Download Protein A gene expression is regulated by DNA supercoiling which

Microbiology (2004), 150, 3807–3819
DOI 10.1099/mic.0.27194-0
Protein A gene expression is regulated by DNA
supercoiling which is modified by the ArlS–ArlR
two-component system of Staphylococcus aureus
Bénédicte Fournier1 and André Klier2
Correspondence
Bénédicte Fournier
[email protected]
Received 29 March 2004
Revised 12 July 2004
Accepted 21 July 2004
1
Laboratoire des Listeria, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15,
France
2
Université Paris 7, UFR de Biochimie, 2 place Jussieu, 75005 Paris, France
Bacterial pathogens such as Staphylococcus aureus undergo major physiological changes
when they infect their hosts, requiring the coordinated regulation of gene expression in response
to the stresses encountered. Several environmental factors modify the expression of S. aureus
virulence genes. This report shows that the expression of spa (virulence gene encoding the
cell-wall-associated protein A) is down-regulated by high osmolarity (1 M NaCl, 1 M KCl or
1 M sucrose) in the wild-type strain and upregulated by novobiocin (a DNA gyrase inhibitor that
relaxes DNA). A gyrB142 allele corresponding to a double mutation in the B subunit of DNA
gyrase relaxed DNA and consequently induced spa expression, confirming that spa expression
is regulated by DNA topology. Furthermore, in the presence of novobiocin plus 1 M NaCl, a
good correlation was observed between DNA supercoiling and spa expression. The ArlS–ArlR
two-component system is involved in the expression of virulence genes such as spa. Presence
of an arlRS deletion decreased the effect of DNA supercoiling modulators on spa expression,
suggesting that active Arl proteins are necessary for the full effect of DNA gyrase inhibitors and
high osmolarity on spa expression. Indeed, evidence is provided for a relationship between
the arlRS deletion and topological changes in plasmid DNA.
INTRODUCTION
Staphylococcus aureus is a major human pathogen, responsible for a large number of nosocomial infections. The
pathogenesis of S. aureus has been attributed to its ability to
produce a diverse range of secreted proteins (haemolysins,
lipases, proteases) and cell-wall-associated proteins (protein
A and adhesins) (Novick, 2000; Projan & Novick, 1997).
Protein A (Spa) is a major surface protein of S. aureus
strains. The biological properties of Spa have been
extensively studied. It has the ability to interact with several
host components (immunoglobulins G, A and E, platelets,
von Willebrand factor), suggesting a role as a virulence
factor in S. aureus infections (Cheung et al., 1997; Hartleib
et al., 2000). In several animal models (peritonitis, subcutaneous model, mastitis and arthritis), the isogenic spa2
mutant causes less severe infections than the wild-type
strain (Hartleib et al., 2000; Palmqvist et al., 2002). The
binding of protein A to IgG antibodies is involved in evading the immune response, perhaps by preventing opsonization (Projan & Novick, 1997).
The regulation of the production of virulence determinants
such as Spa in S. aureus involves several global regulatory
loci. The agr (Morfeldt et al., 1988; Peng et al., 1988) and
0002-7194 G 2004 SGM
sarA (Cheung et al., 1992) loci are the best characterized of
these loci. Several other regulatory loci have also been
described: sar homologues (Cheung et al., 2001; Manna &
Cheung, 2001, 2003; McNamara et al., 2000; Schmidt et al.,
2001; Tegmark et al., 2000), sae (Giraudo et al., 1994), arl
(Fournier et al., 2001) and srr (Yarwood et al., 2001). In
S. aureus, virulence determinant production is modulated in
response to growth conditions (Chan & Foster, 1998a, b;
Lindsay & Foster, 1999; Novick, 2000), reflecting the ability
of S. aureus to adapt and to survive in many different environmental niches. The mechanism by which the
external signals are transduced to modify gene expression is
crucial for understanding how S. aureus adapts to its host.
This adaptation often involves two-component systems,
generally consisting of a sensor (histidine kinase) and a
response regulator (Stock et al., 1989). The sensor is an
integral membrane protein that becomes phosphorylated
in response to environmental signals. The phosphate is
then transferred to a conserved residue in the response
regulator. The response regulator is often a transcription
factor, the affinity of which for DNA is modulated by
phosphorylation (Stock et al., 1989). To date, four different
two-component systems that control virulence have been
identified in S. aureus: Agr, Sae, Srr and Arl. The twocomponent system ArlS–ArlR is involved in several cell
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B. Fournier and A. Klier
activities. Production of a multidrug-resistance efflux
pump, NorA, was increased in an arlS transposon insertion
mutant (Fournier et al., 2000). The arlS mutant formed
a biofilm on polystyrene surfaces, probably because of
altered activity of secreted peptidoglycan hydrolases
(Fournier & Hooper, 2000). The arl locus is also involved
in the regulation of several virulence factors, mainly protein
A, and some secreted proteins (a-toxin, b-haemolysin,
coagulase, lipase). The arl locus probably exerts its effects
on virulence factors mostly via the agr and/or sarA regulatory pathway (Fournier et al., 2001).
