JB Try MCB Online
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ji, G.
Right arrow Articles by Novick, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ji, G.
Right arrow Articles by Novick, R. P.
Journal of Bacteriology, May 2005, p. 3139-3150, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3139-3150.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Staphylococcus intermedius Produces a Functional agr Autoinducing Peptide Containing a Cyclic Lactone

Guangyong Ji,1* Wuhong Pei,1 Linsheng Zhang,1,{dagger} Rongde Qiu,1 Jianqun Lin,1 Yvonne Benito,2 Gerard Lina,2 and Richard P. Novick3*

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, Maryland 20814,1 Centre National de Référence des Staphylocoques, INSERM E0230, IFR62 Laennec, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France,2 Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Ave., New York, New York 100163

Received 31 July 2004/ Accepted 19 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The agr locus, a global regulator of genes involved in pathogenesis and other accessory functions (27), is widely conserved among staphylococci (2) and has homologs in other species (11). It consists of two divergent operons, driven by promoters P2 and P3, respectively (6, 22, 24, 26). The P2 operon contains four genes: agrA, -B, -C, and -D. AgrA and -C comprise a two-component signaling module, of which AgrC is the receptor and AgrA the response regulator (22), which, when activated, upregulates the transcription of both the P2 and the P3 operons. The P3 transcript, RNAIII, encodes delta-hemolysin and is the effector of the agr response (5, 24). The AgrC ligand is an autoinducing peptide (AIP) (9, 14, 15) that is proteolytically processed by AgrB from a propeptide encoded by agrD and probably also secreted by AgrB (9, 37). Although structurally conserved, agrB, -D, and -C have diverged widely among staphylococci, giving rise to multiple specificity groups, in which heterologous AIP-receptor interactions are inhibitory. The mature AIPs are seven to nine amino acids long and have variable amino acid sequences. All of the seven native staphylococcal AIPs thus far analyzed, including the four known Staphylococcus aureus variants plus strains of Staphylococcus warneri, Staphylococcus epidermidis, and Staphylococcus lugdunensis (7, 8, 23, 25), contain a five-amino-acid thiolactone ring, essential for function, in which a conserved cysteine is attached to the C-terminal carboxyl by a thioester linkage. AgrD has been sequenced for ca. 20 other non-S. aureus staphylococci, and all but one are predicted to contain a cysteine at the same position (2), suggesting that the mature AIPs would each contain the same thiolactone ring (8, 9, 16-18, 25). The exception is a strain of Staphylococcus intermedius that is predicted to contain a serine in place of the usual cysteine (2, 20). AIPs with a serine replacing cysteine and therefore having a lactone rather than a thiolactone ring have been synthesized in vitro. These do not activate the cognate receptor, and they inhibit agr activation in heterologous combinations (17, 18). This raises three questions. (i) Do other S. intermedius strains produce serine-containing AIPs and do they produce an AIP, or is the serine the result of a missense mutation resulting in an inactive agrD gene? (ii) If a lactone AIP is produced, is it a functional autoinducer? (iii) Finally, can an S. aureus AgrB that normally processes a cysteine-containing pro-AIP also process a serine-containing pro-AIP and vice versa?

We have analyzed here the serine-producing S. intermedius CCM5739 plus five other S. intermedius strains and have found that all have a serine in the conserved position in AgrD and that culture supernatants of each of the six S. intermedius strains inhibit agr activation in S. aureus, suggesting that each produces an AIP. We determined the specificity and the involvement of S. intermedius AgrB (AgrB-Si) in the processing of S. intermedius AgrD (AgrD-Si) to produce the mature lactone AIP. For strain CCM5739, tandem mass spectroscopy has confirmed that the AIP contains a lactone ring (10), and we show that this AIP and also that produced by ATCC 29663 are self-activators and that all six are cross-inhibitors. In addition, an S. aureus agr group I strain with a mutation in agrD that would replace the conserved cysteine with a serine does not produce any detectable AIP, whereas an S. intermedius strain with cysteine replacing the AIP serine produces a mature thiolactone AIP that retains (weak) self-activation, as well as strong cross-inhibition activity.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and growth conditions. Staphylococcal strains and plasmids used in the present study are listed in Table 1. Escherichia coli strains JM109 and DH5{alpha} were used for cloning.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Staphylococcal strains and plasmids used in this study

 
S. aureus cells were grown in CYGP broth (21) with shaking at 37°C. Supernatants of cultures in late exponential phase or post-exponential phase were centrifuged, filter sterilized, and used as a source of group-specific AIPs. Generally, a 1/10 volume of such supernatants contained sufficient AIP for full activation or inhibition. Overnight cultures on GL plates (21) were routinely used as inocula. Cell growth was monitored with a Klett-Summerson colorimeter with a green (540-nm) filter (Klett, Long Island City, NY). Antibiotics used for plasmid maintenance were erythromycin (10 µg/ml) and tetracycline (5 µg/ml).

Transformation of S. intermedius. Attempts to directly transform S. intermedius with DNA isolated from S. aureus by either electroporation (30) or protoplast transformation (21) failed. S. intermedius was successfully transformed by first amplifying the intact plasmid by using PCR, followed by ligation of the PCR products. This material could then be used for protoplast transformation (21). The success of this method depends on the use of wholly PCR-generated DNA, which seems to be taken up across species barriers, possibly owing to lack of methylation and/or other modifications.

Nucleotide sequencing. The agr locus of S. intermedius ATCC 29663 was sequenced by two methods. First, a PCR product was made with oligonucleotides WP4 and WP6 as primers (oligonucleotides used in the present study are listed in Table 2), and chromosomal DNA prepared from S. intermedius ATCC 29663 as a template. The PCR product was directly sequenced. The DNA sequence was used to design primers for the second sequencing method as follows: chromosomal DNA was digested with both HindIII and EcoRI enzymes. The resulting DNA fragments were separated on agarose gels, followed by Southern blot hybridization with either PCR probe A generated with oligonucleotides WP4 and WP11 as primers and chromosomal DNA as a template or PCR probe B amplified from chromosomal DNA with primers WP14 and WP6. Southern blot hybridization was performed accordingly (28). Two chromosomal DNA fragments (ca. 1.7 and 2.0 kb) were detected by using probe A and probe B, respectively. DNA fragments within either 1.7- or 2.0-kb regions were excised from agarose gels, purified, and then ligated into the HindIII and EcoRI sites of an E. coli cloning vector, pGEM-3zf (Promega). PCR products were then made by using oligonucleotides WP7 and T7 promoter primer (located on the vector) as primers and the ligation mixtures (pGEM-3zf plus 1.7-kb DNA fragment) as a template or by using oligonucleotides WP8 and T7 promoter primer as primers and the ligation mixtures (pGEM-3zf plus 2.0-kb DNA fragment) as templates. Both PCR products were used for DNA sequencing. We note that all PCR products were amplified with PfuTurbo high-fidelity DNA polymerase (Stratagene), and the PCR products from three independent PCRs were generated and sequenced to confirm the lack of PCR errors. Four other S. intermedius agrD genes were sequenced by using oligonucleotides SiB-F3 and SiC-R5, from conserved positions in agrB and agrC, respectively, and rnaIII in S. intermedius CCM5739 was sequenced by using primers SaR3-F1 and SiB-R3.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Oligonucleotides used in this study

