Analysis of plcR regulator gene expression in Bacillus spp. using GFP fusions
and flow cytometry
A Thesis submitted to the Graduate School
in partial fulfillment of the requirements
For the Degree
Master of Science
By
Marina Vukojevic
Thesis Advisor: John L. McKillip, Ph.D.
Thesis Committee Members: Derron Bishop, Ph.D. and Najma Javed, Ph.D.
Ball State University
Muncie, Indiana
July 2014
1
Table of Contents
Pages
I: Abstract……………………………………………………………….............. 3 - 4
II: Introduction
Section I. Bacillus genus
A) General features and environment………………………….
B) Clinical and environmental importance…………………......
C) Virulence factors and their regulation………………………
D) Stress response and effects on virulence gene
expression in Bacillus spp……………………………………
E) Genetic studies on Bacillus spp……………………………..
4-7
7 - 11
11 - 16
16 - 17
17 - 21
Section II. Carvacrol
F) Biological properties and mechanism of action…………… 21 - 23
G) Health benefits and possible side effects………………….. 23 - 25
Section III. Flow cytometry
H) General features…………………………………………….. 25 - 26
I) Use in bacterial studies…................................................... 26 - 28
Section IV. Hypothesis/Rationale for study…................................... 28- 31
III: Methods
A) Chemicals used………………………………………………
B) DNA extraction………………………………………….…….
C) Real-time Reverse Transcriptase Polymerase Reactions
D) Cloning………………………………………………………..
E) Transformation……………………………………………….
F) Plasmid purification………………………………………….
G) Flow cytometry…………………………………………….....
31
32
32 - 33
33 - 34
34 - 35
35 - 36
36
IV: Results
A) Transformation………………………………………………. 37 - 38
B) PCR…………………………………………………………… 38 - 40
C) Flow cytometry………………………………………………. 41 - 44
V: Discussion…………………………………………………………………. 44 - 49
Acknowledgments……………………………………………………………. 50
VI: References…………………………………………………………...….… 51 - 59
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ABSTRACT
THESIS: Analysis of plcR regulator gene expression in Bacillus spp. using GFP fusions
and flow cytometry.
STUDENT: Marina Vukojevic
DEGREE: Master of Science
COLLEGE: Sciences and Humanities
DATE: July, 2014
PAGES: 59
Carvacrol, the key extract of oregano oil, is known for its antimicrobial properties toward
Bacillus cereus and other bacteria. As such, carvacrol may be considered beneficial in
treatment of severe endophthalmitis (ocular infections) caused by B. cereus. However,
in vitro, subinhibitory concentrations of carvacrol (SIC, 1 mM), have been shown to
upregulate mRNA expression of B. cereus hblC and nheA enterotoxin genes by 5%.
This is clinically relevant because B. cereus virulence may be exacerbated if
endophthalmitis treatment involves incorrect/SIC levels of carvacrol. In Bacillus spp.,
hemolytic BL (HBL) and non-hemolytic (Nhe) toxin production is activated by at least
one transcriptional regulator, a phospholipase C regulator (PlcR). This factor also
regulates endotoxin production in the important insect pathogen, B. thuringiensis. Here
we investigated whether plcR expression is upregulated by SIC of carvacrol in B. cereus
ATTC14579 and B. thuringiensis subsp. israelensis as measured by flow cytometry.
Green fluorescent protein (GFP)-expressing B. cereus ATTC14579 and B. thuringiensis
subsp. israelensis were constructed in order to measure the effect of carvacrol on plcR
using flow cytometry. The results show that SIC carvacrol treated transformed B.
3
cereus ATTC14579 and B. thuringiensis subsp. israelensis increased plcR expression
by 25% and 70% respectively, relative to untreated cultures. Our results reveal that plcR
is upregulated to similar levels as nheA and hblC when subjected to SIC carvacrol. In
other words, SIC carvacrol increases virulence of B. cereus and B. thuringiensis subsp.
israelensis. Furthermore, results indicate that hblC and nheA are directly controlled by
plcR regulator expression in response to the SIC carvacrol and do not apparently
necessitate additional regulatory effector proteins. The implications of this study may
further clarify the order of regulatory virulence events in B. cereus and B. thuringiensis.
II: INTRODUCTION
Section I: Bacillus genus
A) General features and environment:
Bacillus bacteria are obligate aerobic (oxygen dependent) - to facultatively
anaerobic (aerobic or anaerobic) Gram positive spore-forming saprophytes [1].
Approximately 200 Bacillus spp. are known to date which live in varied ecosystems
(soil, rocks, dust, water vegetation, food and the gut of insects and other animals)
because of their highly resistant endospores. Endospore formation is a Bacillus
response to starvation and stress, and a strategy for survival in the soil environments in
which these bacteria prevail [2]. Endospores are resilient to temperature extremes,
chemicals, dehydration and UV light. They can remain dormant for extended periods of
time even without any accessible nutrients, but can germinate easily if nutrients become
available.
4
Bacillus diversity in the natural environment is the result of their phenotypic and
genotypic dissimilarities [3].This can be explained by various physiological properties,
such as the ability to degrade cellulose, starch, proteins, agar, hydrocarbons, and
biofuels. Bacillus spp. are characterized by highly variable G+C content (guaninecytosine) of their DNA, which can range from 32-78% across the genus. Moreover,
highly diverse 16S rRNA sequences of Bacillus are commonly used to illustrate their
differences, as well as to identify new isolates. Lastly, Bacillus diversity also arises from
their ability to grow at various pH values (2-10) and optimal temperatures (25-37°C) [4].
Some species can even grow at 75°C or as low as 3°C. In fact, many well-characterized
food associated pathogenic strains have been shown to be psychrotrophic.
The Bacillus genus includes both free-living (non-parasitic or saprophytic)
and pathogenic (parasitic) species [4]. Most Bacillus spp. are nonpathogenic
saprophytes, with a few exceptions [1]. B. subtilis is generally considered as nonpathogenic and usually used as a model organism for learning the main characteristics
of Gram-positive spore-forming bacteria [5]. This species is arguably the most
genetically well-characterized member of the family Bacillaceae, having served as a
cloning host for varied shuttle vectors for many years [6].
Members of the Bacillus cereus group (including Bacillus anthracis. B.
thuringiensis and B. cereus) are opportunistic or obligate pathogens to some insects or
mammals [1]. B. anthracis is known for causing anthrax [7], whereas B. thuringiensis is
as an important obligate insect pathogen used commercially as a biological control
agent since the late 1960’s [8]. B. cereus may elicit foodborne illness if ingested in
heavily contaminated foods [1]. The clinical symptoms of foodborne illness include
5
vomiting (caused by cerulide exotoxin) and diarrheal syndromes (caused by Nhe, HBL
enterotoxins, and Cytotoxin K) [9]. Both syndromes are usually self-limiting. What is
more significant is B. cereus association with numerous opportunistic and hospitalacquired infections, such as severe endophthalmitis and septicemia. These infections
usually occur due to compromised host immunity, malignant disease, or by exposure
during surgical procedures (iatrogenic infections), due to the ubiquity of B. cereus.
Therefore, while members of the B. cereus group mostly consist of soil-dwelling
saprophytes, this species can also cause a wide range of diseases in humans, including
food poisoning, systemic infections and highly lethal forms of anthrax.
Bacillus anthracis, B. thuringiensis and B. cereus are closely related genetically;
they may be differentiated by their plasmid-borne specific toxins genes, PlcR regulation,
and biosynthesis of virulence capsules [10]. Virulence of B. anthracis and B.
thuringiensis is determined in large part by the expression of toxin genes located on
plasmids, while in B. cereus, only the emetic toxin is plasmid-borne [3]. Also, B.
anthracis carries a nonsense mutation in the gene for the global transcriptional regulator
PlcR, and thus virulence is controlled by other regulators. Nevertheless, PlcR is still
primarly responsible for the production of virulence factors in B. cereus and B.
thuringiensis.
The first comprehensive genome study of B. cereus was performed in 2003
on B. anthracis Ames Porton strain in order to elucidate B. anthracis pathogenesis [11].
Chromosomally encoded proteins, such as haemolysins, phospholipases, iron
acquisition, and certain surface proteins were found in B. anthracis and B. cereus,
emphasizing the similarity between these two bacteria. In two other genomic studies, B.
6
cereus American Type Culture Collection (ATCC) 14579 was revealed as the most
distantly related strain from B. anthracis [12], while B. thuringiensis subsp. kurstaki was
characterized as the most similar to B. cereus ATCC 14579 [10]. The type B. cereus
ATCC 14579 was originally an airborne isolate from a cattle pen in the 1880’s [1].
Currently, B. cereus ATCC 14579, B. thuringiensis subsp. kurstaki [13] and B.
thuringiensis subsp. israelensis [14] strains are the most common strains used for
laboratory genetic manipulation.
B) Clinical and environmental importance of Bacillus spp.
