From the Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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ABSTRACT |
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Hyaluronic acid production by group A
streptococci is regulated by transcriptional control. In this study,
transposon mutagenesis of an unencapsulated strain yielded an
encapsulated mutant. Two genes homologous to sensors and response
regulators of bacterial two-component systems were identified
downstream of the transposon insertion. Inactivation of the putative
sensor gene, csrS, in three different unencapsulated
strains yielded encapsulated mutant strains. Electrophoretic mobility
shift assays determined factor(s) in a cytoplasmic extract of an
unencapsulated group A streptococcal strain was binding to a
double-stranded DNA fragment derived from the has operon
promoter. In contrast, similarly prepared cytoplasmic extracts from a
csrS deletion mutant did not shift the fragment. The
putative response regulator, CsrR, was partially purified and was shown
to bind the has operon promoter fragment. The affinity and
specificity of CsrR for the fragment were increased significantly after
incubation with acetyl phosphate. DNase I footprinting determined that
the acetyl phosphate-treated CsrR was binding to key sequences in the
promoter and the coding region of hasA. Therefore, a
two-component system is repressing the production of hyaluronic acid in
group A streptococci using a phosphorylation-dependent
binding interaction between the response regulator CsrR and the
promoter region of the has operon.
The Gram-positive bacteria Streptococcus pyogenes are
the cause for many suppurative infections of the skin and throat, as well as the nonsuppurative sequelae of rheumatic fever and
post-streptococcal glomerulonephritis. Virulence factors produced by
group A streptococci (GAS)1
such as C5a peptidase and pyrogenic exotoxins have been shown to
promote host damage while the surface-exposed M-protein and the
hyaluronic acid capsule have been shown specifically to prevent complement-mediated phagocytosis. Strains of GAS which lack M-protein are more readily destroyed in the presence of phagocytes than strains
with M-protein (1). Similar studies have shown that the presence of a
hyaluronic capsule inhibited phagocytosis and increased the virulence
of GAS during invasive infection (2-4). Increased isolation of mucoid
strains of GAS from human outbreaks of rheumatic fever as well as
severe invasive infections suggests that capsule production may be an
important virulence factor in these diseases (5). In addition,
production of hyaluronic acid by GAS is apparently important in the
initial stages of colonization of the upper respiratory tract of
intranasally inoculated mice (6, 7). This evidence is supported by the
observation that hyaluronic acid can function as an adhesin capable of
binding to CD44 on keratinocytes found on the pharyngeal mucosa and
skin (8).
Hyaluronic acid is a linear glycosaminoglycan that consists of
repeating subunits of The expression of the M-protein as well as many of the secreted
extracellular products of GAS is controlled at the transcriptional level by the trans-activator Mga (16-18). A decrease in
transcription of hasA in a strain of GAS in which the gene
encoding Mga (mga) was inactivated indicated that Mga may be
involved in has operon regulation (19). However, Perez-Casal
et al. (17) reported no effect on hyaluronic production
after inactivation of mga. Recently, Alberti et
al. (20) defined strain-specific cis-acting sequences
within the promoter region of hasA which affected capsule gene expression, thus implying that differences observed in capsule expression among strains of GAS are related to individual promoter structure and strength. However, the data could not completely rule out
that trans-acting factors may play a part in regulation as
well (20). Therefore, no consistent evidence exists correlating a
trans- or cis-acting factor with the
transcriptional regulation of the production of hyaluronic acid.
This report describes the identification of two genes encoding a
putative two-component system that influences the production of
hyaluronic acid by GAS. Inactivation of these genes converted the
previously unencapsulated strains (B931, D471, and GT8760) to
encapsulated strains. Further analysis of these two genes determined that inactivation of the putative sensor in the two-component system
was sufficient for converting an unencapsulated strain of GAS into an
encapsulated strain. In addition, the product of the gene encoding the
putative response regulator was partially purified, and after the
protein was treated with acetyl phosphate it was shown to bind
specifically to regions within the promoter and coding region of
hasA.
Bacterial Strains and Plasmids--
The streptococcal strains
used in this study (B931(T2), D471(T6), S43(T6), B915(T49), 5-19(T3),
GT8760(T49), and WF51(T18)) as well as the strains generated
(B931EnTn916, B931 DNA Purification and Manipulation--
Streptococcal DNA was
prepared as described previously by Dougherty and van de Rijn (12).
Plasmid DNA was prepared from E. coli cultures by the
alkaline lysis procedure (28) or by the Qiagen plasmid kits (Qiagen;
Chatsworth, CA) and were suspended in either water or TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Enzymes required for restriction digests and ligations were acquired from Promega (Madison, WI) and were used according to the manufacturer's suggestions. Pwo polymerase (Boehringer Mannheim) was used
for all PCRs per manufacturer's description. Oligonucleotide primers used in this study are listed in Table
I.
Filter Matings--
The transposon Tn916 was
transferred to GAS via conjugation as described by Caparon and Scott
(29) with modifications. Cultures of the recipient GAS and the donor,
CG110, were grown to an OD650 = 0.8 in chemically defined
medium and were mixed at a ratio of 10:1, respectively. The bacteria
were transferred onto a filter (0.45 µm; Millipore; Bedford, MA) and
then grown on THY agar plates with defibrinated sheep's blood (2%).
The filters were incubated overnight at 37 °C after which the
bacteria were washed from the filter with chemically defined medium,
and aliquots of this suspension were plated on Todd Hewitt yeast blood
plates supplemented with tetracycline (5 µg/ml) and streptomycin (1 mg/ml). Transductants were screened for capsule production by direct
microscopic observation and India ink staining. To confirm that the
transposon insertion was the cause of the observed mutation,
generalized transductions were conducted using the GAS phage A25 as
described by Caparon and Scott (29).