 
agr activation and inhibition reporter gene assays. Two methods were used for these assays, (i) Assays were performed with bacterial cultures in early exponential phase (~2 x 108 cells/ml) by using a ß-lactamase reporter gene readout. For activation assays, 80 µl of cells (9 x 108 cells/ml) were treated in duplicate with various amounts of culture supernatant containing AIP at ca. 60 nM, as estimated by comparing its activity with that of a sample of synthetic AIP of known concentration, and incubated with shaking at 37°C for 60 or 90 min in a THERMOmax microplate reader (Molecular Devices) with monitoring of cell density at an optical density at 650 nm, followed by determination of agr activation by the ß-lactamase-nitrocefin assay (9). Nitrocefin was purchased from Becton Dickinson (Franklin Lakes, NJ). For inhibition assays, 80 µl of cells (9 x 108 cells/ml) was treated in duplicate with AIP-containing culture supernatants in the presence of the group-specific wild-type AIP agonist at a concentration of 100 nM for 90 min, followed by a ß-lactamase assay. An agonist concentration of 100 nM is a saturating but not oversaturating dose of activator, which generates maximal activation against which to test various concentrations of heterologous AIPs. (ii) Assays with S. aureus reporter cells containing plasmid pRN6683 (22) with the S. aureus P3-blaZ fusion were done according to the method described previously (8, 9). The same method was used for the AIP activity assays with S. intermedius reporter cells harboring plasmid pWP1004 (S. intermedius P3-blaZ) (Table 1). In brief, culture supernatants were prepared from either S. intermedius wild-type cells grown in CYGP media at 37°C for 6 h or S. aureus cells containing the cloned S. intermedius agr gene(s) under the control of Pbla promoter grown at 37°C to 70 Klett units, followed by induction with 0.5 mM methicillin at 37°C for 5 h. The supernatants were filtered with 0.22-µm-pore-size filters. To 45 µl of culture of S. intermedius ATCC 29663(pWP1004) (~40 and ~100 Klett units for activation and inhibition assays, respectively), 5 µl of supernatant prepared (or diluted in CYGP medium) was added, and the mixtures were incubated at 37°C for 55 min (activation) or 80 min (inhibition) in a VERSAmax microplate reader (Molecular Devices). Diluted cultures (10% in CYGP plus 5 nM sodium azide) were mixed with equal volumes of nitrocefin solution, and the ß-lactamase activities were measured as described previously (8, 9).

Agr induction by S. intermedius culture supernatants. The stationary-phase filtered bacterial supernatant from an overnight 25-ml culture of S. intermedius strain CCM5739 was lyophilized. Lyophilized brain heart infusion broth was used as a control. From 50 ml of a 1-h culture, 25 ml was poured into a flask containing the lyophilized supernatant and 25 ml was poured into another flask containing the lyophilized brain heart infusion. Then, 1-ml aliquots were drawn at 0, 30, 60, and 90 min, and the cell densities were measured. The 1-ml samples were used to prepare RNA for Northern blot hybridization. Equal masses of cells were used for each time point.

Plasmid constructions. Plasmid pWP1004 was constructed as follows. The putative P2/P3 promoter region of S. intermedius was amplified by PCR using oligonucleotides WP12 and SINT11 as primers and chromosomal DNA as a template. The PCR product was cloned into E. coli cloning vector pGEM-T (Promega) in both orientations. The resulting plasmid pGEM-T-P2P3 was digested with EcoRI and SpeI, and the DNA fragment containing the P2/P3 promoter region was then cloned into the EcoRI/NheI sites of pRN5543, followed by insertion of an EcoRI fragment from pRN6683 containing the staphylococcal blaZ gene into the EcoRI site of the resulting plasmid.

Plasmid pWP1101 was constructed by cloning a PCR product amplified from the chromosomal DNA using primers SINT1 and SINT5 into the XbaI site of pRN5548. Plasmid pWP1102 was made by cloning a PCR product amplified from the chromosomal DNA using primers SINT1 and SINT2 into the XbaI/EcoRI sites of pRN5548. To construct pWP1103, a PCR product was prepared by using oligonucleotides SINT3 and SINT4 as primers and S. intermedius chromosomal DNA as a template. The EcoRI/BspHI-digested PCR product was then cloned into the EcoRI/BspHI sites of pLZ4012 (38). Plasmid pWP1104 was made by cloning a PCR product amplified from chromosomal DNA by using primers SINT1 and SINT5 into the XbaI site of pWP1103.

Site-directed mutagenesis of the agrD-Si gene was done by using PCR primers containing desired mutations and the ExSite PCR-based site-directed mutagenesis method according to the manufacturer's instruction (Stratagene). PCR products were generated by using T4 polynucleotide kinase (MBI Fermentas) phosphorylated oligonucleotides SINT6 and SINT8, or SINT15 and SINT8, as primers and pWP1104 as a template. The PCR products were purified from agarose gels and then ligated, resulting in plasmids pWP1105 and pWP1106, respectively. pLZ4003 carrying the S. aureus group III agrD gene was constructed by cloning a ClaI fragment of pRN6963 (8) into the ClaI site of pRN6441.

Plasmid pLZ4006 that carries the S. aureus group I agrD (agrD-I) gene with the cysteine codon in the AIP coding region changed to a serine codon and with six histidine codons fused at both its 3' and 5' ends was constructed as follows. A PCR product was generated by using primers GJ#56 and GJ#28, with pRN6852 (9) as a template. The PCR product was then digested with XbaI and cloned into pRN5548 XbaI and EcoRI (blunted with Klenow) sites. The resulting plasmid was then used as a template to generate a PCR product using oligonucleotides GJ#45 and CS-1 as primers. The PCR products were digested with ScaI and NcoI and cloned into the ScaI/NcoI sites of pGJ4004 (37).