Two Bacillus species are considered most clinically significant: B. anthracis,
causative agent of anthrax [15], and B. cereus, significant cause of foodborne illness [9].
A third species, B. thurngiensis, is an important insect pathogen that has been used
commercially for nearly 50 years as a biological control agent [16].
B. anthracis is a common disease of livestock and it is the only
obligate pathogen within the Bacillus genus [15]. Anthrax is rarely seen in humans and
is considered to be a contact disease in farmers, field veterinarians and workers who
are exposed to dead animals and animal products. It is only spread by spore ingestion,
inhalation or skin contact. B. anthracis is also known as biological weapon for humans
and animals (World War I and II). Early antibiotic treatment of anthrax (lung, skin, and
intestine forms) is important and requires intravenous and oral antibiotics, such as
ciprofloxacin, doxycycline, and penicillin. Moreover, immunization is provided in all
areas where the frequency of the disease is high.
7
B. cereus is a foodborne and food spoilage bacterium commonly found in soil,
growing plants and gut microflora of animals and insects [1]. B. cereus is easily spread
from the soil environment to different types of row and processed foods and
consequently can cause short duration food-associated illness. Also, if given the
opportunity, B. cereus can enter mammalian tissues causing severe endophthalmitis
[17], wound infections [18] or septicemia [19].
B. cereus foodborne illness is associated with the production of exotoxins that
can cause two distinct intoxication syndromes: emetic and diarrheal [1]. The clinical
indicators of foodborne vomiting (caused by cerulide toxin) and diarrheal syndromes
(caused by Nhe, HBL and cytotoxin K) are usually mild and disappear within 24 hours.
Diarrheal syndrome is characterized by watery diarrhea, abdominal cramps, and pain
occurring 6-15 hours after ingestion [20]. Symptoms associated with emetic syndrome
include nausea and vomiting within 1-6 hours after food ingestion [21].
B. cereus causes ocular infections such as keratitis [22], endophthalmitis [17],
and panophthalmitis [23] due to its ability to induce a permeability of the blood-retinal
barrier [24]. The eye is considered to be an immuno-privileged organ protected by
blood-ocular barriers from the vascular inflammatory tissues of the body (innate
immunity). Once these blood-ocular barriers are disrupted, the cells of the innate
immune system (including macrophages and neutrophils) will cause damage to the eye
architecture. B. cereus endophthamitis usually happens due to post-operative and posttraumatic wounds [25]. HBL enterotoxin is believed to be the main virulence factor in B.
cereus endophthalmitis which can often cause the detachment of the retina and
blindness. In the case of B. cereus induced endophthalmitis, blindness may occur within
8
24 h regardless of the type of therapeutic used [4]. Treatment of B. cereus
endophthalmitis requires a combination of clindamycin, gentamicin and vancomycin, but
no suitable agent exists to decrease the ocular inflammation.
B. cereus local and systemic infections (including gas gangrene-like cutaneous
infections, endocarditis, meningitis, bacteremia and septicemia) are mostly hospitalacquired [4]. They usually happen due to a host’s compromised immunity, malignant
disease, or surgical procedures. B. cereus has been found to contaminate intravenous
catheters [26] due to its ability to form biofilms, and thus leads to blood infections [27].
Moreover, B. cereus and its endospores have been shown to contaminate air filtration
and ventilation equipment [28], fiber-optic bronchoscopy equipment [29], linens [30],
gloves [31], specimen collection tubes and balloons used in manual ventilation [32],
alcohol-based hand wash solutions [33], plaster-impregnated gauze [34], and various
antiseptics such as chlorhexidine and povodone iodine [35].
B. cereus, B. licheniformis and some other saprophytic bacilli can act as food
spoilers usually contaminating rice, chicken, vegetables, spices and dairy products [1].
Spoilage of dairy products is very common due to bacteria surviving the pasteurization
process by highly heat-resistant endospores [36]. Also, fresh vegetables (including
carrots, cucumbers, onions, potatoes, tomatoes) are very important in food spoilage,
which is usually caused by B. licheniformis [37]. B. subtilis is a notable food spoiler as
well, causing rope spoilage in bread and related bakery products [38], while the
incidence of B. cereus is high in milk and its products [39].
B. thuringiensis is an insect pathogen [40] which produces crystalline insecticidal
proteins toxins that are mostly active against Lepidoptera (moths and butterflies),
9
Diptera (flies and mosquitoes) and Coleoptera (beetles) larvae [41]. B. thuringiensis can
be found naturally in the intestines of certain insects as well in the soil, leaf surfaces,
aquatic environments, animal feces, insect rich environments, flour mills, and grain
storage facilities [13]. Genetic engineering uses B. thuringiensis genes in order to
incorporate them into corn and cotton crops and construct genetically modified field
crops resistant to certain insect pests. More commonly, B. thuringiensis subsp. kurstaki
endotoxin spray application to specific crops controls the caterpillar larval stage of the
tomato and tobacco hornworm, cabbage worms, loopers, leaf rollers, bagworms, gypsy
moths, tent caterpillar, fall webworm and others [13]. B. thuringiensis subsp. israelensis
endotoxin kills mosquito larvae in mosquito breeding sites (standing water) [14].
Besides the clinical and environmental relevance of Bacillus spp. the
immunogenic properties of their endospores also need to be mentioned [42]. Spores of
B. pumilus and B. subtilis have been found to induce the proinflammatory cytokine
interleukin-6 (IL-6) in a cultured macrophage cell line. These spores also induced the
proinflammatory cytokine tumor necrosis factor alpha (TNFα) and the Th1 cytokine
gamma interferon (IFNγ) in a mouse model. This could mean that a potential probiotic
effect of B. pumilus and B. subtilis can increase resistance to infection by initiating
macrophages activation (innate immune system) similar to Lactobacillus species [43].
Correspondingly, oral immunization of mice with BiosubtylNT spores (B. cereus
commercial probiotic) has been reported to elicit a modest IgG response, suggesting
that spores possess the ability to induce humoral or adaptive immune response as well.
However, not all Bacillus spp. spores can be used as potential probiotics. Food-
10
poisoning B. cereus spores can cause gastrointestinal disease due to their production of
HBL and Nhe enterotoxins, and therefore are not safe for human use.
Certain Bacillus spp. are considered good producers of antimicrobial substances
such as bacteriocins [44]. Bacteriocins and bacteriocin-like inhibitory substances (not
well characterized antimicrobial substances) are produced by ribosomes in various
bacteria [45]. They are commonly directed against competitive microorganisms, but can
be active against closely related species as well. For instance, B. cereus subsp.
vietnami and B. pumilus strains exhibit bacteriocin characteristics against other Bacillus
species [42]. Similarly, a bacteriocin-like inhibitory substance isolated from B. subtilis is
active against H. pylori [46]. Therefore, bacitracin and bacteriocin-like inhibitory
substances can be used to antagonize certain pathogenic bacteria. Moreover,
bacteriocins can be very important in the food industry (lactic acid bacteria) as food
preservatives to prevent spoilage and pathogenic organisms.
C) Virulence factors of Bacillus spp. and their regulation
Members of the B. cereus group can be distinguished by different virulence
factors which are generally determined by genes located on plasmids [3]. B. anthracis
pathogenicity is the result of the production of plasmid-encoded virulence factors such
as the anthrax toxins and the polyglutamate capsule [7]. These virulence factors are
located on two distinct virulence plasmids pXO1 and pXO2 that are both required for
virulence. The genes that encode for anthrax triple toxin (pagA, lef, cya) are located on
the pXO1 pathogenicity island (PAI). These genes on pXO2 define anthrax toxins, as
well as other genes responsible for the biosynthesis of the polyglutamate capsule. The
11
role of the capsule is to protect the bacteria from being killed by host macrophages, due
to the capsule allowing the bacteria to escape the host immune system [47]. Anthrax
toxin is a triple bacterial toxin which can cause edema and lethality. The three
components of this toxin are: protective antigen (PA), edema factor (EF) or lethal factor
(LF) [7]. PA facilitates target cell entry, while PA and LF are enzymatically active. The
combination of PA and LF produces lethal toxin, whereas PA plus EF generates edema
toxin.
The transcriptional regulator AtxA upregulates anthrax toxin genes in response to
host CO2 and temperature levels [7]. While AtxA is encoded by genes located on pXO1
PAI, it has an influence on the expression of genes involved in capsule synthesis found
on pXO2 as well. Therefore, AtxA regulates virulence gene expression in B. anthracis,
whereas a different virulence regulatory system is found in B. cereus and B.
thuringiensis. The transcriptional regulator PlcR, which is responsible for the majority of
virulence potential in B. cereus and B. thuringiensis, is inactive in B. anthracis due to a
nonsense mutation in the plcR gene [3]. For this reason, the anthrax toxin gene
expression uses a different virulence regulatory mechanism. However, the AtxA
virulence pathway is not the only regulatory mechanism of anthrax virulence. Nutrient
limitation or stationary phase of bacterial growth also activates CodY, the global
regulator of virulence in the low G+C Gram-positive bacteria [48]. Upon activation, CodY
controls metabolism, sporulation and virulence in low G+C Gram-positive bacteria.