Southern Blot Analysis--
Chromosomal DNA was digested with
HindIII, separated on a 0.5% agarose gel, and transferred
to Zeta-Probe® GT Genomic Tested Blotting Membranes
(Bio-Rad) via capillary action. DNA-DNA hybridization between the DNA
blotted on the membrane and the probe DNA was conducted according to
the Genius System (Boehringer Mannheim) with the following
modifications. After incubation with anti-digoxygenin alkaline
phosphatase antibody, the membrane was washed with 1 × Post-SAAP
(0.05 M Tris-HCl, pH 10, 0.1 M NaCl) for 20 min, four times, with 5-min washes with distilled water between each
wash. Hybridization was detected by incubation of the membrane
with disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenylphosphate
(Boehringer Mannheim) for 15 min at 37 °C, and bands were
visualized by exposure of the membrane to BioMax 228 MR film (Eastman
Kodak) at room temperature.
Hyaluronic Acid Detection and Quantitation--
Capsule
production was initially detected by India ink staining (30).
Quantitation of hyaluronic acid production was conducted using a
colorimetric assay described previously by Moses et al. (31)
with the exception that chemically defined medium was used as the
growth medium instead of THY because THY contains interfering substances.
Analysis of Regions Flanking Tn916 Insertion--
Chromosomal
DNA from strains being analyzed was purified, digested with
HindIII, and analyzed by Southern blotting to determine the
size of the two fragments of Tn916 generated. The digested DNA was run on a 0.5% agarose gel, and the desired fragments of DNA
were extracted from the gel using the Qiagen Qia-Quick system. The
extracted DNA was ligated and purified, and primers specific for a site
at the left end of Tn916 (D224; Table I) and the region flanking the HindIII site (D225; Table I) were used to
amplify the DNA adjacent to the insertion site via PCR. The standard
conditions used for PCR in these studies were as follows: 8 µl of 20 mM MgSO4, 5 µl of 10 × PCR buffer
without MgSO4 for pwo polymerase, 5 µl of a 10 µM stock of the oligonucleotide primers, 5 µl of the
ligated DNA, 4 µl of a 10 mM stock of dNTPs, 18 µl of
deionized water, and 0.26 µL of pwo polymerase. Using a
DNA Thermal Cycler (Perkin Elmer Cetus), the region was amplified for
35 cycles using the following conditions: melting temperature
(95 °C) for 45 s, annealing temperature (47 °C) for 30 s, and extension temperature (72 °C) for 2 min. The PCR product was
sequenced at the WFUBMC Sequencing Laboratory using the primer D224
(Table I). The sequence obtained was analyzed using the data base of
the M1 streptococcus DNA sequence at the University of Oklahoma
Advanced Center for Genome Technology ().
Allelic Exchange--
A shuttle vector for allelic replacement
of a portion of the second open reading frame in the potential
two-component system was generated by amplifying two regions,
approximately 1 kb in size, flanking the replacement site (via PCR) and
ligating them into the allelic replacement vector pFW6. This PCR
amplification required the oligonucleotide primers D231and D232 (Table
I) for generation of the region upstream of the replacement site and D233b and D234 (Table I) for generation of the region downstream of the
site. The standard conditions for PCR for these studies were used
except with the following changes: melting temperature (95 °C) for 1 min, annealing temperature (52 °C) for 1 min, and extension
temperature (72 °C) for 1 min; 35 cycles. The upstream and
downstream fragments were cloned into the MCSI and MCSII sites of pFW6,
respectively, and electroporated into streptococci using a method
described previously (32). Transformants were selected on Todd Hewitt
yeast blood plates with spectinomycin 100 µg/ml. Using a fragment
specific for the region targeted for allelic exchange within
csrS, double and single crossovers were confirmed by
Southern analysis. The DNA for this probe was amplified using primers
D239 and D240 (Table I). Standard conditions for PCR were used except
for the following changes: annealing temperature (60 °C) for 1 min
and extension temperature (72 °C) for 1 min; 35 cycles.
Electrophoretic Mobility Shift Assays (EMSA)--
Cytoplasmic
extracts from strains being analyzed were prepared (11) and assayed for
the ability to bind to the promoter region of the has operon
using a protocol described previously by Chodosh (33). The following
modifications were made. Reaction conditions for the crude extracts and
the radiolabeled DNA fragment included (in a final volume of 10 µl)
extract (2-30 µg), 2 fmol of 32P-radiolabeled DNA
fragment, 2 µg of poly(dA-dT), 60 mM Hepes, pH 7.9, 60%
glycerol, 20 mM Tris-HCl, 0.3 M KCl, 5 mM EDTA, and 5 mM dithioerythritol. Reactions
were initiated by the addition of crude extracts to the reaction
mixture and were incubated for 15 min at 15 °C. Next, the
DNA-protein complexes were separated by electrophoresis through a 5%
(w/v) polyacrylamide gel in TBE (90 mM Tris, 90 mM borate, and 2 mM EDTA) buffer. Gels were run for 40 min at 200 volts (constant voltage, 4 °C) and transferred to
and dried upon 3MM Whatman paper (80 °C). Finally, the dried gel was
subjected to autoradiography using BioMax 228 MR film.
The radiolabeled DNA fragment was generated using the primers D48 and
D55 (Table I). The PCR conditions for generating this DNA fragment were
as follows: 8 µl of 20 mM MgSO4; 5 µl of
10 × PCR buffer without MgSO4 for pwo
polymerase; 3 µl of a 10 µM stock of the
oligonucleotide primers; 1 µl of individual 10 µM stocks of dTTP, dCTP, and dGTP; 0.5 µl of a 10 µM stock
of dATP; 1 µl of a 1:10 dilution of B931 chromosomal DNA; 25 µl of
deionized water; and 2 µl of [ T7 Overexpression of csrR in E. coli--
To clone
csrR into the expression vector pET-11a, csrR was
amplified using the oligonucleotide primer D241 to incorporate an
NdeI site at the 5'-end of csrR and the primer
D242 to incorporate a BamHI at the 3'-end. The standard PCR
conditions were used with the following changes: annealing temperature
(58 °C) for 1 min and extension temperature (72 °C) for 55 s. The 696-bp PCR product was subjected to T4 polynucleotide kinase to
phosphorylate the ends to prepare them for ligation into the
EcoRV-digested plasmid pBluescript. Once subcloned,
csrR was removed from pBS-csrR by digestion with
NdeI and BamHI and was then ligated into
NdeI/BamHI-digested pET-11a to create
pET-11a-csrR. Finally, the pET-11a-csrR plasmid was transformed into the E. coli strain BL21 (DE3)(pLysS) to
overexpress the protein using the T7 promoter expression system (25).