All PCR products used for plasmid construction were amplified with PfuTurbo high-fidelity DNA polymerase (Stratagene). The DNA sequences of the cloned wild-type and mutated genes in the constructed plasmids were confirmed.

Whole-cell lysates and Northern blotting for RNAIII expression. Whole-cell lysates were prepared as described previously (13). Cultures were centrifuged, fixed in acetone-ethanol (1:1), and washed in N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-sucrose buffer. Equalized cell samples were incubated on ice for 30 min with lysostaphin (150 µg/ml) in TES-sucrose buffer (20% sucrose, 20 mM Tris [pH 7.6], 10 mM EDTA, 50 mM NaCl) and shaken for 1 h with proteinase K (50 µg/ml; Sigma) and 2% sodium dodecyl sulfate (SDS) at 4°C. For Northern blotting, the same amount of cell lysate was electrophoresed through a 0.66 M formaldehyde-1% agarose gel in morpholinepropanesulfonic acid buffer (28). Nucleic acids were transferred to a nitrocellulose membrane (Amersham) with a VacuGene apparatus (Pharmacia) in 20x SSC (3 M NaCl plus 0.3 M sodium citrate [pH 7]) and fixed under UV light. The membrane was preincubated for 2 h at 52°C in 2x Denhardt solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.05 M EDTA [pH 8], 0.2% SDS, and 5x SSC, with sonicated and heat-denatured salmon sperm DNA at 100 µg/ml) and 10% dextran (Sigma) and then hybridized overnight with a 32P-labeled DNA probe in hybridization solution. 32P-labeled RNAIII probes were prepared by PCR using oligonucleotide primers RNAIII-F and RNAIII-R, and pRN6735 DNA as a template. In labeling reactions, dATP concentration was reduced to 2 µM, and the reaction mixture contained 50 µCi of [{alpha}-32P]dATP (Amersham; 1 Ci = 37 GBq). The blot was exposed to a storage phosphor screen (Molecular Dynamics).

Western blot hybridization. S. aureus cells expressing various genes under the control of Pbla were grown and induced with 0.5 mM methicillin for 4 h at 37°C. The cultures were then mixed with an equal volume of ice-cold solution (50% acetone and 50% ethanol). The fixed cells were washed with 1x SMM (21) and suspended in 1x SMM plus lysostaphin (Sigma). After 1 h of incubation at 37°C, cells were lysed by sonication. Whole-cell lysates were separated by 16% polyacrylamide Tris-Tricine SDS-polyacrylamide gel electrophoresis (29) and electrophoretically transferred onto a Protran nitrocellulose membrane (Schleicher & Schuell). After incubation at 4°C overnight in blocking buffer (Tris-buffered saline plus 0.05% Tween 20 and 5% bovine serum albumin [TBST]) (28), the membranes were incubated in blocking buffer containing anti-T7-tag monoclonal antibody (1:5,000 dilution; Novagen) for 1 h at room temperature. The membranes were washed extensively with TBST buffer, probed with horseradish peroxidase-conjugated goat anti-mouse antibody (Amersham), and detected with ECL Plus Western blotting detection kit (Amersham), followed by exposure to Kodak Biomax MR film.

The S. intermedius agr nucleotide sequence. The nucleotide sequences of the S. intermedius ATCC 29663 agr locus (GenBank accession number AY557375), agrD genes (RN9161, GenBank accession number AY87105; RN9167, GenBank accession number AY87106; RN9169, GenBank accession numberAY87107; and RN9515, GenBank accession number AF346723), and hld coding sequence of S. intermedius CCM5739 (GenBank accession number AY860843) have been deposited in the GenBank.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The S. intermedius agrD sequences. We determined the DNA sequence of agrD genes for three uncharacterized S. intermedius strains kindly provided by Wesley Kloos, two more from our strain collection, and a sixth strain, ATCC 29663, by using the PCR methods described in Materials and Methods. These are shown in Fig. 1A with the four groups of the S. aureus AgrDs for comparison. AgrD-Si is highly conserved and there are two variants among the six strains. It is not known at present whether this variation affects activation specificity. Shown in boldface are the predicted sequences of the S. intermedius AIPs, one of which, that of CCM5739, has been confirmed as a cyclic lactone with the above sequence by mass spectroscopy (10). Note that the C-terminal processing site is absolutely conserved among these strains, as well as among 30 other staphylococci, whereas the N-terminal processing site is not conserved among other staphylococci (2). The latter is clearly conserved, however, among these S. intermedius strains.



View larger version (88K):
[in this window]
[in a new window]
  		FIG. 1. Comparison of the AgrD amino acid sequences from S. aureus and S. intermedius. The amino acid sequences were analyzed by the Clone Manager software (Sci-Ed Software, Durham, NC). Dash lines represent gaps generated by the analysis program. The identical amino acid residues are dark shaded, and the AIP sequences in AgrDs are in boldface. The amino acid sequences of AgrD proteins can be accessed through the NCBI protein database under NCBI accession no. CAA36782 (S. aureus group I, Sa I) (22), AAB63265 (S. aureus group II, Sa II) (8), AAB63268 (S. aureus group III, Sa III) (8), AAG03056 (S. aureus group IV, Sa IV) (7), AY871105 (RN9161), AY871106 (RN9167), AY871107 (RN9169), AAL65836 (CCM5739) (2), AF346723 (RN9515), and AAS66746 (ATCC 29663). The amino acid sequences of AgrD proteins can be accessed through the NCBI protein database under NCBI accession no. CAA36782 (S. aureus group I, Sa I) (22), AAB63265 (S. aureus group II, Sa II) (8), AAB63268 (S. aureus group III, Sa III) (8), AAG03056 (S. aureus group IV, Sa IV) (7), AAC38294 (S. epidermidis, Se) (35), AAA71977 (S. lugdunensis, Sl) (34), and AAS66746 (S. intermedius ATCC 29663, Si).

 
The AgrB-Si sequence. Sequencing of a 2,084-bp segment of the S. intermedius ATCC 29663 agr locus revealed that this portion of the locus was identical to the published partial sequence of the same region of S. intermedius CCM5739 (2). Comparison of the AgrB-Si sequence to that of other staphylococci showed that AgrB-Si was clearly related to other staphylococcal AgrBs (Fig. 1B). Interestingly, the N-terminal region, two regions that are highly hydrophilic and are proposed to be two transmembrane segments (37), and a small C-terminal region were significantly similar. The divergent N-terminal half of AgrC (data not shown) was also clearly a member of the AgrC receptor family, having significant similarities with other AgrC receptor domains (2, 20), but with little overall conservation, providing no obvious indication of how it would be activated by a lactone AIP.