Previous studies involving B. anthracis reported substantial decreases in anthrax toxin
production in codY deletion mutants, as well as significantly lower levels of AtxA protein
[49]. However, changes were not correspondingly noted in atxA mRNA levels,
12
suggesting that CodY indirectly regulates anthrax toxin production through the posttranscriptional accumulation of AtxA.
B. cereus enterotoxins are chromosomally located, while the genes that
determine emetic toxin are plasmid-borne [50]. HBL and Nhe enterotoxins are
composed of three protein components encoded in an operon, whereas Cytotoxin K
(CytK) is encoded in a single chromosomal gene (cytK). Protein components L2, L1 and
B form the HBL enterotoxin and are transcribed from the HBL operon. Proteins NhA,
NheB and NheC form the Nhe enterotoxin complex, and are co-transcribed by the
nheABC operon. All three proteins from both enterotoxins are required in order to
achieve maximum virulence.
Expression of enterotoxin genes in B. cereus is regulated by a transcriptional
phospholipase C regulator (PlcR) [51], CodY [48], redox-sensitive transduction system
(ResDE) [52], the redox-regulator (Fnr) [53] and catabolite control protein (CcpA) (Fig.
1) [54]. PlcR autoactivates and then induces expression of hbl, nhe and cytK upon the
interaction with PapR, a sensor protein [50]. When high cell densities are reached, PlcR
is activated by PapR which enables binding of PlcR to the “PlcR box”, a highly
conserved palindromic DNA operon promoter sequence TATGnAnnnnTnCAT(A)[55].
Binding of PlcR to this region activates gene transcription and production of various
toxins. Therefore, the PapR-PlcR quorum sensing system regulates transcription of
numerous genes that code for extracellular proteins crucial for sensing environmental
host factors and stressors [51]. In this manner, the PlcR regulon ensures survival of B.
cereus (and other species) when bacteria encounter stress. ResDE and Fnr are
activated by low oxygen and redox potential. They directly stimulate Nhe and HBL
13
expressions via their operon promoter regions. CcpA represses expression of both nhe
and hbl operons by binding to their Catabolite Responsive Element (CRE) [54]. CodY is
the positive regulator of B. cereus HBL and Nhe enterotoxin [56], while the initiator of
sporulation, Spo0A represses activation of PlcR [57]. Cody and Spo0A are triggered by
stationary phase of bacterial growth and are both required for cerulide activation as well.
Cerulide toxin is produced by an enzyme, a non-ribosomal peptide synthase
(NRPS), encoded by the ces gene cluster (cesHPTABCD genes) [58]. These ces genes
are located on a pXO1-like plasmid (pCERE01) and their structure and location is
homologue to those found in B. anthracis pXO1 toxin plasmid.
Fructose
Low oxygen
and redox potential
Glucose
CcpA
Fnr
High population density
PapR
ResDE
CodY
Spo0A
AbrB
PlcR
HBL
Signal
Cerulide
Regulators
Nhe
Regulated
genes
stimulation
InhA1
metalloprotease
CytK
inhibition
Figure 1 Regulation of toxin and virulence factor gene transcription in Bacillus cereus.
Fig. 1 was adapted from Ceuppens et al, 2011. Crit. Rev. Microbiol., 37(3): 188-213.
14
Cerulide toxin biosynthesis is under direct regulation of the transition state
regulator AbrB, which is controlled by Spo0A (Fig. 1) [59]. Activated Spo0A binds and
represses AbrB while triggering expression of the ces gene cluster. Transcription of the
ces operon stops as the cells enter stationary phase; production of cerulide is further
regulated by other transcriptional factors. In addition, CodY acts as direct repressor of
cerulide synthetase gene expression (Fig. 1) by being able to bind to the promoter
region of the cesPTABCD operon [56]. This suggests the existence of a co-regulatory
virulence mechanism in production of cerulide toxin similar to one found in B. anthracis.
Therefore, CodY has influence on both, PlcR-PapR quorum sensing and the Spo0AAbrB regulatory circuit system which have been believed to work independently of each
other [56]. Also, codY links B. cereus metabolism and pathogenicity by binding to the
InhA1 metalloprotease, ensuring Bacillus resistance to the immune systems of different
hosts.
B. thuringiensis is generally categorized as insect pathogen and its insecticidal
activity is attributed to Cry and Cyt proteins, encoded by plasmid-borne crystal protein
genes [60]. The production of crystal toxins commonly coincides with the activation of
sporulation triggered by stationary phase [61]. The crystals are comprised of protoxins
which become active after a susceptible insect ingests them. Interestingly, the presence
of crystalline toxin genes is the only characteristic which differentiates B.
thuringiensis from B. cereus. In addition, B. thuringiensis phospholipases, hemolysins,
and enterotoxins are responsible for its potential virulence in humans which can cause
tissue necrosis, pulmonary infection or food-poisoning if given the opportunity [62].
15
The transcriptional regulator PlcR activates numerous virulence factors in B.
thuringiensis [57]. PlcR is growth-phase-dependent and regulated by Spo0A. However,
the production of toxins is also coordinated by FlhA, the integral membrane protein of a
flagellar export apparatus [63]. The activity of FlhA is required for motility and
enterotoxin virulence production in B. thuringiensis during parasite-host interactions.
Furthermore, proteomic studies of different phases of bacterial growth identified CodY
as one of the proteins that may be involved in the activation of B. thuringiensis crystal
genes and spore formation as well [64].
D) Stress response and its effects on virulence gene expression of Bacillus spp.
Bacillus spp. can overcome hostile environmental conditions (temperature,
humidity, pH, and nutrient limitation) by displaying an adaptive stress response [65]. For
instance, in order to survive amino acid limitation, cells will shut off the synthesis of
rRNA and ribosomal proteins. Endospore production and multilayered biofilm formation
are selective adaptations to nutrient limitation within the Bacillus genus, and may
include lack of carbon, nitrogen or phosphorus [66]. Pathogenic bacteria frequently
display unique adaptive responses to stress and starvation [67]. Pathogenic bacteria
become parasites in the host organism in order to survive. Stationary phase of bacterial
growth provides a multifaceted signal that triggers regulatory mechanisms upregulating
expression of virulence genes. These genes will damage the host by inducing protein
synthesis in order to provide necessary nutrients. Bacteria will not only kill the host’s cell
to survive, but will also try to eliminate other competitive bacteria by producing
antibiotics and bacteriocins, Therefore, stress and starvation turn on growth-phase-
16
dependent virulence in pathogenic Bacillus spp. that targeting both, eukaryotic and
prokaryotic organisms.
F) Genetic studies on Bacillus spp. and natural competence
B. subtilis is one of the most studied bacteria at the molecular level next to
Escherichia coli [5]. B. subtilis has been used as a model for differentiation, gene and
protein regulation, metabolism, motility, antibiotic production, and sporulation
mechanisms in the Bacillus genus. This has all been possible because of B. subtilis
natural competence (NA) and potential for transformation [68]. NA is physiological and
genetical ability of certain bacteria strains (including Streptococcus pneumoniae, S.
sanguis, Haemophilus influenza, and Neisseria gonorrhoeae) to uptake foreign DNA.
NA is commonly triggered by nutritional shortage and in B. subtillis occurs
postexponentially. NA requires the expression of specialized proteins in order to create
DNA-uptake complexes necessary for transformation to occur. First group of required
specialized proteins plays a regulatory role (early competence products), whereas
second group is crucial for binding, processing of DNA and establishing of the
competence mechanism (late competence proteins).
Late-competence genes (comDE, comC, comF, comG, and nucA) require the
competence factor ComK for transcription [69]. ComG is necessary for the DNA to pass
the cell wall and reach the cell membrane [70]. ComEA binds the DNA, which is then
taken up by the ComEC permease, ComFA and NucA. Finally, the DNA uptake is
completed via recombination with the help of RecA, SsbB, DprA and YjbF proteins (Fig.
2) [71].
17
DprA
SsbB
YjbF
Figure 2 Schematic presentation of the DNA-uptake and DNA- integration process
active in a competent B. subtilis cell. The number and the structure of the proteins
which form the DNA translocation complex are randomly illustrated (A=NucA;
C=ComC; E=ComE; F=ComF; G=ComG; CW=cell wall; CM=cell membrane;
CYT=cytoplasm). Fig. 2 was adapted from Leendert et al, 2003. Microbiol., 149, 9–17.
