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Southern Illinois University CarbondaleOpenSIUC
Theses Theses and Dissertations
12-2009
Thermoregulation of Capsule Production ofStreptococcus pyogenes Strain HSC5Trilce Michelle GaleasSouthern Illinois University Carbondale, [email protected]
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Recommended CitationGaleas, Trilce Michelle, "Thermoregulation of Capsule Production of Streptococcus pyogenes Strain HSC5" (2009). Theses. Paper 122.
THERMOREGULATION OF CAPSULE PRODUCTION IN Streptococcus
pyogenes STRAIN HSC5
By
Trilce Michelle Galeas Peña
MD, Universidad Autónoma de Santo Domingo, Santo Domingo. Dominican Republic. 2005
A Thesis Submitted in Partial Fulfillment of the Requirements
For the Master of Science Degree
Department of Microbiology
Molecular Biology, Microbiology, and Biochemistry Graduate Program
Southern Illinois University, Carbondale, IL 62901
December, 2009.
THESIS APPROVAL
THERMOREGULATION OF CAPSULE PRODUCTION IN Streptococcus pyogenes STRAIN HSC5
By
Trilce Michelle Galeas Peña
A Thesis Submitted in Partial
Fulfillments of the Requirements
for the Degree of
Master of Science
in the field of Microbiology, Molecular Biology and Biochemistry
Approved by:
Dr. KyuHong Cho, PhD
Dr. Douglas Fix, PhD Chair
Dr. David Clark, PhD
Graduate School
Southern Illinois University, Carbondale.
December, 2009.
AN ABSTRACT OF THE THESIS OF
TRILCE MICHELLE GALEAS PEÑA, for the Master of Science degree in MOLECULAR BIOLOGY, MICROBIOLOGY and BIOCHEMISTRY, presented on JULY 15th , 2009, at Southern Illinois University Carbondale.
TITLE: THERMOREGULATION OF CAPSULE PRODUCTION IN Streptococcus pyogenes STRAIN HSC5.
MAJOR PROFESSOR: Dr. KyuHong Cho
Group A Streptococcus (GAS) is responsible for mild and common
infections like tonsillitis and pharyngitis, and more serious invasive disorders like
necrotizing fasciitis and glomerulonephritis. The ability to invade tissues is
closely linked to the virulence factors expressed by the bacterium. Hyaluronic
acid capsule expression is variable among all the strains in S. pyogenes and
confers the capacity to evade the immune response. In a previous study, it was
found that capsule production in CovR mutants was temperature-regulated,
showing no capsule production at 37 but increased production was observed
at 25. In this study, the objective is to find the elements involved in the
thermoregulation using a genetic approach. First, mutants were created by
knocking-out CovR, the response regulator of the CovRS two-component system
that controls about 15% of GAS genome. Transposon mutants were screened to
find changes in capsular phenotype. Colonies expressing capsule at 37 were
selected for sequencing. The sequencing revealed three different events in
different mutants. Two of them pointed at hypothetical proteins, one of them,
i
SpyM3_1255, was phage associated protein with a DnaD domain and the other
one, SpyM3_1377, encoded cvfA. A third over-producer mutant showed an
insertion in the promoter area of the has operon, the operon that encodes for
hyaluronan synthase production, upstream from other disruptions in the promoter
area that generated non-producing mutants. This suggest that there is more than
one factor involved in thermoregulation of capsule production.
ii
TABLE OF CONTENTS
CHAPTER PAGE
ABSTRACT ........................................................................................................... i
LIST OF TABLES ................................................................................................ vi
LIST OF FIGURES ............................................................................................ viii
INTRODUCTION ................................................................................................. 1
Pathogenesis and Virulence Factors of S. pyogenes ................................ 2
Purpose of this study ................................................................................. 7
Hyaluronic Acid Capsule Structure and Synthesis .................................... 8
The Hyaluronic Acid Synthesis (has) Operon in Streptococcus sp ......... 14
Streptococcus pyogenes gene regulation ............................................... 16
Thermoregulation in bacterial gene expression ...................................... 20
Research Aims ........................................................................................ 23
MATERIALS AND METHODS ............................................................................ 24
Media, Buffers, Solutions and Standard Protocols .................................. 24
Bacterial Strains and Plasmids ............................................................... 24
iii
Plasmid Construction .............................................................................. 24
Creation of CovR mutants ....................................................................... 25
Transposon Mutagenesis ........................................................................ 25
Capsule Quantitation ............................................................................... 26
RESULTS AND DISCUSSION ........................................................................... 29
Wild Types and the generation of CovR Mutants .................................... 29
CovRS complementation ........................................................................ 30
Temperature dependence of capsule expression ................................... 31
Transformation and phenotypification of MGAS315 ................................ 31
Transposon Mutagenesis ........................................................................ 48
Knock-out Experiment ............................................................................. 57
REFERENCES .................................................................................................. 64
APPENDICES
Appendix A - Media, Buffers and Solutions ........................................................ 83
Media ................................................................................................................. 83
Antibiotics ........................................................................................................... 85
iv
Buffers and Solutions .............................................................................. 86
Appendix B - Protocols ...................................................................................... 87
Generation of In-Frame Deletion Strain .................................................. 87
Isopropanol Precipitation ......................................................................... 88
Electroporation of S. pyogenes ............................................................... 90
Electroporation of S. pyogenes with pJRS233 Derivatives ..................... 92
Escherichia coli Transformation .............................................................. 95
Quantitation of S. pyogenes Capsule ...................................................... 96
Storing Cells at -80 Freezer ................................................................ 98
Plasmid Extraction .................................................................................. 99
DNA Purification of PCR Products .......................................................... 99
Preparation of Chromosome of S. pyogenes with Sigma Column .......... 99
Preparation of E. coli Competent Cells ................................................. 100
VITA .................................................................................................................. 102
v
LIST OF TABLES
TABLE PAGE
Table 1: Bacterial Strains ................................................................................... 27
Table 2: Plasmids ............................................................................................... 28
Table 3: Capsule quantitation of wild types and CovR mutants at 37 ............ 33
Table 4: Capsule quantitation of wild types and CovR mutants at 30 ............ 34
Table 5: Capsule quantitation of wild types and CovR mutants at 25 ............ 35
Table 6: Capsule quantitation of HSC5 and CovR mutants at 37 .................. 38
Table 7: Capsule quantitation of HSC5 and CovR mutants at 30 .................. 39
Table 8: Capsule quantitation of HSC5 and CovR mutants at 25 .................. 40
Table 9: Results of capsule quantitation of complementation strain and
controls at 37 .................................................................................... 43
Table 10: Results of capsule quantitation of complementation strain and
controls at 30 .................................................................................. 44
Table 11: Results of capsule quantitation of complementation strain and
controls at 25 ................................................................................. 45
Table 12: Capsule quantitation of transposon mutants at 37 ......................... 54
vi
Table 13: Capsule quantitation of transposon mutants at 25 ......................... 55
Table 14: PCR primers used for hasA promoter area amplification ................... 59
Table 15: Primers used for hasA promoter area knock-out confirmation ........... 59
vii
LIST OF FIGURES
FIGURE PAGE
Figure 1: Draw of Streptococcus .......................................................................... 3
Figure 2: Structure of hyaluronic acid ................................................................ 11
Figure 3: Enzyme functions needed for hyaluronic acid biosynthesis ............... 13
Figure 4: Graphic representation of has operon ................................................ 15
Figure 5: Capsule phenotype ............................................................................. 32
Figure 6: Capsule quantitation of wild types and CovR mutants at 37 ......... 36
Figure 7: Capsule quantitation of wild types and CovR mutants at 30 .......... 36
Figure 8: Capsule quantitation of wild types and CovR mutants at 37 ......... 37
Figure 9: Effect of temperature in wild type and CovR mutants ........................ 37
Figure 10: Capsule quantitation of HSC5 and CovR mutants at 37 ............... 41
Figure 11: Capsule quantitation of HSC5 and CovR mutants at 30 ............... 41
Figure 12: Capsule quantitation of HSC5 and CovR mutants at 25 .............. 42
Figure 13: Effect of temperature on HSC5 and CovR mutants .......................... 42
Figure 14: Capsule quantitation for CovRS complementation strain and
controls at 37 ................................................................................ 46
viii
Figure 15: Capsule quantitation for CovRS complementation strain and
controls at 30 ................................................................................ 46
Figure 16: Capsule quantitation for CovRS complementation strain and
controls at 37 ................................................................................ 47
Figure 17: Effect of temperature in complementation strain and controls .......... 47
Figure 18: Complete sequence of spyM3_1254 ................................................ 51
Figure 19: Partial sequence of spyM3_1376 ..................................................... 52
Figure 20: The hasA promoter region ................................................................ 53
Figure 21: Capsule production of transposon mutants at 37 ......................... 56
Figure 22: Capsule production of transposon mutants at 25 ......................... 56
Figure 23: Gel electrophoresis of nucleic acids for knockout experiments ........ 60
Figure 24: Primer annealing for knock-out confirmation .................................... 61
Figure 25: Graphic representation of transposon insertion ................................ 62
Figure 26: Graphic representation of insertional inactivation ............................. 63
ix
INTRODUCTION
Streptococcus pyogenes is a Gram positive human pathogen that grows in
chains (Figure 1) and is the causative agent of relatively mild diseases such as
tonsillitis, pharyngitis, pyoderma and impetigo or serious invasive infections like
necrotizing fasciitis and streptococcal toxic shock syndrome. In some cases, after
infection with Group A Streptococcus (GAS) post-streptococcal systemic
diseases like rheumatic fever, glomerulonephritis, endocarditis (91) and
Sydenhamʼs chorea (56) could develop. S. pyogenes can be found naturally in
the human skin and throat. It is an aerotolerant anaerobe called β-hemolytic
because of the complete hemolysis pattern formed in blood agar (β-hemolysis)
(67).
In the United States, each year there are over 10,000 cases of invasive
streptococcus infections such as necrotizing fasciitis, bacteremia and toxic shock
syndrome, resulting in 1,000 – 1,800 deaths (76).
Global epidemiology is unknown due to poor report and surveillance
systems in most developing countries. However, it is estimated that there are at
least 517,000 deaths each year linked to severe streptococcal infections and
post-streptococcal diseases. The prevalence of GAS is at least 18.1 million cases
with 1.78 million new cases each year (13) and there are at least 663,000 new
cases with 163,000 deaths each year from invasive GAS infections. That added
to the fact that there are more than 111 million cases of pyoderma and 616
1
million cases of pharyngitis per year, it is easy to conclude that there is a high
prevalence of streptococcal diseases in the clinical practice.
Rebecca Lancefield, in 1928, published a method to serotype
streptococcus based on its M protein, resulting in over 100 serotypes (59). Later,
in 1946, she published another serotyping method based on the T protein, 20
serotypes are known in this system (60), and 4 of the antigens related to T
protein are pili, a structure relevant to attachment to host cells (75).