Agr autoactivation by an S. intermedius culture supernatant. The supernatant of a 6-h (postexponential) CYGP culture of strain CCM5739 was tested for agr activation by Northern blotting for the agr effector transcript, RNAIII. Expression of RNAIII in an untreated culture was compared to that in a culture in which the residue from 25 ml of lyophilized CCM5739 culture supernatant was added to a 25-ml culture of the same strain during early exponential phase. As can be seen in Fig. 2, there was clear induction of RNAIII transcription after the addition of the postexponential supernatant well before RNAIII expression was initiated in the parallel control culture. The amount of postexponential supernatant used here was 10-fold greater than that ordinarily used for agr induction, suggesting that agr induction is weak in this strain, either because the AIP concentration was low or because the response to the AIP was weak. At this concentration the postexponential supernatant had a moderate inhibitory effect on the growth of the bacteria, owing to the relatively high concentration of postexponential products in the added supernatant. Similar self-activation was seen with a culture supernatant of S. intermedius ATCC 29663 (data not shown). In S. aureus, however, previous studies with synthetic lactone analogs of the agr-I and agr-II thiolactone AIPs have shown that neither of these is a self-activator but that both are potent cross-inhibitors of heterologous agr activation involving agr-I, -II, and -III (agr-IV was not tested). Thus, S. intermedius represents the first example of activation by a lactone AIP in the staphylococci. The only other known case of a lactone-AIP is that recently described for an agr analog in Enterococcus faecalis, which is unrelated to the staphylococcal AIPs (19).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. agr self-activation by S. intermedius CCM5739 supernatant. (A) Northern blot analysis. The lyophilized residue from an equal volume of postexponential S. intermedius supernatant (plus S. intermedius) or broth (+BH) was added to an early-exponential-phase culture of CCM5739, and samples were drawn every 30 min for Northern blot hybridization analysis with an RNAIII-specific probe. (B) Growth curves. Cell density monitored turbidimetrically during the 4-h experiment is plotted versus time.

 
Inhibitory activities. Postexponential supernatants of five of the S. intermedius strains were then tested for their effects on rnaIII expression for each of the four S. aureus agr groups. For these experiments, an agr-P3-blaZ transcriptional fusion was introduced into four different derivatives of the agr-null strain, RN6911, each containing a tester plasmid specific for activation by one of the four agr group-specific AIPs. Each of these tester plasmids contains the agrAC segments derived from one of the four groups, driven by the respective agr-P2 promoter, and can be activated only by the cognate AIP supplied exogenously. Activation of the agr-P3-blaZ fusion is not itself group-specific but depends on activation of the coresident agrAC unit, which is group specific. For each test, a fixed amount of an S. aureus culture supernatant containing an AIP was added to an early-exponential-phase culture of the corresponding tester strain, along with an aliquot of a post-exponential-phase supernatant from one of the S. intermedius strains. These cultures were incubated for a further 90 min and then assayed for ß-lactamase activity. For each strain, the ß-lactamase activity obtained in the presence of an S. intermedius supernatant was normalized to that obtained with the cognate activator alone, and the results are plotted in Fig. 3A. As can be seen, all five S. intermedius supernatants inhibited most of the tester strains to a greater or lesser extent. agr-I was generally the most susceptible, and agr-IV was the least susceptible. The strength of the observed inhibition suggests that the S. intermedius AIP was produced in amounts comparable to those produced by standard S. aureus cultures, and therefore the weak response of S. intermedius to its own AIP probably represents weak activation. Similar results were obtained for the sixth S. intermedius strain, ATCC 29663, using a slightly different reporter system (data not shown). Although two of the AIPs have an N-terminal arginine and the other four have an N-terminal lysine, there was no obvious difference in activities that could be attributed to the N-terminal variation. In addition, there was apparent strain-dependent variation in the AIP inhibitory activities despite the identity of the AIPs, perhaps owing to differing amounts of AIP produced or to other strain-specific factors. This result adds to previous observations that non-S. aureus AIPs generally inhibit agr activation in S. aureus. Finally, as shown in Fig. 3B, the AIP-containing supernatants from S. aureus groups I, II, and III had weak inhibitory activities for S. intermedius ATCC 29663.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. (A) agr inhibition by S. intermedius supernatants. To 50-µl aliquots of early-exponential-phase cultures (at cell densities of ~100 Klett) of the four agr group-specific tester strains were added 10 µl of culture supernatants from the respective agr wild-type strains plus 0, 10, or 30 µl of a culture supernatant of the S. intermedius strain to be tested for inhibitory activity. Total volumes were made up to 100 µl with CYGP broth, and the plates were incubated for 90 min with shaking at 37°C. Then, 50 µl was transferred to a new microtiter plate, 50 µl of saturated nitrocefin was added, and the ß-lactamase reaction was monitored kinetically, with the slope of the reaction over the first 5 min used to represent the enzyme activity. The rates in the presence of the S. intermedius supernatants were then normalized to the rate in the presence of activator alone. Hatched bars, 10 µl of S. intermedius supernatant; black bars, 30 µl of S. intermedius supernatant; shaded bars, control, activator only. (B) S. intermedius agr inhibition by S. aureus group I, II, and III AIPs. Culture supernatants were prepared from S. aureus strains (group I, RN6390B; group II, SA502A; and group III, RN8463). AIP activity assays with S. intermedius harboring pWP1004 as reporter cells were performed as described in Materials and Methods.