Plasmid pET-11a was used as a negative control. After the plasmids were transformed into E. coli, a microcolony from each
transformation was transferred to 5 ml of TYPG and grown at 37 °C to
an OD600 = 0.7. A 1-ml sample of this culture was removed
to represent a preinduction culture, while the remaining culture was
induced with 0.4 mM isopropyl
1-thio-
To generate a large preparation of the overexpressed CsrR, 125 ml of
TYPG was inoculated with 1 ml of a culture (OD600 = 0.7) containing BL21 (DE3)(pLysS) (pET-11a-csrR or pET-11a) and
grown to an OD600 = 0.8 before induction with 0.4 mM isopropyl 1-thio- Treatment of CsrR with Acetyl Phosphate--
The partially
purified CsrR and control extracts from E. coli (0.2-6
µg) were treated with acetyl phosphate as described by McCleary (35).
Briefly, extracts were treated with 32 mM acetyl phosphate
in a phosphorylation buffer (50 mM Tris-HCl, pH 8, 10 mM MgCl2, 3 mM dithioerythritol)
for 75 min at 37 °C and then were analyzed using EMSA as described
above. The final volume for each reaction was 6 µl prior to the
addition of 4 µl of the EMSA mixture buffer (described above).
DNase I Footprinting of the has Operon Promoter--
To
footprint the promoter of the has operon with the partially
purified CsrR, the 220-bp fragment extending from
For each reaction, 15,000-20,000 cpm of the DNA fragment was used.
First, 0.5 µg of each extract was reacted with or without acetyl
phosphate as described above. Next, the 6-µl reaction containing the
acetyl phosphate-treated protein was added to 44 µl of a reaction buffer containing 1× EMSA buffer, 2 µg of poly(dA-dT), and the radiolabeled DNA fragment. The binding reactions were conducted using
the same conditions as described for EMSA. After incubation, 50 µl of
a solution containing 10 mM MgCl2 and 5 mM CaCl2 was added to each reaction. A 1:100
dilution of RQ1 RNase-Free DNase (Promega) was added to each tube and
incubated for 2 min at 37 °C. After 2 min, 100 µl of a stop
solution (1% SDS, 20 mM MgCl2, 2 mM EDTA, pH 8, 4 µg/µl salmon sperm DNA) was
immediately added to each tube and mixed. Finally, each sample was
purified via phenol chloroform extraction and concentrated via ethanol
precipitation. After drying, the DNA was suspended in a 1:1 dilution of
the stop solution from the Sequenase 2.0 kit (Amersham Pharmacia
Biotech). Samples were heated for 10 min at 65 °C and run on a
sequencing gel adjacent to a sequencing ladder of the region being
analyzed. The sequencing was conducted using Sequenase 2.0 (Amersham
Pharmacia Biotech). The gel was transferred to and dried upon 3MM
Whatman paper and subjected to autoradiography using BioMax 228 MR film.
Generation of an Encapsulated Strain of GAS by Tn916
Mutagenesis--
As shown previously by Crater and van de Rijn
(15), the has operon is controlled at the
transcriptional level. To determine if unencapsulated strains of GAS
may have trans-acting factors that repress the expression of
the has operon, an unencapsulated GAS strain, B931, was
mutagenized using Tn916 transposon mutagenesis. Using the
enterococcal strain CG110 as the donor and a streptomycin-resistant derivative of GAS strain B931 as the recipient, the conjugative transposon Tn916 was transferred via filter matings. Of the
resulting transconjugants, one transconjugant (B931EnTn916)
was encapsulated and was isolated for further study. This mutant
excluded the capsule stain India ink, and this exclusion was lost in
the presence of the enzyme hyaluronidase. Unencapsulated
transconjugants were also analyzed and did not exclude India ink. To
confirm that the capsule produced by the transconjugant
B931EnTn916 was hyaluronic acid, the hyaluronic acid content
of both the parent strain and the mutant strain was examined. The
actual amount of a cell-associated and total (bound plus secreted)
hyaluronic acid was determined, and the values are presented in Table
II. As shown, mutant strain B931EnTn916 produced hyaluronic acid during the exponential
phase of growth (61 fg/cfu) compared with the parent strain B931 (<10 fg/cfu).
Analysis of Tn916 Insertion in B931En--
To determine the number
and location of Tn916 insertions in the chromosome of strain
B931EnTn916, Southern analysis was employed using a
digoxigenin-11-dUTP-labeled probe derived from the plasmid pAM620 which
contains Tn916. The results indicated that a single insertion of Tn916 was present in the genome of
B931EnTn916. Two bands were detected (14 and 8 kb) with
strain B931EnTn916, but no bands were detected from strain
B931. To link the Tn916 insertion to the encapsulated
phenotype observed with the mutant, transduction using the GAS
bacteriophage A25 was utilized. Lysates from this infection were then
used to infect the unencapsulated parent strain (B931) in order to
transduce the insertion into its genome. Transductants were selected
for resistance to tetracycline and screened for the production of
hyaluronic acid. The frequency of transduction was 4 × 10 Sequence Analysis of Regions Flanking the Tn916 Insertion--
To
characterize the sequences flanking the Tn916 insertion in
B931EnTn916, PCR was employed using oligonucleotide primers derived from the known sequence of Tn916. As determined by
Southern analysis, HindIII digestion of the chromosomal DNA
from strain B931EnTn916 yielded two fragments (14 and 8 kb)
which contained the arms of Tn916 and a portion of the DNA
flanking the insertion. To isolate the fragments for sequencing, they
were separated on an agarose gel, eluted, and religated to form closed,
circular DNA. Next, PCR amplification of the ligated DNA from the large fragment (14 kb) yielded a 1.6-kb fragment of chromosomal DNA flanking
the Tn916 insertion. Finally, the fragment yielded
approximately 400 bp of DNA sequence that was analyzed using the data
base of the M1 streptococcus DNA sequence at the University of Oklahoma Advanced Center for Genome Technology. The sequence submitted matched
exactly with a portion of the M1 streptococcus sequence, including a
portion of an unidentified open reading frame. Upon further analysis,
as shown in Fig. 1, two open reading
frames (684 and 1,500 bp) were identified 220 bp downstream of the
Tn916 insertion site using the sequencing analysis software
GCG. No open reading frames were observed upstream of the insertion
site after scanning 800 bp upstream. The two open reading frames were 5 bp apart and showed homology to genes found in two-component regulatory
systems. Since the completion of this work, Levin and Wessels (36) also
identified these two open reading frames and named them csrR
and csrS for capsule synthesis
regulator Regulator component and
Sensor component, respectively.
