if increased Ad5 mediated c-di-GMP production can improve
protection.

123

CHAPTER 6 – Conclusions
Conclusions
While there are numerous studies describing the role c-di-GMP plays in
regulating the physiology of many bacteria, the regulatory inputs to this almost
ubiquitously utilized second messenger are currently under-characterized. I have
demonstrated that the growth environment effects intracellular c-di-GMP
concentrations in V. cholerae, and that these differences can be as great as 20-fold
(Fig. 5). Furthermore, I have developed a novel method to differentiate the impact
of c-di-GMP synthesis and hydrolysis on overall intracellular c-di-GMP. This
method can be applied to other bacterial systems to determine the effects of specific
environmental factors on c-di-GMP, the effects of deleting DGCs and PDEs, and
differences in c-di-GMP synthesis and hydrolysis between different organisms.
The intestinal environment is a tropism for many human pathogens. To
efficiently cause disease, pathogens recognize specific host-derived cues and
subsequently produce virulence factors. It has been proposed that the intracellular
c-di-GMP of V. cholerae is elevated in the marine environment, and reduced in the
human host. In contrast, my research suggests that the regulation of intracellular cdi-GMP in V. cholerae in the human host is more complex than previously
appreciated. I have demonstrated that bile and bicarbonate inversely modulate
intracellular c-di-GMP, where bile increases c-di-GMP approximately 4-fold and
bicarbonate quenches this induction (Fig. 8, 18). As c-di-GMP signaling is widely
distributed amongst bacteria, it is possible that other enteric bacteria employ
similar responses as V. cholerae to bile and bicarbonate in order to adapt to
124

different microenvironments within the small intestine. Thus, this study may
provide insights about the role c-di-GMP plays in the biology of both human
pathogens and normal members of the human intestinal microbiota.
Growth rate regulation of bacteria has been a focus of study for the last 50
years, and yet there are still many questions regarding how cell division is
governed. I have demonstrated that intracellular c-di-GMP concentration and
growth rate are negatively correlated in V. cholerae (Fig. 20, 21, 22). I have also
shown that rRNA is decreased upon ectopic DGC expression (Fig. 25). Determining
the mechanism governing the c-di-GMP mediated growth rate inhibition is
important, as this phenotype could have large implications in core bacterial
processes. Understanding the relationship between growth rate regulation and c-diGMP is also informative for drug discovery studies targeting compounds that effect
c-di-GMP synthesis and hydrolysis, as these compounds may have unanticipated
effects on bacterial growth rate.
As new and re-emerging infectious diseases continue to take a toll on our
population and burden our healthcare system, vaccine and adjuvant development
offer promising solutions to combat this problem. I have shown that a DGC
delivered into the cytoplasm of a eukaryotic cell can synthesize c-di-GMP and
activate innate immunity by inducing the production of 15 cytokines and
chemokines (Fig. 26, 27; Table 2, 3). Furthermore, I have provided evidence that
using adenovirus to deliver a DGC gene can be utilized as a vaccine adjuvant to
enhance an adaptive immune response (Fig. 29). As c-di-GMP initiates a potent
125

innate response, our novel adenovirus construct is a promising candidate for clinical
applications to enhance the efficacy of existing vaccine antigens. Additionally, the
Ad5-VCA0956 vector is capable of efficiently producing high levels of c-di-GMP in
eukaryotic cells, and thus can be used as a tool elucidate the different c-di-GMP
response mechanisms of various eukaryotic cell types.
There are still many questions regarding regulatory inputs to c-di-GMP
signaling networks, as well as what behaviors c-di-GMP regulates in response to
these inputs. Discerning how bacteria interact with and perceive their changing
environments is paramount to understanding the ecology and behaviors of these
organisms, as well as developing new clinical applications to protect ourselves
against devastating human pathogens. I have demonstrated here that the role of cdi-GMP as a second messenger is to translate environmental signals into
physiological changes that allow V. cholerae to adapt and persist in various niches.
As c-di-GMP is ubiquitously utilized amongst bacteria, we are only beginning to
appreciate the role this molecule plays in enabling bacteria to transition between
different lifestyles, the physiological changes associated with different levels of
intracellular c-di-GMP, and the subsequent impact on the pathogenesis of bacteria.