 
The specificity of the S. intermedius AgrB-Si. It is well established that AgrB determines the specificity of AgrD processing in S. aureus (8). Accordingly, we determined the specificity of the interaction between S. intermedius AgrB-Si and AgrD-Si, by cloning these two genes on separate plasmids. Various combinations of AgrBs and AgrDs from S. aureus group I, II, and III, as well as from S. intermedius, were expressed in S. aureus GJ2035, and the AIP activities were then measured (Fig. 4). Neither S. aureus AgrB-I nor AgrB-II could process S. intermedius AgrD-Si, and the S. intermedius AgrB-Si could only process its cognate AgrD-Si to generate mature AIP-Si. These results indicated that the interaction between AgrB-Si and AgrD-Si was specific.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 4. Interaction between AgrB and AgrD. Conditioned media were prepared from S. aureus GJ2035 expressing various combinations of AgrB and AgrD, and the AIP activities were measured by using S. intermedius containing pWP1004 as reporter cells. (A and B) Test for activation of S. intermedius agr by AIPs prepared from cells coexpressing S. aureus AgrB-I and AgrB-II or S. intermedius AgrB-Si and the S. intermedius AgrD-Si (A) and test for inhibition of S. intermedius agr by AIPs prepared from bacteria coexpressing the S. intermedius AgrB-Si and S. aureus AgrD-I, AgrD-II, or AgrD-III (B). Reporter cells grown in the absence of AIP were used as controls. Values are means from three independent experiments with standard errors as indicated.

 
Cross-processing of S-to-C and C-to-S mutants of AgrD. Since AgrB determines the specificity of AIP processing (8), it is very likely that AgrB catalyzes the formation of the cyclic thiolactone bond in the S. aureus AIPs and, therefore, also the cyclic lactone bond in the S. intermedius AIPs. We therefore tested the possibility that AgrB-I can catalyze the formation of the AIP-I lactone analog by mutationally replacing the cysteine codon (TGT) in agrD-I with a serine codon (AGT), yielding agrD-I (C28S). Since the synthetic AIP-I lactone is a potent cross-inhibitor, and possibly a very weak self-activator, culture supernatants of the mutant strain were then tested for inhibition of agr activation in the other S. aureus agr groups, as well as for self-activation of agr-I. As shown in Fig. 5, no inhibitory activity was detected, nor did this supernatant activate the agr-I reporter or either of the other two. For these tests, we used only the group I, II, and III reporter strains, since these would have detected any AIP produced. Note that activation of the group I reporter was seen only in track C, containing the wild-type agrD-I and agrB-I plasmids. No activation was seen with either the group II or III reporters. Conversely, inhibition of either the group II or group III reporters (tracks C) was seen only with the wild-type agrD-B combination. We therefore conclude that AgrB-I cannot process a serine-containing AgrD-I.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5. Inability of AgrB-I to process a serine-containing AgrD-I mutant. Using a cloned agrD-I derivative, the cysteine codon, TGT, was replaced by a serine codon, AGT, and the mutant agrD was cloned into a vector between N- and C-terminal His6 tags. The resulting construct was tested in vivo in the presence or absence of an agrB-I- containing plasmid and compared to the native agrD-I, also containing the N- and C-terminal His tags, for the production of agr-activating or -inhibiting substances by using agr reporter strains with a ß-lactamase readout. (A) For activation tests, the culture was grown for 90 min in the presence of the supernatant to be tested for activation and then assayed. (B) For inhibition tests, a sample of a cognate supernatant (activator) (i.e., group II supernatant for the group II reporter and group III supernatant for the group III reporter) was added at the same time as the supernatant to be tested for inhibition, and the culture then grown for 90 min and assayed for ß-lactamase. For each of the five sets of tests shown, the columns are labeled as follows: A, wild-type AgrD; B, AgrD, C28S; C, wild-type AgrD plus AgrB-I; D, AgrD, C28S plus AgrB-I [the reporters were RN6390B(pRN6683) for group I, SA502A(pRN6683) for group II, and RN8463(pRN6683) for group III as described previously (8, 9)].

 
We next prepared and tested the corresponding S27C mutant in AgrD-Si, in which the serine was replaced by a cysteine and also an S27A mutation. As noted, S. intermedius AgrB-Si is quite similar to the other known AgrBs (2), so that its divergent sites may be informative with respect to the pro-AIP processing mechanism and, especially, with regard to the ability to process a serine-containing pro-AIP. As shown in Fig. 6, the AgrD-Si serine-to-cysteine mutant, AgrD-Si(S27C), could be processed by AgrB-Si to generate a mature mutant AIP, as determined by AIP activity assays (Fig. 6A to D). However, activation of the S. intermedius agr response by the mutant AIP-Si was much weaker than that by wild-type AIP-Si. The AgrD-Si serine-to-alanine mutant, AgrD-Si(S27A), did not generate any functionally detectable AIP (Fig. 6). The inhibitory activities of the S27C mutant AIP-Si (presumably a thiolactone molecule) on the agr responses in group II but not in group I and III S. aureus strains were stronger than those of the wild-type AIP-Si (Fig. 6B to D).




View larger version (59K):
[in this window]
[in a new window]
  		FIG. 6. Processing of the wild-type and the serine-to-cysteine mutant AgrD-Si by AgrB-Si. (A to D) AIP activity assays. S. aureus cells were grown and induced. After centrifugation, the culture supernatants were used as the conditioned media (either concentrated or diluted with CYGP medium) to perform either AIP activation assays with S. intermedius containing pWP1004 as reporter cells (A) or AIP inhibition assays with RN6390B(pRN6683) (S. aureus group I) (B), SA502A(pRN6683) (S. aureus group II) (C), and RN8463(pRN6683) (S. aureus group III) (D) reporter cells. Undiluted conditioned media were equal to 100% AIP. Conditioned media were prepared from cultures of cells coexpressing AgrB-Si and the wild-type AgrD-Si (•), AgrD-Si(S27C) ({blacktriangleup}), or AgrD-Si(S27A) ({blacksquare}). Values are means from three independent experiments with the standard errors as indicated. (E) Western blot hybridization analysis. S. aureus cells coexpressing AgrB-Si and wild-type AgrD-Si, AgrD-Si(S27C), or AgrD-Si(S27A) were grown and induced, and the cell cultures were mixed with ethanol-acetone (1:1). The mixtures were centrifuged, and the cells were washed and lysed. Whole-cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were then probed with an anti-T7 tag monoclonal antibody.