In response to high cell density, ComK, the major regulator of competence, is
released by the anti-adaptor protein ComS, which is induced by the quorum-sensing
pathways [72]. The subsequent binding of ComK to its own promoter region depresses
transcription which increases levels of ComK in the bacteria and makes them
competent. ComK activates late-competence genes by binding to specific sequences,
the K-boxes [73]. The boxes are composed of two AT-boxes with the consensus
sequence AAAA-N5-TTTT, as well as separated by several helix turn positions. These
positions ensure the boxes are located on the same side of the DNA-helix. The
induction of ComK is controlled at the transcriptional and post-translational levels [74].
The transcription of comK is repressed by AbrB, Rok and CodY transcriptional
18
regulators, but is also activated by the DegSU two-component signal transduction
system, SinR and by ComK itself.
The natural DNA uptake in the Bacillus genus has been described for B. subtilis
[75], B. licheniformis [76] and B. amyloliquefaciens [77], but not for the B. cereus group.
However, screening of the genome sequences in Bacillus spp. has revealed the
presence of the competence regulator proteins and competence-related structural
proteins throughout the whole genus (Fig. 3) [78]. Moreover, homologues of most
structural proteins required for transformation in B. subtilis have been found in B.
cereus, B. thuringiensis and B. anthracis, suggesting that these strains have a potential
for developing natural competence.
Figure 3 Presence of competence regulator proteins (a) and competence-related
structural proteins (b) in Bacillus spp. and closely related species required for natural
competence. Genesis 1.6 software was used to help visualize BLAST searches. White
19
color signifies absence of proteins (with e-value of E-0), while dark blue indicates
presence of proteins (e-value < E-20). Protein names are indicated on the right (b).
Bacterial species which were analyzed (a): Bsu, B. subtilis; Bam, B. amyloliquefaciens;
Bli, B. licheniformis; Bpu, B. pumilus; Ban, B. anthracis; Bce, B. cereus; Bth, B.
thuringiensis; Bwe, B. weihenstephanensis; Oih, O. iheyensis; Gka, G. kaustophilus;
Gth,G. thermodenitrificans; Bcl, B. clausii; Bha, B. halodurans. Ban, Bce, Bth, and Bwe
are all considered within the same genetic subgroup. Figure 2 was adapted from
Kovacs et al, 2009. Environ. Microbiol., 11(8):1911-1922.
Interestingly, two homologues of the B. subtilis ComK protein (ComK1 and
ComK2) have been found in the pathogenic B. cereus group. ComK1 shows 62%
similarity to the B. subtilis ComK, while the ComK2 shared 44-48% resemblance and
was C-terminally shorter by 22-32 amino acids. The function of these two B. subtilis
ComK homologues is yet to be determined.
In a previous study, B. cereus has shown ability to take up DNA [79]. Since the
function of B. cereus ComK1 and ComK2 is still unknown, the competence was induced
by overexpression of B. subtilis ComK in B. cereus. The results revealed low
transformation efficiency with chromosomal and plasmid DNA. This indicated that gene
structure in B. cereus is adequate to uptake and integrate foreign DNA. Also, it was
suggested that other regulatory or structural factors than overexpressed B. subtilis
ComK can increase DNA uptake more efficiently in B. cereus, which would explain low
levels of transformation. Furthermore, it is believed that similar induction of functional
20
DNA uptake can be achieved in B. thuringiensis and B. anthracis as well, which will
significantly facilitate molecular genetic studies with these organisms.
Section II: Carvacrol
Biological properties and mechanism of action
Oil of oregano, the distilled product of oregano, is well known for its antimicrobial
properties [80]. It is believed that carvacrol, the compound which represents about
68.5% of oil of oregano’s total volume, is responsible for these antimicrobial effects [81].
Among 43 species of oregano, only Oregano onites (67-82% of carvacrol) and Oregano
vulgare (79% of carvacrol) possess significant levels of carvacrol, which makes them
medically important [82]. When consumed on the tongue, carvacrol activates transient
receptor potential channel 3 (TRPV3) and causes a short-term warming feeling in the
mouth [83]. TRPV3 is a type of skin temperature receptor activated via the
phospholipase C pathway. Carvacrol also activates peroxisome proliferator-activated
receptors (PPARs) and suppresses the Cyclooxygenase-2 (COX-2) enzyme [84]. COX2, the enzyme involved in prostaglandin biosynthesis, plays a key role in inflammation
and circulatory homeostasis. COX-2 is controlled by PPARs and vice versa. By being
able to suppress COX-2 activity, carvacrol can be considered as a promising antiinflammatory agent.
Carvacrol can kill several bacteria strains, e.g. E. coli [85], B. cereus [86] and S.
aureus [87]. It is believed that carvacrol is able to kill bacteria by disruption of their cell
membrane [88]. This affects pH homeostasis and stability of K+/H+ ions inside the cell
obstructing cell division and causing death. In addition, the antimicrobial property of
21
carvacrol can be explained chemically by the hydroxyl group located at the para position
(Fig. 4) [89]. The removal of the hydroxyl group and the other aliphatic ring substituents
leads to a substantial lower antimicrobial activity [90].
In order to enter the cell, carvacrol causes the bacterial cell membranes to swell
frequently with the help of its precursor molecule p-cymene [88]. P-cymene does not
contain a hydroxyl group at the para position and is not critically required for carvacrol to
enter the cell. However, p-cymene’s presence increases the efficiency of this process.
It is believed that once the carvacrol is inside the cell, the hydroxyl group removes
protons inside the cell and collects potassium ions, as well as removes potassium ions
outside the cell when it leaves (Fig. 4).
Figure 4 Hypothesized model of carvacrol activity. It is believed that undissociated
carvacrol dissociates and releases its proton to cytoplasm when enters the cell. Then
carvacrol leaves the cytoplasm undissociated while carrying a potassium ion across the
22
cytoplasmic membrane to the external environment. Fig. 3 was adapted from Ultee et
al. 2002. Appl. Environ. Microbiol., 4:1561-1568.
In the cell, potassium activates cytoplasmic enzymes, and keeps osmotic
pressure and pH within the neutral range. Carvacrol disrupts these potassium activities
and consequently reduces ATP in the cell which is vital for its metabolism. Furthermore,
carvacrol concentrations of 2 mM have been found to decrease ATP levels noticeably in
B. cereus within 7 minutes, whereas SIC carvacrol (1 mM) lowers membrane fluidity
[91]. Additionally, studies from our lab have shown that the minimum bactericidal
concentration of carvacrol (11 mM) was necessary to decrease the degree of B. cereus
infection in vitro.
Health benefits and possible side effects
Carvacrol has a wide range of health benefits, including antimicrobial [86],
antitumor [92], antioxidant [93], hepatoprotective [94], anti-inflammatory properties [95],
and more. Carvacrol can protect DNA from damage and thus has been shown to have
anticarcinogenic potential [96]. One recent study conducted on human hepatoma cells
(HepG2) and human colonic cells (Caco-2) subjected to various concentrations
of carvacrol showed significant DNA protection from the potent oxidant hydrogen
peroxide. Another study has even shown that carvacrol can cause apoptosis in MDAMB 231 cancer cells by decreasing mitochondrial membrane potential of the cell [97].
This resulted in release of cytochrome c from mitochondria, caspase activation, and
23
cleavage of poly ADP- ribose polymerase (PARP) involved in apoptosis. However, the
mechanism by which this is done is yet to be determined.
Antioxidant properties of carvacrol have been shown in several studies. For
instance, experiments with various concentrations of carvacrol incorporated in chitosan
films (biodegradable biocompatible polymers) revealed that all of the chitosan-carvacrol
film extracts inhibited erythrocyte hemolysis [98]. Furthermore, carvacrol may also be
beneficial in liver regeneration, similar to silymarin (active ingredient of milk thistle) as
well as being hepatoprotective via anti-inflammatory properties [94]. Additionally,
carvacrol has been shown to have a moderate anticoagulant property (similar to aspirin)
by being able to decrease the thromboxane A2 production in platelets. This restricted
expression of the GPIIb/IIIa platelet receptor for fibrinogen which is involved in platelet
activation [99].
No known toxic effects of carvacrol have been recorded when consumed by
humans. It has been shown that carvacrol is metabolized and excreted 24 h following
ingestion, so accumulation in tissues is not an issue [100]. Studies conducted in our lab
have shown that SIC of carvacrol upregulated mRNA expression of B. cereus hblC and
nheA genes by 5% in vitro. This is clinically significant because the B. cereus virulence
may increase if the SIC of carvacrol is administered into an eye of endophthalmitis
patient. Hence, we hypothesize that SIC carvacrol can intensify the eye damage of the
endophthalmitis patient by increasing expression levels of nhe and hbl. We believe that
this happens directly through the plcR pathway. Therefore, carvacrol can kill B. cereus,
but it can also be harmful if used at inappropriate concentrations. Also, applying
carvacrol into an eye can cause certain discomfort, similar to the warming sensation
24
when applied on the tongue. In this manner, one primary concern would be disruption of
normal H+/K+ concentrations gradients locally by carvacrol due to the activation of
TRPV3. Furthermore, there is the possibility that carvacrol’s anticoagulant properties
can increase bleeding time in individuals that already use some anti-coagulant
medication. Finally, additional studies completed in our laboratory have shown that eyes
of BALB /c mice were irritated and red after being subjected to carvacrol, although this
condition generally disappeared in 5-10 min. An analgesic was applied to the eyes of
the mice to reduce these side-effects of carvacrol. Hence, the use of an analgesic can
be beneficial if carvacrol is shown to be successful as an anti-inflammatory agent in
treating B. cereus endophthalmitis.