126

APPENDICES
APPENDICES

127

Strain

Genotype

Escherichia coli

Reference
Invitrogen

DH10B

Escherichia coli

[103]

S17

Vibrio cholerae

WT

C6706

Vibrio cholerae
∆vpsL
Vibrio cholerae
∆hapR
Vibrio cholerae
∆luxO
Vibrio cholerae
∆cdgA
Vibrio cholerae
∆toxR
Vibrio cholerae
∆toxT
Vibrio cholerae

[78]

VC0934 (vpsL)
[72]
knockout mutant
hapR knockout
[67]
mutant
VC1021 (luxO)
[68]
knockout mutant
VCA0074 knockout
Chapter 2
mutant
VC0984 (toxR)
Chapter 3
knockout mutant
VC0838 (toxT)
Chapter 3
knockout mutant
VC1372 knockout
Chapter 3
∆VC1372
mutant
Vibrio cholerae
VC1376 knockout
Chapter 3
∆VC1376
mutant
Vibrio cholerae
VC1067 knockout
Chapter 3
∆VC1067
mutant
VC1372, VC1376
Vibrio cholerae
double knockout
Chapter 3
∆VC1372 ∆VC1376
mutant
Vibrio cholerae
VC1067, VC1372,
∆VC1372 ∆VC1376
VC1376 triple
Chapter 3
∆VC1067
knockout mutant
Vibrio cholerae
VC1295 knockout
Chapter 3
∆VC1295
mutant
VC1295, VC1067,
Vibrio cholerae
VC1372, VC1376
∆VC1295 ∆VC1372
Chapter 3
quadruple knockout
∆VC1376 ∆VC1067
mutant
Clostridium difficile
NAP-1, Ribotype
Unpublished
CD2015
027
Table A1. Bacteria strain list. All bacteria strains used in this
dissertation, and their references.

128

Plasmid Name

Description

Reference

pTL17
pKD3
pEVS141
pVCA0956
pCMW121
pBBRlux
6:C9-lux
vpsT-lux

Inducible FLP recombinase vector
Contains cat flanked by FRT sites
Backbone for DGC expression plasmids, vector control
IPTG inducible VCA0956 GGDEF expression vector
IPTG inducible VC1086 EAL expression vector
Backbone for luminescence reporter plasmids
VC1673 – c-d-GMP inducible lux reporter vector
VC2647 - c-d-GMP inducible lux reporter vector
qrgB expression plasmid, pMMB67eh backbone
(Pursley, B.R.)
IPTG inducible qrgB expression vector
IPTG inducible qrgB* active site mutant expression
vector
IPTG inducible VC1153 (tfoX) expression plasmid
(Chan, C.)
IPTG inducible VC0900 GGDEF expression vector
IPTG inducible VC1029 GGDEF expression vector
IPTG inducible VC1067 GGDEF expression vector
IPTG inducible VC1104 GGDEF expression vector
IPTG inducible VC1185 GGDEF expression vector
IPTG inducible VC1216 GGDEF expression vector
IPTG inducible VC1353 GGDEF expression vector
IPTG inducible VC1367 GGDEF expression vector
IPTG inducible VC1370 GGDEF expression vector
IPTG inducible VC1372 GGDEF expression vector
IPTG inducible VC1376 GGDEF expression vector
IPTG inducible VC1593 GGDEF expression vector
IPTG inducible VC1599 GGDEF expression vector
IPTG inducible VC2224 GGDEF expression vector
IPTG inducible VC2285 GGDEF expression vector
IPTG inducible VC2454 GGDEF expression vector
IPTG inducible VC2697 GGDEF expression vector
IPTG inducible VCA0049 GGDEF expression vector
IPTG inducible VCA0074 GGDEF expression vector
IPTG inducible VCA0165 GGDEF expression vector
IPTG inducible VCA0217 GGDEF expression vector
IPTG inducible VCA0557 GGDEF expression vector
IPTG inducible VCA0560 GGDEF expression vector
IPTG inducible VCA0697 GGDEF expression vector
IPTG inducible VCA0848 GGDEF expression vector
IPTG inducible VCA0939 GGDEF expression vector

[86]
[84]
[195]
[79]
[72]
[69]
[107]
[196]

pBRP2
pCMW75
pCMW98
pANDA2
pVC0900
pVC1029
pVC1067
pVC1104
pVC1185
pVC1216
pVC1353
pVC1367
pVC1370
pVC1372
pVC1376
pVC1593
pVC1599
pVC2224
pVC2285
pVC2454
pVC2697
pVCA0049
pVCA0074
pVCA0165
pVCA0217
VCA0557
pVCA0560
pVCA0697
pVCA0848
pVCA0939

Chapter 3
[72]
[72]
Chapter 3

Table A2.
A2. Plasmid list. All plasmids used in this dissertation, and their references.