Bacterial DNA is maintained in a negatively supercoiled
state, which favours DNA recombination, replication, transcription and transposition. As the level of DNA supercoiling varies with the cellular energy charge, it can change
rapidly in response to nutritional and environmental conditions. Several independent studies have provided evidence
that DNA supercoiling plays a role in the transduction of
environmental signals to the bacterial nucleoid. In particular, changes in temperature, osmolarity and oxygen
availability have been shown to change DNA supercoiling
(Dorman, 1991; Hsieh et al., 1991). In Escherichia coli, it
has been estimated that roughly 50 % of the supercoiling
is constrained by proteins bound to DNA. The remaining
supercoils are maintained actively at the cost of ATP
hydrolysis, via topoisomerase activities (Hatfield & Benham,
2002). Thus, in E. coli, factors responsible for modifying
DNA supercoiling fall into three general categories: those
affecting DNA topoisomerases, those altering DNA structure, and those influencing cellular energetics (Drlica, 1992).
In this study, we showed that protein A gene expression is
regulated by DNA supercoiling in response to environmental
conditions such as high osmolarity, and that the Arl system
may interact with the mechanism by which modulators of
DNA topology (high osmolarity or DNA gyrase inhibitors)
control spa expression. We also provide evidence for a
relationship between the arlRS deletion and topological
changes in plasmid DNA conformation.
METHODS
Bacterial strains and plasmids. The bacterial strains and
plasmids used in this study are listed in Table 1. Staphylococci were
grown in trypticase soy broth (TSB) at 37 uC. Cultures of 150 ml in
500 ml flasks were inoculated at a starting OD600 value of 0?025
prior to growth at 37 uC. When required, antibiotics were added
at the following concentrations: 7 mg chloramphenicol l21; 20 mg
erythromycin l21; 10 mg kanamycin l21; 3 mg tetracycline l21.
DNA transformation. Plasmid DNA was isolated and used to
transform S. aureus by electroporation as previously described
(Novick, 1991). Chromosomal DNA was isolated from S. aureus as
described by Stahl & Pattee (1983). Transformation with high
molecular mass chromosomal DNA was performed as previously
described (Stahl & Pattee, 1983).
Plasmid construction. A transcriptional spa : : lacZ reporter fusion
had previously been constructed by cloning PCR fragments into
pBF50. pBF50 is an integrative promoterless transcriptional vector
(Table 1) (Fournier et al., 2001). It contains the attP site of staphylococcal phage L54a and is capable of integrating specifically into
the chromosomal attB site in the geh gene, which encodes lipase
(Lee et al., 1991).
b-Galactosidase activity. b-Galactosidase activity was determined
as previously described (Fournier et al., 2001). Briefly, cells were
assayed for b-galactosidase (LacZ) activity using the chemiluminescent
reporter assay system (Galacton-Light Plus, Applied Biosystems).
Bacterial cells were grown to different OD600, harvested, washed and
resuspended in lysis buffer supplemented with 50 mg lysostaphin ml21
Table 1. Strains and plasmids used in this study
Em, erythromycin; Tet, tetracycline; Km, kanamycin; Ap, ampicillin.
Strain/plasmid
Strains
RN4220
RN6390BF
BF21
MT5
BF41
BF42
Plasmids
pE194
pT181
pUB110
pBF50
pBFSpa
3808
Genotype/description
8325-4, nitrosoguanidine-induced restriction mutant used as a
primary recipient for plasmids propagated in E. coli
Wild-type derivative of 8325-4
RN6390BF arlR : : cat
8325 gyrB142 hisG15 pig-131
RN6390BF gyrB142
RN6390BF arlR : : cat gyrB142
3?7 kb S. aureus plasmid, Emr
4?4 kb S. aureus plasmid, Tetr
4?5 kb S. aureus plasmid, Kmr
12 kb shuttle promoterless transcriptional lacZ fusion vector carrying
the attP site of phage L54a; Apr (E. coli), Tetr (S. aureus)
300 bp fragment containing the spa promoter cloned upstream of
the lacZ gene of pBF50
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Reference
Kreiswirth et al. (1983)
Fournier et al. (2001); Peng et al. (1988)
Fournier et al. (2001)
Fournier & Hooper (1998)
This study
This study
Shivakumar et al. (1980)
Khan & Novick (1983)
Jalanko et al. (1981)
Fournier et al. (2001)
Fournier et al. (2001)
Microbiology 150
DNA supercoiling in S. aureus
60 000
40 000
20 000
0.5
1
1.5
2
2.5
3
OD600
)
(b)
M
TSB medium with appropriate antibiotics to an OD600 of 0?25–2?0.
After centrifugation, cell pellets were stored at 220 uC. Cells were
thawed and plasmid DNA extracted. Plasmid DNA was separated on
1 % agarose gels containing 80 or 140 mg ml21 chloroquine, a DNA
intercalator, in TBE buffer (90 mM Tris/borate, 2 mM EDTA).
Chloroquine was removed by soaking the gels in distilled water for
at least 4 h before staining with ethidium bromide (3 mg ml21). The
gels were scanned and subjected to densitometry using National
Institutes of Health Image, a public domain image-processing
program. The experiment was repeated three to four times and a
representative set of values is shown.