Allelic Exchange to Generate a Deletion Mutant of
csrS--
Because the Tn916 inserted 220 bp upstream of the
two open reading frames, it was uncertain if csrR and
csrS encoded products that were involved in the regulation
of hyaluronic acid synthesis. To analyze the genes in this potential
two-component system, allelic exchange was employed to disrupt
csrS (the second gene of the two-component system, which has
homology to sensors from other two-component systems). A 1-kb portion
of the 5'-end of csrS was replaced with the gene for
spectinomycin resistance using allelic exchange with a nonreplicating
suicide vector (pFW6
The plasmid pFW6
The amount of hyaluronic acid produced by all of the transformants was
assayed and is shown in Table II. As shown, the null mutants
B931
As shown in Table II, there is approximately a 10-20-fold difference
between the amount of hyaluronic acid produced by
D471 EMSA and Overexpression of CsrR--
Because the disruption of the
csr locus allowed for hyaluronic acid production, the
hypothesis was formed that a repressor was controlling the expression
of the has operon in GAS. This theory was supported by
preliminary RNA studies conducted by Heath et al. (37) in
which a similar Tn916 insertion upstream of the csr locus led to an increase in has operon
mRNA production and capsule production over wild type levels.
Because csrR has homology to genes encoding DNA-binding
proteins (38), a radiolabeled double-stranded DNA fragment derived from
the promoter region of the has operon (11) was used in EMSA
assays to determine if a binding activity could be obtained from crude
cytoplasmic extracts derived from the GAS strains tested in this study.
Analysis of cytoplasmic extracts derived from an exponential phase
culture of strain B931 demonstrated an increase in the binding of the radiolabeled DNA fragment with increasing amounts of crude extract (Fig. 3, lanes 2-6). In
contrast, similarly prepared crude extracts from the
B931
To test the hypothesis that CsrR was binding to the DNA fragment,
csrR was cloned into an expression vector and overexpressed in E. coli. Cytoplasmic extracts from E. coli
with and without the overexpressed CsrR were analyzed using
SDS-polyacrylamide gel electrophoresis. A band at approximately 25 kDa
was identified in extracts with CsrR but not in the control extracts
lacking CsrR (data not shown). The predicted size for CsrR is 26 kDa. The CsrR was partially purified with ammonium sulfate fractionation and
analyzed using EMSA. The partially purified CsrR increasingly bound the
has promoter DNA fragment with increasing amounts of protein
(data not shown). In comparison, the similarly prepared control
extracts from E. coli without CsrR did not hinder the mobility of the DNA fragment (data not shown). Therefore, CsrR was
binding close to or within the promoter region of the has operon.
Treatment of CsrR with Acetyl Phosphate--
Once it was
determined that CsrR was binding directly to the promoter region of the
has operon, the question of why strain B931 Competition Assays to Determine Specificity of CsrR--
To test
if the binding reaction was specific, a competition assay was conducted
using increasing amounts of nonradioactive specific (original
has promoter DNA fragment) and nonspecific competitor (a DNA
fragment derived from ~700 bp upstream of the promoter). Acetyl
phosphate-treated extracts were used in the competition studies. As
seen in Fig. 5 (lanes 3 and
4), with increasing amounts of the specific competitor, the
binding activity of acetyl phosphate-treated CsrR is lost compared with
the control. In comparison, the nonspecific competitor (Fig. 5,
lanes 5 and 6) retained the binding activity of
the control fraction without any competitor (Fig. 5, lane
2). These results indicate that the acetyl phosphate-treated CsrR
is binding specifically to the promoter region of the has operon. Without acetyl phosphate, CsrR did not demonstrate a loss in
gel shift activity after the specific competitor was added, indicating that the specificity of CsrR binding to the has
operon promoter is dependent on treatment with acetyl phosphate (data not shown).