Section III: FLOW CYTOMETRY
General features
Flow cytometry (FCM) is a powerful analytical tool designed to identify and
calculate specific cellular elements in suspension by measuring cell-surface and/or cellinner markers [101]. In order to recognize and analyze characteristics of an individual
cell, FCM uses beams of laser light to generate fluorescent emission coming from the
cells labeled with antibodies conjugated with fluorochrome (fluorescently-tagged
antibody - marker). The most common fluorophores used are
fluorescein isothiocyanate (FITC) that emits yellow-green color at 519 nm and Rphycoerythrin (PE), which emits red color at 578 nm. Moreover, the green florescent
protein (GFP) from Aequorea victoria, which emits green color at 509 nm, is frequently
used as a reporter of gene/protein expression as well [102]. The use of GFP
25
gene/protein reporter in microbiology can even allow quantification of the several
bacterial genes within the cell through proper choices of filter sets and combinations of
GFP variants. This is particularly appropriate for the study of intracellular bacterial
pathogenicity.
Different cell types can be identified within diverse cell populations and studied
separately or via functional intercellular interactions. FCM is commonly applied to
inspect morphology, viability (quiescent, activated, growing, differentiating, proliferating,
dying, and dead cells) and intrinsic elements (cell cycle and cell-signaling) of the normal
and abnormal cells [103]. In addition to cell analysis, subcellular organelles and even
chromosomes can be analyzed by FCM [104], as well as the various cell elements
present in lysates or biological fluids [105]. Thus, FCM is frequently applied in the fields
of immunology, hematology, pathology, and cancer to sensitively quantify cellular
phenomena [101].
FCM use in Bacillus spp. studies
FCM technology is not exhaustively used within microbiology, even though there
is a great potential for it [106]. Multiparameter data acquisition and multivariate data
analysis collected for each individual cell, high-speed analysis and cell sorting
possibilities (fluorescence-activated cell sorting or FACS) are advantages of FCM from
which microbiology, for example food safety, can greatly benefit. Although a number of
techniques (serological and molecular) can be used to detect Bacillus spp. [107], it is
believed that development of FCM-based methods could provide a better technical and
quantitative approach compared to other more conventional methods [108]. For
26
instance, FCM-based approaches for detection of B. cereus would be more rapid, more
specific (antibodies or probes used for B. cereus), and could use specific markers
(GFP) and nucleic acid probes in order to measure physiological status of vegetative
cells and/or endospores. Additionally, FCM is already used to enumerate bacterial cells
during growth experiments in liquid media and to even obtain data on DNA content as
well as physiological status on single cells [109]. Bacterial growth rates have been
assessed under different conditions, allowing construction of growth curves [110]. FCM
can be also used to analyze the responses of individual cells to antimicrobial
compounds or treatments [111]. It is even possible to study the mechanism of action of
such treatments by staining treated B. cereus cells with numerous known fluorescent
dyes viable and nonviable bacteria [106].
Studies related to food safety are also focused on investigating the production of
toxins under different environmental conditions [112, 113], in an effort to relate structure
of toxins to their function [114, 115]. Recently, multiplex bead array assays were
developed in order to simultaneously detect and quantify assorted proteins within the
sample [116]. This kind of assay would be very useful in order to explain how various
toxins interact to initiate disease such as endophthalmitis, caused by B. cereus [108].
FCM has much to offer in Bacillus spp. endospore resistance research as well.
[117]. For instance, recent FCM studies have used fluorescently-tagged antibodies
which stain certain proteins in endospores lacking a cortex component in order to
investigate the mechanism of sporicidal compounds or treatments. For this purpose, the
endospore cortex was removed due to the fact that it possesses the resistance factor
which prevented fluorescent antibodies from labeling the desired intrinsic proteins. FCM
27
could also be used to screen for mutants, e.g. resistance to UV light, or to detect
oxidative damage to the DNA all of which contributes to the characterization of genes
responsible for resistance. Also, if florescent dyes specific to different structural
components of the endosporium become available, FCM could perform real-time
analysis of sporulation processes more efficiently and rapidly compared to the present
technique [118]. Current approaches use GFP-transformed strains of Bacillus spp. in
order to elucidate the genetic control mechanisms of sporulation as measured via FCM
[119].
Since FCM measurements are made on single cells, heterogeneity within the
population can be identified and quantified as well [120]. Furthermore, FCM is able to
measure up to 12 parameters per cell in order to collect phenotypic data [108]. These
data can be used to further define and classify strains and thus contribute to the
taxonomic studies. Although there are still few obstacles that need to be overcome in
the area of microbial FCM, it is clear that FCM may greatly improve and provide more
comprehensive studies of B. cereus growth and physiology than more conventional
approaches allow.
Section IV.
Hypothesis
PlcR directly regulates expression of the virulence factor gene expression during
subinhibitory carvacrol stress in B. cereus and B. thuringiensis subsp. israelensis.
28
Rationale for study
B. cereus causes severe nosocomial eye infections [25], which may cause
blindness within 24 h regardless of the type of therapy used [4]. The blindness is the
result of eye tissue destruction from B. cereus disrupting blood-ocular barriers, which
naturally protect the eye from the inflammatory cells of the innate immune response
(including macrophages and neutrophils) [24]. Once they reach blood-ocular barriers,
these inflammatory cells will irreversibly damage the eye tissue. It is believed that the
HBL enterotoxin is the main virulence factor in B. cereus endophthalmitis. Additionally,
the transcriptional regulator of virulence in B. cereus, PlcR, is likewise considered a key
factor in B. cereus endophthalmitis pathogenesis as well [121]. However, it is still
unknown whether PlcR directly regulates enterotoxin production or perhaps indirectly
via some intermediate global effector protein.
Treatment of B. cereus endophthalmitis requires a combination of clindamycin,
gentamicin and vancomycin, but no suitable agent exists to decrease the ocular
inflammatory response [4]. Carvacrol’s anti-inflammatory properties (COX2 inhibitor)
may significantly reduce the damage of the eye’s architecture caused by B. cereus [95].
However, a recent study conducted in our laboratory showed an increase in nheA and
hblC expression by 5% in response to SIC carvacrol in vitro. In other words, SIC
carvacrol can potentially increase B. cereus virulence toward within the host. As
previously mentioned, the beneficial or harmful properties of carvacrol are usually
concentration-dependent in terms of B. cereus. Hence, SIC carvacrol can act as a
sublethal bacterial stressor capable of initiating virulence mechanisms in B. cereus. As
29
such, SIC carvacrol can be used as a valuable instrument in order to reveal the
molecular hierarchy of the virulence gene regulation in B. cereus and B. thuringiensis.
Here we proposed to investigate the relationship that SIC carvacrol may have
with the enterotoxins HblC and NheA through the plcR transduction pathway in B.
cereus and B. thuringiensis subsp. israelensis. To do this, GFP-expressing strains of
Bacillus spp. were constructed in order to assess the effect of carvacrol on plcR using
flow cytometry. The B. cereus ATTC 14579 plcR region was cloned into pAD 123, an E.
coli-B. subtilis shuttle vector, for construction of the GFP-expressing strains. Newly
constructed pAD 123: plcRGFP was to be transformed into B. cereus, B. subtilis, B.
thuringiensis, and E. coli in order to quantify the level of GFP expression using flow
cytometry in the presence of SIC carvacrol.
B. subtilis [68] and E. coli [122] were already shown to be competent for
transformation in contrast to B. cereus and B. thuringiensis strains. A previous study
revealed that B. cereus can be naturally competent, which suggests that B.
thuringiensis can be competent as well [78, 79]. This conclusion was based upon the
almost identical genomic sequences of type strains of these two bacteria. Therefore, the
B. subtilis ‘natural competence’ protocol was used to make B. subtilis competent for
transformation, as well as B. cereus ATTC 14579 and B. thuringiensis subsp.
israelensis. It was an important to transform into multiple Bacillus spp. in order to reveal
the true nature of PlcR-mediated virulence in B. cereus and B. thuringiensis. The
implication of this study may further contribute to the development of improved
treatment options for B. cereus endophthalmitis. Furthermore, our work will demonstrate
30
that successful transformation of multiple members of the B. cereus subgroup is a
practical option for recombinant strain generation and subsequent physiological studies.