 
To demonstrate that AgrB-Si was involved in the proteolytic processing of AgrD-Si, we performed Western blot hybridization analysis with an anti-T7 monoclonal antibody as a probe. We note that AgrD-Si used in these experiments was doubly tagged with a T7 epitope at the N terminus and a six-histidine stretch (His6) at the C terminus, which would facilitate the detection of AgrD-Si, as well as of its potential processing intermediate(s), by Western blot analyses with commercially available antibodies. We note that cells expressing the doubly tagged AgrD-Si and AgrB-Si produced an amount of AIP-Si comparable to those expressing the wild-type AgrD-Si and AgrB-Si, indicating that the addition of these tags had no effects on the AgrD-Si processing and AIPSi secretion (data not shown). As shown in Fig. 6E, a band with a molecular mass of ca. 9 kDa corresponding to the calculated molecular mass of the doubly tagged AgrD-Si (9,205 Da) was detected in the lane containing a lysate of cells lacking agrB-Si or containing AgrD-Si(S27A) and producing the tagged AgrD-Si. In the presence of the wild-type AgrB-Si or AgrD-Si(S27C), an anti-T7 antibody-responding band with a molecular mass of ca. 6 kDa was detected. Although this band probably corresponds to a processing intermediate previously observed with S. aureus AgrD-I (18), we have not been able to obtain sufficient material for confirmation by mass spectroscopy. These results suggested that AgrB-Si could process both the serine and the cysteine containing AgrD-Si to generate AIPs that had the ability to activate the S. intermedius and inhibit the S. aureus agr responses.

Hemolytic activities and agr function. agr is widely conserved among the staphylococci (2), although its function has been defined in only two species, S. aureus and S. epidermidis (9, 36). Given that the S. intermedius lactone AIP is an agr autoinducer, it follows that agr is likely to be functional in S. intermedius, which would make S. intermedius the third. A very preliminary picture of the hemolytic activities of S. intermedius supports this, although a definitive conclusion would require the construction of an agr knockout. The production of delta-hemolysin, which is encoded by rnaIII, is generally an indication of agr self-activation. We have determined the DNA sequence of agr rnaIII in an S. intermedius strain, CCM5739, and identified a typical staphylococcal delta-hemolysin reading frame (3). An alignment of delta-hemolysin sequences from staphylococci is shown in Table 3. The NCBI accession numbers are as follows: AAW55662 (S. intermedius), CAA11542 (S. simulans) (33), LESAD (S. aureus) (3), CAA11541 (S. epidermidis) (33), CAA11543 (S. warneri 1) (33), and CAA11544 (S. warneri 2) (33).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Alignment of delta-hemolysin sequences

 
The results of a simple test for the production of delta-hemolysin and other hemolysins, involving cross-streaking on sheep blood agar against RN4220, are shown in Fig. 7. At least four of the five S. intermedius strains shown—RN9161, RN9167, 9168, and 9515—produce delta-hemolysins that are detectable by this test. The fifth strain, CCM5739 (shown as RN9423 in the picture), is also likely to produce delta-hemolysin, since agr is autoinducible in this strain and encodes delta-hemolysin, although it is not identifiable in the CCM5739 hemolytic pattern. Hemolysins are typically agr upregulated, but the group of S. intermedius strains is rather heterogeneous with respect to hemolytic activities. Thus, three produce beta-hemolysin, although in considerably different amounts, and none produces detectable amounts of alpha-hemolysin, but CCM5739 has the strongest hemolytic activity of any of the five, probably producing at least two different hemolysins, neither of which is typical of the S. aureus hemolysins as characterized by this simple test. These results are consistent with agr functionality but certainly do not prove it. Experiments are in progress to isolate an agr knockout to obtain a definitive view of what is regulated by agr in S. intermedius.



View larger version (120K):
[in this window]
[in a new window]
  		FIG. 7. Hemolytic patterns. Cultures to be tested were grown overnight on GL agar, cross-streaked on sheep blood agar against a culture of RN4220, incubated overnight at 37°C, and then incubated for 6 h at 4°C. The patterns can be interpreted as follows: the "hot-cold" beta-hemolysin appears as a partially turbid zone, as seen with RN4220, which produces only beta-hemolysin. Delta-hemolysin is synergistic with beta-hemolysin and is seen as a clearing where the two hemolysins intersect; this is best seen with RN9515 (top left). Coproduction of beta-hemolysin and delta-hemolysin is seen as a clearing next to the streak within a wider beta-hemolysin zone (best illustrated with RN9169, bottom left) and, less strongly, with RN9167 (bottom right). RN9161 produces only delta-hemolysin and quite weakly. RN6734 produces a very strong alpha-hemolysin zone, as shown by the characteristic antagonism between alpha-hemolysin and beta-hemolysin. It also produces delta-hemolysin, as shown by the clearer zone where the beta-hemolysin and delta-hemolysin zones intersect. RN7206 produces a very weak alpha-hemolysin zone, as shown by its inhibition by beta-hemolysin. The CCM5739 (labeled RN9423) pattern could represent a beta-hemolysin zone equivalent to that of RN9169 plus a very strong delta-hemolysin zone, which obscures that of beta-hemolysin. The interaction of CCM5739 (labeled RN9423) with the RN4220 beta-hemolysin zone is very atypical and is not interpretable according to our understanding of the activities of the S. aureus hemolysins.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since S. intermedius was classified as a new staphylococcal species in 1972, this bacterium has been isolated from various animals (12). S. intermedius produces toxins, hemolysins, coagulase and maybe other virulence factors that have been suggested to cause food poisoning in humans (1) and diseases ranging from abscesses to mastitis and endocarditis in dogs (4, 31). Occasionally, S. intermedius causes infection in humans (32). Although S. intermedius is, in most respects, a typical staphylococcal species, it differs strikingly in one respect: it contains a serine in place of the cysteine that is absolutely conserved in the AIPs of all of the 14 other staphylococcal species thus far analyzed. All of the six S. intermedius strains studied produce a nonomeric agrD lactone AIP. The predicted AgrB-Si, AgrD-Si, and the N-terminal portion of AgrC-Si from the sequenced S. intermedius agr locus were clearly members of the widespread family of agr gene products. The AgrB-Si specifically processed AgrD-Si to produce mature AIP-Si, and it also had one interesting feature that was different from other AgrBs, i.e., its abilities to process both the wild-type and a mutant AgrD-Si, in which the serine residue in the AIP-Si region was replaced by a cysteine residue, to produce mature AIPs. In contrast, the AgrB-I could process only the native cysteine containing AgrD-I but not cysteine-to-serine mutants to generate mature AIP. We note that chemically synthesized linear AIPs have neither activation nor inhibition activities (9, 17). These results imply that AgrB-Si can catalyze the formation of both ester and thioester bonds in AIP-Si.

The agr autoinducing activity has been demonstrated for two of the strains, and the production of a cyclic lactone was confirmed. Cross-inhibition has been demonstrated for all six strains. Four of the six strains clearly produce delta-hemolysin, and the fifth probably does so as well, and the overall hemolytic patterns are consistent with agr functionality. Given that the only agr autoinducing and cross-inhibiting substances yet identified are the cyclic AIPs, we suggest that S. intermedius produces lactone AIPs that are active as autoinducers and cross-inhibitors and that agr has a functional role in S. intermedius.