DNase I Footprinting to Identify the Binding Site of CsrR--
To
locate where the acetyl phosphate-treated CsrR (CsrR-Pi)
was binding in the promoter region of the has
operon, DNase I footprinting was employed. Using an
end-labeled radioactive DNA fragment derived from the noncoding strand
of the has operon promoter, footprinting indicated that CsrR
treated with acetyl phosphate protected several regions including the
Hyaluronic acid production by GAS is under the control of
transcriptional mechanisms as shown by Crater and van de Rijn (15). The
goal of the experiments in this report was to identify one or more of
the control mechanisms. Transposon mutagenesis of an unencapsulated
strain of GAS yielded an encapsulated mutant producing hyaluronic acid
(Table I). Confirmation of the transposon causing the capsule phenotype
was obtained by transduction of the mutation into a fresh background of
the parent unencapsulated GAS strain. Sequence analysis confirmed that
there were two genes flanking the insertion site of the transposon
which were homologous to genes encoding two-component regulatory
systems (Fig. 1). These two genes, csrR and csrS,
are identical to those identified in an abstract by Heath et
al. (37) and recently by Levin and Wessels (36). In their studies,
Tn916 mutagenesis and allelic exchange were used to
inactivate the entire csr locus or csrR alone in order to generate a hyaluronic acid-producing mutant from a single strain of GAS. In this report, three encapsulated GAS mutants were
generated from three different unencapsulated strains by inactivation
of the gene for the putative sensor (csrS). In addition, EMSA showed that crude cytoplasmic extracts from an unencapsulated strain of GAS were able to bind a DNA fragment derived from the promoter region of the has operon. In contrast, it was shown
that crude cytoplasmic extracts from a strain of GAS in which
csrS was inactivated did not bind the fragment. Partially
purified CsrR, the putative response regulator, was assayed using the
same EMSA, and it was determined that CsrR was capable of binding the promoter fragment. The affinity and specificity for this fragment increased after CsrR was treated with the phospho-donor acetyl phosphate. DNase I footprinting of the binding site revealed that CsrR
treated with acetyl phosphate was binding to the The requirement for bacteria to adapt to rapidly changing environments
has led to the evolution of efficient regulatory systems that utilize
signals from the environment and from within the bacteria to modulate
the expression of specific gene products. These regulatory systems
often require a minimum of two components, a sensor and a response
regulator (39). In most examples studied, the sensor possesses a
histidine kinase that contains conserved histidine residues that are
phosphorylated after autophosphorylation in the presence of ATP (39).
Because most sensors are membrane-bound, transferring the phosphate to
a response regulator present in the cytoplasm is required for
transmission of the signal received (40). Once a conserved amino acid
within the response regulator is phosphorylated, the response regulator
will undergo a conformational change that affects its ability to bind
regulatory regions of the targeted genes. Depending on the function and
phosphorylation state of the response regulator, genes are either
repressed or activated once the regulatory regions are bound.
As shown in Fig. 1, the data demonstrate that the transposon
Tn916 inserted upstream of the putative Compared with the binding activity of the wild type strain B931, the
inability of cytoplasmic extracts derived from exponentially grown
cells of strain B931 Although the binding of CsrR to the promoter region of hasA
does not directly demonstrate that CsrR is repressing the transcription of the has operon, the binding sites of CsrR within the
promoter of hasA suggest that this protein could affect the
binding of RNA polymerase. As shown in Fig. 6, protection of regions
within the The transcriptional control of the has operon by the
csr two-component system may be influenced by many factors.
First, the overall strength of the hasA promoter for each
strain studied may be variable. As shown in Table II, strain
D471 The ability for acetyl phosphate to increase the binding activity of
CsrR is similar to studies involving other response regulators such as
PhoB of E. coli (35). Although the Kd for
the binding of CsrR to the promoter of hasA is not presently
known, this report demonstrates that an increase in affinity was
observed (Fig. 4). An increase in specificity for the hasA
promoter fragment was also observed after CsrR was treated with acetyl
phosphate. This specificity was not observed with the non-acetyl
phosphate-treated CsrR, indicating that phosphorylation may be
essential for specific binding. In the case of PhoB, the presence of
acetyl phosphate increased the affinity of the protein for its binding
site dramatically. For PhoB, the Kd for the
phosphorylated PhoB showed a 10-fold increase in affinity over the
non-phosphorylated protein (35). The phosphorylation of PhoB by acetyl
phosphate leads to dimerization that allows for it to bind more tightly
to a consensus sequence (pho-box) found in the promoters it
activates (35). Other than the sequences outlined in this report, no
knowledge of the binding sites of CsrR is presently known. In addition, the mechanism by which acetyl phosphate transfers a phosphate group to
the response regulator as well as whether or not acetyl phosphate is
relevant to the signals affecting CsrS is unknown. Structure analysis
of a fully purified CsrR and its binding sites should address questions
regarding the conformational changes, if any, which occur after
phosphorylation and their affect on binding affinity and the activity
of the has operon promoter.
INTRODUCTION
Top
Abstract
Introduction
References
1,4-linked disaccharides of glucuronic acid
1,3-linked to N-acetylglucosamine. Its structure is
chemically indistinguishable from hyaluronic acid found in the
connective tissues of its hosts. Production of hyaluronic acid by GAS
occurs only at the exponential phase of growth and is lost at the
stationary phase. This loss of production has been correlated with a
loss of synthase activity from membranes of GAS isolated at the
stationary phase (9). The enzymes required for hyaluronic acid
production are encoded in the has operon. These enzymes are
hasA (encodes the hyaluronic acid synthase) (10-12),
hasB (encodes UDP-glucose dehydrogenase) (13), and
hasC (encodes UDP-glucose pyrophosphorylase) (14).
Expression of the has operon is under the control of a single promoter identified upstream of hasA (11, 15). In
addition, this operon is highly conserved among GAS regardless of
capsule phenotype (15). As shown by Crater and van de Rijn, the
simultaneous detection of the has operon mRNA transcript
and hyaluronic acid during the exponential growth phase of encapsulated
strains but not unencapsulated strains suggests that transcriptional
mechanisms control hyaluronic acid production (15).
EXPERIMENTAL PROCEDURES
csrS,
D471
csrS, and GT8760
csrS) were grown in
chemically defined medium (21) or Todd Hewitt yeast (THY) broth.
Escherichia coli strains (JM109 (22), BL21 (DE3) (23) with
and without the plasmid pLys-S (23)) were grown in TYPG (16 g of
tryptone, 16 g of yeast extract, 5 g of NaCl, 2.5 g of
K2PO4, and 5 g of glucose/liter of
H2O) or LB broth as indicated. Plasmids used in this study
include pBluescript (Stratagene; La Jolla, CA), pFW6 (24), pET-11a
(25), pAM620 (26), and pDL278 (27).