III: METHODS
Chemicals. The following chemicals were used at specific concentrations in the
methodology described below and obtained from several laboratories in the Biology
Department, Ball State University: Tris-EDTA buffer (cat. # 93283), isopropanol (cat. # I9518), 20 mg/ml lysozyme (cat. # L-6876), 2 mM EGTA (cat. # E3889), 50 µg/ml
ampicillin (cat. # A 9518), 1% agarose (cat. #19539), 10 mM EDTA (cat. # E1161), 1%
SDS (cat. # L3771), NaOH (cat. # 32041), potassium acetate (cat. # P-3542), acetic
acid pH 4.8 (cat. # A8976), 1 M magnesium sulfate (cat. # 63126) and 1 mM carvacrol
(cat. # W224502, Sigma Aldrich, St. Luis, MO); chloroform-isoamyl alcohol (cat. # K169)
and proteinase K (cat. # E-195 AMRESCO, Solon, OH); 3 M sodium acetate (cat. # S210 Fisher Scientific, Waltham, MA); Solution A (50mM glucose (cat. # G7528), 10 mM
EDTA and 25 mM Tris (cat. # BP152, Fisher Scientific)); Solution B (NaOH and 1%
SDS); Solution C (3 M potassium acetate and acetic acid pH 4.8); SPC and SPII media
(1 M magnesium sulfate, 50% glucose, 10% yeast extract (cat. # 212750) and 10% beef
extract (cat. # 0884-17, Becton-Dickinson, Sparks, MD)); trypticase soy agar (TSA, cat.
# T0502, Teknova, Hollister, CA); trypticase soy broth (TSB, cat. # 221715, BectonDickinson); SYBR Green (cat. # 50513, Lonza, Hopkinton, MA) and 2X SensiMix (cat. #
QT255-20, Bioline, Taunton, MA).
31
Bacterial Cultures. The following bacteria were used in the experimental
procedure: Bacillus cereus ATTC 14579 (American Type Culture Collection, Manassas,
VA), B. subtilis 1012 (Bacillus Genetic Stock Center, Columbus, OH), B. thuringiensis
subsp. israelensis (ATCC 35646), E. coli (ECE166, Bacillus Genetic Stock Center) and
the shuttle vector pAD 123 (ECE165, Bacillus Genetic Stock Center).
DNA Extraction. The total DNA isolation procedure was performed according to
Phelps and McKillip [123]. In order to obtain fresh bacterial genomic DNA, bacteria
were subcultured in TSB every 48 h at 32°C for 6 days while shaking at 165 rpm. Each
experiment was repeated in triplicate.
Real-Time Polymerase Chain Reaction (PCR) Gene Expression. Extracted
bacterial DNA yield and purity was assessed via spectrophotometry by reading
absorbance at 260 and 280 nm. The plcR gene (Accession # AY776145) was used for
primer design:
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
atgcacgcag
caaaaacggt
gcggtatacc
attcattttt
caaattatta
gagttgaaga
gtagctgctt
ttgctcaatc
attgcaaaca
atattaaaac
aatcatgcaa
aaagccattg
caaaaaggtg
gaaaaagcgt
aaaaaaatga
aaaaattagg aagcgaaata aagaaaatta gggtgatgag aggattaaca
tatccgataa tatatgtcac caatcggaag tgagccgaat tgaatcgggc
caagtatgga tatattgcaa ggtattgcgg cgaaattaca agttcccatt
atgaggtact catttattct gatattgaga ggaataagca gttaaaagat
tgctttgtaa gcaaaagaaa tataaagaaa tttataatag ggtatggaat
aggaagaata tcatcccgag cttgagcaat ttcttcaatg gcaatatcat
acatattgaa aaaaatcgat tacgattatt gtattttaga attaaagaaa
aacaattggt aggaatagat gtatatcaga atctttatat tgaaaacgca
tttatgctga aaatggctat tttaagaaga gtattgagtt atttgaaaat
aattagaggc attgcatgat aataaagagt ttgatgtgaa ggtgagacat
aagcattata cttagataat caatatgaag aagcgctttg tcacgtaaat
aactatcgtg tcaaattaat agtatgacat tgattggaca gttatactat
aatgcctaga aaagctagag tgtgatagag cagaaattga agatgcttat
gcttcttttt cgatatatta ggaatccatg cattaaaaga atcacttata
agaaataa (specific primer annealing sites highlighted)
Reverse primer sequence: 5’ ACT AAG GTC CTT ATTC TGC TGA TTT TAT TTA C- 3’
and forward primer sequence: 5’ ACT AGG ATC CAT GCA AGC AGA GAA ATT AG –
32
3’ (91897266/67, Integrated DNA Technologies, San Jose, CA) were used at a
concentration of 100 pmol/µl. Samples were amplified via real-time PCR (Rotor-Gene,
R-3000) during 42 amplification cycles using the following conditions: denaturation at 94
°C, annealing at 51°C and extension at 70 °C. 2X Sensi Mix was used in order to
provide deoxynucleotide triphosphates (dNTPs). The negative control was the sample
without DNA. The amplification of plcR was confirmed by SYBR Green real-time melt
curve detection on RotorGene and measured by cycle threshold values (Ct value). PCR
reaction components and volumes used are presented in the table below:
Table 1
PCR reaction Components
SYBR Green
DNA
plcRF primer
plcRR primer
SensiMix
H20
Volumes used
0.5 µl
at concentration of 5 µg/µl
1 µl
1 µl
12.5 µl
to the final volume of 25 µl
PCR products were analyzed for amplification by electrophoresis on 1% agarose gel.
Experiment was repeated in triplicate.
Construction of Recombinant Plasmid. The B. cereus ATTC 14579 plcR
region was cloned into pAD 123, an E. coli-B. subtilis shuttle vector for construction of
the GFP construct according to Dunn and Handelsman (Fig. 5) [124]. The digestion of
plcR and the pAD123 expression vector was performed by using BamHI (BP80051,
Fisher Bioreagents, Pittsburg, PA). Reactions were incubated at 37 °C for 2 h. pAD 123
was ligated with plcRGFP using a T4 DNA Ligase kit according to the manufacturer’s
33
instructions (M0202S, New England BioLabs, Ipswich, MA). This kit did not require
previous dephosphorylation step of vector. Reaction was incubated for 2 h at 16 °C.
Figure 5 B. subtilis-E coli shuttle vector for construction of GFP fusions. Gfpmut3a
indicates promoter-less gene encoding a variant of GFP from plasmid pFPV25. Rep
signifies replication initiation protein from cryptic rolling circle plasmid pTA1060 from B.
subtilis “natto”. Cat encodes chloramphenicol acetyl transferase- selectable for E. coli
and B. subtilis (chloramphenicol 5µg/ml). Bla translates β-lactamase-only selectable in
E. coli (ampicillin 100µg/ml) pAD 123 replicates. Fig. 5 was adapted from Dunn and
Handelsman 1999. Gene, 226(2): 297-305.
‘Natural Competence’ Protocol for Bacillus spp. B. cereus, B. subtilis and B.
thuringiensis subsp. israelensis were separately grown and each subcultured for 48 h in
TSB at 32°C while shaking at 165 rpm. Cultures were diluted with sterile saline 1:2 and
their OD600 was measured. The desired OD600 is less than 1. Bacterial cultures were
added to the SpC media, then incubated while shaking for 2 h at 32°C. This mixture was
then added to the SpII media prewarmed at 32°C which is differentiated from SpC by
34
containing more MgSO4 and less yeast extract and caseamino acids. Finally, mixtures
were incubated while shaking at 165 rpm at 32 °C for 90 min, after which bacteria
cultures were centrifuged at 10,000 xg for 3 min.
Transformation Protocol Using Heat Shock for E. coli. E. coli was made
competent for transformation according to Froger and Hall [125]. This bacterium served
as the positive control in order to test the plasmid vector/transformation protocol on a
known competent cell strain for which the shuttle vector was designed.
Transformation of Competent Bacillus spp. In order to perform
transformation, 100 µl of competent bacteria was added to 100 µl of 1xT-base 2 mM
EGTA. Ligation products were added to this mixture at a concentration of 100 ng and
reactions were incubated while shaking for 60 min at 32 °C. Finally, the entire volume
was added to prewarmed 32 °C TSB and incubated while shaking for additional 2.5 h at
32 °C. Transformed bacteria (100 μl) were plated on TSA plates containing ampicillin at
a final concentration of 50 µg/ml. Plated transformed cultures were incubated inverted at
37 °C overnight in order to screen clones.
Plasmid Purification. In order to purify the plasmid vector from transformants,
colonies were picked with sterile inoculating needle and grown overnight in TSB with
ampicillin at a final concentration of 50 µg/ml. Broth cultures were then centrifuged at
7,000 xg for 7 min at ambient temperature. Cells were washed with 50 µl Solution A
(50mM glucose, 10 mM EDTA, and 25 mM Tris), followed by addition of 20 mg/ml
lysozyme, 60 µl Solution B (NaOH and 1% SDS) and 50 µl Solution C (3 M potassium
acetate and acetic acid pH 4.8). In between solutions A, B and C, cells were incubated
on ice for 10 min. Then, the cell lysates were treated with 60 µl ammonium acetate,
35
centrifuged at 12,000 xg for 15 min at room temperature, subjected to 150 µl
isopropanol addition and finally centrifuged for 20 min at 13,000 xg. The supernatant
was discarded and pellets were air dried. The following day, after spectrophotometry
analysis, the plasmid vector was screened for the plcR insert using real-time PCR.
Once the anticipated recombinant plasmid was confirmed, the transformed bacteria
were grown overnight at 32°C in the presence of ampicillin at a final concentration of 50
µg/ml and presence and absence of 1 mM carvacrol (0.75 µl). The nontransformed
bacterial cultures served as a negative control, grown with and without the presence of
carvacrol. Experiments were completed in triplicate.
Flow Cytometry. 30 minutes prior to the flow cytometry analysis (BD Accuri C6
model), 1 ml of transformed overnight bacterial cells was pelleted (3 min. at 13,000 xg),
washed once with 500 µl room temperature sterile saline, pelleted again, and
resuspended in 1 ml saline. Green fluorescence was measured using a 509 nm band
pass filter (GFP channel) and data were collected from 6,000 events for each sample.
The nontransformed bacterial cultures were also used as a negative control, grown with
and without the presence of SIC carvacrol. All flow cytometric analyses were completed
in triplicate and analyzed by analysis of variance (ANOVA) using p < 0.05 to denote
whether groups were significantly different.
36
IV: RESULTS
Transformation of pAD123:plcRGFP into Bacillus spp.
Figure 6 The colony morphology of transformed B. cereus ACCT 14759 grown on
tryptic soy agar for 24 h at 37°C. By macroscopic assessment, colonies appeared
smooth, round, white and small. 100 µl of transformed bacteria (see details in Methods
and Materials) were plated on tryptic soy plates containing ampicillin at a final
concentration of 50 µg/ml. Average number of colonies detected from B. cereus was 18
counted from 6 plates.
We wanted to examine whether B. cereus and B. thuringiensis subsp. israelensis
could be transformed with a recombinant GFP-expressing vector. Therefore, the
‘natural competence’ protocol was used for B. subtilis, B. cereus and B. thuringiensis.
This protocol is based upon nutritional shortage. However, in order to transform E. coli,
37
the heat shock procedure was used. Transformed cultures were incubated at 37 °C
overnight for 24 h in order to screen clones. Unfortunately, we were not successful in
transforming into B. subtilis and E. coli. None of the colonies were observed after
repeated attempts. However, successful transformation of B. cereus was achieved and
these colonies/clones served as the samples for the recombinant GFP/flow cytometry
experiments. Successful transformation of B. thuringiensis subsp. israelensis was also
obtained and used for flow cytometric analyses.
Real-time PCR analysis of cloned plcR
A)
dRFU/dT (melt peak vs. T)
1
2
3
Temperature °C
38
B)
1
Fluorescence
2
3
Cycle number
Figure 7 Melting peak vs. temperature plot (A) and fluorescence plot (B) obtained from
transformed Bacillus cereus ATTC 14759 (2) and B. thuringiensis subsp. israelensis (1)
amplified by real-time PCR. The real-time PCR analysis showed melting peaks
corresponding to the specific B. cereus and B. thuringiensis subsp. israelensis PCR
products at 75.5°C (1, 2). No signal was observed for the negative control (3). Samples
were amplified by using Rotor-Gene, R-3000 during 42 amplification cycles under the
following conditions: denaturation at 94 °C, annealing at 51°C and extension at 70 °C.
PCR reaction comprised of 2X SensiMix, SYBR Green (12.5 µl), primers (1µl each)
(plcRR: 5’ACTAAGGTC CTT ATTC TGC TGA TTT TAT TTA C-3’ and plcRF: 5’ACT AGG
ATC CAT GCA AGC AGA GAA ATT AG-3’), DNA (0.5 µg/µl) and PCR graded H2O to the
final volume of 25 µl.
39
Real-time PCR was used in order to confirm the cloning process. As we
expected, real-time PCR analysis revealed two primer melting temperature Tm peaks
(A) at 75.5 ºC displayed in different colors (B. thuringiensis subsp. israelensis - green,
B. cereus - blue). This corresponded to the amplicon length of the plcR region of 858 bp
within the recombinant plasmid pAD123 (5952 bp). In addition to melting peak vs.
temperature, the fluorescence plot (B) also revealed substantial changes in
fluorescence signals at amplification cycles 17 and 19. Finally, samples lacking the
cloned plcR did not display Tm at all and served as negative control (designated as
number 3 on plots and displayed in red).
40
Flow cytometry analysis
A) Samples without SIC caravacrol
B) Samples subjected to SIC carvacrol
A1)
B1)
A2)
B2)
A3)
B3)
A4)
A5)
B4)
B5)
41
C)
D
12
Fluorescence (%)
10
8
6
4
2
A
C
B
B
B
B
B
0
-2
Figure 7 Flow cytometric analyses of nontransformed (A) and transformed (B) Bacillus
spp. subjected to SIC carvacrol revealed substantial increase in fluorescence from
transformed B. cereus ATTC 14579 and B. thuringiensis subsp. israelensis. Bacillus
cereus, B. subtils and B. thuringiensis subsp. israelensis were grown overnight at 32 °C
in 5 ml of tryptic soy broth in the presence of SIC carvacrol or without carvacrol. 30
minutes prior to the flow cytometry analysis, 1 ml of overnight cultures was pelleted (3
min. at 13,000 xg), washed once with 500 µl of sterile saline, pelleted again and
resuspended in 1 ml of saline. Data were collected from 6,000 events for each sample
and measured using 509 nm band pass filter for green color detection (GFP channel).
The percentage of fluorescence was presented on plots by means of M1 values (A, B).
Flow cytometer BD Accuri C6 model was used. Samples were assessed in triplicate.
42
Differences were determined to be statistically significant at p< 0.05 by one-way
analysis of variance (C).
Analysis of nontransformed B. cereus, B. subtilis, and B. thuringiensis subsp.
israelensis, treated and untreated with SIC of carvacrol (A1, A2, A3, B1, B2, and B3),
showed detectable fluorescence measured by the green detection filter, GFP. Flow
cytometric representative data are given in the form of fluorescence plots (A and B),
while the average percent values of all assessed replicates are presented in the bar
graph (C). M1 values revealed 1.2 % fluorescence collected from B. cereus sample
subjected to SIC carvacrol (B1) and 2.4 % from the untreated sample (A1). We detected
an increase of M1 value (0.8%) for carvacrol-treated B. thuringiensis subsp. israelensis
(B3) as well as lower fluorescence (0.2%) from the sample lacking carvacrol (A3).
However, B. subtilis showed almost identical M1 values for treated (1.1 %) (B2) and
untreated (1.2%) samples (A2). Therefore, untransformed B. cereus showed higher
fluorescence in the absence of SIC carvacrol compared to the samples that were
subjected to this stressor. In nontransformed B. thuringiensis subsp. israelensis, the
higher fluorescence was generated from the sample subjected to SIC carvacrol, while in
B. subtilis we were not able to detect any significant differences in fluorescence
between carvacrol - treated and untreated samples.
However, significant increases in fluorescence were recorded from transformed
B. cereus ATTC 14579 and B. thuringiensis subsp. israelensis subjected to SIC
carvacrol (B4, B5, and C) compared to carvacrol untreated transformed samples, as
well as untransformed control samples. M1 values revealed 4.6 % fluorescence
43
collected from transformed B. cereus sample subjected to SIC carvacrol (B4), whereas
1.2% of fluorescence was detected from the untreated sample (A4). Interestingly,
transformed B. thuringiensis subsp. israelensis displayed 9.5% of fluorescence (B5)
compared to untreated sample, which had an M1 value of 1.0% (A5). Therefore,
transformed B. thuringiensis subsp. israelensis subjected to SIC carvacrol showed
higher fluorescence values than transformed B. cereus, but both displayed significantly
greater fluorescence than other experimental samples. Finally, average data of all
replicates (C) displayed an increase in the plcR expression by 25% in transformed B.
cereus ATTC14579 subjected to SIC carvacrol, as well as 70% in the treated B.
thuringiensis subsp. israelensis, compared to untreated cultures.
V: DISCUSSION
SIC carvacrol-treated B. cereus ATTC14579 and B. thuringiensis subsp.
israelensis increase plcR expression by 25% and 70% respectively, relative to untreated
cultures.