129

[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]

pVCA0960
pVCA0965
pVCA1082
pVC0072
pVC0130
pVC0398
pVC0653
pVC0658
pVC0703
pVC1934
pVC2750
pVCA0080
pVCA0785
pVC1652
pVCA1083
pBK34
pBK35
pBK36
pBK37
pBK38
pBK39
pBK40
pBK41
pBK42
pBK43
pBK45
pBK46
pBK47
pBK48
pCMW122
pCMW123
pCMW124

Table A2 (cont’d)
cont’d)
IPTG inducible VCA0960 GGDEF expression vector
IPTG inducible VCA0965 GGDEF expression vector
IPTG inducible VCA1082 GGDEF expression vector
IPTG inducible VC0072 GGDEF/EAL expression
vector
IPTG inducible VC0130 GGDEF/EAL expression
vector
IPTG inducible VC0398 GGDEF/EAL expression
vector
IPTG inducible VC0653 GGDEF/EAL expression
vector
IPTG inducible VC0658 GGDEF/EAL expression
vector
IPTG inducible VC0703 GGDEF/EAL expression
vector
IPTG inducible VC1934 GGDEF/EAL expression
vector
IPTG inducible VC2750 GGDEF/EAL expression
vector
IPTG inducible VCA0080 GGDEF/EAL expression
vector
IPTG inducible VCA0785 GGDEF/EAL expression
vector
IPTG inducible VC1652 EAL expression vector
IPTG inducible VCA1083 EAL expression vector
IPTG inducible VC1087 HD-GYP expression vector
IPTG inducible VC1295 HD-GYP expression vector
IPTG inducible VC1348 HD-GYP expression vector
IPTG inducible VC2340 HD-GYP expression vector
IPTG inducible VC2497 HD-GYP expression vector
IPTG inducible VCA0210 HD-GYP expression vector
IPTG inducible VCA0681 HD-GYP expression vector
IPTG inducible VCA0895 HD-GYP expression vector
IPTG inducible VCA0931 HD-GYP expression vector
IPTG inducible VC0137 EAL expression vector
IPTG inducible VC1592 EAL expression vector
IPTG inducible VC1851 EAL expression vector
IPTG inducible VCA0101 EAL expression vector
IPTG inducible VCA0536 EAL expression vector
IPTG inducible VC1211 EAL expression vector
IPTG inducible VC1641 EAL expression vector
IPTG inducible VC1710 EAL expression vector
130

[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
[79]
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
Chapter 3
[72]
[72]
[72]

pVC1372*
pVC1376*
pVC1067*
pBK51
pCMW27
pCMW28
pCMW33
pCMW34
pCWM35
pCMW36
pCMW37
pCMW38
pCMW39
pCMW40
pCMW41
pCMW42
pCMW47
pCMW48
pCMW49
pCMW50
pCMW51
pCMW52
pCMW53
pCMW54
pCMW55
pCMW56
pCMW57
pCMW58
pCMW59
pCMW60
pCMW61
pCMW62
pCMW63
pCMW64
pCMW65
pCMW66
pCMW67
pCMW68
pCMW69

Table A2 (cont’d)
IPTG inducible VC1372* active site mutant
expression vector
IPTG inducible VC1376* active site mutant
expression vector
IPTG inducible VC1067* active site mutant
expression vector
toxT knockout vector, pKAS32 backbone
VC0072-gfp transcriptional fusion
VC0653-gfp transcriptional fusion
VC0130-gfp transcriptional fusion
VC0900-gfp transcriptional fusion
VC1029-gfp transcriptional fusion
VC1104-gfp transcriptional fusion
VC1185-gfp transcriptional fusion
VC1216-gfp transcriptional fusion
VC1353-gfp transcriptional fusion
VC1367-gfp transcriptional fusion
VC1372-gfp transcriptional fusion
VCA0074-gfp transcriptional fusion
VC1370-gfp transcriptional fusion
VC1376-gfp transcriptional fusion
VC1593-gfp transcriptional fusion
VC1599-gfp transcriptional fusion
VC1934-gfp transcriptional fusion
VC2224-gfp transcriptional fusion
VC2285-gfp transcriptional fusion
VC2370-gfp transcriptional fusion
VC2454-gfp transcriptional fusion
VC2697-gfp transcriptional fusion
VC2750-gfp transcriptional fusion
VCA0049-gfp transcriptional fusion
VCA0080-gfp transcriptional fusion
VCA0165-gfp transcriptional fusion
VCA0217-gfp transcriptional fusion
VCA0557-gfp transcriptional fusion
VCA0560-gfp transcriptional fusion
VCA0697-gfp transcriptional fusion
VCA0785-gfp transcriptional fusion
VCA0848-gfp transcriptional fusion
VCA0939-gfp transcriptional fusion
VCA0956-gfp transcriptional fusion
VCA0960-gfp transcriptional fusion
131