Pathogenesis and virulence factors of S. pyogenes
There are several virulence factors in S. pyogenes that enable the
bacterium to attach to host cells, invade deep tissues favoring spreading and
avoid immune responses. Among those is the hyaluronic acid capsule which is
the subject of this research and will be discussed in detail later.
M protein, which is a major factor in phagocytosis evasion uses its ability to bind
two different components of the immune response (C4BP and IgA) inhibiting
opsonization and phagocytosis (14). It is also related to the autoimmune
response in rheumatic fever and rheumatic heart disease derived from post-
streptococcal infection by eliciting a molecular mimicry mechanism and activating
opsonic antibodies and lymphocytes (42). Another relevant characteristic of the
well studied M protein is its interaction with peptides derived from the fifth domain
of beta-2 glycoprotein I (beta-2-GPI), a human heparin binding plasma protein,
which has antibacterial activities against Gram-positive and Gram-negative
bacteria. Also, protein H and M1 protein, released from the bacterial cell wall by
2
FIGURE 1: Photomicrograph of Streptococcus bacteria, 900x magnification,
showing its typical spherical shape and chain growth. (CDC Public Health Image
Library). http://phil.cdc.gov/phil/details.asp
3
polymorphonuclear-derived proteases, bind to, and inhibit the activity of,
beta(2)GPI-derived antibacterial peptides. Taken together, it is suggested that the
interaction between the streptococcal proteins and beta(2)GPI or beta(2)GPI-
derived peptides presents a novel mechanism to resist an antibacterial attack by
beta(2)GPI-cleavage products (77).
Another virulence factor is F protein, which facilitates adherence to host
cells (9), and is linked to severe soft tissue infections (80). It also mediates
invasion in HeLa cells and enables the bacteria to be internalized, proving that M
protein is essential for adhesion, but F protein is essential for internalization (82).
F protein shall not be confused with streptococcal pyrogenic exotoxin SpeF,
previously referred as mitogenic factor, which, in Toxic Shock-like Syndrome
caused by Streptococcus, causes permeabilization of lung vessels, leading to
Acute Respiration Distress Syndrome (70). Moreover, F protein has
superantigenic properties. Fibronectin-binding properties of S. pyogenes are also
mediated by protein F (50). While M protein of S. pyogenes mediates the binding
of the bacterium to keratinocytes, protein F directs the adherence of the
organism to Langerhans' cells (81).
Many of the virulence factors of GAS are not present in all clinical
isolates, however, Streptolysin O (SLO), an oxygen-labile (thiol-activated)
cytolysin, is present in most and is a powerful toxin that is partially responsible for
hemolytic pattern and is highly cardiotoxic (49, 46). When absent, S. pyogenes
shows a significant decrease in lethality and ability to cause disease (63). A
4
recent study found this pore-forming cytolytic streptolysin O as responsible and
sufficient for apoptosis induction of macrophages and neutrophils involving
caspase-1. This requires internalization of the bacterium by the phagocyte,
contributing to GAS virulence and immune evasion (102). This toxin has the
potential to establish a novel class of suicide gene therapeutic reagents (114).
Membrane damage by SLO is analogous to that mediated by previously studied
channel formers, namely, the C5b-9 complement complex and staphylococcal
alpha-toxin (8). On capsule production, capsular and acapsular M3 strains were
tested and the result was that the cytotoxic effects of streptolysin O protect GAS
from phagocytic killing and enhance bacterial virulence, particularly of strains that
may be relatively deficient in hyaluronic acid capsule (96).
Another key virulence factor is streptolysin S (SLS), that is partially
responsible, along with SLO, for the beta-hemolytic phenotype. It is encoded by
the sagA gene (28). Despite a long time since its discovery, its chemical structure
remains unknown (74). SLS is not immunogenic; thus, no neutralizing antibodies
are evoked during the course of natural infection, but recently, a synthetic peptide
containing aminoacids 10 to 30 from the sagA gene coupled with keyhole limpet
hemocyanin elicited an antibody response in a rabbit and reversed the hemolytic
phenotype, giving cues to develop a new vaccine (28).
GAS also secretes streptokinase which binds and activates plasminogen
(7), the enzyme that destroys fibrin clots. Interactions between bacteria,
fibrinogen, streptokinase and plasminogen resulted in acquisition of cell-
associated enzymatic activity that can lyse fibrin clots despite the presence of the
5
major physiological plasmin inhibitor, alpha 2-antiplasmin (107), and this
interaction promotes bacterial invasion of tissues (101). It is strongly believed
that streptokinase plays a mayor role in acute post-streptococcal
glomerulonephritis by initiating the nephritis process by glomerular deposition,
which leads to local activation of the complement cascade. Detection of
streptokinase in kidney tissue increased with the degree of glomerular
hypercellularity. Thus, the severity of the pathological process may be a reflection
of the degree of streptokinase deposition (78, 79).
Virtually all strains of the human bacterial pathogen Streptococcus
pyogenes express a highly conserved extracellular cysteine protease known as
streptococcal pyrogenic exotoxin B (SpeB) (65) that is used by GAS to kill
phagocytes, as demonstrated by Lukomski et al, where a mutant lacking
expression of SpeB was unable to resist phagocytosis and subsequent
dissemination (66). SpeB also affects directly the production of hyaluronic acid
capsule, when inactivated, extracellular hyaluronic acid levels decreased (112). It
also degrades IgG, IgM, IgA, IgD and IgE (23). The mechanism to inactivate IgG
is by cleaving IgG into Fc and Fab fragments, therefore, increasing bacterial
survival (24, 37). Another proposed mechanism of phagocytic evasion by SpeB is
by mitochondrial damage to the polymorphonuclear cells at an early infectious
stage (18). As reviewed so far, SpeB cleaves or degrades host serum proteins
such as human extracellular matrix, immunoglobulins, complement components,
and even GAS surface and secreted proteins. Destruction of both host and
6
bacterial proteins makes SpeB the key virulence factor in GAS pathogenesis
(19).
To invade host tissues, S. pyogenes uses hyaluronidase as one of the
many virulence factors involved in invasion and dissemination. This chromosomal
and phage-associated enzyme degrades hyaluronic acid from host tissues and
increase adherence to epithelial host cells (6). A recent study found that
hyaluronidase digests tissue hyaluronic acid and facilitates spread of large
molecules but is not sufficient to cause subcutaneous diffusion of bacteria or to
affect lesion size (98). Along with hyaluronidase, GAS also produces hyaluronic
acid for its capsule, and another interesting finding was that hyaluronidase is not
sufficiently active to remove capsule during peak synthesis (98).
Purpose of this Study
Human pathogens are exposed to changes in the environmental
conditions, such as temperature, osmolarity and pH. In order to survive, bacteria
have to regulate their gene expression according to environmental factors and,
thus, ensure a successful colonization that allows them to thrive in the highly
hostile human body.
Temperature is one of the most relevant environmental factors. Several
thermoregulation studies have been done in other bacteria, demonstrating how
differential gene expression may be temperature-dependent, especially in
pathogenesis, where body temperature is a significant cue for the bacterium to
start the virulence machinery.
7
In the specific case of Group A Streptococcus, where colonization of both
skin surface and blood is possible, there must be a thermoregulation in the
capsule production that allows the bacteria to switch from one place to another
while evading phagocytosis and internalization.
Also, the products of temperature-regulated genes with central roles in
physiology and virulence could be targets for novel therapeutics or mutation to
generate defined live attenuated vaccines.
Hyaluronic acid capsule structure and synthesis
The most relevant virulence factor for this study is the hyaluronic acid (HA)
capsule produced in the surface of the bacterial cell that plays a key role in
virulence and invasiveness in mucoid strains (109). The capsule is also a
mechanism to avoid oxygen damage by isolating the cell from the environment,
making oxygen diffusion through the capsule much more slow, providing the
bacterium with a shield from oxygen metabolites (22). The virulence of HA
capsule is derived from its capacity to confer phagocytosis resistance to the
bacterium (110) and is not necessary to be heavily expressed to confer full
virulence, as demonstrated by Ashbaugh, where moderately encapsulated
strains and heavily encapsulated strains posed the same invasiveness and
virulence (4). The capsule in Streptococcus pyogenes is also required for biofilm
formation by helping in the process of maturation into a tridimensional structure
(20)
8
The composition of hyaluronic acid is shared by bacteria and mammalians
alike, therefore the immune system is unable to recognize encapsulated bacteria
leading to failed opsonization and phagocytosis (27). Encapsulated strains are
very invasive in soft tissue infections, as oppose to uncapsulated strains that are
internalized by keratinocytes (93) resulting in a noninvasive disease, and
evidencing the fact that in order to spread, GAS needs to avoid phagocytosis and
internalization and this is accomplished by capsule production (30). Introduction
of GAS into the pharynx or deep tissues activates capsule production in
capsulated strains (44), however, the underlying mechanism remains unknown,
although several putative environmental triggers have been studied with little
success. In this regard, a recent publication tried to identify the trigger, after
noticing how mucoid strains on primary culture lost their encapsulated phenotype
after laboratory passage, suggesting that the trigger must be present in the host
environment and absent in most laboratory culture media. They found that
cathelicidin LL-37, a human antimicrobial, had the ability to stimulate CovRS-
regulated virulence factors (45).
HA is synthesized at the protoplast and its structure is a polymer of
disaccharides, themselves composed of D-glucuronic acid and D-N-
acetylglucosamine, linked via alternating β-1,4 and β-1,3 glycosidic bonds.
Hyaluronic acid can be 25,000 disaccharide repeats in length. Polymers of
hyaluronan can range in size from 5,000 to 20,000,000 Da in vivo (99, 73, 100)
(Figure 2). The average molecular weight of extracellular HA is 2 X 106, and its
production is only effective until the cell reach stationary phase, where production
9
is halted, possibly because of changes in membrane conformation (106). HA
release after formation is mediated by an ABC transport system, coded adjacent
to the HA synthase gene with a reading frame in opposite direction (84).
Synthesis of hyaluronic acid requires much energy and takes a high
percentage of sugar, therefore, the obvious advantages of its synthesis is not
widespread among pathogenic bacteria. The enzyme responsible for hyaluronic
acid synthesis is Hyaluronic Acid Synthase. The gene encoding this enzyme was
first discovered in 1993 from GAS (31) and after that, it has been described in
different species in bacteria and mammals as well. These various hyaluronic acid
synthases share many common features and aminoacid sequence regions
identity or similarity (108). In Streptococcus pyogenes, the HA synthase is known
as HAs Class I.
The first work identifying the HA synthase was in 1950 by Dorfman. In his
paper, he described the enzyme as membrane-bound that needed Mg++ and
used two sugar-nucleotide substrates, uridine diphospho-glucuronic acid (UDP-
GlcA) and uridine diphospho-H-acetylglucosamine (UDP-GlcNAc) to polymerize
a hyaluronic acid chain (69) adding one after another at the reducing end (103).