These data support our hypothesis that SIC carvacrol can increase virulence of
B. cereus ATTC14579 and B. thuringiensis subsp. israelensis. Additionally, global
regulation of Bacillus spp. virulence factors, such as exotoxins, seems to involve a
direct interaction between PlcR and effector sites on virulence gene operons, and does
not apparently necessitate additional global regulatory effector. This level of PlcR
regulator expression is consistent with earlier work in our lab showing hblC and nheA
enterotoxin expression in SIC-treated B. cereus ATCC14579 to be proportional in vitro
44
(the enzyme-linked immunosorbent assay, ELISA and the reverse passive latex
agglutination, RPLA), and in a Caenorhabditis elegans in vivo bioassay model. The
objective of these previous studies was to monitor carvacrol effects on B. cereus
virulence in order to identify carvacrol as a potential alternative treatment for severe
endophthalmitis caused by this bacterium. However, data showed that inappropriate
concentrations of carvacrol, such as SIC, could induce even more damage to the
patient’s eye by increasing B. cereus virulence factors expression.
Similarly, our earlier ELISA analysis revealed a 46.8% increase in NheA toxin
production in response to SIC carvacrol exposure, while RPLA analysis showed a 50%
increase in HblC toxin expression. Therefore, it was suggested that the bacteria were
experiencing sublethal stress when subjected to SIC carvacrol. These findings were
important since they determined that SIC of carvacrol may increase the production of
HblC and NheA enterotoxins. This is clinically relevant because the virulence of B.
cereus may increase if SIC is administered to an endophthalmitis patient, which would
theoretically facilitate dissemination within the host. However, it was also found that a 2
mM or 8 mM concentration of carvacrol inhibited expression of HblC or NheA proteins,
suggesting that beneficial or harmful effects of carvacrol are concentration-dependent.
A Caenorhabditis elegans bioassay model was previously used in our laboratory
in order to validate in vitro findings. This was the first time that effects of carvacrol on
virulence gene expression were measured using the nematode C. elegans as a model
system. As the result of this study, SIC of carvacrol significantly increased mortality (by
72%) of C. elegans 96-120 h after treatment compared to other experimental groups.
These data were consistent with the previous discoveries and supported the hypothesis.
45
B. cereus endophthalmitis is very serious condition that can cause blindness due
to its ability to induce inflammatory responses within the eye that irreversibly damages
the retinal photoreceptor cells. Moreover, high doses of antimicrobial agents given to
treat infection contribute to the seriousness of this condition [126, 127]. Therefore, it is
necessary to find a new treatment option that can rapidly kill B. cereus, neutralize its
toxin production, as well as decrease the inflammatory response in the ocular tissues
protecting the retina. Since carvacrol has been earlier shown to kill B. cereus [88], as
well as to exhibit anti-inflammatory properties (acts as a COX-2 inhibitor) [95], it is
strongly believed that it can be beneficial for the treatment of endophthalmitis if used
properly.
Our study was focused on understanding the PlcR regulation of the B. cereus
virulence factors in order to find potential gene targets for antimicrobial drugs. The
transformation of the virulence regulator PlcR into several Bacillus spp. was crucial in
order to reveal the true nature of the PlcR-mediated virulence in B. cereus and B.
thuringiensis, which are genetically almost identical. B. cereus and B. thuringiensis were
not generally considered to be naturally competent for transformation. However, they
possess the most important structural proteins required for natural transformation such
as those found in B. subtilis [78]. Moreover, a prior study by others reported successful
induced transformation in B. cereus, which motivated us to try something similar.
Fortunately, we were able to transform our cloned plcR into B. cereus and B.
thuringiensis subsp. israelensis, which was confirmed by real-time PCR analysis.
However, we were unable to transform the construct into B. subtilis and E. coli, which
was unanticipated due to the fact that pAD123 was designed as an E. coli-B. subtilis
46
shuttle vector for creation of the GFP construct. This could be explained by the
possibility that these two bacteria were not competent enough to result in measureable
transformation efficiency. Nevertheless, B. subtilis and E. coli served as transformation
controls. The more important event was that cloned plcR was transformed into the
favored B. cereus and B. thuringiensis subsp. israelensis strains which allowed us to
make informative conclusions about the plcR expression levels in response to the
stressor carvacrol.
Some would say that choosing flow cytometry technology to measure effects of
carvacrol on GFP-expressing bacteria over fluorescence microscopy or another
approach was atypical. However, we were encouraged to try this novel approach by an
analogous study that measured bacterial responses to antibiotics [128]. Investigators
showed that flow cytometry was sensitive enough to detect changes in bacterial
morphology before and after exposure to antibiotics. Certainly flow cytometry is
sensitive enough to detect small changes in expression levels from the GFP-labeled
plcR subjected to SIC carvacrol and thus represents an ideal means of performing
relative quantification of these expression comparisons. Additionally access and
familiarity to flow cytometry factored into our decision to select this tool as our method of
analysis.
Flow cytometric results revealed that B. cereus and B. thuringiensis subsp.
israelensis displayed significantly greater fluorescence than nonstressed control
samples when subjected to SIC carvacrol. Furthermore, these data were significantly
higher than the background fluorescence obtained from nontransformed experimental
samples (Figure 6; A1, A2, A3, B1, B2, and B3). In other words, plcR expression was
47
increased when bacteria were stressed which corresponded to the previous findings
that virulence of HBL and Nhe increases in response to SIC carvacrol. Interestingly, B.
thuringiensis subsp. israelensis showed twice as much of fluorescence (9.5%) than B.
cereus (4.6%). Perhaps this could be explained by B. thuringiensis subsp. israelensis
harboring more copies of plcR. Therefore, our data suggested that flow cytometry is
highly applicable in measurements of GFP-labeled gene expressions in B. cereus and
B. thuringiensis.
Here we show that PlcR apparently directly regulates expression of hbl and nhe
enterotoxin virulence genes in B. cereus and B. thuringiensis subsp. israelensis.
Therefore, PlcR can be a potential drug target in order to treat B. cereus
endophthalmitis more efficiently. However, there is one more issue that needs to be
considered for the future studies. Other possible future experiments could investigate
whether PlcR expression is actually upregulated by CodY, a nutrient-responsive
regulator of Gram-positive bacteria [48]. As we previously mentioned in the Introduction,
nutrient limitation or stationary phase of bacterial growth activates CodY, which controls
metabolism, sporulation and virulence in low G+C Gram-positive bacteria. It has been
shown that CodY has a major role in regulation of virulence in B. cereus, B.
thuringiensis and B. anthracis. It was suggested by others that CodY indirectly regulates
anthrax toxin production through the post-transcriptional accumulation of AtxA [49].
CodY was also found to be the positive regulator of B. cereus HBL and Nhe
enterotoxins, while it negatively regulates cerulide synthetase gene expression [56].
Furthermore, proteomic studies of different phases of bacterial growth identified CodY
48
as one of the proteins that may be involved in the activation of B. thuringiensis virulence
crystal proteins and spore formation as well [64].
Therefore, CodY has influence on both, PlcR-PapR quorum sensing (enterotoxin
production) and Spo0A-AbrB regulatory circuit system (cerulide production) in B.
cereus, which have been believed to work independently of each other (Fig. 1) [56]. In
addition, deletion of codY causes significant decreases in B. cereus virulence.
Moreover, the activity of Nhe was practically abolished in codY mutant supernatants
and untraceable in biotoxicity assay. Collectively, these results suggest that CodY might
indirectly stimulate PlcR quorum sensing-dependent genes in B. cereus. However, the
overlapping nature of the CodY and PlcR regulons in their activation of enterotoxins as
well as the impact of CodY on the PlcR-PapR complex are yet to be fully explained. Our
findings further contribute to the clarification of the pathogenesis of B. cereus induced
endophthalmitis infections. Finally, PapR, the signal peptide that activates PlcR and that
is by some means controlled by CodY, can be also drug targeted and investigated as a
potential form of endophthalmitis treatment.
49
Acknowledgements
This research project would not have been possible without the support of many
people. I would like to express my deepest gratitude to my thesis advisor Dr. John L.
McKillip who was tremendously helpful and offered invaluable research assistance,
support and guidance. Without Dr. Mckillip’s patience, motivation and enthusiasm, I
would have never successfully accomplished my educational goal. I would also like to
thank Dr. Heather Bruns for her assistance with flow cytometry analysis, Dr. Susan
McDowell and Dr. James Mitchell for sharing some of the reagents and equipment from
their laboratories, and to Yenling Ho for his invaluable assistance during research.
Furthermore, my deepest gratitude is also due to the committee members, Dr. Derron
Bishop, Dr. Najma Javed and Dr. Jagdish Khubchandani, without whose understanding,
support and flexibility, this study would not have been possible. I would like to express
my sincere love and gratitude to my beloved sister Ana and her husband John Mann
who generously shared their home with my husband, my son and me, as well as
financially supported us through the entire period of my studies. I would like to thank my
dear husband Goran and beloved son Milosh for their understanding, forbear and
endless love throughout this entire demanding process. Finally, I am very grateful for
my parents, Milenko and Olgica Cice without whose love, encouragement and spiritual
support I would not have finished this thesis and graduate program.
50
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