80 000
N
o
N
N aC
aC l
l(
1
Plasmid supercoiling analysis. Strains were grown at 37 uC in
(a)
b-Galactosidase activity
(RLU per g protein)
and 15 mg DNase ml21. The mixture was incubated for 30 min
at 37 uC, and the b-galactosidase and protein concentrations of
the supernatant were then determined. b-Galactosidase activity
is expressed in relative light units (RLU). Assays were performed
on duplicate samples and the mean values were determined. The
results presented here are representative of at least two independent
experiments.
Protein A analysis. Cultures were grown to an OD600 of 2?0
and centrifuged. Cell-wall-associated proteins were extracted with
lysostaphin in a hypertonic medium (30 % raffinose), as described
previously (Cheung & Fischetti, 1988; Fournier et al., 2001). Briefly,
cell-wall-associated proteins (30 mg) were resolved on a 10 % SDSpolyacrylamide gel, electroblotted onto nitrocellulose Hybond-C
Pure and probed with rabbit anti-staphylococcal protein A antibody
(Sigma) at a 1 : 15 000 dilution. Bound antibody was detected with
donkey anti-rabbit immunoglobulin G conjugated to peroxidase
(1 : 20 000) and the ECL Western blotting detection system
(Amersham). Quantification of signals from Western blots was
performed by densitometric analysis of the autoradiograms using
NIH Image. Arbitrary units (AU) correspond to the integrated
density measured by the program. The experiment was performed
twice and a representative set of values is shown.
Increasing negative
supercoiling
(c)
Increasing negative supercoiling
Measurement of transformation efficiency. pT181 and pE194
were extracted from strain RN4220 and the same amount of DNA
was then transformed into different strains by electroporation.
Bacteria were grown on TSA plates containing either tetracycline
(pT181) or erythromycin (pE194). The competence of each S. aureus
recipient was assessed by counting the number of transformants.
Data are representative of at least four independent determinations.
No NaCl
RESULTS AND DISCUSSION
NaCl (1 M)
Repression of spa expression in the presence
of high osmolarity
Several studies have demonstrated that NaCl, sucrose,
alkaline pH, glucose, temperature and several ions modify
the expression of virulence genes (Chan & Foster, 1998a, b;
Regassa & Betley, 1992; Regassa et al., 1992). A transcriptional spa : : lacZ fusion was previously inserted as a single
copy into the chromosome of the wild-type strain
RN6390BF (Fournier et al., 2001).
The presence of 1 M NaCl dramatically repressed spa
expression (Fig. 1a). The expression level of spa was about
four- to sevenfold lower in the presence of 1 M NaCl
than in its absence (Fig. 1a). To determine whether NaCl
repression is due to a general osmotic stress effect or
specifically to sodium or chloride ions, we studied the
effect on spa transcription of other osmolytes: another
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Fig. 1. Effect of high osmolarity on the expression of the
spa : : lacZ fusion (a) and on plasmid DNA supercoiling (b, c).
(a) Expression of the spa : : lacZ fusion in the wild-type strain
(RN6390BF) in the presence of different osmolytes: 1 M NaCl
($), 1 M KCl (%) and 1 M sucrose (&); #, absence of osmolyte. b-Galactosidase activity is expressed in relative light units
(RLU) per g protein. OD600, rather than time, was plotted on
the x axis because the presence of certain additives slows
down growth. (b) Plasmid DNA supercoiling in the absence
and presence of 1 M NaCl. Strain RN6390BF (pE194) was
grown to an OD600 of 2?0. After plasmid extraction, topoisomers were separated in agarose gels containing 80 mg chloroquine ml”1 in Tris/borate buffer (in these conditions, less
negatively supercoiled topoisomers migrate more rapidly).
(c) Scan analysis of topoisomer distribution in the plasmids
separated in the gel shown in (b).
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3809
B. Fournier and A. Klier
ionic osmolyte similar to NaCl (KCl) and a non-ionic
osmolyte (sucrose). Sucrose (1 M) and KCl (1 M) similarly repressed spa expression (Fig. 1a). Thus, the repression effect on spa is likely specific for the osmotic stress
response.
Expression of several virulence genes of S. aureus, such
as those encoding a-toxin, protein A and toxic-shock
syndrome toxin-1, have been shown to be dependent on
osmolarity (Chan & Foster, 1998a, b; Regassa & Betley,
1992; Regassa et al., 1992). One of the distinguishing
characteristics of S. aureus is its considerable tolerance of
NaCl. S. aureus is the most halotolerant, non-halophilic
eubacterium known. It is generally accepted that the lowest
water activity that allows the growth of this organism is
0?86 (equivalent to 3?5 M), and the reduction of water
activity is commonly used as a method of food preservation. This organism is probably the most common cause
of food-poisoning outbreaks (Graham & Wilkinson, 1992).
(b)
(a)
It is also noteworthy that the pathogenicity of S. aureus
involves the colonization of a high-salt environment, the
mammalian skin. Thus, response to NaCl could be an
important factor for adaptibility and pathogenicity in S.
aureus.
Effect of high osmolarity on DNA supercoiling
High osmolarity alters the degree of plasmid DNA supercoiling (Dorman, 1991). Thus, we examined the effect of
high osmolarity on the topoisomer distribution of pE194
(Shivakumar et al., 1980) isolated from RN6390BF. Strains
were grown at 37 uC in TSB medium with appropriate
antibiotics to an OD600 of 2?0 and plasmid DNA extracted.
As expected, plasmid preparations isolated from cells
grown in high-osmolarity medium contained more supercoiled topoisomers than those grown in low-osmolarity
conditions (Fig. 1b, c).
Increasing negative supercoiling
_
Novobiocin (ng ml 1)
5
20 40
Increasing negative supercoiling
0
(c)
Novobiocin
concentration
_
(ng ml 1)
0
_
Novobiocin (ng ml 1)
0
40
5
Relaxed
Supercoiled
20
40
3810
Fig. 2. Effect of novobiocin on plasmid
DNA supercoiling. (a) Effect of novobiocin
on pE194 supercoiling in the wild-type
strain; the concentration of chloroquine in
the gel was 80 mg ml”1. The numbers above
the lanes indicate the concentration of novobiocin (5, 20 and 40 ng ml”1) in the culture
medium. (b) Scan analysis of topoisomer
distribution in the plasmids separated in the
gel shown in (a). (c) Effect of novobiocin on
pE194 supercoiling in the wild-type strain;
the concentration of chloroquine in the gel
was 7 mg ml”1.
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Microbiology 150
DNA supercoiling in S. aureus
As the expression of the protein A gene is dramatically
repressed by high salt concentrations (1 M NaCl) and as
high osmolarity alters the degree of plasmid DNA supercoiling, we looked at the effect of changing the degree of
DNA supercoiling on protein A gene expression. We first
determined the concentration of novobiocin, a DNA gyrase
inhibitor, which relaxes DNA. Strain RN6390BF containing
pE194 was grown at 37 uC in TSB medium containing
different concentrations of novobiocin and the topoisomer
distribution of pE194 was examined. No differences in
DNA supercoiling were observed between the peak of
distribution of plasmids from bacteria grown without
antibiotic and those grown with 5 ng novobiocin ml21
(Fig. 2a, b). However, DNA relaxed slightly with 20 ng
novobiocin ml21 and even more at 40 ng novobiocin
ml21 (Fig. 2a, b). With a lower concentration of chloroquine (7 mg ml21) in the gel, we also observed an increase
of relaxed DNA (Fig. 2c). Thus, as expected, 20 and 40 ng
novobiocin ml21 resulted in plasmid relaxation.
We then tested the effect of novobiocin on the expression of
the protein A gene in cells harbouring the spa : : lacZ fusion.
The wild-type strain was grown in TSB medium in the
absence and presence of novobiocin. spa expression was
similar in the absence and presence of 40 ng novobiocin
ml21 at the beginning of the exponential phase (OD600 0?1)
(Fig. 3a). Subsequently, novobiocin induced spa expression
at all growth stages. At the end of the exponential phase
(OD600 2?0), spa expression was 26-fold higher when
bacteria were grown with 40 ng novobiocin ml21 (Fig. 3a).
The highest concentration of novobiocin used (40 ng
ml21) corresponds to one-quarter of the minimal inhibitory concentration (160 ng ml21), and did not affect the
growth rate (data not shown). Thus, the novobiocininduced increase in protein A gene expression was not
simply due to alterations in the growth rate. We also found
that the level of production of the cell-wall-associated
protein A was much higher (15-fold) in the presence of
novobiocin than in its absence (Fig. 3b).
Effect of gyrB mutations on DNA supercoiling
and protein A gene expression
DNA supercoiling is regulated by several enzymes. DNA
gyrase is the bacterial topoisomerase that introduces
negative supercoils into DNA by using the free energy
created by ATP hydrolysis. This enzyme consists of two
proteins, A and B; the active enzyme is an A2B2 complex. The
A protein (GyrA) is responsible for the breakage-reunion
of DNA (N-terminal domain) and for the DNA–protein
interactions (C-terminal domain). The N-terminal domain
of the B protein contains the ATPase activity. Coumarins
such as novobiocin or coumermycin inhibit the ATPase
reaction (Maxwell, 1993). In S. aureus, a gyrB142 allele
corresponding to a double mutation (Ile102Ser and
http://mic.sgmjournals.org
(a)
b-Galactosidase activity
(RLU per g protein)
Protein A gene expression is modified by
novobiocin
400 000
300 000
200 000
100 000
0.5
1
1.5
2
2.5
3
OD600
(b)
_
Novobiocin (ng ml 1)
0
40
0.9
14
AU per µg protein
Fig. 3. Effect of novobiocin on the expression of the spa : : lacZ
fusion and the production of protein A. (a) Expression of spa in
the absence (#) and presence ($) of 40 ng novobiocin ml”1
in the wild-type strain RN6390BF. b-Galactosidase activities
were measured as in Fig. 1. (b) Western blot analysis of protein A in cell-wall-associated extracts of the wild-type strain
RN6390BF grown to an OD600 of 2?0 in the absence and
presence of 40 ng novobiocin ml”1. Quantification of signals
from Western blots performed by densitometric analysis (AU,
arbitrary units) is indicated below the corresponding figure.
Arg144Ile) confers a high level of resistance to novobiocin
(Fournier & Hooper, 1998). These mutations are located
in the N-terminal domain responsible for the ATPase
activity. The Arg144 residue in the S. aureus protein is at
the same position as Arg136 in the E. coli homologue. DNA
supercoiling activities of the mutants Arg136-His, -Cys and
-Ser are reduced to a greater or lesser extent relative to the
wild-type enzyme (Contreras & Maxwell, 1992). Thus, it
is assumed that, similarly to what is observed in E. coli,
the presence of the gyrB142 allele of S. aureus will induce
DNA relaxation by modifying the ATPase activity. We
determined the effect of the gyrB142 double mutation
(Ile102Ser and Arg144Ile) (Fournier & Hooper, 1998) on
DNA supercoiling. We analysed the topoisomer distribution of plasmid preparations from the wild-type strain
and the gyrB142 mutant (Fig. 4a, b). The topoisomer
distribution of pE194 in the wild-type strain differed
from that in the gyrB142 mutant, ranging from two to
three shifts (Fig. 4a, b). Thus, in S. aureus strains carrying
the gyrB142 allele, a relaxation of plasmid DNA was
observed. Therefore, we used this mutant to study the
relationship between DNA supercoiling and spa expression in S. aureus.
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3811
B
B. Fournier and A. Klier
The transcriptional spa : : lacZ fusion was introduced into
S. aureus BF41, a derivative of strain RN6390BF carrying
the gyrB142 allele on the chromosome (Table 1). The
introduction of the gyrB142 allele into the wild-type strain
resulted in a significant increase (sevenfold) in protein A
gene expression at the beginning of the exponential phase
(OD600 0?1) (Fig. 4c). This sevenfold increase was observed
until an OD600 of 0?5. At the end of the exponential phase
(OD600 2?0), no significant difference in spa expression was
observed between the wild-type strain and its gyrB142
mutant (Fig. 4c). These results indicate that the gyrB142
allele increases protein A gene expression mainly during
the exponential phase. As this allele also induces DNA
relaxation, these results reinforce the hypothesis that there
is a relationship between DNA relaxation and an increase
in protein A production. Although we measured DNA
supercoiling using plasmid DNA, the fact that novobiocin
also affected expression from the supercoiling-sensitive
chromosomal spa promoter strongly suggests a similar
change in chromosomal DNA supercoiling. Thus, this result
strongly suggests that protein A gene expression is regulated
by DNA supercoiling in the wild-type background.
gy
r
W
T
(a)
Increasing negative
supercoiling
(b)
Increasing negative supercoiling
WT
Both the presence of novobiocin and the gyrB142 allele
induce DNA relaxation. However, spa expression is differently modified by these two DNA supercoiling modulators.
In the presence of novobiocin, expression of spa was mainly
increased at the end of the exponential phase (Fig. 3a),
whereas in the presence of the gyrB142 allele, spa expression was mainly increased at the beginning of the exponential phase (Fig. 4c). Novobiocin has to be added to the
culture medium before it can enter the cell. Conversely, the
gyrB142 allele is already efficient in the cell at the beginning
of the growth. This also suggests that the modification of
DNA supercoiling is dependent on the growth phase, as spa
expression was not modified at the end of the exponential
phase with the gyrB142 allele (Fig. 4c).
gyrB
(c)
b-Galactosidase activity
(RLU per g protein)
600 000
500 000
400 000
300 000
Combined effect of high osmolarity and
novobiocin on spa expression and DNA
supercoiling
200 000
100 000
0.5
1
1.5
OD600
2
2.5
3
Fig. 4. Effect of the gyrB142 allele on plasmid DNA supercoiling (a, b) and on the spa : : lacZ fusion (c). (a) Effect of the
gyrB142 allele on pE194 supercoiling. Chloroquine gel analysis
was performed as in Fig. 1b, except that bacteria were grown
to an OD600 of 0?5. (b) Scan analysis of topoisomer distribution in the plasmids separated in the gel shown in (a). (c)
Expression of spa in the wild-type strain (RN6390BF, #) and
gyrB142 mutant (BF41, $). b-Galactosidase activities were
measured as in Fig. 1. WT, Wild-type.
3812
To observe the combined effect of high osmolarity and
novobiocin on protein A gene expression, the wild-type
strain harbouring the spa : : lacZ fusion was grown in TSB
medium containing 1 M NaCl in the presence and absence
of novobiocin. In parallel, we also determined the DNA
topological state of pE194. The concentration of novobiocin used was lower (10 ng ml21) in the presence of NaCl
than in its absence (40 ng ml21) because cells cannot grow
with a higher concentration.
Addition of 10 ng novobiocin ml21 in the presence of 1 M
NaCl increased protein A gene expression by up to fourfold
at the end of the exponential phase (OD600 2?0) (Fig. 5a).
Thus, we determined the effect of novobiocin on NaClinduced DNA supercoiling (Fig. 5b, c). Increasing concentrations of novobiocin relaxed plasmid DNA, even in the
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Microbiology 150
DNA supercoiling in S. aureus
(a)
(c)
Increasing negative
supercoiling
b-Galactosidase activity
(RLU per g protein)
80 000
60 000
40 000
20 000
No NaCl
0.5
1
1.5
2
2.5
3
OD600
NaCl (1 M)+
_
0 5 10 ng novobiocin ml 1
Increasing negative supercoiling
No NaCl
(b)
1 M NaCl
1 M NaCl+
_
5 ng novobiocin ml 1
1 M NaCl+
_
10 ng novobiocin ml 1
presence of high concentrations of NaCl. This result
correlates well with the induction of spa expression when
novobiocin was added to the medium. Furthermore, spa
expression of bacteria grown in 1 M NaCl and 10 ng
novobiocin ml21 was lower than that of bacteria grown
in the medium alone (Fig. 5a). The peak of distribution of
plasmids from bacteria grown with NaCl and novobiocin
was more negatively supercoiled than that from bacteria
grown in the medium alone (Fig. 5b, c). Thus, there is
a good correlation between DNA supercoiling and spa
expression.
We found that the expression of spa was repressed by high
osmolarity and induced by increasing concentrations of
novobiocin. Furthermore, the presence of the gyrB142
allele also increased protein A gene expression. Thus, protein A gene expression appears to be at least regulated by
DNA supercoiling. Previous studies on S. aureus (Chan &
Foster, 1998a; Lindsay & Foster, 1999) have suggested that
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Fig. 5. Effect of combined high osmolarity
and novobiocin on spa expression (a) and
DNA supercoiling (b, c). (a) Expression of
the spa : : lacZ fusion in different conditions:
#, absence of NaCl; $, 1 M NaCl; %, 1 M
NaCl and 10 ng novobiocin ml”1. Bacteria
were grown as in Fig. 1. b-Galactosidase
activity was measured as in Fig. 1. (b)
Plasmid DNA supercoiling in the same conditions. Chloroquine gel analysis was performed as in Fig. 1. (c) Scan analysis of
topoisomer distribution in the plasmids separated in the gel shown in (b).
the expression of the genes encoding the a-toxin (hla), the
toxic shock syndrome toxin-1 (tst) and protein A (spa) is
not regulated by DNA supercoiling because the expression
of these genes is not affected by 0?5 ng novobiocin ml21.
This novobiocin concentration is much lower than that
used here. We found that 1?2 ng novobiocin ml21 did not
modify protein A gene expression or DNA supercoiling.
Thus, we speculate that the discrepancy observed between
the two studies is due to the novobiocin concentrations
used. However, another gene (eta) is strongly induced
when S. aureus is grown in the presence of subinhibitory
concentrations of novobiocin (Sheehan et al., 1992). These
results suggest that the regulation of virulence genes such
as spa and eta is dependent on DNA supercoiling.
DNA superhelicity is known to exert a wide variety of effects
on prokaryotic gene expression. Increasing or decreasing
DNA supercoiling modifies the transcription initiation
reaction of certain promoters and has consequences for
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B. Fournier and A. Klier
other topological perturbations of DNA (e.g. looping or
bending) that affect transcription (Dorman, 1991). The
relative abundance for 88 proteins has been measured in
E. coli strains carrying non-lethal mutations in genes
encoding topoisomerase I or gyrase (which raise and
lower the level of supercoiling, respectively). The abundances of many proteins are maximal at wild-type supercoiling levels, whereas others exhibit maximal abundance
at relaxed levels of DNA supercoiling and a smaller number
show maximal abundance at elevated levels of DNA supercoiling (Steck et al., 1993). RNA polymerase-mediated
initiation of transcription requires the transient melting
of the DNA duplex at the transcription initiation start site.
The untwisting stresses imposed by negative supercoiling
can facilitate this energetically costly process, which can,
in turn, increase the rate of the transcription intitiation
reaction (Sheridan et al., 2001). However, similarly to gyrA
expression in E. coli, spa expression in S. aureus is repressed
by elevated levels of DNA supercoiling. It has been suggested
that the DNA supercoiling-mediated repression of transcription from the gyrA promoter is facilitated by the
superhelical realignment of specific promoter recognition
elements into an unfavourable arrangement, either through
DNA duplex twisting or through bending (Sheridan et al.,
2001; Wang & Syvanen, 1992). The protein A gene is
expressed during the exponential phase and it is transcriptionally down-regulated as cells enter the post-exponential
phase (Fig. 1a) (Projan & Novick, 1997). Two regulators,
SarS and SarA, have been shown to directly bind to the spa
promoter (Chien et al., 1999; Tegmark et al., 2000). SarS is
an activator, whereas SarA is a repressor of spa expression.
During the exponential phase of growth, there is optimal
expression of SarS, which binds to the SarS binding site
and displaces the SarA repressor complex from the spa
promoter. When cells enter the post-exponential phase,
SarS levels fall. Therefore, SarA occupies binding sites and
spa transcription is inhibited (Gao & Stewart, 2004).
Furthermore, binding of Sar proteins can alter the shape
of DNA, inducing bending or overwinding of DNA
(Schumacher et al., 2001). Since spa transcriptional
regulation involves several regulatory sites, it is possible
that negative superhelical tension similarly affects the spa
promoter of S. aureus and the gyrA promoter of E. coli.
Effect of the Arl system on the regulation of
spa expression by DNA supercoiling
The Arl system regulates protein A gene expression through
the sarA locus (Fournier et al., 2001). Thus, we studied the
combined effect of the arl mutation and the modulation of
DNA supercoiling by high osmolarity, novobiocin and the
gyrB142 allele on spa expression. We analysed protein A
gene expression in the arlRS mutant in the presence and
absence of novobiocin. At the beginning of the exponential
phase (OD600 0?1), spa expression in the presence of 40 ng
novobiocin ml21 was less than twofold lower than that in
the absence of novobiocin. The presence of novobiocin
increased protein A gene expression by 10-fold in the arlRS
3814
mutant at the end of the exponential phase (OD600 2?0)
(Fig. 6a). The presence of the gyrB142 mutation in the
arlRS mutant slightly increased (2?5-fold) protein A gene
expression at the beginning of the exponential phase (OD600
0?1) (Fig. 6b). At higher OD600, the gyrB142 mutation in
the arl mutant altered spa expression by less than twofold.
Thus, spa expression is not induced by the gyrB mutation
in the arl mutant background, whereas it is induced in the
wild-type background. We also analysed spa expression in
the arlRS mutant in the presence of high osmolarity. In the
arlRS null mutant, the expression of spa was less than
twofold lower in the presence of 1 M NaCl (or 1 M KCl
and 1 M sucrose) than in its absence at all growth stages
(Fig. 6c). Thus, spa expression was dramatically repressed
by high osmolarity in the wild-type strain but not in the
arl mutant.
spa expression in the presence of novobiocin was increased
by 10-fold in the arl mutant and by 26-fold in the wildtype strain. Similarly, the gyrB142 mutation, which increased spa expression in the wild-type strain (sevenfold), did
not modify spa expression to the same extent in the presence of the arl mutation (2?5-fold). Furthermore, in the
presence of 1 M NaCl, spa expression was decreased in the
wild-type strain (four- to sevenfold), whereas it was
unchanged in the arl mutant (less than twofold). Thus,
absence of arlRS decreases the efficiency of DNA supercoiling modulators by a factor of two- to threefold,
suggesting that active arl genes are necessary for the full
action of DNA gyrase inhibitors and high osmolarity on
spa expression. It seems that the Arl system interacts with
the mechanism by which modulators of DNA topology
(high osmolarity or inhibitors of DNA gyrase) control spa
expression. Increased DNA supercoiling due to salt shock
is largely prevented by a concentration of coumermycin
that had no detectable effect on supercoiling in the absence
of salt, suggesting that gyrase is the source of the increased
supercoiling associated with salt shock (Hsieh et al., 1991).
When E. coli was shifted from a medium lacking salt to one
containing 0?5 M NaCl, both the [ATP]/[ADP] ratio and
negative supercoiling of plasmid DNA increased within a
few minutes (Hsieh et al., 1991). Since in vitro the [ATP]/
[ADP] ratio influences the level of supercoiling generated
by gyrase (Westerhoff et al., 1988), the physiological response of supercoiling to salt shock could be explained by
the sensitivity of gyrase to changes in the intracellular
[ATP]/[ADP] ratio (Hsieh et al., 1991; Jensen et al., 1995).
Thus, DNA gyrase activity is essential for the transmission
of the osmotic signals that change DNA supercoiling levels
in bacteria (Bhriain et al., 1989). Furthermore, novobiocin
and the gyrB142 allele inhibit the ATPase activity of the
DNA gyrase. These observations suggest that DNA gyrase
is probably an important enzyme in the modulation of spa
expression. The Arl system may act in different ways. It is
possible that the Arl system acts indirectly by modulating
the expression of several genes in response to modification
of DNA supercoiling. Alternatively, the arl mutation itself
may affect DNA supercoiling.
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Microbiology 150
DNA supercoiling in S. aureus
Fig. 6. Effect of the arlRS mutation on the spa : : lacZ fusion in
the presence of different modulators of DNA supercoiling. (a)
Expression of spa in the arlRS mutant (BF21) in the absence
(#) and presence ($) of 40 ng novobiocin ml”1. (b)
Expression of spa in the arlRS mutant (BF21, #) and arlRS
gyrB142 mutant (BF42, $). (c) Expression of spa in the presence of different osmolytes ($, 1 M NaCl; %, 1 M KCl; &,
1 M sucrose) in the arlRS mutant (BF21, #). b-Galactosidase
activities were measured as in Fig. 1.
strain was determined (Fig. 7a). Significant and reproducible differences in DNA supercoiling were detected in the
arlRS mutant. The distribution peak was shifted approximately one to three topoisomers in the arlRS mutant relative to the wild-type strain (Fig. 7a). The plasmid isolated
from the arlRS mutant migrated more slowly than that
from the wild-type strain on the gel containing 80 mg
chloroquine ml21. This indicates that more highly supercoiled topoisomers were present in plasmid DNA isolated
from the arlRS null mutant than in that from strain
RN6390BF. To confirm that the Arl system plays a role in
DNA supercoiling, we used pUB110 as a DNA supercoiling
reporter. pUB110 from the arlRS mutant contained more
highly supercoiled topoisomers (two to six shifts) than
that from the wild-type strain (Fig. 7b). Thus, deletion of
the arlRS locus results in changes in the level of DNA
supercoiling.
Effect of the arlRS mutation on genetic
competence
The arl mutation affects DNA supercoiling
To determine whether the Arl system can modulate DNA
supercoiling, strains RN6390BF and BF21 (Table 1) containing pE194 were grown in parallel at 37 uC in TSB
medium. The topoisomer distribution of DNA from each
http://mic.sgmjournals.org
To analyse further the role of the arl null mutation in DNA
supercoiling, we measured the transformation efficiency.
Indeed, the development of genetic competence is related
to intracellular DNA supercoiling (Ashiuchi et al., 2002;
Chandler & Smith, 1996). We previously showed that
the gyrB142 allele, corresponding to a double mutation
(Ile102Ser and Arg144Ile), decreases plasmid DNA supercoiling (Fig. 4a, b). Thus, we analysed the genetic competence of strains with different mutations or grown in
different conditions by measuring the transformation
efficiency with pE194 and pT181. Plasmid DNA isolated
from strain RN4220 was introduced into these strains
by electroporation. The transformation efficiencies with
pE194 and pT181 were 11- and 21-fold higher, respectively,
in the arlRS null mutant than in the wild-type strain
(Table 2). In the wild-type background, the gyrB142 allele
decreased the transformation efficiency by 25-fold for
pE194 and by threefold for pT181. Cells were grown
with 40 ng novobiocin ml21, which has similar effects to
the gyrB142 allele, and then transformed with pT181 by
electroporation. The transformation efficiency was ninefold lower in the presence of novobiocin, confirming
the results obtained with the gyrB142 allele. Finally, the
combined effects of the arlRS deletion plus either the
gyrB142 allele or 40 ng novobiocin ml21 were similar to
those obtained with the arlRS mutation alone (Table 2),
suggesting that the arlRS mutation inhibits the effects of
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B. Fournier and A. Klier
Increasing negative supercoiling
(a)
arlRS
WT
WT
Increasing negative
supercoiling
arlRS
(b)
WT
Increasing negative supercoiling
arlRS
Increasing negative
supercoiling
Fig. 7. Effect of the arlRS mutation on
plasmid DNA topology in different strains.
Wild-type (WT; RN6390BF) and the arlRS
mutant (BF21) strains carrying different
plasmids: pE194 (a), and pUB110 (b).
Strains were grown to an OD600 of 2?0.
After plasmid extraction, topoisomers were
separated in agarose gels containing
80 mg ml”1 (a) or 140 mg ml”1 (b) chloroquine. Scan analyses of topoisomer distribution in the plasmids separated in the gel are
presented on the right.
WT
arlRS
Table 2. Effect of mutations and growth conditions on the transformation efficiency of two plasmids
Transformation competence is expressed as number of transformants per c.f.u.
S. aureus recipient
Genotype
3816
Not determined.
Competence and ratio using:
”9
RN6390BF
+Novobiocin (40 ng ml21)
BF21
+Novobiocin (40 ng ml21)
BF41
BF42
ND,
Wild-type
pE194 (610 )
Ratio
pT181 (610”8)
Ratio
50
1
35
4
750
1000
9
1530
1
0?1
21
25
0?3
44
ND
arlRS
550
11
ND
gyrB142
arlRS gyrB142
2
620
0?04
12
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Microbiology 150
DNA supercoiling in S. aureus
Increasing negative supercoiling
(a)
4.
0
1.
0
0.
25
Increasing negative supercoiling
OD600
0.25
1.0
4.0
(b)
Increasing negative supercoiling
4.
0
1.
0
0.
25
Increasing negative supercoiling
OD600
0.25
1.0
4.0
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Fig. 8. Plasmid DNA supercoiling at different growth stages. pUB110 DNA supercoiling was measured at different growth stages
(OD600 0?25, 1?0 and 4?0). (a) Wild-type
strain RN6390BF. (b) arlRS mutant strain
BF21. Chloroquine gel analysis was performed as in Fig. 1. Scan analyses of topoisomer distribution in plasmids separated in
gels are presented on the right.
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B. Fournier and A. Klier
novobiocin and the gyrB142 allele. Interestingly, we
previously showed that the presence of the arlRS deletion
decreases the effect of DNA supercoiling modulators such
as high osmolarity, novobiocin and gyrB142 allele on spa
expression (Fig. 6). Thus, we confirmed that the arlRS
deletion also decreases the effect of DNA supercoiling
modulators on genetic competence.
As stated above, DNA topology influences genetic competence (Chandler & Smith, 1996). S. aureus is competent
at very early stages of exponential growth (Novick, 1991).
The transformation efficiency of the arl mutant was
dramatically increased relative to the wild-type strain.
However, the presence of the gyrB142 allele or treatment
by novobiocin decreased genetic competence. These two
results are consistent with each other and suggest that
the genetic competence of S. aureus is decreased by the
relaxation of DNA. This also strongly suggests the role of
the Arl system in the modulation of DNA supercoiling.
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