Different from proteoglycans, hyaluronic acid chain initiation does not need a
protein backbone, the sole presence of the nucleotide sugar precursors are
enough to elicit initiation and elongation (84).
10
FIGURE 2: Structure of hyaluronic acid. Hyaluronic acid consists of repeating
disaccharides composed of N-acetylglucosamine (GlcNAc) and glucuronic acid
(GlcA).
11
HA synthase is very unique; it has two different functions as
glycosyltransferase. The product of the first sugar addition is the substrate for the
next addition (108). The overall reaction for the synthesis of one hyaluronan
disaccharide unit is:
Although this reaction seems simple, it is believed that at least six different
reactions take place to complete the cycle. Before it is released, the chain can
grow to more than 40,000 monosaccharides, corresponding to a mass of more
than 8 million Da. The sugar-nucleotide substrates are produced and used by the
synthase inside the cell, and the hyaluronan chain is continuously transferred
(translocated) so that it is extruded into the exterior of the cell to form the capsule
(Figure 3). It was also believed that in absence of precursors, capsule would not
be able to be synthesized and that hyaluronidase may play a role in solving this
problem. To demonstrate a possible nutritional role for hyaluronidase, GAS was
shown to grow with N-acetylglucosamine but not d-glucuronic acid (both
components of HA) as a sole carbon source. However, only hyaluronidase (+)
strains could grow utilizing HA as a sole carbon source, suggesting that
hyaluronidase may permit the organism to utilize host HA or its own capsule as
an energy source (98).
As bacterial hyaluronic acid is very similar to human and other vertebrates
molecule, different strains of Streptococcus is being use for production and later
use for purpose of human health and cosmetic industry (113). Also recombinant
12
FIGURE 3: Enzyme functions needed for hyaluronic acid biosynthesis. The
diagram shows the membrane-bound hyaluronan synthase and the six
independent activities required for the enzyme to make a disaccharide unit and
extend the growing hyaluronic acid chain. (Image used with permission of
Glycoforum. www.glycoforum.gr.jp).
13
HA from E. coli (115), B. subtillis (111) and Agrobacterium sp. (68) is used
for commercial purposes.
The hyaluronic acid synthesis (has) operon in Streptococcus species.
Hyaluronic acid production is coded in an operon containing three genes:
hasA, hasB, and hasC, which encode hyaluronan synthase, UDP-Glc
dehydrogenase and UDP-Glc pyrophosphorylase, respectively (Figure 4). hasA
codes for the enzyme responsible for the overall hyaluronic acid synthesis
reaction (35), hasB is necessary to make UDP-GlcA, one of the substrates
needed for HA production, from UDP-Glc in an oxidation that requires 2 NAD+
(34), and hasC codes for the enzyme that creates UDP-Glc from UTP and Glc-1-
phosphate (25). Deletion analysis of the operon by Ashbaugh demonstrated that
hasC gene, although it is part of the transcript, is not necessary for hyaluronic
acid production, therefore hasAB are the only genes necessary for capsule
production (5) and probably the hasC function is shared by other metabolic
pathways in the cell. This operon is quite unique for Streptococcal species and
recent phylogenetic studies show that there is no evidence of detectable inter-
species lateral gene transfer suggesting intra-species genetic rearrangement as
the origin of has operon (34). It also shows that the hasA is highly conserved
among streptococcal strains, different from the M protein that is highly variable
(32). Even when all streptococcal strains posses the operon, not every strain
have the capsular phenotype. In acapsular phenotype, no operon mRNA
14
HAS OPERON: 3606 base pairs (bp)
hasA (1259 bp hasB (1208 bp) hasC (915 bp) spyM3_1854 recF
FIGURE 4: General representation of has operon and its genomic context. The
P represents the promoter area and the T is the terminator.
15
P T
transcript is expressed (26). Further characterization of the operon showed a
promoter consensus sequence upstream the hasA gene, and no terminator
sequence was found on hasA, indicating that the has operon promoter could
regulate transcription of the entire operon, thus producing a polycistronic mRNA
(26), therefore, mutations in the has operon promoter or in the hasA gene lead to
a polar effect.
The level of capsule production sometimes differs among individual strains
of the same M-type. It is likely that strain-specific factors also affect capsule
production along with the intrinsic strength of the has promoter conferred by the
serotype-specific has promoter sequence (1).
Streptococcus pyogenes gene regulation
Gene regulation in Streptococcus pyogenes is mediated by several
regulators that either activate or repress certain genes in response to growth
phase or environmental triggers this is achieved by transcriptional regulator
proteins, like Mga (12), a regulator of gene expression, which is responsible for
the activation of expression during exponential growth of genes involved in
adhesion, internalization and immune evasion and influences the expression of
over 10% of all GAS genome, primarily genes and operons involved in
metabolism and sugar utilization (53). Mga functions as a DNA-binding protein
that interacts with sites both proximal and distal to the start of transcription for the
16
genes that it regulates (2). Ribardo et al (89) described an open reading frame
named amrA that is required for maximal activation of the Mga regulon during
exponential phase and is strongly conserved across different streptococcal M
types. Inactivation of amrA in an M3 serotype also resulted in reduction of the
gene coding M protein, emm, transcripts, therefore, the role of amrA does not
appear to be serotype specific. Although the specific function of AmrA is
unknown, its putative membrane localization and homology to transporters
involved in cell wall synthesis suggest a link between growth and Mga (89).
Many virulence regulators in other bacteria respond to carbon catabolite
repression, a potential catabolite-responsive element important for binding by the
catabolite control protein A (CcpA) was found in the promoter area of Mga
upstream of its distal P1 start of transcription, supporting the role for CcpA in the
early activation of the Mga promoter and establishing a link between carbon
catabolite repression and Mga regulation in GAS (3). A link in a regulatory
pathway has been found between CovRS and Mga, this is mediated by a novel
two-component system regulator, which is CovR repressed, named TrxR that
regulates the expression of Mga regulon genes, therefore, connecting CovR
expression with Mga, affecting pathogenesis in GAS (61).
Gene regulation in S. pyogenes is also achieved by two-component
systems like CovRS, the global response regulator that mediates expression of
about 15% of all GAS genome (43).
Two-component regulatory systems constitute an extensive superfamily of
pairs of functionally linked proteins that modulate the activity of biochemical
17
pathways and regulate the expression of virulence factors in many bacteria. The
sensor component typically consists of a membrane-bound protein that responds
to an environmental signal by phosphorylating a conserved histidine residue in a
cytoplasmic transmitter domain of the sensor protein. The phosphate group is
subsequently transferred from the sensor protein to a conserved aspartic acid
residue in the receiver domain of the response regulator, a cytoplasmic protein.
Regulator proteins of this type influence expression of target genes by acting as
transcriptional activators or repressors, and their activity at binding sites
upstream of target genes depends upon the phosphorylation state of the protein.
Phosphorylation alters the DNA binding properties of the response protein
regarding either affinity or target specificity.
Levin and Wessels described the effect of the system on hyaluronic acid
capsule production and named the system csrRS for “capsule synthesis
regulator” and found that csrR was the negative response regulator and csrS was
the sensor component and are very conserved among Streptococcus (62).
Federle found five different sites of CovR binding to the has operon promoter
area and concluded that, except for the almost overlapping binding site pair CB-1
and -2, binding of CovR to each of the five sites surrounding the hasA promoter
is independent of binding at the other sites. However, binding at all of these sites
is needed for complete repression of this promoter in vivo (38).
CovRS protein products are members of the OmpR/EnvZ two-component
signal transduction family (29). It is widely accepted that CovS mediates CovR
phosphorylation, however, further studies gave evidence that in some
18
exceptional cases CovS may not be needed for CovR phosphorylation and
consequent activation, even when these two proteins are cotranscribed, there
could be another element in the cell able to phosphorylate CovR in absence of
CovS (29). Phosphorylation of CovR by acetyl phosphate results in dimerization
(48) significantly enhancing repression of one group of genes (e.g. speA, hasA,
ska) while reducing repression of a second group (e.g. speB, grab, spd3) (105).
CovS is required for growth under stress conditions like high temperature,
high salt concentration and low pH (29). CovRS has been described as a
double-negative regulator, where CovR represses transcription of the covRS
operon by promoter occlusion (47), so as long as CovR is active, very little new
CovR protein is made.
The consensus region of CovR binding at the promoter area of both covR
and has operons is ATTARA, where R could be any purine. In this sequence the
T residues key in CovR binding (40). Another consequence of stress-induced
inactivation of CovR is derepression of CovR synthesis. It can be expected that
under stress conditions, more inactive CovR accumulates so that when stress is
relieved and CovR becomes active, there is a rapid repression response. The
design of this system thus makes it very sensitive to environmental change, like
temperature. Since CovR-regulated promoters may vary in their degrees of
sensitivity to CovR phosphorylation, CovR-regulated genes may exhibit
differential expression under both normal and stress conditions and may respond
to various degrees when the GAS encounters stress conditions (29).
19
CovR is also regulated by RocA, named for “regulator of Cov”, which plays
an important role in regulation of the operon by increasing its expression and
activation of covR transcription is increased about threefold. As expected, a rocA
mutant is mucoid and produces more transcript from the has promoter since this
promoter is repressed by CovR. This regulator is present in GAS but not in other
species like Group B Streptococcus or Group G Streptococcus (10).
There are other regulators than the previously described CovRS and
Mga. For instance, the RofA-like proteins (RALP) including RofA and Nra (39),
regulate genes involved in persistence; FasA, which is involved in growth phase
regulation of virulence gene expression (58) and Rgg (RopB), which activates
expression of extracellular proteins such as SpeB (16). Rgg has also been
shown to play a role in regulating the metabolic activities of GAS (17).
Thermoregulation in bacterial gene expression
Temperature is a relevant stress factor sensed by bacteria and used to
regulate gene expression. Depending on the induction mechanism, two different
pathways have to be distinguished, namely the heat shock response and the
high temperature response. While the heat shock response is induced by
temperature increments and is transient, the high temperature response needs a
specific temperature to become induced and proceeds as long as cells are
exposed to that temperature. The heat shock response is induced by denatured
proteins and aimed to prevent definitive denaturation and formation of protein
aggregates by refolding, and the high temperature response is mainly used by
20
pathogenic bacteria to detect entry into a mammalian host followed by induction
of their virulence genes (94). Thus, thermoregulation in gene expression has
been described in several bacterial species. Several pathogenic bacteria
undergo temperature shift when going from one host to another or from one
environment to another, e.g. skin to deep tissues. Genes are up or down-
regulated depending on different temperatures; this difference, in some cases,
has been linked to promoter sequence, as for maRAB and acrAB in Salmonella
thyphimurium (51). Another report on Salmonella found a strong thermosensing
for virulence phenotype where bacteria grown at 30 or lower were unable to
activate the intracellular type III secretion system even under strong inducing
signals such as synthetic medium, contact with macrophages, and exposure to
the murine gut (36).