Chapter 3
Chapter 3
[79]
Chapter 3
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]

pCMW70
pCMW71
pCMW72
pCMW73
pCMW78
pCMW79
pCMW80
pCMW81
pCMW82
pCMW83
pCMW84
pCMW85
pCMW86
pCMW87
pCMW88
pCMW89
pCMW90
pBBRluxVC1295
pBBRLuxVC1348
pBBRluxVC2340
pBBRluxVC2497
pBBRluxVCA0210
pBBRluxVCA0681
pBBRluxVCA0895
pBBRluxVCA0931
pShuttle-CMV
pShuttleCMV-

qrgB

Table A2 (cont’d)
VCA0965-gfp transcriptional fusion
VC0658-gfp transcriptional fusion
VC1067-gfp transcriptional fusion
VC0703-gfp transcriptional fusion
VCA1082-gfp transcriptional fusion
VC0398-gfp transcriptional fusion
VC0137-gfp transcriptional fusion
VC0515-gfp transcriptional fusion
VC1086-gfp transcriptional fusion
VC1211-gfp transcriptional fusion
VC1592-gfp transcriptional fusion
VC1641-gfp transcriptional fusion
VC1652-gfp transcriptional fusion
VC1710-gfp transcriptional fusion
VC1851-gfp transcriptional fusion
VCA0101-gfp transcriptional fusion
VCA0536-gfp transcriptional fusion

[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]
[72]

VC1295-lux transcriptional fusion

[66]

VC1348-lux transcriptional fusion

[66]

VC2340-lux transcriptional fusion

[66]

VC2497-lux transcriptional fusion

Chapter 3

VCA0210-lux transcriptional fusion

[66]

VCA0681-lux transcriptional fusion

[66]

VCA0895-lux transcriptional fusion

[66]

VCA0931-lux transcriptional fusion

[66]

Backbone for DGC eukaryote expression vectors

[165]

qrgB expression vector under CMV control

Chapter 5

pShuttleCMV-

qrgB* active site mutant expression vector under

qrgB*

CMV control

pShuttleCMVVCA0848
pShuttleCMVVCA0848*

Chapter 5

VCA0848 expression vector under CMV control

Chapter 5

VCA0848* active site mutant expression vector under
CMV control

Chapter 5

132

Table A2 (cont’d)
pShuttleCMVVCA0956
pShuttleCMVVCA0956*

VCA0956 expression vector under CMV control

Chapter 5

VCA0956* active site mutant expression vector under
CMV control

Chapter 5

133

Primer Name

Description

Sequence

VCA0074KO1
VCA0074KO2
VCA0074KO3
VCA0074KO4
342-us.2ecoR1
342-ds.2bamH1
VC1087fwecoR1
VC1087rvbamh1
VC1295fwecoR1
VC1295rvbamh1
VC1348fwecoR1
VC1348rvbamh1
VC2340fwecoR1
VC2340rvbamh1
VC2497fwecoR1
VC2497rvbamh1
VCA0210fwecoR1
VCA0210rvbamh1
VCA0681fwecoR1
VCA0681rvbamh1