Oligonucleotide primers used for PCR and sequencing reactions
-32P]dATP (3,000 Ci
mmol
1, 10 µCi µl
1; ICN; Costa Mesa,
CA). The DNA fragment was amplified for 35 cycles using the following
conditions: melting temperature (95 °C) for 30 s, annealing
temperature (53 °C) for 35 s, and extension temperature
(72 °C) for 25 s. After completion of amplification free
unlabeled radionucleotide was removed using a G-50 spin column (5' - 3', Inc.; Boulder, CO). The radioactivity of the DNA fragment was
measured in a liquid scintillation counter, and the DNA was stored at
70 °C until used.
-D-galactopyranoside. This culture was incubated
for 3 additional h, and a 1-ml sample was taken. The pre- and
postinduction samples were sedimented at 13,000 × g
for 10 s, and the pellet was suspended in 150 µl of TE. A
nonreducing sample buffer was added to the samples and boiled for 10 min in a water bath. Aliquots of each sample were removed and separated on a 10% SDS-polyacrylamide gel via electrophoresis (28) to confirm
expression of CsrR.
-D-galactopyranoside. The induced culture was grown for 3 h postinduction, and then the
cells were sedimented at 13,000 × g for 10 min. The
cells were suspended in 10 ml of the following buffer: 0.05 M NaH/KH2 phosphate buffer, pH 6.9, 0.15 M NaCl, 10 mM MgCl2, 5 mM dithiothreitol, and 10% glycerol. Cells were lysed with
a French press (12-14,000 p.s.i.), and the membranes were sedimented
at 30,000 × g. The supernatant was retained
(cytoplasmic extract) and stored at
70 °C. The protein
concentration was determined using the Bradford protein assay. For the
EMSA analyzing the binding activity of the CsrR, ammonium sulfate
precipitation was used as described by Scopes (34) to partially purify
CsrR from the E. coli cytoplasmic extracts. Each fraction
was dialyzed at 4 °C with the above buffer using Spectrapor®
membrane tubing (Spectrum Medical Industries, Los Angeles) with a
molecular mass cutoff of 12,000-14,000 kDa. Extracts were stored at
4 °C after dialysis.
80 to +140 (strain
B931) of hasA was used. To label the DNA fragment, the 220-bp fragment was digested with EcoRI, and the end was
labeled with the Klenow fragment in the presence of
[
-32P]dATP. Free, unlabeled radionucleotide was
removed using a G-50 spin column (5' - 3', Inc.). The DNA fragment was
purified by phenol chloroform extraction and concentrated via ethanol
precipitation. To ensure purity, the DNA fragment was electrophoresed
on a 5% acrylamide gel, excised, and eluted from the acrylamide in a
solution of 0.5 M ammonium acetate and 1 mM
EDTA, pH 8. Ethanol precipitation was used to concentrate the DNA
fragment after elution. The radioactivity of the DNA fragment was
measured in a liquid scintillation counter, and the DNA was stored at
70 °C until used.
RESULTS
Hyaluronic acid production by parent, transposon mutant, and
allelic exchange mutants
8 plaque-forming units/ml, and all transductants were
encapsulated. Southern analysis showed that the transductants had the
bands of the same size as those of the original B931EnTn916.
These results confirmed that the Tn916 insertion directly
affected the synthesis of hyaluronic acid in strain B931.
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Fig. 1.
Panel A, schematic of the
csrR/csrS locus. The location of the Tn916
insertion site is 220 bp upstream of the start codon of
csrR. The putative 10 and
35 sites of the possible
promoter for the csr locus are shown flanking the insertion
site. The striped area within csrS indicates the
1-kb region knocked out by the plasmid pFW6. The lines
marked by MCS 1 and MCS 2 indicate the regions
used to generate the deletion of csrS in pFW6. Panel
B, Southern blot confirming mutation of csrS.
HindIII-digested chromosomal DNA prepared from the wild type
strains (lanes 1, 3, and 5) and from
the
csrS mutants (lanes 2, 4, and
6) were assayed with the digoxygenin-labeled probe derived
from the region being targeted for allelic exchange. The lane
assignments are as follows: lane 1, B931; lane 2,
B931
csrS; lane 3, D471; lane 4,
D471
csrS; lane 5, GT8760; lane 6,
GT8760
csrS.
csrS) which contained two 1-kb
portions flanking the csrS deletion site (Fig. 1A). As shown, the region upstream of the area targeted for
allelic exchange included all of csrR and a portion of the
region upstream, whereas the region downstream included the last third
of csrS and extended approximately 500 bp 3'.
csrS was electroporated into
unencapsulated strains of GAS (B931, D471, and GT8760), and mutants
were selected for spectinomycin resistance and screened for hyaluronic
acid production. One encapsulated colony was isolated from strains B931, D471, and GT8760. However, there were approximately 20 unencapsulated transformants isolated for every one encapsulated.
Because the possibility existed that a single crossover event could
occur via integration of the entire plasmid into the chromosome without disrupting either csrR or csrS, genomic DNA
(digested with HindIII) from all transformants (encapsulated
and unencapsulated) was analyzed via Southern analysis using a
digoxigenin-11-dUTP-labeled DNA fragment containing the csrS
region that was inactivated. All unencapsulated transformants produced
a signal on a Southern blot, indicating that a single crossover event
had occurred and that the region had not been exchanged (data not
shown). However, the encapsulated transformants yielded no signal,
indicating that the targeted region was replaced successfully with the
spectinomycin resistance gene (Fig. 1B, lanes 2,
4, and 6). Chromosomal DNA from the wild type
strains tested displayed the same band observed in the unencapsulated
transformants (Fig. 1B, lanes 1, 3,
and 5). In addition, PCR analysis using primers specific for
the region being deleted in csrS yielded product with all
unencapsulated transformants but not with the encapsulated
transformants (data not shown).
csrS, D471
csrS, and
GT8760
csrS produced hyaluronic acid during exponential
phase of growth (50, 101, and 12 fg/cfu; respectively) compared with
the parent strains B931, D471, and GT8760 (<10 fg/cfu). When total
hyaluronic acid produced was measured (Table II), the mutant strains
B931
csrS, D471
csrS, and
GT8760
csrS produced 87, 176, and 10 µg/ml, whereas the
parent strains all produced <10 µg/ml. These results, therefore,
indicated that the product of csrS is involved in regulation
of the synthesis of hyaluronic acid in GAS.
csrS and GT8760
csrS. This difference
suggested that there are additional factors involved in regulating
capsule besides those encoded by the csr locus. Because it
was suggested recently by Alberti et al. (20) that there are
cis-acting elements that determine the strength of the
has operon promoter, the hypothesis was formed that the
difference observed in capsule production in the
csrS-inactivated strains may be caused by variations in the
has promoters of these strains. To test this hypothesis, the
promoter regions from the csrS-inactivated strains tested as
well as other GAS strains were sequenced and analyzed. As shown in Fig.