Three different macromolecules have been identified being able to sense
temperature: DNA, proteins and mRNA. For example, DNA supercoiling can
control gene expression of pVY virulence plasmid in Yersinia enterocolítica (90)
as well as virB expression in Shigella (87). Protein regulation that are
temperature-dependent are found in the expression of flagella gene in Listeria
species (95) and the two-component signal transduction BvgAS in Bordetella
pertussis (55). RNA regulation is conferred by riboswitches and an example is
the LcrF production in Yersinia pestis (52) that induces expression of several
virulence factors. An example of how temperature regulates the expression of a
two-component signal transduction is BvgSR in Bordetella pertussis, the
causative agent of whooping cough (57). This system is transcribed and up-
21
regulated at 37°C and down-regulated at 25°C (88) and the mechanism is very
similar to CovR activation by phophorylation (55). Microarray studies with
Francisella tularensis showed that after shifting temperature from 26°C to 37°C
about 11% of the bacteriumʼs genes were differentially regulated and 40% of
those genes are linked to pathogenesis (54). In group B streptococcus (GBS)
changes in the genome occurred after incubation in blood at a temperature
similar to those in patients with infection (about 40°C) (72), proving adaptative
changes mediated by temperature in response to invasive infection. In a recent
study in E. coli the effect of temperature on colicin K production was investigated
and the results showed that expression was higher at 37°C than at 22°C and is
hypothesized that SOS response indirectly regulates thermoregulation of colicin
K (11). Another example of bacteria that uses temperature as a regulatory cue for
pathogenicity is Borrelia burgdorferi, the causative agent of Lyme disease. It
regulates expression of RevA in response to temperature, with the protein being
synthesized by bacteria cultivated at 34 but not by those grown at 23 (15).
In some cases, a change in DNA topology, instead of promoter sequence, can
lead to thermoregulation, as reported for virB transcription in Shigella flexneri,
where a reduction in negative superhelicity at the promoter area was identified
(104).
As for Streptococcus pyogenes, previous studies have related some gene
expression and temperature. At least 9% of the genes are expressed
differentially at 29°C relative to 37°C, as shown in microarray analysis. These
genes include transporter proteins, proteins involved in iron homeostasis,
22
transcriptional regulators, phage-associated proteins, and proteins with no known
homologue. Relatively few known virulence genes were differentially expressed
at this threshold (97). However, at 40°C more gene expression was shifted,
showing that there is thermoregulation in gene expression in GAS.
Research Aims
The aim of this study was to identify the gene(s) or protein(s) responsible
for thermoregulation and which areas of the has operon promoter are involved.
For this purpose transposon mutagenesis was performed.
23
MATERIALS AND METHODS
Media, Solutions and Standard Protocols
All the recipes for media, buffers and solutions used in this research are
provided in Appendix A. The protocols are described in Appendix B.
Bacterial Strains and Plasmids
The bacterial strains used in this research and their relevant
characteristics are described in Table 1. Plasmid vectors are listed in Table 2.
Molecular cloning experiments were performed with Top10 E. coli cells (64) which
were cultured in Luria-Bertani broth at 37°C with agitation at 250 rpm. For routine
culture of S. pyogenes, Todd-Hewitt medium (BBL) supplemented with 0.2%
yeast extract (Difco) (THY medium) was employed. For growth in liquid media, S.
pyogenes was cultured at 37°C in sealed tubes without agitation. To produce
solid media, Bacto agar (Difco) was added to a final concentration of 1.4% (wt/
vol). When appropriate, antibiotics were added to the media (see Appendix A).
Plasmid construction
The plasmids used in this work are listed in Table 2. Gene manipulation
and cloning were carried out according to standard protocols (Sambrook et al,
1989). Plasmid constructs were verified by PCR and gel electrophoresis.
24
Creation of CovR mutants
CovR is the response regulator in the CovRS two-component system that
regulates capsule expression. CovR mutants were created by two methods. One
was an insertional disruption on the covR gene that was achieved by using
pSpc18 vector that confers spectinomycin resistance. The plasmid is a 2.94 kb
suicide vector with a multicloning site and an origin of replication for E. coli. A
fragment of the covR gene was inserted to create the disruption in the target loci
of the chromosome. Also, an in-frame deletion mutant was created using the
temperature induced plasmid pJRS233; no resistance is conferred after plasmid
excision. Both transformations were performed as described in Appendix B.
Transposon mutagenesis
Electroporation was performed to introduce the transposon into competent
S. pyogenes cells prepared as described in Appendix B. Mutants were plated in
THY plates with the appropriate antibiotic and incubated at 37°C as described in
standard methods (21). Transformants with insertions of pMGC8, a plasmid
carrying a transposon, were then recovered following overnight incubation at
37°C on THY medium plates supplemented with kanamycin. Insertion was
confirmed with PCR. For mutants displaying the desired phenotypes
(overproducers or non-producers) the transposon insertion site was identified by
chromosomal DNA sequencing. The strain used for transposon mutagenesis was
CovRIFD which does not produce capsule at 37°C but produces it at 25°C.
25
Capsule quantitation assay
As different strains expressed capsule differently, a method to measure
the amount of capsule was developed and optimized using Stains-all reagent; the
complete protocol can be found in Appendix B. Stains-all is known to bind to acid
polysaccharides. As hyaluronic acid is a highly acidic polysaccharide, Stains-all
was used to quantitate the capsule amount produced by cells. The
spectrophotometric readings were done using a 640 nm wavelength. The first
method included a resuspension of THY medium pellet along with the colony to
be measured. This method was discontinued because the media caused
interference with the spectrophotometer readings. The new method included only
the colony in the resuspension. Another problem was the continuous growth of
cells when incubating at room temperature for 24 hrs after the initial 37
incubation. To solve this problem, readings were done the same day using
different sets of cells for each temperature to be measured.
A standard curve was created by measuring the absorbance of hyaluronic
acid concentrations from 1 to 5 µL at 640 nm wavelength. The values obtained
were plotted. The X axis represents optical density and Y axis the hyaluronic acid
concentration. This generated an Y/X slope that was used to multiply the OD of
the samples, the resulting number was then multiplied considering the dilution
factor, which in this case was 2. The subsequent value was divided between the
total cell number of the colonies and the result was converted from µg/cfu to fg/
cfu. All samples were duplicated and the values were averaged.
26
TABLE 1: Bacterial Strains
Bacterial strains Relevant characteristics SourceE. coli Top10 hsdR, mcrA, lacZΔM15, endA1,
recA1(64)
S. pyogenes strains: HSC5
OPTM18
OPTM 2
OPTM7
ΩCovR
CovRIFD
MGAS315
MGAS315ΩCovR
Wild type, M14 serotype
Transposon generated mutant with a disruption in has operon promoter area created with pMGC8. Overproducer phenotype
Transposon mutant with a disruption in a hypothetical protein created by pMGC8. Over-producer phenotype
Transposon mutant with a disruption in a hypothetical protein created by pMGC8. Overproducer phenotype.
HSC5 with a mutation in the covR gene created with pSPC18
HSC5 with an in-frame deletion in the covR gene created with pJRS233
Wild type
MGAS315 with an in-frame deletion in the covR gene created with pSPC18
(50)
This study
This study
This study
(84)
This study
(65, 85)
This study
27
TABLE 2: Plasmids
Plasmid Relevant characteristics SourcepMGC8
pJRS233
pSPC18
pCIV2
pLZ12
Transposon containing km gene that confers kanamycin resistance.
Temperature-sensitive shuttle vector. Confers erythromycin resistance.
pUC18-based suicide vector containing aad9 (spectinomycin resistance gene from Enterococcus faecalis).
pUC18-based streptococcal integration vector; contains aphA3 conferringkanamycin resistance
Self replicating plasmid. Confers chloramphenicol and kanamycin resistance.
(41)
(86)
(20)
(21)
(29)
28
RESULTS AND DISCUSSION
Wild types and the generation of CovR mutants
One of the wild type strains used in this study was the non-mucoid HSC5,
which shows no capsule production at any temperature. To study the
thermoregulation of capsule production, CovR, the response regulator of the
CovRS two-component system was mutated, conferring a less regulated capsule
expression with an obvious temperature-dependent production. The CovR
mutants presented a clear temperature-dependent phenotype where no capsule
was produced at 37 and there was capsule production after incubation at 25
(Figure 5). Because HSC5 is not fully sequenced, MGAS315 was used for
genomic and phenotypical comparison. It showed the same non-mucoid
phenotype as HSC5.
To generate ΩCovR, the wild type was transformed with pSPC18, a
plasmid carrying spectinomycin resistance that creates a disruption and partial
duplication.
An in-frame deletion CovR mutant was also created using the temperature
regulated plasmid pJRS233 carrying erythromycin resistance. The resulting
phenotype was similar to the ΩCovR mutant. For transposon mutagenesis
experiments, this was the strain used because the in-frame deletion generates
no partial duplicates, as opposed to insertional disruption (Figures 25 and 26).
And because no double antibiotic resistance genes are carried (for each plasmid
insertion).
29
HSC5, MGAS315 and CovR mutant strains showed a low capsule reading
at 37, around 40 - 130 fg/μL. The difference in capsule production between
37 and 25 was not significant, showing always low values. However,
MGAS315 wild type usually had a higher values in capsule quantitation,
compared to HSC5, but macroscopically, it showed no characteristic coat of
hyaluronic acid, but only slightly larger colonies. For CovR mutants, the
difference was significant (Tables 3-5) showing an increased production of
hyaluronic acid capsule at 25 (Figures 6-9). Only in one measurement, HSC5
showed capsule production (Tables 6-8, Figures 10-13) but further attempts to
obtain the same phenotype failed, suggesting that the capsule production
observed in HSC5 was a consequence of a technical or human error.
Measurements at 30 were performed to determine if there was an
intermediate phenotype at an intermediate temperature. The results for this
reading showed that CovR mutants start their capsule production slowly after
removal from the 37 environment.
CovRS complementation
The phenotype of CovR mutants shows a temperature dependent capsule
expression. To rule out a possible secondary mutation causing the phenotype a
complementation experiment was performed. The plasmid used was pLZ12
containing a fragment of the CovRS two-component system. After
electroporation, revertants were obtained, showing the same phenotype
30
observed in the wild type. Capsule quantitation assay demonstrated a lack of
capsule production in the complementation mutants (Tables 9-11, Figures 14-17).
Temperature dependence of capsule expression
The mutant strains ΩCovR and CovRIFD showed a temperature-
dependent expression of capsule. At 37, the strain showed no hyaluronic acid
production, but at 25 an evident coat of hyaluronic acid capsule was produced
(Figure 5). In order to study the temperature dependence in HSC5, capsule
measurements at 30 were performed. It was observed that the amount of
capsule produced at this intermediate temperature was lower than the amount
produced at 25, but higher than the amount at 37 (Tables 4, 7 and 10.