For ∆cdgA
strain
For ∆cdgA
strain
For ∆cdgA
strain
For ∆cdgA
strain

TCGATGTGTCTGATCTGCGTATCCG
CGTTGGTAT
GAAGCAGCTCCAGCCTACACGGGG
CAAAGTTTACCAT
TAAGGAGGATATTCATATGGGGCTT
CTTATGAATCAAAAT
GGTTGAGAAGTAGAACACAATCGAT
ATCCACACG
ATAGAATTCCTCTGCGCTCCTCATA
TTCT
ATAGGATCCGACAGAGAAAGTTAAG
AAAC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGCAAATGGATAACAT
ATAGGATCCTTATCCTATATAAACG
GGGATCGGC
ATAGAATTCAGGAGCTAAGGAAGCT
AAATTGACCATTTGGGTTCT
ATAGGATCCTTATGCGGCATCTTTA
AAGTGTGCT
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGGCAACCGCCAATAT
ATAGGATCCTCAGCCTGACGCTTGT
TGAC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGCAGATGCAACCCAA
ATAGGATCCTCAGTGGGTGATTCCC
TGGTC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAGTGGCAAGCATTAAAAT
ATAGGATCCTTACTCTTCGCTGTCA
AAGAAGTATG
ATAGAATTCAGGAGCTAAGGAAGCT
AAATTGAAGTGGTTTAAATA
ATAGGATCCCTAGTCAGGCAGCGAA
GCAC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGAGATGGTCAGAAAT
ATAGGATCCTTATGCCCATTCGGTT
GGGCTTT

For pBRP2
For pBRP2
For pBK34
For pBK34
For pBK35
For pBK35
For pBK36
For pBK36
For pBK37
For pBK37
For pBK38
For pBK38
For pBK39
For pBK39
For pBK40
For pBK40

Restriction
Site

EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI

Table A3. Primer list. All primers used in this dissertation and their sequence, and any
restriction sites included for cloning applications.

134

VCA0895fwmfeI
VCA0895rvbglII
VCA0931fwecoR1
VCA0931rvbamh1
VC0137fwecoR1
VC0137rvbamH1
VC0515fwecoR1
VC0515rvbamH1
VC1592fwecoR1
VC1592rvbamH1
VC1851fwecoR1
VC1851rvbamH1
VCA0101fwecoR1
VCA0101rvbglII
VCA0536fwecoR1
VCA0536rvbamH1
CMW464

For pANDA2

CMW465

For pANDA2

For pBK41
For pBK41
For pBK42
For pBK42
For pBK43
For pBK43
For pBK44
For pBK44
For pBK45
For pBK45
For pBK46
For pBK46
For pBK47
For pBK47
For pBK48
For pBK48

For
pVC1372*
For
VC1376-ASM
pVC1376*
For
VC1067-ASM
pVC1067*
For pBBRlux2497-fw-SacI
2497
VC1372-ASM

Table A3 (cont’d)
ATACAATTGAGGAGCTAAGGAAGCT
AAATTGCAACCTAGCCAAGA
ATAAGATCTCTAGTAATCTTCCCGT
AAATACGCC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGAGTGTTGCACAAAA
ATAGGATCCTTAGGTCTTATCTGAA
GAGTGTGGTA
ATAGAATTCAGGAGCTAAGGAAGCT
AAATTGGTTCGATGTTTATG
ATAGGATCCCTAAATTAATCGATTG
ATGTCCTGGC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGGATAGTTTCATAGC
ATAGGATCCCTATTTAACTTCTTTC
ATTAAAGTTCGAC
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGAATCACATCCACCC
ATAGGATCCCTATAACGCAGACATA
CGTGGAG
ATAGAATTCAGGAGCTAAGGAAGCT
AAATTGAAGTACTCGTATGT
ATAGGATCCTTAGGTTTGTACACCA
AGAAGATCG
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGGCTAAGGAAAAGAC
ATAAGATCTTTATATTTTGCTTGTG
GCAAAGTTTAATG
ATAGAATTCAGGAGCTAAGGAAGCT
AAAATGGCGTGCGTCAAAAG
ATAGGATCCTTAGTTAGCATTTGCT
ACCTTAGCG
GAATTCGGGGAACGTGATTAAAGG
GGATCCACGCTGCTGACAACTTTCT
AAC
TCGCGAACTCTTCAGCGGCATATCG
AGCGACC
ATATTTTTGCCCGTTTCGCCGCTGA
TGAGTTTGCCATCTT
GGGCGCAGCTATCGGATTGCTGCG
G
GAGCTC
TCTTGGTTACTTGAAAGAGGCC
135

Mfe1
BglII
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
BglII
EcoRI
BamHI
EcoRI
BamHI

SacI

2497-rvBamHI
VC1372KO-1
VC1372KO-2
VC1372KO-3
VC1372KO-4
VC1376KO-1
VC1376KO-2
VC1376KO-3
VC1376KO-4
VC1067KO-1
VC1067KO-2
VC1067KO-3
VC1067KO-4
VC1295KO-1
VC1295KO-2
VC1295KO-3
VC1295KO-4
toxRKO-1
toxRKO-2
toxRKO-3
toxRKO-4