2, the cis-elements
(boldfaced with asterisks above) identified by
Alberti et al. (20) in the strong promoter of an M-type 18 (highly encapsulated) were found in unencapsulated strains of GAS. As
shown, strain B931 had the same cis-elements of the M18
promoter with the exception of an additional cytosine at position +4.
In addition, D471 does not have the M18 cis-elements (Fig.
2), but when csrS is inactivated in D471, a significant
amount of hyaluronic acid is produced by this strain (Table II).
Together these data suggest that there are variations in the
has operon promoters of GAS which do not correlate with the
variations in capsule production observed in the
csrS-inactivated strains.
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Fig. 2.
Sequences of the has
promoters of GAS. The has promoters of the
strains studied in this report were sequenced. The 10,
35, and
transcription start are marked and boldfaced. An
asterisk (*) indicates regions identified previously by
Alberti et al. (20) which influence the strength of the
has promoter. The black dots indicate areas
lacking nucleotides found in other has promoter sequences.
The underlined region in the row containing the sequence for
B931 is one of the regions protected by the binding of CsrR to this
region (see Fig. 6). The sequences for M13 and M3 are as reported by
Alberti et al. (20). The (+) or (
) indicates the presence
or absence of hyaluronic acid, respectively.
csrS knockout mutant did not show any binding
activity (Fig. 3, lanes 7-11). However, it was uncertain if
the binding activity observed with strain B931 was a result of CsrR
(the product of the first gene of the two-component system) binding or
another protein controlled by the csr locus.
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Fig. 3.
EMSA of the hasA promoter
and cytoplasmic extracts from strain B931. A radiolabeled 220-bp
DNA fragment derived from the hasA promoter ( 80 to +140)
was reacted with increasing amounts of cytoplasmic extracts from an
exponential phase culture of B931 (lanes 2-6) or
B931
csrS (lanes 7-11). The amount of extract
contained in each of the lanes is as follows: lane 1, 0 µg; lanes 2 and 7, 2 µg; lanes 3 and 8, 5 µg; lanes 4 and 9, 10 µg;
lanes 5 and 10, 15 µg; lanes 6 and
11, 30 µg.
csrS lost its activity was addressed. As seen in
other two-component systems (39), the phosphorylation of the response
regulator is important in determining its functionality. To determine
if phosphorylation of CsrR would increase its affinity and/or
specificity for the radiolabeled DNA fragment, CsrR was treated with
acetyl phosphate and then analyzed immediately for binding activity
using an EMSA. After treatment with acetyl phosphate, the binding
activity of the partially purified CsrR increased 50-100-fold over the activity obtained from the same amount of CsrR without treatment with
acetyl phosphate (Fig. 4, lanes
2-5). A gel shift was observed with non-acetyl phosphate-treated
extracts only after the minimal amount of extract needed for gel shifts
with acetyl phosphate was increased more than 25-fold (Fig. 4,
lane 9).
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Fig. 4.
EMSA of CsrR binding to the hasA
promoter with or without treatment with acetyl phosphate.
Increasing amounts of the partially purified CsrR were incubated in the
presence (lanes 2-5) or absence (lanes 6-9) of
acetyl phosphate and analyzed via an EMSA using the 220-bp
hasA promoter DNA fragment. The amount of CsrR contained in
each of the lanes is as follows: lane 1, 0 µg; lanes
2 and 6, 0.2 µg; lanes 3 and 7,
0.9 µg; lanes 4 and 8, 2.5 µg; lanes
5 and 9, 5.5 µg. The concentration of acetyl
phosphate used per reaction is 32 mM.
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Fig. 5.
EMSA of CsrR, treated with acetyl phosphate,
binding to the sequence of the hasA promoter in the
presence of a specific or nonspecific competitor. Each reaction
contains 0.3 µg of the partially purified CsrR treated with acetyl
phosphate. The ratio of cold competitor to the radiolabeled 220-bp
hasA promoter fragment in each lane is as follows:
lane 1, no protein or competitor; lane 2, protein
only; lane 3, 50:1 specific competitor; lane 4,
100:1 specific competitor; lane 5, 50:1 nonspecific
competitor; lane 6, 100:1 nonspecific competitor.
10, transcription start, and a portion of the coding region of the
NH2 terminus of hasA (Fig.
6A, lane 5). In
addition, areas of hypersensitivity were observed flanking portions of
these protected sites as well as the
35 site (lane 5).
These regions are shown schematically in Fig. 6B. CsrR not
treated with acetyl phosphate did not footprint (Fig. 6A,
lane 4), even at levels high enough to cause a gel shift (data not shown). Control extracts from E. coli did not
protect any regions regardless of the presence of acetyl phosphate
(Fig. 6A, lanes 2 and 3).
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Fig. 6.