Figures 7, 11 and 15 ).
Transformation of MGAS315
The strain used in this study, HSC5, is not fully sequenced and in order to
have genomic profiles of the disrupted genes we used the sequence of
MGAS315, a strain with the closest similarity to HSC5. The capsular phenotype
of MGAS315 was the same as HSC5. An ΩCovR mutant using MGAS315 was
created and the phenotype corresponded to that of the ΩCovR mutant with
HSC5 background. Capsule quantitation assays showed only a slight increment
in capsule amount (Tables 3-5), but the thermoregulated pattern of the MGAS315
CovR mutant was maintained (Figures 6-9).
31
a) b)
c)
FIGURE 5: Capsule phenotype. a) no capsule production. b) and c) capsule
production.
32
TABLE 3: Capsule quantitation of wild types and CovR mutants at 37
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/
cfu) Average Standard deviation
HSC5-1 0.017 720,000 140.90
HSC5-2 0.018 730,000 147.14 144.02 4.41
MGAS315-1 0.020 700,000 170.50
MGAS315-2 0.020 780,000 153.01 161.75 12.36
ΩCovR-1 0.023 396,000 301.38
ΩCovR-2 0.021 394,000 257.48 279.43 31.04
MGAS315ΩCovR-1 0.020 480,000 285.94
MGAS315ΩCovR-2 0.017 500,000 250.63 268.28 24.96
33
TABLE 4: Capsule quantitation of wild types and CovR mutants at 30
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu) Average Standard
deviation
HSC5-1 0.020 440,000 264.51
HSC5-2 0.020 430,000 270.66 267.58 4.34
MGAS315-1 0.030 480,000 363.70
MGAS315-2 0.030 510,000 342.31 353.00 15.12
ΩCovR-1 0.100 244,000 2384.92
ΩCovR-2 0.090 236,000 2219.19 2302.05 117.18
MGAS315ΩCovR-1 0.120 370,000 1887.31
MGAS315ΩCovR-2 0.110 330,000 1939.73 1913.52 37.07
34
TABLE 5: Capsule quantitation of wild types and CovR mutants at 25
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/
cfu)
Average Standard deviation
HSC5-1 0.05 3,600,000 80.82
HSC5-2 0.05 3,700,000 78.64 79.73 1.54
MGAS315-1 0.04 3,300,000 70.54
MGAS315-2 0.04 3,200,000 72.74 71.64 1.55
ΩCovR-1 0.15 350,000 2,493.94
ΩCovR-2 0.13 33,000 2,292.41 2,393.18 142.50
MGAS315ΩCovR-1 0.09 180,000 2,909.60
MGAS315ΩCovR-2 0.09 190,000 2,756.46 2,833.03 108.28
35
FIGURE 6: Capsule quantitation of wild types and CovR mutants at 37.
FIGURE 7: Capsule quantitation of wild types and CovR mutants at 30. Note
the different capsule amount in the CovR mutants, showing that in those strains
the capsule expression starts after leaving the 37 incubation.
0
100
200
300
400
HSC5 MGAS315 ΩCovR MGAS315ΩCovR
0
750
1500
2250
3000
HSC5 MGAS315 ΩCovR MGAS315ΩCovR
36
fg/cfu
fg/cfu
FIGURE 8: Capsule quantitation of wild types and CovR mutants at 25.
FIGURE 9: Effect of temperature in wild type and CovR mutants. Note how CovR
mutants present a thermoregulated capsule phenotype, as opposed to wild types
HSC5 and MGAS315
0
750
1500
2250
3000
HSC5 MGAS315 ΩCovR MGAS315ΩCovR
0
750
1500
2250
3000
HSC5 MGAS315 ΩCovR MGAS315ΩCovR
37 30 25
37
fg/cfu
fg/cfu
TABLE 6: Capsule quantitation of HSC5 and CovR mutants at 37
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu) Average Standard
deviation
HSC5-1 0.004 4,000,000 58.09
HSC5-2 0.004 4,500,000 51.63 54.86 4.56
CovRIFD-1 0.004 6,500,000 35.75
CovRIFD-2 0.005 6,600,000 44.00 39.87 5.84
ΩCovR-1 0.005 7,800,000 37.23
ΩCovR-2 0.005 7,900,000 36.768 37.00 0.33
38
TABLE 7: Capsule quantitation of HSC5 and CovR mutants at 30
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu)
Average Standard deviation
HSC5-1 0.05 1,170,000 251.08
HSC5-2 0.04 1,090,000 215.60 233.34 25.08
CovRIFD-1 0.09 1,370,000 385.96
CovRIFD-2 0.07 1,280,000 321.30 353.63 45.72
ΩCovR-1 0.10 2,070,000 283.83
ΩCovR-2 0.09 1,970,000 268.41 276.12 10.90
39
TABLE 8: Capsule quantitation of HSC5 and CovR mutants at 25
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu)
Average Standard deviation
HSC5-1 0.17 1,270,000 786.44
HSC5-2 0.18 1,350,000 783.36 784.90 2.18
CovRIFD-1 0.18 1,590,000 665.12
CovRIFD-2 0.17 1,470,000 679.44 672.28 10.13
ΩCovR-1 0.20 1,670,000 703.62
ΩCovR-2 0.22 1,600,000 807.84 755.73 73.69
40
FIGURE 10: Capsule quantitation of HSC5 and CovR mutants at 37.
FIGURE 11: Capsule quantitation of HSC5 and CovR mutants at 30.
0
15
30
45
60
HSC5 CovRIFD ΩCovR
0
100
200
300
400
HSC5 CovRIFD ΩCovR
41
fg/cfu
fg/cfu
FIGURE 12: Capsule quantitation of HSC5 and CovR mutants at 25.
FIGURE 13: Effect of temperature on HSC5 and CovR mutants. Note that HSC5
wild type showed increased capsule production at 30 and 25, which is not the
usual behavior.
0
225
450
675
900
HSC5 CovRIFD ΩCovR
37 30 25
0
200
400
600
800
HSC5 CovRIFD ΩCovR
42
fg/cfu
fg/cfu
TABLE 9: Results of capsule quantitation of complementation strain and controls
at 37.
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu)
Average Standard deviation
HSC5-1 0.003 422,000 45.97
HSC5-2 0.003 427,000 45.43 45.70 0.38
ΩCovR-1 0.006 820,000 47.31
ΩCovR-2 0.007 1,030,000 43.94 45.63 2.38
ΩCovR(pCovRS)-1 0.005 820,000 39.43
ΩCovR(pCovRS)-2 0.006 860,000 45.11 42.27 4.01
43
TABLE 10: Capsule quantitation for complementation strain and controls at 30
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu)
Average Standard deviation
HSC5-1 0.006 730,000 47.05
HSC5-2 0.006 760,000 45.19 46.12 1.31
ΩCovR-1 0.177 1,790,000 566.06
ΩCovR-2 0.171 1,680,000 582.68 574.37 11.75
ΩCovR(pCovRS)-1 0.009 1,040,000 49.54
ΩCovR(pCovRS)-2 0.009 1,030,000 50.02 49.78 0.34
44
TABLE 11: Capsule quantitation for complementation strain and controls at 25
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu) Average Standard
deviation
HSC5-1 0.008 1,030,000 44.46
HSC5-2 0.007 1,060,000 37.80 41.13 4.71
ΩCovR-1 0.240 2,070,000 663.72
ΩCovR-2 0.243 1,980,000 693.89 678.81 21.33
ΩCovR(pCovRS)-1 0.010 1,240,000 46.17
ΩCovR(pCovRS)-2 0.009 1,360,000 37.88 42.02 5.86
45
FIGURE 14: Capsule quantitation for CovRS complementation strain and
controls at 37.
FIGURE 15: Capsule quantitation for CovRS complementation strain and
controls at 30.
0
11.5
23.0
34.5
46.0
HSC5 ΩCovR(pCovRS) ΩCovR
0
150
300
450
600
HSC5 ΩCovR(pCovRS) ΩCovR
46
fg/cfu
fg/cfu
FIGURE 16: Capsule quantitation for CovRS complementation strain and
controls at 25.
FIGURE 17: Effect of temperature in complementation strain and controls.
0
200
400
600
800
HSC5 ΩCovR(pCovRS) ΩCovR
0
175
350
525
700
HSC5 ΩCovR(pCovRS) ΩCovR
37 30 25
47
fg/cfu
fg/cfu
Transposon mutagenesis
The efficiency is expected to be high in this kind of experiments because
the insertion is random and the mobile element is very stable. After
electroporation and incubation, several mutants were screened and selected
mutants showing anomalous capsule expression were tested for proper insertion
and sequencing. The strain used in these experiments was CovRIFD. The
capsular phenotypes observed in the transposon mutants were a) non-producer,
which didnʼt produce capsule at room temperature, and b) overproducer that
produced capsule at 37, as opposed to the background strain CovRIFD.
Approximately 20 non-producer colonies were examined and all of them showed
insertions in either hasA gene or hasA promoter area. Of particular interest were
the overproducer phenotype, and those were sequenced to identify the disrupted
area. The mutants were named OPTM for “overproducing transposon mutant”.
The data obtained from the transposon mutagenesis experiments
revealed more than one chromosomal location involved in the thermoregulation
of capsule production and that a mutation in this system confers an increased
capsule production at 37, thus, giving the potential of more virulence and
invasiveness, adding more evidence to the fact that virulence regulation is
complex in S. pyogenes and that it could respond to more than one molecular
trigger.
Four mutants with three different insertion sites that resulted in massive
capsule expression at 37, were analyzed. The strains were called OPTM2,
48
OPTM7, OPTM17 and OPTM18. All were sequenced to obtain the insertion
location.
OPTM2
The first mutant sequenced was named OPTM2. Blasting the sequence
against an MGAS315 genomic database showed an insertion in the gene
spyM3_1254 (Figure 18). There was a possibility that this disruption caused
polarity effect on downstream genes and gave an overproducer phenotype.
There are about 14 genes downstream of spyM3_1254 that are also hypothetical
proteins encoding phage associated proteins whose products have not been well
characterized. Inactivation in any of these genes by polar effect may lead to an
overproducing phenotype from a gene different from the one disrupted.
OPTM7 and OPTM16
Two other overproducers, OPTM7 and OPTM16 showed an insertion in
the cvfA (spym3_1377) gene (Figure 19). Downstream of cvfA there is a gene
encoding a guanylate kinase. CvfA is a predicted HD superfamily hydrolase that
in previous studies it has been found to be linked to virulence in S. pyogenes, as
well as virulence in B. subtillis and S. aureus (71). As part of the continued
research, protein work is being performed in order to elucidate the possible
interaction between cvfA and thermoregulation of capsule production.
49
OPTM18
Another over-producer mutant, OPTM18, showed a different insertion site.
Identification of the transposon insertion loci revealed that this mutant had an
insertion in the intergenic area, upstream from the insertion site at the promoter
area of hasA, found in non-producers (Figure 20).