For pBBRlux2497
For ∆VC1372
strain
For ∆VC1372
strain
For ∆VC1372
strain
For ∆VC1372
strain
For ∆VC1376
strain
For ∆VC1376
strain
For ∆VC1376
strain
For ∆VC1376
strain
For ∆VC1067
strain
For ∆VC1067
strain
For ∆VC1067
strain
For ∆VC1067
strain
For ∆VC1295
strain
For ∆VC1295
strain
For ∆VC1295
strain
For ∆VC1295
strain
For ∆toxR
strain
For ∆toxR
strain
For ∆toxR
strain
For ∆toxR
strain

Table A3 (cont’d)
GGATCC
AATCTTAATTACATCTGAGTGAATA
ACC
CGTCTTGTTGATATTGATGATTGCG
TGCGACCAGT
GAAGCAGCTCCAGCCTACACGCTTG
GTCGTCCTGAATGCACAAA
TAAGGAGGATATTCATATGGCTCAG
ATTGTTTATAGGATGGGAGC
CATTTACAGGTGGAAGACAAAACTC
TACTCGCCAC
GCGCTTGGCCAGTCACTTGACCGGT
TAG
GAAGCAGCTCCAGCCTACACGAAAA
ATCATGATGAAG
TAAGGAGGATATTCATATGAATCGA
ATTATGCCTTCAA
CCAACGCGGATGGCACGATTGATCC
GC
GGCCATTTTGGCGATAGCGCCGTAA
GTCG
GAAGCAGCTCCAGCCTACACAGAC
GTTGAATAATGGTATG
TAAGGAGGATATTCATATGTCAAAC
TGGTGAAATGC
CCCTTCCCAACTAAGCAGCCATCGT
GGC
GCCAGCGGCATTGAACACACC
GAAGCAGCTCCAGCCTACAC
TTGCAGCACACTTTAAAGATGC
TAAGGAGGATATTCATATG
TAAGTTACGGCTAGTTAAGTAT
GCTCCTGTGAAAACAGGAACTGATC
CTAAAGTTCGACATCCCCTTGC
GAAGCAGCTCCAGCCTACACGATCT
TGCTATGCAAAATAGACACA
TAAGGAGGATATTCATATGCTAATG
TCCCAGTATCTCCCTG
GTTGAGCGGCAGATCGTTTGAG
136

BamHI

toxTpkas1pBK51
toxTpkas2pBK51
toxTpkas3pBK51
toxTpkas4pBK51

qrgB-CMVfw

qrgB-CMV-rv
VCA0848CMV-fw
VCA0848CMV-rv
VCA0848ASM
VCA0956CMV-fw
VCA0956CMV-rv
VCA0956ASM

For ∆toxT
strain
For ∆toxT
strain
For ∆toxT
strain
For ∆toxT
strain
For
pShuttleCMV
-qrgB
For
pShuttleCMV
-qrgB
For
pShuttleCMV
-VCA0848
For
pShuttleCMV
-VCA0848
For
pShuttleCMV
-VCA0848*
For
pShuttleCMV
-VCA0956
For
pShuttleCMV
-VCA0956
For
pShuttleCMV
-VCA0956*

Table A3 (cont’d)
ATAGAGCTCCCTCATATCAAAGTTT
ATGAAGGGAC
ATAGCGGCCGCTGCGTTCTACTCTG
AAGATATATAAATAA
ATAGCGGCCGCATGGAATACGTTTA
CTTGATCCTATTTTC
ATAGGTACCCAAAGAGTCTAGCGTT
TCAGAAATAG

SacI
NotI
NotI
KpnI

ATACTCGAGTTAAGAAACCTTTTGG
ATTCTTAAGTTG

KpnI

ATACTCGAGTTAAGAAACCTTTTGG
ATTCTTAAGTTG

XhoI

ATAGGTACCCCACCATGAATGACAA
AGTGCT

KpnI

ATACTCGAGTTAGAAAAGTTCAACG
TCATCAGAA

XhoI

GTCTTCTCAACTATTTCGCTTTGCT
GCTGAAGAGTTCGTGATTATTTTTT
ATAGGTACCCCACCGTGATGACAAC
TGAAGATTTCA

KpnI

ATACTCGAGTTAGAGCGGCATGACT
CGAT

XhoI

TGACAGCTTATCGTTATGCCGCTGA
AGAGTTTGCACTGAT

137

REFERENCES

138

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