Panel A,DNase I footprinting of the
noncoding strand of the 220-bp promoter region of hasA ( 80
to +142) using partially purified CsrR with or without acetyl
phosphate.In each reaction, 0.5 µg of extract was used. Each sample
was treated with 32 mM acetyl phosphate (lanes
1, 3, and 5) or without (lanes 2 and 4). The lane assignments are as follows: lane
1, free fragment; lanes 2 and 3, 35%
fraction of E. coli extracts without CsrR; lanes
4 and 5, partially purified CsrR. The solid
lines mark the regions that were protected by CsrR. The
arrows indicate regions of hypersensitivity. The sequence of
the noncoding strand of the hasA promoter is shown to the
right of the footprints. Panel B, the
hasA promoter showing the CsrR binding sites. The regions of
CsrR protection are shown with solid lines. The areas of
hypersensitivity are marked with the circle-head arrows. The
sequence of the promoter is of the coding strand. The
10,
35, and
transcription start are in boldface and labeled.
DISCUSSION
10 transcription start site and portions of the coding region of hasA.
Therefore, this report demonstrates that the regulation of hyaluronic
acid production is dependent on both genes encoded in the
csr locus and that CsrR will bind to key regions of the
has operon promoter after it is treated with a phospho-donor
such as acetyl phosphate or perhaps phosphorylated by CsrS.
10 and
35 sites
upstream of csrR and csrS in the encapsulated
mutant GAS strain B931EnTn916. As described in this report,
the inactivation of csrS alone led to hyaluronic acid
production in three different strains of GAS (Table II). Recently,
other evidence has been reported which demonstrates the presence of
Tn916 upstream of the two csr genes or that the inactivation of csrR alone results in a loss of the mRNA
encoding the two genes and an increase in the production of the
has operon mRNA (36, 37). Therefore, one can conclude
that both genes, csrR and csrS, are important in
regulating capsule gene expression. Because the COOH terminus of
csrS shows high homology with histidine kinases and its
inactivation alone leads to hyaluronic acid production, a functional
CsrS may regulate hyaluronic acid production by phosphorylating CsrR
and/or other proteins involved in regulation of hyaluronic acid production.
csrS to bind the DNA fragment derived from the has promoter suggests that CsrS is required for a
protein(s) to bind to this region (Fig. 3). This loss of binding
activity along with the increases in hyaluronic acid production
suggests that CsrS may control the activity of a repressor. Because
csrR had high homology to other genes for response
regulators in the OmpR subfamily of response regulators which bind to
specific regions within targeted promoters, CsrR was partially purified
and assayed for its ability to bind to the hasA promoter
fragment. This report demonstrates that CsrR is capable of binding the
promoter region of hasA (Fig. 4). In addition, a significant
increase in the affinity and specificity of CsrR for key regions within
and around the promoter of hasA was observed after CsrR was
treated with the phospho-donor acetyl phosphate. Although it was not
demonstrated in this report, this increase in binding activity after
treatment of CsrR with acetyl phosphate indicates that CsrR was
phosphorylated and may have caused conformational changes in CsrR which
increase its binding activity. This hypothesized dependence of CsrR to be phosphorylated in order for it to bind to the has
promoter is supported by the loss of gel shift activity observed in the CsrS deletion mutant (Fig. 3). However, it was not determined if the
loss of binding activity in the CsrS deletion mutant was caused by a
loss of the phosphorylation of CsrR. Nevertheless, taken together, this
evidence strongly suggests that a phosphorylated CsrR can repress the
has operon (Figs. 4-6). Therefore, CsrS may phosphorylate
CsrR in vivo when the appropriate signals are received allowing for CsrR-Pi to bind to the promoter region of
hasA.
10, around the transcription start site, and portions of the coding region as well as the observation of areas of
hypersensitivity at the
35 site demonstrates that multiple sites
could be bound by CsrR. Whether or not these sites are bound all at
once by CsrR with varying amounts of CsrR is unknown. In addition, the
number of sites bound by the protein as well as the observation that increased amounts of this protein directly decreased the mobility of
the promoter fragment in the EMSA suggests that complexes consisting of
multimers of CsrR are binding the promoter. This additive property of
the binding interaction indicates that the level of expression could be
tightly regulated allowing for varying degrees of expression of the
has operon.
csrS showed a two-fold increase over the amount of
hyaluronic acid produced by strain B931
csrS and a
10-20-fold increase over the amount of hyaluronic acid produced by
GT8760
csrS. However, as shown in Fig. 2, these variations
in capsule production could not be linked to differences in
cis-acting elements identified previously by Alberti
et al. (20) to influence capsule production. In addition, Alberti et al. (20) stated in their report that their
evidence suggested that trans-acting factors were likely
influencing the activity of the has promoters. Another
factor involved is the signal received by the sensor to regulate the
production of hyaluronic acid. The possibility exists that strains of
GAS may experience conditions in vivo promoting the need for
the expression of the hyaluronic acid capsule which are not found
in vitro. Levin and Wessels (36) demonstrated that the
strains in which the csr locus or csrR alone was
inactivated were more virulent and less easily phagocytized in a murine
model than its parent strain. Although they admitted that they could
not rule out the possibility the products of the csr locus
were regulating other virulence factors besides hyaluronic acid, their
evidence certainly suggests that the factors affecting this locus are
relevant to the overall virulence of GAS.
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ACKNOWLEDGEMENTS |
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We are indebted to Kelley Bellomo for technical assistance and to Drs. Dan Wozniak, Ian Blomfield, Dinene Crater, Riccardo Manganelli, and Volker Nickel for support and thoughtful discussions.
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FOOTNOTES |
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* This work was supported in part by Public Health Service Grant AI37320 from the National Institutes of Health (to I. v. d. R.) and by Grant CA12107 from the Oligonucleotide Core Laboratory of the Comprehensive Cancer Center of Wake Forest University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF110765.
Recipient of Predoctoral Fellow Grant P98102N from the North
Carolina affiliate of the American Heart Association.
§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-2263; Fax: 336-716-4204; E-mail: ivr{at}wfubmc.edu.
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ABBREVIATIONS |
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The abbreviations used are: GAS, group A streptococci; THY, Todd Hewitt yeast broth; PCR, polymerase chain reaction; kb, kilobase(s); EMSA, electrophoretic mobility shift assay(s); bp, base pair(s); cfu, colony-forming units.
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REFERENCES |
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