There are five binding sites for CovR at the promoter area; none of these
was affected by the transposon insertion, but this particular finding may imply
that part of the sequence upstream from the promoter region of the has operon is
involved in the thermoregulation in GAS, along with other elements found in the
other mutants obtained in this study.
Capsule was measured in mutants and wild type strains. OPTM18 showed
an increment in capsule production at 37 compared with the wild type,
reaching values higher than 900 fg/μL at 37 (Figures 21 and 22).
However, insertional inactivation of the same promoter area region by site
directed mutagenesis did not lead to any observable defect on capsule
production.
50
spyM3_1254:
ATGATGAAGAAGAAAGGAGAAGCATTGGACAAAAAAGAACTGTTTGATAATTT
CCAAATAATTGGATGCGTCTCTTATCACCATTTGAGATTGAAGACATTAATAAG TGGATTGACGAAGAGAAGATGCCTGTTGAAGTTGTTAACGAGGCACTAAAAT
CGACAATTCTATACAACGCACCAAACCTTAGATACTTAAACAGAGTCTTGAAC
AACTGGAAGCGACAAGGGATTGATACAGTCGAGAAAGTCGAATTTGCTAGGT
TGCAATTTGAAAATAAAAAGCTCAGTCAAAATAAAAATCATCAATCCAACGTCC
CAAGCTGGTCGAATCCAGACTACAAAGAACCAGATTTAAAAGAATTTGCACTA stopGGAAGCATAGACGGTATAGAAGATGGATCAGGAGATTTTTAA
FIGURE 18: Complete sequence of spyM3_1254. The insertion site of the
transposon is shown with a vertical arrow. The gene is 414 nucleotides long. This
insertion generated OPTM2.
51
spyM3_1376:
a b ATGGTTAATATTATTTTATTAATTGTTTCTGCCCTCATTGGTTTAATATTAGGTTATGCACTTATTTCGATTAGACTCAAGTCTGCGAAGGAAGCTGCAGAGTTGACTCTTTTAAACGCTGAACAAGAAGCTGTTGATATTCGTGGCAAAGCAGAAGTAGATGCTGAACACATCAAAAAAACAGCTAAACGTGAAAGTAAAGCAAATCGTAAAGAATTACTTTTAGAAGCAAAAGAAGAGGCAAGAAAATATCGTGAAGAGATTGAACAAGAATTTAAGTCTGAAAGACAAGAGCTTAAACAACTCGAGACACGCTTAGCGGAGCGCTCCTTAACTCTTGACCGTAAAGATGAAAACCTATCAAGTAAAGAAAAGGTACTAGATAGTAAAGAACAAAGTCTGACCGATAAATCTAAACACATTGATGAGCGGCAACTTCAAGTAGAAAAACTTGAAGAGGAGAAAAAAGCAGAACTGGAAAAAGTTGCTGCGATGACGATTGCAGAAGCGCGTGAAGTGATTTTAATGGAGACGGAAAACAAACTGACCCATGAAATTGCGACGCGCATTCGAGATGCCGAACGTGACATCAAGGACCGAACAGTTAAAACCGCCAAGGACTTGTTAGCGCAAGCCATGCAACGCCTTGCTGGTGAGTATGTGACTGAACAAACTATTACCAGTGTCCATCTCCCAGACGACAACATGAAGGGCCGAATTATTGGACGTGAAGGCCGTAATATTCGTACTTTAGAGAGCTTGACTGGCATTGACGTTATTATTGACGATACTCCTGAAGTTGTTATCTTATCAGGATTTGATCCTATTCGACGTGAAATTGCTCGTATGACCTTGGAATCTCTGATTGCTGATGGTCGCATCCATCCAGCTCGTATCGAGGAATTGGTTGAGAAAAATCGTCTTGAAATGGATAATCGTATTCGTGAGTACGGTGAAGCTGCAGCCTATGAGATTGGTGCACCAAACCTTCATCCTGATTTGATTAAAATCATGGGACGCCTGCAATTCCGTACCTCGTTTGGTCAAAATGTCCTACGTCACTCTGTTGAGGTTGGTAAGTTAGCTGGTATTTTAGCTGGTGAGTTAGGTGAAAATGTTGCTCTTGCCCGCCGTGCTGGTTTCTTGCATGATATGGGTAAAGCTATTGACCGTGAGGTTGAAGGTAGTCACGTTGAGATTGGAATGGAATTTGCACGTAAATACAAAGAACATCCAGTTGTTGTCAACACTATTGCTAGCCACCACGGAGATGTGGAGCCAGATTCTGTGATTGCTGTGCTAGTAGCTGCAGCAGACGCCCTCAGTTCGGCTCGTCCAGGCGCTCGTAATGAGTCAATGGAGAATTACATCAAGCGTCTTCGTGATTTAGAAGAAATCGCGACAAGTTTTGATGGGGTACAAAATAGTTTTGCTCTACAAGCTGGACGTGAGATTCGTATTATGGTTCAACCTGAAAAAATTTCAGATGATCAGGTTGTCATTTTGTCGCATAAAGTAAGAGAAAAAATTGAAAACAATCTAGATTACCCAGGAAATATTAAAGTAACTGTTATTCGTGAGATGAGAGCGGTTGATTATGCCAAGTAG stop
FIGURE 19: Partial sequence of spym3_1376, the cvfA gene. The gene is 1608
nucleotides long. Here only the first 259 nucleotides are shown. The vertical
arrows represent the insertion site of the transposon. Arrow “a” points the
insertion site found in OPTM16, arrow “b” shows insertion site found in OPTM7.
52
5ʼ - spym3_2199
TCTTAATCAGATGAAGTTGTACTCCCTGAACAATTTTCATAATGTTTCCTTAATA
AATAGTGTGATCCCTACGATTATACCATTTTTATTCAGGTTAGGTGAGTGATAA
CTTTTTTTGACTTAATTGTCGGGATTCTAGTGTTAGTAAAATATAGTTTTCTACA
ATATATCCTTTACCAGTTATCATATTTCTTGATATTTTTTAAAAATCAAAATATATC aTATTTTTTCATAAAATTAGATAAAACTCTTGGAAAAGTTTACTAAAATAATTTATA
ACAATTCAATTATCCTGATTTTTCTTTTTTCGGGGGAATTTTTTTTATTGAAACA -35 CAATTTTATTAAAAATATCTCTATGTCTAGTTGACATTATTTCTTATTTATATTAG -10 +1 b RBS AATATTGAGGTCCTTTCTTTCAAGGAAATTAAAAAAGAAAGAGGTGTAATT
5ʼ -hasA
GTGCCTATTTTTAAAAAAACTTTAATTGTTTTA
FIGURE 20: The hasA promoter region. The start of hasA transcription is shown
as +1. Promoter elements (-10 and -35 region) and the ribosome-binding site
(RBS) are indicated in bold. 5ʼ-ends of hasA and spy_2199 are indicated by
horizontal arrows. Five CovR binding sites are underlined. The two vertical
arrows indicate transposon insertion sites showing opposite phenotypes. The
transposon insertion close to the transcription start site (vertical arrow b)
generated a capsule non-producer. The transposon insertion 178 bases
upstream of the start codon (vertical arrow a) generated a capsule overproducer
at 37˚C (OPTM18).
53
TABLE 12: Capsule quantitation of transposon mutants at 37
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu)
Average Standard deviation
MGAS315-1 0.004 340,000 85.95
MGAS315-2 0.005 420,000 86.97 86.46 0.72
MGAS315ΩCovR-1 0.006 470,000 93.27
MGAS315ΩCovR-2 0.006 440,000 99.62 96.45 4.50
OPTM2-1 0.648 4,600,000 1,029.16
OPTM2-2 0.618 4,100,000 1,101.22 1,065.19 50.95
OPTM7-1 0.547 4,800,000 832.56
OPTM7-2 0.532 4,300,000 903.88 868.22 50.43
OPTM18-1 0.597 4,600,000 948.17
OPTM18-2 0.666 4,200,000 1,158.49 1,053.33 148.72
54
TABLE 13: Capsule quantitation of transposon mutants at 25
Strain (duplicated) OD640
Colony number(cfu/mL)
Capsule conc (fg/cfu)
Average Standard deviation
MGAS315-1 0.007 620,000 82.48
MGAS315-2 0.008 680,000 85.95 84.22 2.45
MGAS315ΩCovR-1 0.191 1690000 800.16
MGAS315ΩCovR-2 0.193 1650000 807.48 803.82 5.18
OPTM2-1 0.735 5,500,000 976.32
OPTM2-2 0.771 5,900,000 954.71 965.51 15.28
OPTM7-1 0.697 6,200,000 821.31
OPTM7-2 0.713 6,400,000 813.91 817.61 5.23
OPTM18-1 0.803 6,100,000 961.73
OPTM18-2 0.796 5,900,000 985.66 973.70 16.92
55
fg/cfu
FIGURE 21: Capsule production of transposon mutants at 37. Note the
increased amount of capsule in transposon mutants.
fg/cfu
FIGURE 22: Capsule production of transposon mutants at 25. Note how wild
type MGAS315 does not change the capsule amount, contrasting to the other
strains.
0
375
750
1125
1500
MGAS31
5
MGAS31
5ΩCovR
OPTM
2OP
TM7
OPTM
18
0
250
500
750
1000
MGAS
315
MGAS
315Ω
CovR
OPT
M2
OPT
M7
OPT
M18
56
Knock-out experiment
Knock-out experiments were done for phenotype confirmation of
OPTM18. The target promoter area was amplified by PCR (Table 14) and cloned
into PCIV2, a 4.195 kb plasmid harboring kanamycin resistance. After
transformation, the phenotype of the transformant was similar to the background
strain, no capsule production at 37 and capsule production at 25. The
insertion mechanism of insertional inactivation is different from transposon
mutagenesis and this could explain the difference in phenotype. While PMGC8
was inserted into the target using transposase (Figure 25), PCIV2 was integrated
causing an insertional disruption with a fragment of the target gene, generating
partial duplicates of certain target sequences that would be inactive (Figure 26)
but this could generate a partial binding site or recognition sequence.
The knock-out was confirmed using a confirmation primer that contains a
sequence in the target gene area, OPhasAP-confirm, and a set of primers that
anneal to the vector in two different sites in a reverse and forward direction, M13-
f and M13-r, respectively (Table 15). The insertion is confirmed when an
amplicon of 782 bp is present in a PCR reaction with the confirmation primer and
one of the M13 primers. In this experiment, bands were present with the M13-r
set, showing a reverse direction insertion (Figures 23 and 24).
However, knock-out experiments failed to mimic the same overproducer
phenotype. This may be partially explained by the different insertion mechanisms
for both, plasmid and transposon. While in transposon mutagenesis the
continuity of the DNA sequence is disrupted by the insertion of the mobile
57
element, in insertional disruption a partial duplication is created (figures 25 and
26).
The influence of temperature in pathogen gene expression has been
widely studied, and many thermoregulated genes are known. Several
mechanisms are described in different species and specific genomic changes
have been determined by microarray analysis in GAS, however, most of the
differential expression revealed was in hypothetical proteins (97).
The results presented in this study could throw some light upon the
hypothetical function of uncharacterized proteins and help to develop a model of
thermoregulation in S. pyogenes capsule production, leading to better
understanding of virulence and physiology in this species.
58
TABLE 14: PCR primers used for hasA promoter area amplification
Primer name Primer sequence
5OPhasASph 5ʼ - AAAAGCATGCTCTGTTTCTAGCATTCAAATGTC C - 3ʼ
3OPhasASph 5ʼ - AAAAGCATGCTCTGTTTCTAGCATTCAAATGTC C - 3ʼ
The first 4 As of the sequence are overhangs, GCATGCT is the recognition site
of the restriction enzyme SphI. The optimum PCR condition for amplification of
the hasA promoter area was 55 for annealing temperature and one minute of
extension.
TABLE 15: Primers used for hasA promoter area knock-out confirmation
Primer name Primer sequence
OPhasAP-confirm 5ʼ - TACTCCCTGAACAATTTTCATAATG - 3ʼ
M13 - f 5ʼ -CGCCAGGGTTTTCCCAGTCACGAC - 3ʼ
M13 - r 5ʼ - AGCGGATAACAATTTCACACAGGA - 3ʼ
The optimum PCR condition to check the right insertion was 52 of annealing
temperature and one minute of extension.
59
a.
b.
1 2 3 4 5 6 7
FIGURE 23: Gel electrophoresis of nucleic acids for knockout experiments. a)
amplification of the promoter area of has operon. b) Bands confirming the
insertion of PCIV2 in a reverse direction and consequent disruption of a region of
the has operon promoter area. Lanes 1-5 are transformation products. Lane 6 is
a positive control and lane 7 is a negative control.
60
782 bp
CFhasA M13-f
M13-r
FIGURE 24: Primer annealing for knock-out confirmation. The black bold lines
represent the chromosome region of hasA. The grey line in between represents
the inserted plasmid. The fine line below the chromosome with plasmid insertion
is the extension obtained in this experiment, confirming a reverse insertion. The
green rectangles are the primers in their annealing site.
61
FIGURE 25: Graphic representation of transposon insertion. The green circles
are transposase. The transposon is inserted randomly into the genome, causing
a wide variety of mutations on each transformation, derived from gene disruption
at the insertion site.
62
FIGURE 26: Graphic representation of insertional inactivation. The plasmid is
inserted into the chromosome by homologous recombination between a fragment
of the target gene and the chromosomal target gene loci. The insertion creates a
disrupted gene and a partial duplication of the target sequence.
63
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APPENDICES
APPENDIX A: Media, Buffers and Solutions
I. Media
1) Luria-Bertani Broth (per liter):
10 grams NaCl
10 grams Tryptone (Fisher Scientific)
5 grams Yeast extract (Difco)
Adjust pH to 7.6 with NaOH solution. Autoclave at 20 psi for 30
minutes.
2) THY broth (per liter):
30 grams Todd Hewitt (BBL)
2 grams Yeast extract (Difco)
Autoclave at 20 psi for 30 minutes.
3) TP broth (per liter):
30 grams Todd Hewitt (BBL)
2 grams Yeast extract (Difco)
20 grams Peptone (Bacto brand)
Autoclave at 20 psi for 30 minutes
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4) THY agar plates (per liter, approximately 40 plates):
30 grams Todd Hewitt
2 grams Yeast extract
14 grams Agar (Bacto brand)
Autoclave at 20 psi for 30 minutes. Let cool down to 55°C. Add
appropriate antibiotic if needed before pouring plates.
5) LB agar plates (per liter, approximately 40 plates):
10 grams NaCl
10 grams Tryptone
5 grams Yeast extract
14 grams Agar (Bacto brand)
Autoclave at 20 psi for 30 minutes. Let cool down to 55°C. Add
appropriate antibiotic if needed before pouring plates. Add X-Gal if
needed.
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II. Antibiotics
Antibiotic AbbreviationSolvent Concentration of stock solution
Storage
Chloramphenicol Cam, Cm 50% EtOH 10 mg/ml 4°CErythromycin for
E. coli
Erm, Em 100% EtOH 75 mg/ml 4°C
Erythromycin for
S. pyogenes
Erm, Em 100% EtOH 1 mg/ml 4°C
Kanamycin Kan, Kn ddH2O, filter 100 mg/ml 4°CSpectinomycin Spc ddH2O, filter 100 mg/ml 4°C
For E. coli:
Antibiotic Final concentration (µg/ml)
Volume of stock solution (µL)10 ml 100 ml
Chloramphenicol 100 15 150Erythromycin 750 10 100Kanamycin 50 5 50Spectinomycin 100 10 100
For S. pyogenes:
Antibiotic Final concentration(µg/ml)
Volume of stock solution (µL)10 ml 100 ml
Chloramphenicol 3 3 30Erythromycin 1 10 100Kanamycin 700 70 700Spectinomycin 150 15 150
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III.Buffers and solutions:
1) PBS buffer (10X)
80 grams NaCl
2 grams KCl
2 grams K phosphate monobasic
11.5 grams Na phosphate dibasic heptahydrate
pH 7.2
2) TAE Buffer (10X)
48.8 grams Tris Base
11.42 mL Glacial acetic acid
2 mL 0.5M EDTA pH 8.0
q.s. to 1 Liter with ddH2O
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APPENDIX B: Protocols
I. Generation of In-Frame Deletion Strain
1. Once Erm resistant colonies are obtained (which have been grown at
30°C) pick colonies and grow them in 10 ml of THY+Erm1 at 30°C.
2. Next morning, make freezer stocks of the overnight cultures and
inoculate 10 µL of overnight cultures into 10 ml of THY+Erm1 and
incubate at 39°C. This shift of temperature will select for the integration
of the plasmid into the chromosome, as pJRS233 cannot replicate at
39°C.
3. Next morning, inoculate 10 µL of overnight culture into 10 ml of THY
+Erm1 and incubate at 39°C. Passing twice at 39°C in presence of
Erm1 ensures integration of the plasmid.
4. Next morning, inoculate 10 µL of overnight culture into 10 ml THY
without Erm and incubate at 30°C. This temperature shift selects for
the excision of the plasmid, which could leave either the wild type or
deletion allele of the gene.
5. Repeat step 4) twice. Three times passing of the cultures at 30°C
without Erm is necessary to get sufficient percentage of Erm sensitive
cells in the culture.
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6. After the final passage, make freezer stock of 2 cultures and plate,
and dilutions on THY agar in the absence of Erm, and incubate
overnight at 30°C in an anaerobic conditions.
7. Pick colonies and replica-plate (by patching with sterile wooden sticks)
on THY+ Erm1 and THY plates. Patch 100 colonies. Incubate
overnight in anaerobic conditions at 30°C.
8. Grow up those patches which were Erm sensitive at 37°C and make
freezer stocks and confirm chromosomal structure by PCR after
chromosomal extraction, as these strains could have either wild type or
deleted chromosomal structure. Test 10 colonies. If all the 10 colonies
have the wild type genotype, try again from the beginning.
9. Make freezer stock of wild type revertant as a control and another
stock of settled down strains.
II. Isopropanol precipitation
1. Transfer the sample to an Eppendorf tube. Sample volume in a tube
should be less than 800 µL. if sample volume is over 800 µL split in
two tubes.
2. Add 1/10 volume of 3M Sodium Acetate solution (pH5.2) to the sample.
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3. Add 70% of the final volume of room temperature isopropanol to the
DNA solution and mix well.
4. Centrifuge the sample immediately at 10,000 – 15,000 x g for 1 hour at
4°C.
5. Carefully decant the supernatant without disturbing the pellet.
6. Add 1 ml of 70% EtOH to a tube gently.
7. Centrifuge at 10,000 – 15,000 xg for 15 minutes at 4°C.
8. Carefully decant the supernatant without disturbing the pellet.
9. Remove the residual ethanol solution with a pipette very carefully.
10. Dry the pellet at 50°C for 5 – 20 minutes (depending on the size of the
pellet).
11. Add DW. Added amount of DDW depends on the final DNA
concentration you want to get. Usually 50 µL is enough.
12. Tap the tubes vigorously to dissolve DNA for about 5 minutes per tube.
13. Examine DNA concentration by gel electrophoresis or nanodrop.
14. Store sample at -20°C.
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III.Electroporation of S. pyogenes
1. Prepare DNA
2. Prepare THY plates containing appropriate antibiotics.
3. Add 100 µL of filter sterilized 2M glycine solution to 10 ml TP medium
and add appropriate antibiotic.
4. Inoculate S. pyogenes and incubate them overnight at 37°C.
5. Next morning, add 1 ml of filter sterilized 2M glycine solution to 200 ml
of TP medium plus appropriate antibiotic.
6. Take out 2 ml for blanks (1 ml per cuvette) for spectrophotometry and
add 2 ml of overnight culture to 98 ml of TP+ glycine and add
appropriate antibiotic.
7. Incubate the culture at 37°C until the culture reaches OD600=0.24.
8. Distribute cultures into 4 tubes of 50 ml falcon tubes.
9. Harvest cells by centrifugation (7,000 x g for 10 minutes at room
temperature). Resuspend total cell pellets in 4 ml of the supernatant.
10. Incubate cells at 43°C for 9 minutes.
11. Add 6 ml of 15% glycerol solution and centrifuge the cells (7,000 x g)
for 10 minutes at room temperature.
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12. Discard the supernatant and wash the cells three times with 10 ml of
15% glycerol solution.
13. Remove the supernatant as much as possible.
14. Resuspend the cell pellet in 800 µL of 15% glycerol solution.
15. Keep competent cells at room temperature until electroporation.
16. Place tubes of 10 ml of THY media and electroporator cuvettes on ice.
17. Set the electroporator condition (Voltage: 2100 V, Capacitor: 25 µF,
Resistance: 200 Ohms, electroporator cuvette: 2 mm).
18. Place DNA in an ice-cold 2 mm electroporator cuvette:
Suicide vectors: 10 – 20 µL
Shuttle vectors: 0.5 – 2 µL
Transposon vectors: 10 – 20 µL
pJRS233: 10 – 20 µL
19. Add 200 µL of competent cells into the cuvettes ad pipette up and
down to mix well.
20. Give electric shock.
21. Immediately, remove cells from the cuvette and add the cells into the
cold 10 ml THY media.
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22. Record the time constant.
23. Incubate cells for 30 minutes on ice, then 1 hour and 30 minutes at
37°C. Dry the plates during this time.
24. Harvest cells by centrifugation (7,000 x g) for 10 minutes at room
temperature and resuspend cells in 500 µL of THY medium.
25. Plate 100 µL of cells on each plate.
26. Incubate overnight at 37°C in anaerobic conditions.
IV. Electroporation of S. pyogenes with pJRS233 derivatives
1. Prepare pJRS233 using a Maxi prep kit (initial volume: 300 ml of
overnight culture, final volume: 50-100 µL in DDW).
2. Prepare THY agar plates with no antibiotic (24 ml THY agar/plate)
3. Add 100 µL of filter sterilized 2M glycine solution to 10 mL of TP
medium.
4. Inoculate S. pyogenes and grow overnight at 37°C.
5. Next morning, add 1 ml of filter sterilized 2M glycine solution to 100
mL of TP medium.
6. Take blanks for spectrophotometry (2 mL) and add 2 mL of overnight
culture to 98 mL of TP+Glycine medium containing proper antibiotic.
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7. Incubate the culture at 37°C until the culture reaches OD= ≈ 0.24.
Usually it takes 2 hours for wild type.
8. Turn on the heat block containing a 15 ml tube block and set
temperature at 43°C.
9. When the culture reaches OD= ≈0.24, distribute cultures into two 50
mL falcon tubes and harvest cells by centrifugation (7,000 x g for 10
minutes at 14°C).
10. Resuspend total cell pellets in 2 mL of the supernatant and transfer to
a 15 mL falcon tube.
11. Incubate cells at 43°C for 9 minutes. Use the heat block.
12. Add 8 mL of 15% glycerol solution and centrifuge the cells (7,000 x g
for 10 minutes at 14°C).
13. Discard the supernatant and wash the cells twice with 10 mL of 15%
glycerol solution (centrifugation at 14°C).
14. During the final wash, place tubes of 10 mL of THY media and
electroporator cuvettes on ice. The tube number are transformation
number plus one (for negative control). The cuvette number is the
same as the transformation number.
15. Remove supernatant as much as possible after the final wash.
16. Resuspend the cell pellet in 600 µL of 15% glycerol solution.
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17. Keep the competent cells at room temperature, not on ice. Perform a
Electroporation as soon as possible.
18. Set the electroporator (voltage: 2100V, Capacitor: 25µF, Resistance:
200 Ω, electroporator cuvette: 2 mm).
19. Place DNA in an ice-cold 2 mm electroporator cuvette.
a. pJRS233 derivative: 10-20 µg
b. DNA concentration after Maxi-prep using 300 mL of 20-24 hrs
culture: 3-4 µg/µL
20. Add 200 µL of competent cells into the cuvettes and pipette up and
down to mix well.
21. Press the red button on the electroporator.
22. Immediately, remove cells from the cuvette and add the cells into the
cold 10 mL THY media.
23. Record the time constant.
24. Incubate cells for 30 minutes on ice, then 1 hour in the 30°C incubator.
You can dry the agar plates during this time if plates are not dry.
25. Harvest cells by centrifugation (7,000 x g for 10 minutes at room
temperature) and resuspend cells in 300 µL of THY medium.
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26. Add 300 µL of cells to 15 mL of THY soft agar (cooled down to 43°C),
mix gently and pour 3 mL of cells-soft agar mix onto a THY + Erm1
plate.
27. Incubate the plates in an anaerobic jar at 30°C at least 2 hours and no
more than 3 hours.
28. Remove the plates from the anaerobic jar. Overlay with an additional 3
mL THY soft agar containing erythromycin (4 µg/mL)
29. Incubate plates at 30°C for 48 hours in an anaerobic jar.
V. E. coli transformation:
1. Thaw frozen Top10 competent cells on ice.
2. To each Top10 cell aliquot add 1 – 200 ng of transforming DNA in a
volume of 10 µL or less. For a ligate, use 10 µL (maximum amount).
Include a positive control (the undigested vector for the cloning) and no
DNA control.
3. Place cells on ice for 30 minutes.
4. Heat shock cells for 30 seconds at 43°C.
5. Add 1 ml of LB broth to each tube and incubate at 37°C for one hour,
shaking intermittently or in a 37°C shaker incubator.
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6. Dry agar plates.
7. Plate 200 µL of cells on selective agar plates (6 plates for
transformation and 1 plate for control cells).
8. Incubate plates overnight at 37°C.
VI. Quantitation of S. pyogenes capsule:
1. Grow S. pyogenes overnight in 10 ml THY media with appropriate
antibiotic.
2. Next morning, dilute cells to 10-5, 10-6, and 10-7 with sterile PBS buffer
and plate 100 µL of cells on THY agar plates. Incubate the plates at
37°C in anaerobic conditions. Make triplicate dilutions and place each
set in a different jar (or as many as the temperature that will be tested)
3. 24 hours later, take out the jars and place one at 25°C (if more
temperatures will be tested, then move each jar to each temperature)
and save one jar for measurement at 37°C. Measure the capsule
amount produced by the 37°C jar.
4. 4-5 hours later, measure the capsule amount produced by colonies on
the plates incubated at 25°C.
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Capsule Quantitation using Stains-All reagent solution
Analyze in duplicate. Do not use PBS buffer in the Stains-All assay
because it inhibits the reaction
5. 2X Stains-All reagent: 20 mg of Stains-All and 60 µL of glacial acetic
acid in 50 mL of 50% formamide. Wrap the reagent bottle with foil
because itʼs degraded by light. Store at 4°C. The reagent is effective
within a month.
6. Take colonies carefully using a spatula and place this colony into a
tube with 10 ml of DDW. Duplicate the samples.
7. Vortex the tubes about 1 minute to resuspend cells and capsule.
8. Take 100 µL of the resuspension and dilute it in 900 µL of PBS buffer.
Save for later serial dilution and plating. This will be the first tube of the
serial dilution.
9. Centrifuge the 10 ml tubes to precipitate cells (6,000 x g) for 10
minutes at room temperature.
10. Take 500 µL of supernatant into a spectrophotometry cuvette.
11. Mix with 500 µL of 2X Stains-all reagent.
12. Prepare hyaluronic acid solutions for a standard curve. Add 1 µL, 2 µL,
3 µL, 4 µL and 5 µL of 1 mg/ml hyaluronic acid in 500 µL of DDW in a
spectrophotometer cuvette. Mix the solution very well with a P1000
97
pippetman. This mixing step is very important because hyaluronic acid
does not dissolve in the Stains-all reagent. Add 500 µL of the Stains-all
reagent to each cuvette and mix well.
13. Prepare blanks with 500 µL of DDW mixed with 500 µL of Stains-all.
14. Measure OD640
15. Dilute resuspended cells to 10-3, 10-4 and 10-5 with PBS buffer and
plate 100 µL of cells on THY agar plates. Make the dilution and plating
in duplicate. Next morning count appropriately spread colonies and
calculate cell concentration (cell number/ml).
16. Using the standard curve and cell concentration, calculate the amount
of capsule produced per CFU.
17. 5 hours later, measure the capsule amount produced by colonies on
the plates at room temperature the same way it was done before,
make dilutions, plate and then count colonies to calculate cell
concentration and capsule production.
VII. Storing cells at -80°C freezer
1. Grow cells overnight in THY media with antibiotics if needed.
2. Transfer 0.7 mL of culture into a cryogenic tube and add 0.3 mL of
80% glycerol solution. Mix well.
98
3. Label the tube and store at -80°C.
VIII. Plasmid extraction
All plasmids were propagated in E. coli Top10 cells and extracted using
Biotech Gerard Maxi Prep Kit according to manufacturerʼs protocol,
except that final volume was 2 mL per sample. Initial volume: 300 mL
of LB broth.
IX. DNA Purification for PCR products
PCR products were purified using Qiagen PCR Purification Kit
following the protocol provided by the manufacturer.
X. Preparation of Chromosome of S. pyogenes with Sigma column
1. Grow 10 ml of overnight culture in THY broth with 100 µL of 20mM
glycine and add appropriate antibiotic if necessary.
2. Harvest cells by centrifugation at 7,000 rpm for 10 minutes.
3. Wash twice with 3 ml of TE buffer.
4. Add 200 µL of lysozyme (100 mg/µL), 2 µL of mutanolysin (100 µ/
µL), 80 µL of RNAse and 520 µL of Gram positive solution.
5. Incubate 1 hour at 37°C with intermittent shaking.
99
6. Follow the remaining steps provided by the manufacturer.
XI. Preparation of E. coli competent cells
1. Grow Top10 cells in 10 mL of LB broth at 37°C overnight.
2. Dilute 1 mL of cells into 200 mL of LBB in a 500 mL flask.
3. Make solutions and keep them on ice.
4. Grow cells with vigorous shaking until they reach 10-8 cells/mL. It
takes approximately 2 hours.
5. Divide 200 mL culture into four 50 mL conical tubes.
6. Harvest cells at 5,000 x g for 5 minutes at 4°C. From this point on,
cells must be kept ice cold.
7. Gently resuspend each pellet in 10 mL ice cold, sterile solution I.
8. Recover cells by centrifugation at 5,000 x g, 5 minutes, 4°C.
9. Discard supernatant. Gently resuspend each pellet in 10 mL of
solution II. Pipette up and down. Do not vortex.
10. Place tubes on ice for 15 minutes.
11. Recover cells by centrifugation, 5,000 x g, 5 minutes, 4°C. gently
resuspend all pellets in 10 mL of solution II containing 10% (v/v)
glycerol.
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12. Divide cells in 200 µL aliquots in eppendorf tubes (approximately 50
tubes) and freeze at -80°C.
Solution I 10 mM MOPS (KOH) pH 7.010 mM RbCl
Solution II 100 mM MOPS (KOH) pH 6.550 mM CaCl2
10 mM RbClStock solutions .1 M MOPS pH 7.0
.1 M RbCl1.0 M MOPS pH 6.50.5 M CaCl2
Solution I 32 mL ddH2O4 mL 0.1M MOPS pH 7.04 mL 0.1 RbCl
Solution II 35 mL ddH2O5 mL 1.0 M MOPS pH 6.55 mL 0.1 M RbCl5 mL 0.5 M CaCl2
Solution II containing 10%
glycerol
10 mL solution II1.43 mL of 80% glycerol
101
VITA
Graduate School
Southern Illinois University
Trilce Michelle Galeas Peña Date of Birth: April 10th, 1979.
1211 W Schwartz St Apt 2. Carbondale, Illinois 62901
Calle Camaguey # 21. San Salvador, El Salvador.
Universidad Autónoma de Santo Domingo
Doctor in Medicine, June 2005.
Special Memberships and Awards:
Fulbright Scholarship recipient
United Nations University – Program for Biotechnology for Latin America and the Caribbean Scholarship recipient
Fundación Carolina Scholarship recipient
American Society for Microbiology member 2007 – present
American Association for Advancement of Science member 2008- present
Thesis Title:
Thermoregulation of Capsule Production in Streptococcus pyogenes strain HSC5.
Major Professor: Dr. KyuHong Cho
102