An enduring question is why evolution has favored the development of cyclic AIPs in staphylococci and enterococci, since cyclic peptides are not used in any of the other known peptide-based receptor-ligand signaling systems described to date. We do not have any compelling hypothesis for this. It was initially thought that activation of the receptor would involve the formation of a covalent bond with the AIP. Although this has been ruled out, the possibility of a transient covalent bond has not been ruled out (14). It has also been suggested that the cyclic configuration could confer stability toward environmental proteases; this would have to be tested by a comparison of linear peptides in other systems with the staphylococcal and enterococcal cyclic ones. Since lactone- and thiolactone-containing peptides would have very different intrinsic stabilities, it is difficult to argue that intrinsic stability is relevant here.

There is increasing evidence that AgrB is necessary and sufficient for the processing and secretion of the agr AIPs (8, 9, 37-39) and that the thiolactone configuration is necessary for activation of AgrC. Given that at least one of the S. aureus AgrBs cannot process a serine-containing variant of its pro-AIP, the ability of the S. intermedius AgrBs to do so and the ability of S. intermedius AgrC to be activated by the lactone AIP have interesting implications regarding the evolutionary paradigm that we have favored to account for the apparent covariation in AgrB, -D, and -C, i.e., that the sequence of the AIP, the processing activity of AgrB, and the receptor specificity of AgrC would have to evolve in concert, so as to preserve the three-way specificity of the system. Several experiments presented here and elsewhere address this point. We have shown here that a cysteine to serine mutation (in AgrD-I) inactivates the system because AgrB-I cannot process the propeptide and, previously, that neither the synthetic AIP-I nor -II lactone can activate the cognate AgrC (17, 18). We have observed, however, that a mutant S. intermedius AgrD propeptide in which the serine has been replaced by a cysteine can be processed to generate an AIP, as demonstrated by testing culture supernatants for AIP activity. This AIP, which would contain a cysteine in place of the native serine, is weakly active as an autoinducer, although potent as a cross-inhibitor of S. aureus agr.

Given that the replacement of a cysteine by a serine or vice versa can occur by a single point mutation, the spontaneous occurrence of a C-to-S or S-to-C switch would have a reasonable probability. If we assume that the S. intermedius propeptide arose by a C-S point mutation and that the S. intermedius agrB and -C subsequently evolved to accommodate the change, it seems reasonable to imagine that S. intermedius AgrB "remembers" how to process a cysteine-containing propeptide and that S. intermedius AgrC "remembers" how to respond to a thiolactone AIP. On the other hand, AgrB-I of S. aureus would never have encountered a serine-containing AgrD and AgrC-I of S. aureus would never have encountered a self-coded lactone AIP. Were a C-S mutation to occur in S. aureus, the rest of the locus would have to evolve so as to restore activity to the system. Since most or all S. intermedius strains have a lactone AIP, one would have to postulate that the divergence, giving rise to S. intermedius, which is the canine equivalent of S. aureus, would probably have occurred after the putative C-to-S mutation in the AIP gene. According to Kloos' hypothesis of coevolution of staphylococci and mammals (W. E. Kloos, unpublished data), S. intermedius would probably have diverged from S. aureus coincidentally with the divergence of the line leading to the carnivores from that leading to the primates, ca. 55 million years ago.


   ACKNOWLEDGMENTS
 
This study was supported by NIH grants RO1AI30138 and RO1AI42783 to R.P.N. and by RO1AI46445 to G.J.

We are grateful to Brian Weinrick for measuring the inhibitory activities of the S. intermedius supernatants.


   FOOTNOTES
 
* Corresponding author. Mailing address for R. P. Novick: Departments of Microbiology and Medicine, Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Ave., New York, NY 10016. Phone: (212) 263-6290. Fax: (212) 263-5711. E-mail: novick{at}saturn.med.nyu.edu. Mailing address for G. Ji: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. Phone: (301) 295-9621. Fax: (301) 295-1545. E-mail: gji{at}usuhs.mil. Back

