From the Departments of Pathology,
Internal
Medicine, and
Biological Chemistry,
University of California, Davis, Davis, California 95616, the
§ Department of Human Microbiology, Sackler School of
Medicine, Tel Aviv University, Tel Aviv 69978, Israel, and the
** Department of Biochemistry, University of Cambridge, 80 Tennis Court
Road, Old Addenbrooke's Site,
Cambridge CB2 1GA, United Kingdom
Received for publication, June 21, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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Staphylococcus aureus can cause
disease through the production of toxins. Toxin production is
autoinduced by the protein RNAIII-activating protein (RAP) and by the
autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting
peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful
vaccine target site, and RIP and inhibitory AIPs as therapeutic
molecules to prevent and suppress S. aureus infections.
Development of therapeutic strategies based on these molecules has been
hindered by a lack of knowledge of the molecular mechanisms by which
they activate or inhibit virulence. Here, we show that RAP specifically
induces the phosphorylation of a novel 21-kDa protein, whereas
RIP inhibits its phosphorylation. This protein was termed target of RAP
(TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is
not activated by RAP in the trap mutant strain, suggesting
that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid
analysis shows that TRAP is histidine-phosphorylated, suggesting that
TRAP may be a sensor of RAP. AIPs up-regulate the synthesis of RNAIII
also in trap mutant strains, suggesting that TRAP and AIPs
activate RNAIII synthesis via distinct signal transduction pathways.
Furthermore, TRAP phosphorylation is down-regulated in the
presence of AIP, suggesting that a network of signal transduction
pathways regulate S. aureus pathogenesis.
Staphylococcus aureus is a Gram-positive bacteria that
can cause many different types of diseases, ranging from minor skin infections to pneumonia, endocarditis, and toxin shock syndrome (1). Many of the diseases caused by S. aureus have been
associated with the toxins the bacteria produce (2-4). Toxic
exomolecules include proteases, hemolysins, enterotoxins, and toxic
shock syndrome toxin (TSST-1) (1) that not only cause disease but also
contribute to the survival of the bacteria in the host (5).
In culture, the bacteria produce toxic exomolecules only when in higher
densities, at the post-exponential phase of growth. In the early
exponential phase, when in lower densities, the bacteria express
surface molecules, such as fibronectin-binding proteins and
fibrinogen-binding protein, that allow the bacteria to adhere to and
colonize host cells. The ability of the bacteria to switch between
expression of surface adhesion molecules and toxin exomolecules (1) is
regulated primarily by an RNA molecule termed RNAIII (6-8). It is
hypothesized that RNAIII enables the bacteria to adhere to host
cells when in low numbers but to disengage and spread when too crowded,
thus allowing dissemination and establishment of the infection.
RNAIII is encoded by the agr locus (6) and regulates at
least 15 genes coding for potential virulence factors. agr
mutants are nonpathogenic and show a decreased synthesis of
extracellular toxins and enzymes, such as The synthesis of RNAIII is regulated by a quorum sensing mechanism
(13). Molecules produced and secreted by the bacteria (autoinducers)
accumulate, and when they reach a threshold concentration, RNAIII is
synthesized. The autoinducers of RNAIII that have been described to
date are the RNAIII-activating protein (RAP) (14-16) and the
agr-encoded AIPs (10, 17, 18). RAP is a ~38-kDa protein
containing the NH2-terminal sequence IKKYKPITN (16). The AIPs are octapeptides encoded by the agr, are processed
from AgrD, and activate RNAIII by inducing the phosphorylation of their receptor AgrC. Interestingly, AIPs produced by some S. aureus strains inhibit the expression of agr in other
strains, and the amino acid sequences of peptide and receptor
(AgrC) are markedly different between such strains, suggesting a
hypervariability-generating mechanism (18). Biochemical analysis of
AIPs has suggested that they contain an unusual thiol ester-linked
cyclic structure, which is absolutely necessary for full biological
activity (10).
RNAIII synthesis can be inhibited by antibodies directed against RAP.
Mice vaccinated with RAP were shown to be protected from a S. aureus infection. The protection level correlated with the titer
of anti-RAP antibodies, suggesting that RAP is a promising vaccine
candidate (15). RNAIII synthesis can be inhibited by AIPs of nonself
(10, 18) and by RNAIII-inhibiting peptide (RIP) (14-16, 20). The RIP
is produced by coagulase negative staphylococcus (suggested to be
Staphylococcus warnerii or Staphylococcus xylosus) (16, 19) and has the sequence YSPXTNF,
where X can be a cysteine, a tryptophan, or a modified amino
acid. Both native RIP and a synthetic analogue YSPWTNF are extremely
effective in inhibiting RNAIII synthesis in vitro and in
suppressing S. aureus infections in vivo (15,
20). RIP (native or synthetic) has been shown to prevent S. aureus SD cellulitis in mice (15). Synthetic RIP has been shown to
prevent keratitis (tested in rabbits against S. aureus
8325-4), osteomyelitis (tested in rabbits against S. aureus
MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections), and septic arthritis (tested
in mice against S. aureus LS-1) (20). These findings strongly evidence the potential value of RIP as a therapeutic agent.
The therapeutic potential of inhibiting RNAIII synthesis was confirmed
by Mayville et al. (10), who demonstrated that peptides (AIPs) that inhibit RNAIII synthesis in vitro do in
fact inhibit S. aureus infections in vivo.
Although the therapeutic potential of RAP, RIP, and inhibitory AIPs is
not in dispute, many questions remain unanswered and hinder the
development of vaccines and therapeutics. These include understanding the detailed mechanism by which RAP, RIP, and AIPs regulate RNAIII synthesis; the precise mutual interactions between RAP,
RIP and AIPs; and the signal transduction pathway that leads to
RNAIII synthesis and concludes in virulence.
Because of the sequence similarity between the NH2-terminal
sequence of RAP and RIP (YKPITN as compared with YSPXTN),
and because RIP has been shown to compete with RAP on the activation of
RNAIII synthesis, we hypothesized that RAP and RIP may bind to the same
receptor, one as an agonist (RAP) and the other as an antagonist (RIP).
To test this hypothesis, RIP derivatives were synthesized according to
the putative NH2-terminal sequence of RAP and tested for
their ability to inhibit RNAIII synthesis in vitro and for
their ability to prevent S. aureus cellulitis in
vivo. The results of these experiments indicate that the peptides most successful in inhibiting RNAIII synthesis and cellulitis were
those that most resembled the NH2 terminus of RAP and
contained the sequence YKPITN (16). These results further suggest that RAP and RIP may in fact act as an agonist (RAP) and an antagonist (RIP)
to the same receptor.
Here, we show that RAP activates and RIP inhibits the phosphorylation
of a 21-kDa protein. We termed this protein target of RAP (TRAP). Amino
acid sequence analysis of TRAP indicates that it is a 167-amino acid
polypeptide that is unique to S. aureus. RAP does not
activate RNAIII synthesis in a trap- mutant,
suggesting that RAP activates RNAIII synthesis via TRAP. We also show
here that the phosphorylation of TRAP is inhibited by AIP of self,
which uses the agr signal transduction system to activate
RNAIII synthesis. Taken together, our results indicate that the
trap and the agr signal transduction systems
interact with one another, resulting in up-regulation of RNAIII
synthesis and in a coordinated production of virulence factors.
Bacterial Strains--
The bacterial strains used were as
follows: wild type S. aureus strain RN6390B (ATCC 55620);
agr-null S. aureus mutant strain RN6911 (6);
RIP-producing, coagulase negative Staphylococcus strain
RN833 (ATCC 55619) (14-16, 19); S. aureus strain RN4220, a
mutant of the wild type S. aureus strain 8325-4 that is
capable of accepting foreign DNA (21); and S. aureus strain
OU20, containing a disrupted trap, grown at 42 °C in the
presence of 10 µg/ml erythromycin. Unless mentioned otherwise, all
bacteria were grown in CY broth supplemented with Preparation of RAP--
To purify RAP, RN6390B cells were grown
to the post-exponential phase of growth. Growth culture was centrifuged
at 6000 × g for 10 min at 4 °C. The supernatant was
collected and filtered through a 0.22 µm filter to remove residual
cells. The supernatant was lyophilized (FlexiDry MP lyophilizer) and
resuspended in water to one-tenth of the original volume (total, 10×).
15 ml of 10× supernatant was applied to a 10-kDa cutoff membrane
(Centriprep 10 (Amicon)). This enabled us both to concentrate the
material further and to remove material that was smaller than 10 kDa. 1 ml of concentrated material greater than 10 kDa was washed twice in PBS
by resuspending it in 15 ml of PBS and reconcentrating it on the
Centriprep 10, and the material greater than 10 kDa was collected
(>10); this material is usually at a concentration of
25×-40×. This material contained no AIP and was used for
RAP/RIP or RAP/AIP competition assays (see below), as well as for the further purification of RAP. To further purify RAP, 600 µl of >10
were fractionated on a gel filtration column (Superose 12, Amersham Pharmacia Biotech) in 1 mM phosphate
buffered saline, pH 7.2 (0.1× PBS), at a flow rate of 0.5 ml/min, and
1-ml fractions were collected. Fractions were concentrated to one-tenth
of their original volume by lyophilization and tested for RNAIII by
Northern blotting, as described below. Active gel filtration fraction
(1 ml) was collected, and RAP was further purified by anion exchange chromatography (HPLC SynchroPak Q300, Keystone Scientific, Inc.) in
water, pH 7.2. Bound material was eluted by a salt gradient of 0-1
M NaCl in water in 1-ml fractions. 38-kDa RAP eluted at 0.75 M NaCl. Active fraction was lyophilized and
resuspended in 100 µl of water (10× active fraction, or RAP). To
test for activity, 50 µl of 10× active fraction (RAP) was applied to
450 µl of early exponential cells (containing about 2 × 109 cells) as described below.
Preparation of AIP and RIP--
To partially purify AIP from
RN6390B post-exponential culture supernatants or native RIP from RN833
post-exponential supernatants, cells were grown to the post-exponential
phase of growth. Growth culture was centrifuged at 6000 × g for 10 min at 4 °C. The supernatant was collected and
filtered through a 0.22 µm filter to remove residual cells. The
supernatant was lyophilized (FlexiDry MP lyophilizer) and
resuspended in water to one-tenth of the original volume. 15 ml of 10×
concentrated supernatant was applied to a 3-kDa cutoff membrane
(Centriprep 10 (Amicon)), and material smaller than 3 kDa
(flow-through) was collected and used to test for activity. To test for
activity, 50 µl of the flow-through was applied to 450 µl of early
exponential cells (containing about 2 × 109 cells) as
described below.
In Vivo Phosphorylation Assays--
S. aureus RN6390B
cells (4 ml) were grown in CY/GP from log phase of growth
(A650 of 0.03) until early exponential
phase of growth of A650 of about 0.2 (equivalent
to about 1 × 109 cells/ml). Cells were collected by
centrifugation at 3000 × g for 30 min or at
12,000 × g for 2 min. Supernatants were
discarded, and the cell pellet was resuspended in 0.9 ml of
phosphate-free buffer (PFB) (20 mM KCl, 80 mM
NaCl, 20 mM NH4Cl, 0.14 mM
Na2SO4, 100 mM Tris, pH 7.4, 2.5 mM MgCl2, 0.1 mM CaCl2,
2 µM FeCl2, 0.4% glucose, 9 µg/ml
thiamine, 0.8 mM potassium phosphate buffer, pH 7.4, 0.25 mM L-arginine, 0.21 mM
L-histidine, 0.62 mM L-lysine, 0.13 mM L-glutamic acid, 0.056 mM
glycine, 0.32 mM L-alanine, 0.46 mM
L-valine, 0.36 mM L-isoleucine,
0.82 mM L-proline, 0.016 mM
L-phenylalanine, 0.50 mM L-serine,
0.34 mM L-threonine, 0.016 mM
L-tyrosine, L-0.27 mM cystein, 0.21 mM L-methionine, 0.13 mM L-asparagine, 0.029 mM nicotinic acid, 0.13 mM L-glutamine) and 28 µCi of radiolabeled
orthophosphate (32P) (ICN Biochemicals). Cells were grown
with shaking for 40 min at 37 °C in the presence of one of the
following (prepared as described above): 50 µl of total
post-exponential supernatant (total 10×, containing 80% AIP and 20%
RAP (16)), 50 µl of AIP, 50 µl of RAP, and 50 µl of native RIP,
with synthetic RIP (10 µg RIP/4 × 106 cells) or
with 50 µl control buffer. For growth phase experiments, cells in PFB
and 32P were grown for the times indicated. Cells were
collected by centrifugation for 2 min at 12,000 × g,
supernatants were removed, and cells pellets were washed once in PBS to
remove unincorporated 32P. Radiolabeled cells were
resuspended in 20 µl of 50 µg/ml lysostaphin in 10 mM
Tris, pH 8.0, 1 mM EDTA for 10 min at room temperature, Laemmli sample buffer was added (without boiling), and the sample (total cell homogenate) was separated by 15% SDS-PAGE. The gel was autoradiographed, and the density of the bands was determined. The
gel was then stained in Coomassie to ensure that equal amounts of
protein were in fact loaded on the gel.
For RAP/RIP or RAP/AIP competition experiments, 450 µl of cells in
PFB and 32P prepared as described above were incubated with
50 µl of RAP (40× >10), which was partially purified from
post-exponential supernatants (see above), together with 50, 25, and
12.5 µl of native RIP or AIP (prepared as described above), and the
volume was adjusted to 550 µl with CY.
Activation of RNAIII Synthesis--
Early exponential 6390B (450 µl) was grown with 50 µl of the sample in question (prepared as
described above: RAP, AIP, total 10×, native RIP, CY, or PBS) for 30 min at 37 °C. Cells were collected by centrifugation (2 min at
12,000 × g), and RNA was purified as described below.
If cells were incubated with synthetic RIP, the amount of peptide used
was 10 µg/4 × 106 cells.
Detection of RNAIII and TRAP Transcript: RNA Purification and
Northern Blotting--
Equal number of cells were resuspended in 50 µl of lysostaphin in TES buffer (200 µg/ml lysostaphin (Sigma) in
100 mM Tris, pH 7.2, 1 mM EDTA, 20% sucrose)
and incubated for 10 min at room temperature. 50 µl of 2% SDS
containing proteinase K (100 µg/ml) was added and vigorously vortexed
for 1 min followed by 10 min of incubation at room temperature. The
sample was frozen and thawed twice. 15 µl of RNA sample was mixed
with 11% deionized glyoxal, 16 mM phosphate buffer, pH
7.0, and 55% Me2SO (final concentrations) and
incubated for 1 h at 65 °C. RNA loading buffer (Ambion) was added, and a sample (of about 5 × 108 cells) was
applied to a 1% agarose gel in 10 mM phosphate buffer, pH
7.0, supplemented with 5 mM iodoacetic acid (Sigma). Gel
was Northern blotted by dry transfer and membrane-stained in
methylene blue to view RNA and ensure that the same amounts of
RNA were transferred. The membrane was prehybridized using
Rapid-Hyb (Amersham Pharmacia Biotech) followed by hybridization
with PCR-radiolabeled RNAIII-specific DNA (6) or with PCR-radiolabeled
3' trap (nt 400-550). The gels were autoradiographed, and
the intensity of the band determined using a quantitative analysis
program (Molecular Analyst).
Phosphorylated TRAP (TRAP-P) Purification--
A cell
pellet of 500 ml of early exponential S. aureus cells (grown
as described above) was resuspended in 30 ml of PFB, 560 µCi of
32P, and 3 ml of RAP and grown for 1 h with shaking at
37 °C. Cells were collected by centrifugation, washed in PBS,
resuspended in 2.5 ml of water containing 100 µg/ml lysostaphin
(Sigma), and incubated on ice for 20 min. 7.5 ml of water was added,
and cells were further incubated on ice for 20 min and disrupted by
extensive sonication (Sonic Dismembrator, Fisher Scientific, microtip
probe) (three times for 10 s each time on ice). Sonicated material
was centrifuged (10 min at 12,000 × g), and soluble
material was collected. Soluble material was concentrated by
lyophilization (Savant, Speedvac Plus SC110A) to 800 µl. Material
was fractionated on a gel filtration HPLC column (Bio-Sil SEC-125
300 × 7.8 mm, Bio-Rad) in 1 mM phosphate-buffered saline, pH 8.0, at a flow rate of 1 ml/min. 1-ml fractions were collected and tested for radioactivity in a scintillation counter (Beckman, LS600 multipurpose scintillation counter). Samples containing high cpm in comparison with the rest of the fractions were selected, separated by 15% SDS-PAGE, and gel autoradiographed. The
fraction containing the radiolabeled 21-kDa protein (phosphorylated
TRAP) was used to further purify TRAP by anion exchange chromatography (HPLC SynchroPak Q300, Keystone Scientific, Inc.) in 0.1× PBS, pH 7.5. Bound material was eluted by a salt gradient of 0-1 M NaCl
in 0.1× PBS, pH 7.5. Fractions containing high cpm were separated by
15% SDS-PAGE and gel autoradiographed to determine the fraction containing TRAP. Phosphorylated TRAP eluted at 0.75 M NaCl. The 21-kDa radioactive band was cut and submitted
for amino acid sequencing (see below).
Amino Acid Sequence Analysis--
The anion exchange
TRAP-containing fraction was applied to SDS-PAGE, and
the gel was stained in Coomassie and autoradiographed. The protein band
corresponding to phosphorylation was cut and NH2-terminally
sequenced or subjected to tryptic digestion for acquiring internal
sequences. Specifically, the gel was dried, rehydrated in 50 mM ammonium bicarbonate, pH 7.8, and incubated with 0.5 µg of trypsin overnight at 37 °C. Peptides were extracted by 70%
acetonitrile/5% formic acid and fractionated on a C18 HPLC (Vydac,
4.6 × 25 cm) in 0.1% TFA. Peptides were eluted on a
115-min gradient of 0-70% acetonitrile. Peptides were collected and
amino acid sequenced commercially by Edman degradation chemistry (ABI 477 sequencer, Protein Structure Laboratory, UC Davis). Sequences were
compared with the S. aureus Genome Sequencing Project data base, and the sequence of TRAP was determined. Primers corresponding to
the 5'- and 3'-ends of the gene were constructed, trap was amplified by PCR, and the DNA sequence was confirmed.
Inactivation of trap--
An internal 317-bp fragment ()
of the trap gene was amplified by PCR using the following
primers: 1) 5'-CGCGCGGATCCCAACTATTCCAATTTTCAG-3' (containing the
BamHI site), and 2) 5'-CGCGAAGCTTCTTAAAGTCTTCGTATG-3' (containing the HindIII site). The amplified PCR fragment
was cloned into the BamHI/HindIII sites of the
pAUL-A vector (kindly provided by S. Del-Cardayre), which is a shuttle
vector between S. aureus and Escherichia coli.
This plasmid contains an erythromycin resistance marker and carries a
temperature-sensitive mutation at the S. aureus origin of
replication, and therefore it is capable of replication in S. aureus cells only at the permissive temperature of 30 °C.
S. aureus strain RN4220 cells (a restriction-deficient derivative of strain 8325-4 that is therefore capable of accepting foreign DNA (21)) were transformed with the above construct by
electroporation as described (22), and transformants were grown on NYE
agar (23) in the presence of 10 µg/ml erythromycin at the permissive
temperature of 30 °C overnight. Transformants were grown on NYE agar
in the presence of 10 µg/ml erythromycin at the restrictive
temperature of 42 °C overnight. Colonies were analyzed for
integration of the plasmid into the chromosome at the trap
site via a Campbell insertion process. The analysis was done by PCR,
employing primers that are homologous to the plasmid region (universal
reverse primer 5'-GTAAAACGACGGCCAGT-3') and absent in the chromosome
and a primer that is homologous to the pre-5'-end of the
trap gene and is not present on the plasmid construct
(5'-GTGGTAATGACTAGTTTATCATCGT-3' (nucleotides -59 to -34). A DNA
fragment of 500 bp was generated using the above primers, indicating
the integration of the plasmid and disruption of trap. The
S. aureus containing the disrupted trap gene was
termed OU20. Of note is the fact that the trap gene was also
inactivated in S. aureus 8325-4 and termed YG1. The
phenotype of S. aureus, YG1, had a similar phenotype to that
of OU20.2
Phosphoamino Acid Analysis--
Purified radiolabeled TRAP-P was
applied to SDS-PAGE. Slices of acrylamide, containing labeled TRAP-P,
were excised and submerged in 3 N KOH at 105 °C for
5 h. The resulting hydrolysate was diluted 25-fold with water
containing internal standards of phosphoserine and phosphotyrosine.
Phosphoamino acids were separated by ion-exchange chromatography (24).
O-Phthalaldehyde was added to the eluate, and the resulting
fluorescence was detected on-line (24). Radioactivity was quantified by
liquid scintillation counting. Phospholysine and phosphohistidine were
synthesized as described previously (24). All other standards were
purchased from Sigma. In this system, phosphoarginine and phospholysine
elute before phosphoserine, phosphothreonine elutes close to
phosphoserine, and phosphohistidine elutes between phosphoserine and
phosphotyrosine (24).
RAP Activates and RIP Inhibits RNAIII Synthesis and the
Phosphorylation of a 21-kDa
Protein--
Early exponential wild type S. aureus cells
were incubated for 40 min in the presence of RAP, synthetic RIP
(Genemed Synthesis, Inc. CA), or PBS only as a control. Cells were
collected, RNA purified, and Northern blotted, and membranes were
incubated with radiolabeled RNAIII-specific DNA as a probe. As
previously demonstrated (14, 16) and as shown in Fig.
1, RAP activates and RIP inhibits RNAIII
synthesis. The pathway by which the autoinducer RAP activates and the
peptide RIP inhibits RNAIII synthesis was not known, but it was
hypothesized that RAP and RIP interact with the same receptor, one as
an agonist (RAP), the other as an antagonist (RIP). Therefore, it
seemed reasonable to assume that, like other quorum sensing molecules,
they would regulate a bacterial two component system by phosphorylation
(25). To identify the signal transduction pathway regulated by RAP and
RIP, in vivo phosphorylation assays were performed. Early
exponential wild type S. aureus were incubated in
phosphate-free buffer supplemented with radiolabeled orthophosphate, together with RAP in PBS, with PBS, or with RIP (native or synthetic). After a 40-min incubation period, the cells were collected by centrifugation and treated with lysostaphin followed by the addition of
sample buffer; without boiling, total cell homogenate was applied to
both 7.5 and 15% SDS-PAGE, and the gel was stained in Coomassie or
autoradiographed. As shown in Fig. 2, RAP
activates and RIP inhibits the specific phosphorylation of a 21-kDa
protein that we termed TRAP. Endogenous RAP is produced as the cells
grow (14), probably contributing to the positive signal in the control
PBS group (Fig. 2, lane 1). To determine whether RIP
competes with RAP on TRAP phosphorylation, cells were incubated with
RAP together with increasing amounts of native RIP and in
vivo phosphorylation assays were carried out. As demonstrated in
Fig. 3, the higher the amount of RIP
present, the lower the amount of TRAP phosphorylation, suggesting that
RIP competes with RAP on the phosphorylation of TRAP.
To determine whether RAP induces the synthesis of TRAP and not only its
phosphorylation, the in vivo phosphorylation assays were
carried out in the presence of 100 µg/ml chloramphenicol, to inhibit
potential translation processes. The results of these experiments (not
shown) indicate that RAP activates TRAP phosphorylation also in the
presence of chloramphenicol, suggesting that RAP activates TRAP
phosphorylation and not synthesis.
Structure of TRAP--
To purify TRAP, wild type early exponential
S. aureus cells were in vivo phosphorylated,
cells were disrupted by extensive sonication, and soluble material
containing TRAP-P was fractionated on an HPLC gel filtration column.
Positive fractions (determined by peak radioactivity and confirmed by
separating a sample by SDS-PAGE) were applied to an HPLC anion exchange
column, and bound material was eluted by a 0-1 M NaCl
gradient. The positive fraction containing TRAP-P eluted at ~0.75
M NaCl.
To determine the amino acid sequence of TRAP, purified TRAP was
internally digested by trypsin, and peptide digests were amino acid
sequenced. Acquired sequences were compared with the S. aureus data base, and the sequence of TRAP was determined to be a
167-amino acid polypeptide (Fig. 4)
(GenBankTM accession number AF202641). The sequence
of TRAP (Fig. 4, A and B) is unique to S. aureus and shows no significant sequence homology to known
proteins or genes but for 5'-end of the Bacillus subtilis
penicillin-binding protein gene (pbpF), with which it shares
28% identity (26). Two-dimensional proton NMR spectra (not shown)
reveal a folded protein made up of both RAP Does Not Activate RNAIII Synthesis in the trap Mutant
Strain--
The trap gene was inactivated by gene
disruption. An internal 317-bp fragment of the trap gene
lacking regions of about 100 bp from the 5'- and 3'-ends of the gene
was cloned into pAUL (Fig. 5A), a temperature-sensitive
shuttle vector (kindly provided by S. Del Cardayre), and plasmid was
used to transform S. aureus RN4220 cells. Transformants were
analyzed for integration of the plasmid into the chromosome at the
trap site via a Campbell insertion process. The analysis was
done by PCR, employing primers that are homologous to the vector and to
the 5' sequences of the pre-trap gene not present on the
plasmid construct. A DNA fragment of about 500 bp was generated,
indicating the integration of the plasmid and the disruption of
trap, resulting in a trap- mutant
strain (S. aureus OU20). Sequence analysis of OU20 indicated the replacement of the 3'-end of the trap gene (from nt 390)
with pAUL DNA. S. aureus OU20 was in vivo
phosphorylated in the presence of RAP, total cell homogenate was
applied to SDS-PAGE, and TRAP-P was detected by autoradiography. As
shown in Fig. 5B, RAP activated the phosphorylation of TRAP
in the trap+ strain (lane 1), but it
did not activate phosphorylation in the trap-
OU20 strain (lane 2), suggesting that in fact the
trap gene was disrupted.
To test whether RAP activates the synthesis of RNAIII via TRAP, early
exponential S. aureus trap+ and
trap- cells were incubated together with RAP;
after 40 min, cells were collected, RNA was extracted, and RNAIII was
analyzed by Northern blotting using radiolabeled RNAIII-specific DNA as
a probe. As shown in Fig. 5C, although RNAIII synthesis was
activated by RAP in the parent trap+ strain, it
was not activated in the trap- strain,
suggesting that the presence of an intact trap gene is necessary for RAP to activate RNAIII synthesis. To test whether RNAIII
can be synthesized in the absence of TRAP during bacterial growth,
trap+ and trap- cells
were grown for several hours from the early exponential phase of
growth, and RNAIII and TRAP tested by Northern blotting. As shown in
Fig. 5D, RNAIII synthesis was greatly reduced in the trap- mutant strain but was not abolished.
These results suggest that trap is important for the
activation of RNAIII synthesis but that the synthesis of RNAIII can
nevertheless be activated at a later stage in the absence of TRAP,
possibly by alternate pathways, such as sar (28, 29). As
also shown in Fig. 5D, trap transcription is in
fact absent in the trap- strain and is
constitutive in the trap+ strain. Of note is the
fact that the translation of TRAP also appears to be constitutive in
the wild type trap+ strain (data not shown). The
fact that trap is constitutively transcribed and translated
while its phosphorylation is regulated further supports our results
indicating that RAP regulates TRAP phosphorylation and not synthesis.
TRAP Is Histidine-phosphorylated--
Two-component systems act
through phosphorylation of the substrate domain of the sensor protein
and subsequent transfer of the phosphate to an aspartate residue in the
regulator protein. The initial phosphorylation is catalyzed by a
protein histidine kinase domain in the sensor protein and results in an
N-phosphorylated histidine residue, which is stable in alkaline
conditions but not in acidic conditions (24).
To test whether TRAP may be histidine-phosphorylated, we tested the
sensitivity of phosphorylated TRAP to acidic and basic conditions.
Phosphorylated TRAP was incubated at pH ranging from 1 to 10 for 10 min
at room temperature. The mixture was then applied to SDS-PAGE, and the
gel was autoradiographed. As shown in Fig. 6A, phosphorylation of TRAP
was found to be stable at pH greater than 8.0 but labile at lower pH
values, consistent with a possible N phosphorylation of a histidine.
Phosphoamino acid analysis indicates that in fact TRAP-P contains
phosphohistidine. Purified radiolabeled TRAP-P was applied to SDS-PAGE.
The gel band containing TRAP-P was subjected to alkaline hydrolysis
followed by chromatography (24). The labeled phosphoamino acid eluted
at the position of phosphohistidine, which is distinct from
phosphoarginine, phospholysine, phosphothreonine, phosphoserine, or
phosphotyrosine (Fig. 6B). Histidine phosphorylation
indicates that TRAP may in fact be a sensor of RAP.
Regulation of TRAP Phosphorylation--
RNAIII is produced
only from the mid-exponential phase of growth, whereas TRAP is
continuously transcribed (Fig. 5D). If RAP activates RNAIII
via TRAP phosphorylation, it seemed reasonable to assume that TRAP
phosphorylation and RNAIII synthesis should be coupled. To determine
when TRAP is phosphorylated during bacterial growth, wild type S. aureus were grown from early to late logarithmic phase of growth
in the presence of 32P. Cells were collected at time
intervals and assayed both for TRAP phosphorylation and for RNAIII
synthesis. As shown in Fig. 7,
A and B, peak phosphorylation of TRAP was reached
at the mid-exponential phase of growth. Peak phosphorylation of TRAP
directly correlates with RNAIII synthesis, supporting our hypothesis
that RAP regulates RNAIII synthesis via TRAP phosphorylation.
As shown in Fig. 7, A and B, TRAP reached its
peak phosphorylation by the mid-exponential phase of growth but was
dephosphorylated by the late logarithmic phase of growth. The RNAIII
gene, on the other hand, once activated, remained up-regulated
throughout growth (Fig. 7B). The fact that TRAP was
dephosphorylated by late log indicates that TRAP phosphorylation is
necessary only for the induction of the RNAIII gene but not for its
ongoing transcription.
AIP Activates RNAIII Synthesis but Inhibits TRAP
Phosphorylation--
RNAIII production has been shown to be
autoinduced also by AIP, an octapeptide encoded by the agr
itself. AIP activates RNAIII synthesis by inducing the phosphorylation
of a two-component system, also encoded by the agr.
Specifically, once the agr is activated in the
mid-exponential phase of growth, an octapeptide is produced (processed
from AgrD), inducing the phosphorylation of a 46-kDa protein, AgrC
(11), which is hypothesized to phosphorylate AgrA (12), leading to
up-regulation of RNAIII synthesis. To determine the interaction of the
agr and the TRAP signal transduction systems, we tested
whether TRAP can be phosphorylated also in an agr-null strain.
An agr-null S. aureus strain RN6911 (a mutant
strain that contains a tetM gene instead of agr
(6)) was grown from early to late logarithmic phase of growth in the
presence of 32P. Cells were collected at time intervals,
applied to SDS-PAGE, and autoradiographed. As shown in Fig.
7C, TRAP is phosphorylated also in the agr-null
strain. As in the wild type, peak phosphorylation was reached at the
mid-exponential phase of growth. However, unlike in the wild type
strain, TRAP was not dephosphorylated by the late logarithmic phase of
growth, suggesting that the agr itself, once activated in
the mid-exponential phase, produces a factor that down-regulates TRAP phosphorylation.
To determine whether AIP is the dephosphorylating,
agr-encoded factor, we incubated wild type cells with AIP,
with RAP, or with culture supernatants containing both RAP and AIP in a
RAP:AIP ration of 20:80 (16). TRAP phosphorylation was tested by
in vivo phosphorylation assays, and RNAIII synthesis was
tested by Northern blotting. As shown in Fig.
8, A and B, whereas
RAP activates RNAIII synthesis and activates TRAP phosphorylation, AIP
activates RNAIII synthesis but inhibits TRAP phosphorylation.
To test whether AIP competes with RAP on TRAP phosphorylation, cells
were grown in the presence of RAP together with increasing amounts of
AIP. As shown in Fig. 8C, the level of TRAP-P is dependent on the ratio between the two autoinducers. The more AIP in the culture
supernatant as compared with RAP, the less TRAP phosphorylation occurred. These results can explain why TRAP is dephosphorylated from
the mid-exponential phase of growth, which is when agr is activated and when AIP is produced.
RAP and AIP Activate RNAIII Synthesis via Different Signal
Transduction Pathways--
RAP does not activate RNAIII synthesis in a
trap- strains (Fig. 5C), suggesting
that RAP activates RNAIII via TRAP phosphorylation. To test whether AIP
and RAP activate RNAIII synthesis by interacting with the same signal
transduction pathway, we tested whether AIP can activate RNAIII
synthesis in a trap- strain.
trap+ and trap- strains
were grown in the presence of RAP or AIP and tested for RNAIII
synthesis. As shown in Fig. 8D, RAP did not activate RNAIII in the trap - strain, whereas AIP activated
RNAIII synthesis both in the trap+ and
trap - strain. These results suggest that AIP
does not activate RNAIII via TRAP and that RAP and AIP activate RNAIII
synthesis via different signal transduction pathways. Whereas AIP
activates RNAIII synthesis via the agr system
(phosphorylation of AgrC (11)), RAP activates RNAIII synthesis via the
TRAP system (phosphorylation of TRAP).
Our work demonstrates that the autoinducer of virulence RAP
activates and the inhibitor of virulence RIP inhibits the
phosphorylation of a 21-kDa protein termed TRAP. Amino acid sequence
analysis of TRAP indicates that the 167 amino acid polypeptide is
unique to S. aureus. RAP does not activate RNAIII synthesis
in a S. aureus strain containing a disrupted
trap, suggesting that an intact trap gene is
necessary for the activation of RNAIII synthesis by RAP. Phosphoamino
acid analysis of TRAP-P indicates that TRAP is
histidine-phosphorylated, indicating that TRAP may be a sensor of RAP.
Secondary structure predictions suggest it, however, to be globular
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-,
-, and
-hemolysin, leucocidin, lipase, hyaluronate lyase, and proteases,
and at the same time an increased synthesis of adhesion molecules,
coagulase, and protein A (2, 9). The agr locus contains two
divergent transcription units, RNAII and RNAIII, driven by the
promoters P2 and P3, both of which are active only from the
mid-exponential phase of growth (9). RNAII contains four open reading
frames: agrA, agrB, agrC, and agrD. The
agrA and agrC genes encode a classical
two-component signal transduction pathway composed of the AgrC signal
receptor and the AgrA response regulator. The agrD gene
product is a propeptide that is processed and secreted through AgrB,
which is an integral membrane protein. The resultant mature
autoinducing peptide (AIP)1
(10) is the ligand that binds to and activates the phosphorylation of
AgrC (11), which in turn is thought to phosphorylate AgrA, leading to
up-regulation of RNAIII synthesis (12).
EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-glycerophosphate
(21) at 37 °C with shaking from early exponential phase of growth.
RESULTS
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DISCUSSION
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Fig. 1.
RNAIII synthesis is activated by RAP and
inhibited by RIP. Early exponential S. aureus cells
were incubated for 40 min together with synthetic RIP (lane
1), with PBS as a control (lane 2), or with RAP
(lane 3) as described under "Experimental Procedures"
("Activation of RNAIII Synthesis"). RNA was purified, equal amount
of RNA were applied to the gel, and the gel was Northern blotted.
RNAIII was detected using radiolabeled RNAIII-specific DNA as a probe.
The membrane was autoradiographed, and the density of the bands was
determined.
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Fig. 2.
The phosphorylation of TRAP is activated by
RAP and inhibited by RIP. Wild type early exponential S. aureus cells were in vivo phosphorylated in the
presence of PBS only as a control (lane 1), synthetic RIP
(lane 2), or RAP (lane 3). Cells were collected,
and total cell homogenate applied to 15% SDS-PAGE (A and
C) or to 7.5% SDS-PAGE (B). The gel was
Coomassie-stained (A) or autoradiographed (B and
C). The approximate molecular mass is indicated in
kDa.
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Fig. 3.
RIP competes with RAP of TRAP
phosphorylation. In vivo phosphorylation: RAP/RIP
competition experiment. Wild type early exponential cells in PFB and
32P (450 µl) were incubated with 50 µl of RAP (>10)
that was partially purified from post-exponential supernatants
(lane 1), with PBS as a control (lane 2), with 50 µl of native RIP (lane 3), or with 50 µl of partially
purified RAP (>10) together with 50 µl (lane 4),
25 µl (lane 5), or 12.5 µl (lane 6) of native
RIP, and the total volume was adjusted to 550 µl with CY (lanes
4-6). After 60 min, cells were collected, the total cell
homogenate was applied to SDS-PAGE, and the gel was
autoradiographed.
-helices and
-sheet
secondary structure elements, in agreement with sequence and threading
analysis (Fig. 4C) generated by the PHD package and
threading analysis (27).
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Fig. 4.
A and B, amino acid sequence
of TRAP (A) and DNA sequence of trap
(B). C, secondary structure prediction of TRAP
generated by the PHD package (27). Cylinders and
arrows denote -helix and
-sheet, respectively.
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Fig. 5.
A, pAUL-A containing trap
nucleotides 73-390. B, in vivo phosphorylation
of S. aureus containing a disrupted trap gene.
Early exponential S. aureus trap- and
trap+ cells were in vivo
phosphorylated in the presence of RAP. After 40 min, cells were
collected, total cell homogenate was applied to SDS-PAGE, and the gel
was autoradiographed. Lane 1, trap+.
Lane 2, trap-. C, the
synthesis of RNAIII is not activated by RAP in the trap
mutant strain. Early exponential S. aureus
trap- and trap+ cells were
incubated for 40 min with PBS (RAP-) or with RAP (RAP +). RNA was
purified, equal amounts of RNA (10 µg) were applied to the gel, and
the gel was Northern blotted. RNAIII was detected using radiolabeled
RNAIII-specific DNA as a probe, and the membrane was autoradiographed.
D, the production of RNAIII is reduced in the
trap- strain. Early exponential (1 × 109 cells/ml) S. aureus trap- and
trap+ cells were grown for 1-4 h. Equal number
of cells were collected, RNA was extracted, the RNAIII and TRAP
transcript was tested by Northern blotting, and the membrane was
autoradiographed. Lanes 1-4, cells were grown for 1-4 h,
respectively.
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Fig. 6.
A, TRAP-P is stable in alkaline
conditions. Phosphorylated TRAP was incubated for 10 min at room
temperature at increasing pH values, applied to SDS-PAGE, and
autoradiographed, and the density of the bands was determined. Results
are presented as percentage of maximum phosphorylation observed
(% of max). B, TRAP-P contains phosphohistidine.
An alkaline hydrolysate of radiolabeled TRAP-P was analyzed by
chromatography. The labeled phosphoamino acid eluted at the position of
phosphohistidine (P-His), distinct from phosphoarginine,
phospholysine, phosphothreonine, phosphoserine (P-Ser), or
phosphotyrosine (P-Tyr).
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Fig. 7.
A and B, TRAP reaches peak
phosphorylation at the mid-exponential phase of growth. Wild type
S. aureus was grown from early to late log phase of growth
in PFB together with 32P. Cells were collected at time
intervals and tested both for RNAIII (by Northern blotting) and for
TRAP phosphorylation (by SDS-PAGE). A, cells were
resuspended in sample buffer and applied to 15% SDS-PAGE, and the gel
was autoradiographed. Approximate molecular mass is indicated
in kDa. B, TRAP phosphorylation versus RNAIII and
cell number (cell #). Results are presented as percentage of
maximum phosphorylation, RNAIII, or cell number observed (% of
max). C, TRAP is phosphorylated in agr-null
strains. Mutant agr-null S. aureus cells RN6911
were grown from early to late log phase of growth in PFB together with
32P. Cells were collected, the total cell homogenate was
applied to SDS-PAGE, and the gel was autoradiographed.
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Fig. 8.
The phosphorylation of TRAP is activated by
RAP and inhibited by AIP. A, wild type early
exponential S. aureus cells were incubated for 1 h in
PFB, 32P, together with RAP (40×, >10) containing no AIP,
with post-exponential total supernatant (total, containing 20% RAP and
80% AIP (16, 19)), with AIP (containing no RAP), or with PBS as a
control. Cells were collected and applied to SDS-PAGE, and the gel was
autoradiographed. The autoradiogram was scanned, and the density of the
bands was determined. B, in parallel, cells (in CY) were
incubated in the presence of RAP, PBS, AIP, and total supernatant for
40 min, and cells were assayed for RNAIII by Northern blotting. The
density of the bands was determined, and results are presented as
percentage of maximum RNAIII observed (% of max).
C, in vivo phosphorylation: RAP/AIP competition
experiment. Wild type early exponential cells in PFB and
32P (450 µl) were incubated with 50 µl of RAP (40×,
>10) that was partially purified from post-exponential supernatants
(lane 1), together with 25 µl of AIP and 25 µl of CY
(lane 2) or with 50 µl of AIP (lane 3). This
gave estimated RAP:AIP ratios of 1:0 (lane 1), 1:0.5
(lane 2), and 1:1 (lane 3). After 40 min, cells
were collected and applied to SDS-PAGE, the gel was autoradiographed,
and the density of the bands was determined. D, RNAIII
synthesis is activated by AIP but not by RAP in a trap
mutant strain. Early exponential (1 × 109 cells/ml)
S. aureus trap- and
trap+ cells were grown for 40 min in the
presence of RAP, PBS, or AIP. Equal numbers of cells were collected,
RNA was extracted, RNAIII was tested by Northern blotting, and the
membrane was autoradiographed.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-helix
-sheet protein that does not have a transmembrane region
that is classical for a histidine kinase. TRAP may therefore either be
part of a nonclassical histidine kinase (30) or be bound to a yet to be
identified, membrane-associated molecule (Fig.
9).
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Fig. 9.
Interaction of TRAP and AgrC. As
the colony multiplies, the autoinducer RAP accumulates and induces the
phosphorylation of its target molecule TRAP (A), resulting
in the production of RNAII (B). Once agr is
activated (in the mid-exponential phase of growth), AIP and its
receptor AgrC are produced (9). AIP down-regulates TRAP phosphorylation
and up-regulates the phosphorylation of its receptor, AgrC
(B) (11), which is hypothesized to phosphorylate AgrA
(C), which then acts as a transcription activator to
activate P3 (12), leading to the production of RNAIII. Production of
RNAIII, in parallel with up-regulation of sar and
sae, causes the expression of toxic exomolecules and the
suppression of adhesion molecules (C) (6, 32, 33), resulting
in dissemination and in disease.
TRAP reaches its peak phosphorylation by the mid-exponential phase of growth but is dephosphorylated by late logarithmic phase of growth. The gene for RNAIII, on the other hand, once activated, remains up-regulated throughout. The fact that TRAP is dephosphorylated by late log indicates that TRAP phosphorylation is necessary only for the induction of the RNAIII gene but not for its ongoing transcription.
TRAP is phosphorylated also in the agr-null strain. As in the wild type, peak phosphorylation is reached at the mid-exponential phase of growth. However, unlike in the wild type strain, TRAP is not dephosphorylated by late log, suggesting that the agr itself, once activated in the mid-exponential phase, produces, or regulates the production of, a factor, which down-regulates TRAP phosphorylation.
One of the factors produced by the agr is the octapeptide AIP that also activates RNAIII synthesis. However, the agr locus is temporally regulated, and therefore the AIP is only produced from the mid-exponential phase of growth (17). We show here that whereas RAP activates RNAIII synthesis as well as TRAP phosphorylation, the AIP activates RNAIII synthesis but inhibits TRAP phosphorylation. Furthermore, whereas RAP does not activate RNAIII synthesis in a trap- strain, AIP does up-regulate RNAIII synthesis in a trap- strain, suggesting that RAP and AIP activate RNAIII synthesis via different signal transduction pathways. The fact that TRAP phosphorylation is down-regulated in the presence of the AIP may explain why TRAP is dephosphorylated at the mid-exponential phase of growth, coinciding with AIP production. The interplay between the two signal transduction pathways suggests that TRAP and the agr gene products could be part of a phosphorelay system. The phosphorelay is an extended and more complex version of the two component system, involving multiple activation signals processed by at least two histidine kinases (30, 31). We propose that whereas RAP (signal A) activates the TRAP signal transduction (kinase A), the AIP (signal B) activates the agr system (kinase B), which probably leads to activation of a phosphatase and to the dephosphorylation of TRAP (Fig. 9).
It has been suggested that RNAIII synthesis is only regulated by the peptides encoded by the agr (AIPs) and that RIP is part of the AIP family of peptides (19). Although this may be the case, several experimental data do not support this hypothesis (16). 1) AIPs must contain a thiolactone structure to be active, whereas the RIPs are synthesized without a cysteine and a thiolactone structure and are active as linear peptides. 2) Both RIP and AIP of self inhibit TRAP phosphorylation, but whereas RIP inhibits RNAIII synthesis, AIP of self activates RNAIII synthesis. 3) AIP activates RNAIII synthesis in a trap- strain, whereas RAP does not, suggesting that AIP and RAP activate RNAIII synthesis via different signal transduction pathways. Furthermore, RIP prevents infections cause by various strains of S. aureus in different infection models, suggesting that unlike the AIPs, RIP is not strain-specific in its inhibitory activity.
In summary, we propose (Fig. 9) that autoinduction of virulence occurs
in a two-step process. As the colony multiplies, the autoinducer RAP
accumulates and induces the phosphorylation of its target molecule
TRAP, resulting in up-regulation of agr to produce
RNAII.2 Once agr
is activated (in the mid-exponential phase of growth), AIP and its
receptor AgrC are produced. AIP up-regulates the phosphorylation of its
receptor, AgrC (11), leading to phosphorylation of AgrA, to
up-regulation of RNAIII synthesis (9), and to down-regulation of TRAP
phosphorylation. Production of RNAIII, in parallel with up-regulation
of sar (32) and sae (33), causes the expression of toxic exomolecules and the suppression of adhesion molecules, resulting in dissemination and in disease. In the presence of anti-RAP
antibodies, RIP, or inhibitory AIPs, RNAIII is not produced, and the
pathogenic potential of the bacteria is greatly reduced (10, 15, 16,
20).
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ACKNOWLEDGEMENTS |
---|
We thank Andrea Carbone (University of California, Davis) for assistance in phosphoamino acid analysis, Dr. Ilya Borovok for helpful discussions, and Profs. Gerald Cohen and Yair Aharonowitz for their generosity. We thank S. Del-Cardayre for kindly providing the pAUL-A plasmid. We thank Dr. Young Moo Lee and Tara Martinez (Protein Structure Laboratory, University of California, Davis) for their assistance in resolving the amino acid sequence of TRAP. We thank B. A. Roe, Yudong Qian, A. Dorman, F. Z. Najar, S. Clifton, and J. Iandolo (University of Oklahoma), who, with funding from the National Institutes of Health and the Merck Genome Research Institute, are carrying out the S. aureus Genome Sequencing Project.
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FOOTNOTES |
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* This project was supported by University of California Davis Health System, InterMune Pharmaceuticals, American Heart Association, and The Council for Tobacco Research.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) AF202641.
¶ To whom correspondence should be addressed: Dept. of Pathology, Research Bldg. 3, University of California, Davis, Medical Center, Sacramento, CA 95817. Tel.: 916-734-3218; Fax: 916-734-2698; E-mail: nbalaban@ucdavis.edu.
§§ Authors listed in alphabetical order.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005446201
2 Y. Gov, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: AIP, autoinducing peptide; bp, base pair(s); HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PFB, phosphate-free buffer; RAP, RNAIII-activating protein; RIP, RNAIII-inhibiting peptide; TRAP, target of RAP; TRAP-P, phosphorylated TRAP.
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