{dagger} Present address: Division of Pediatric Oncology, The Johns Hopkins University, 1650 Orleans St., Baltimore, MD 21231. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Becker, K., B. Keller, C. von Eiff, M. Bruck, G. Lubritz, J. Etienne, and G. Peters. 2001. Enterotoxigenic potential of Staphylococcus intermedius. Appl. Environ. Microbiol. 67:5551-5557.[Abstract/Free Full Text]
  2. Dufour, P., S. Jarraud, F. Vandenesch, T. Greenland, R. P. Novick, M. Bes, J. Etienne, and G. Lina. 2002. High genetic variability of the agr locus in Staphylococcus species. J. Bacteriol. 184:1180-1186.[Abstract/Free Full Text]
  3. Fitton, J. E., A. Dell, and W. V. Shaw. 1980. The amino acid sequence of the delta haemolysin of Staphylococcus aureus. FEBS Lett. 115:209-212.[CrossRef][Medline]
  4. Girard, C., and R. Higgins. 1999. Staphylococcus intermedius cellulitis and toxic shock in a dog. Can. Vet. J. 40:501-502.[Medline]
  5. Janzon, L., and S. Arvidson. 1990. The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus. EMBO J. 9:1391-1399.[Medline]
  6. Janzon, L., S. Lofdahl, and S. Arvidson. 1989. Identification and nucleotide sequence of the delta-lysin gene, hld, adjacent to the accessory gene regulator (agr) of Staphylococcus aureus. Mol. Gen. Genet. 219:480-485.[CrossRef][Medline]
  7. Jarraud, S., G. J. Lyon, A. M. Figueiredo, L. Gerard, F. Vandenesch, J. Etienne, T. W. Muir, and R. P. Novick. 2000. Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus. J. Bacteriol. 182:6517-6522.[Abstract/Free Full Text]
  8. Ji, G., R. Beavis, and R. P. Novick. 1997. Bacterial interference caused by autoinducing peptide variants. Science 276:2027-2030.[Abstract/Free Full Text]
  9. Ji, G., R. C. Beavis, and R. P. Novick. 1995. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA 92:12055-12059.[Abstract/Free Full Text]
  10. Kalkum, M., G. J. Lyon, and B. T. Chait. 2003. Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry. Proc. Natl. Acad. Sci. USA 100:2795-2800.[Abstract/Free Full Text]
  11. Kleerebezem, M., L. E. Quadri, O. P. Kuipers, and W. M. de Vos. 1997. Quorum sensing by peptide pheromones and two-component signal-transduction systems in gram-positive bacteria. Mol. Microbiol. 24:895-904.[CrossRef][Medline]
  12. Kloos, W. E. 1980. Natural populations of the genus Staphylococcus. Annu. Rev. Microbiol. 34:559-592.[CrossRef][Medline]
  13. Kornblum, J. S., S. J. Projan, S. L. Moghazeh, and R. P. Novick. 1988. A rapid method to quantitate non-labeled RNA species in bacterial cells. Gene 63:75-85.[CrossRef][Medline]
  14. Lyon, G. J., P. Mayville, T. W. Muir, and R. P. Novick. 2000. Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC. Proc. Natl. Acad. Sci. USA 97:13330-13335.[Abstract/Free Full Text]
  15. Lyon, G. J., J. S. Wright, A. Christopoulos, R. P. Novick, and T. W. Muir. 2002. Reversible and specific extracellular antagonism of receptor-histidine kinase signaling. J. Biol. Chem. 277:6247-6253.[Abstract/Free Full Text]
  16. Lyon, G. J., J. S. Wright, T. W. Muir, and R. P. Novick. 2002. Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus. Biochemistry 41:10095-10104.[CrossRef][Medline]
  17. Mayville, P., G. Ji, R. Beavis, H. Yang, M. Goger, R. P. Novick, and T. W. Muir. 1999. Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl. Acad. Sci. USA 96:1218-1223.[Abstract/Free Full Text]
  18. McDowell, P., Z. Affas, C. Reynolds, M. T. Holden, S. J. Wood, S. Saint, A. Cockayne, P. J. Hill, C. E. Dodd, B. W. Bycroft, W. C. Chan, and P. Williams. 2001. Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus. Mol. Microbiol. 41:503-512.[CrossRef][Medline]
  19. Nakayama, J., Y. Cao, T. Horii, S. Sakuda, and H. Nagasawa. 2001. Chemical synthesis and biological activity of the gelatinase biosynthesis-activating pheromone of Enterococcus faecalis and its analogs. Biosci. Biotechnol. Biochem. 65:2322-2325.[CrossRef][Medline]
  20. Novick, R. P. 2003. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Microbiol. 48:1429-1449.[CrossRef][Medline]
  21. Novick, R. P. 1991. Genetic systems in staphylococci. Methods Enzymol. 204:587-636.[Medline]
  22. Novick, R. P., S. J. Projan, J. Kornblum, H. F. Ross, G. Ji, B. Kreiswirth, F. Vandenesch, and S. Moghazeh. 1995. The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. Mol. Gen. Genet. 248:446-458.[CrossRef][Medline]
  23. Novick, R. P., H. F. Ross, A. M. S. Figueiredo, G. Abramochkin, and T. W. Muir. 2000. Activation and inhibition of the staphylococcal Agr system. Science 287:391a.[Free Full Text]
  24. Novick, R. P., H. F. Ross, S. J. Projan, J. Kornblum, B. Kreiswirth, and S. Moghazeh. 1993. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J. 12:3967-3975.[Medline]
  25. Otto, M., R. Sussmuth, G. Jung, and F. Gotz. 1998. Structure of the pheromone peptide of the Staphylococcus epidermidis agr system. FEBS Lett. 424:89-94.[CrossRef][Medline]
  26. Peng, H. L., R. P. Novick, B. Kreiswirth, J. Kornblum, and P. Schlievert. 1988. Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus. J. Bacteriol. 170:4365-4372.[Abstract/Free Full Text]
  27. Recsei, P., B. Kreiswirth, M. O’Reilly, P. Schlievert, A. Gruss, and R. P. Novick. 1986. Regulation of exoprotein gene expression in Staphylococcus aureus by agar. Mol. Gen. Genet. 202:58-61.[CrossRef][Medline]
  28. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  29. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379.[CrossRef][Medline]
  30. Schenk, S., and R. A. Laddaga. 1992. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol. Lett. 73:133-138.[Medline]
  31. Smith, A. N., S. T. Finn-Bodner, and A. R. Dillon. 2000. Left ventricular outflow tract to left atrial fistula associated with endocarditis in a dog. J. Am. Anim. Hosp. Assoc. 36:133-136.[Abstract]
  32. Tanner, M. A., C. L. Everett, and D. C. Youvan. 2000. Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human. J. Clin. Microbiol. 38:1628-1631.[Abstract/Free Full Text]
  33. Tegmark, K., E. Morfeldt, and S. Arvidson. 1998. Regulation of agr-dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase-negative staphylococci. J. Bacteriol. 180:3181-3186.[Abstract]
  34. Vandenesch, F., S. J. Projan, B. Kreiswirth, J. Etienne, and R. P. Novick. 1993. Agr-related sequences in Staphylococcus lugdunensis. FEMS Microbiol. Lett. 111:115-122.[CrossRef][Medline]
  35. Van Wamel, W. J., G. van Rossum, J. Verhoef, C. M. Vandenbroucke-Grauls, and A. C. Fluit. 1998. Cloning and characterization of an accessory gene regulator (agr)-like locus from Staphylococcus epidermidis. FEMS Microbiol. Lett. 163:1-9.[Medline]
  36. Vuong, C., F. Gotz, and M. Otto. 2000. Construction and characterization of an agr deletion mutant of Staphylococcus epidermidis. Infect. Immun. 68:1048-1053.[Abstract/Free Full Text]
  37. Zhang, L., L. Gray, R. P. Novick, and G. Ji. 2002. Transmembrane topology of AgrB, the protein involved in the post-translational modification of AgrD in Staphylococcus aureus. J. Biol. Chem. 277:34736-34742.[Abstract/Free Full Text]
  38. Zhang, L., and G. Ji. 2004. Identification of staphylococcal AgrB segment(s) responsible for group-specific processing of AgrD by gene swapping. J. Bacteriol. 186:6706-6713.[Abstract/Free Full Text]
  39. Zhang, L., J. Lin, and G. Ji. 2004. Membrane anchoring of the AgrD N-terminal amphipathic region is required for its processing to produce a quorum sensing pheromone in Staphylococcus aureus. J. Biol. Chem. 279:19448-19456.[Abstract/Free Full Text]

Journal of Bacteriology, May 2005, p. 3139-3150, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3139-3150.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ji, G.
Right arrow Articles by Novick, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ji, G.
Right arrow Articles by Novick, R. P.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS