• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. Jul 2009; 77(7): 2849–2856.
Published online Apr 20, 2009. doi:  10.1128/IAI.01405-08
PMCID: PMC2708582

The Staphylococcus aureus GGDEF Domain-Containing Protein, GdpS, Influences Protein A Gene Expression in a Cyclic Diguanylic Acid-Independent Manner[down-pointing small open triangle]

Abstract

Staphylococcus aureus is an important human pathogen that is the principal cause of a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. The success of the organism as a pathogen and its ability to cause such a wide range of infections are due to its extensive virulence factors. In this study, we identified the role of the only GGDEF domain protein (GdpS [GGDEF domain protein from Staphylococcus]) in the virulence of S. aureus NCTC8325. Inactivation of gdpS results in an alteration in the production of a range of virulence factors, such as serine and cysteine proteases, fibrinogen-binding proteins, and, specifically, protein A (Spa), a major surface protein of S. aureus. The transcript level of spa decreases eightfold in the gdpS mutant compared with the parental NCTC8325 strain. Furthermore, the transcript level of sarS, which encodes a direct positive regulator of spa, also decreases in the gdpS mutant compared with the wild type, while the transcript levels of agr, sarA, sarT, and rot display no apparent changes in the gdpS mutant, suggesting that GdpS affects the expression of spa through interaction with SarS by unknown mechanisms. Furthermore, the complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C-terminal GGDEF domain with conserved GGDEF, suggesting that GdpS functions in S. aureus by an unknown mechanism independent of 3′,5′-cyclic diguanylic acid signaling.

Staphylococcus aureus is a well-known human pathogen which is the most common cause of a broad range of infections in humans involving all organ systems, ranging from localized skin infections to life-threatening systemic infections (3, 30). Production of a wide range of virulence factors is thought to be a key to this organism's ability to colonize, infect, and eventually cause disease in its host tissue (4, 27). These factors include secreted proteins, such as serine and cysteine proteases, nuclease, hemolysins, enterotoxins, lipase, and coagulase, and proteins exposed on the cell surface, such as protein A (Spa) and fibrinogen-, fibronectin-, and collagen-binding proteins (14, 34). Most of the studies of the mechanisms of staphylococcal pathogenesis have focused mainly on the regulatory mechanisms involved in the virulence factor gene expression in order to institute a more efficient infection control model (4, 10, 33).

Among the many virulence factors, Spa is a cell wall-associated exoprotein that binds to the Fc regions of immunoglobulin Gs (IgG) of diverse mammalian species and is thought to be an important component of the immune evasion machinery of this pathogen (16, 21, 29, 45). Previous studies have shown that strains of S. aureus with a high Spa content are more resistant to phagocytosis by human neutrophils in vitro than strains with smaller amount of Spa, possibly owing to the IgG Fc-binding property of Spa. The role of Spa in the pathogenesis of staphylococcal infections has been investigated in animal models. In a murine septic arthritis model, the wild-type strain was observed to produce a more severe disease condition than the Spa-deficient strain, indicating that protein A is an important virulence factor in arthritis (36, 38).

spa expression has been suggested to be regulated in a complicated way by a variety of factors (9, 20, 37). Among these factors, the first regulatory component identified was Agr, which is a repressor of spa transcription (22). The Agr system generates two divergent transcripts, RNAII and RNAIII, and RNAIII has been identified to be responsible for the downregulation of spa expression, not only at the transcriptional level but also by the RNAIII-mediated inhibition of translation and degradation of the stable spa mRNA by RNase III (24, 50). Expression of spa is also controlled by SarA. Like Agr, SarA also represses the transcription of the spa gene (9). Recently, additional regulatory components, including the SarA homologs SarS, SarT, and Rot, have been confirmed to be associated with this regulation (11, 41, 42, 48). Interestingly, it has been revealed that SarS, which is encoded by a gene located immediately upstream of spa, appears to be a key regulator in this regulatory network and to be responsible for the Agr- and SarA-dependent repression of protein A synthesis (11, 48).

In recent years, the 3′,5′-cyclic diguanylic acid (c-di-GMP) signaling system has drawn much attention. c-di-GMP was initially described as an allosteric activator of cellulose synthase in Gluconacetobacter xylinus and is now recognized as a second messenger ubiquitous in bacteria and involved in the regulation of a number of complex physiological processes (13, 26, 40, 46). This novel second messenger is synthesized by a class of enzymes containing GGDEF domains and hydrolyzed by EAL or HD-GYP domain proteins. Although the role of c-di-GMP as a second messenger has been extensively studied in diverse bacteria, its role in some low-GC gram-positive bacteria still remains obscure (5, 19, 27). The genome (CP000253) of S. aureus NCTC8325 encodes only one GGDEF domain-containing protein and another protein with a modified GGDEF domain, whereas no EAL domain-containing proteins are encoded. A recent study focusing on the GdpS protein in Staphylococcus epidermidis showed that inactivation of gdpS resulted in impaired biofilm formation capacity, and this function was independent of c-di-GMP signaling. In addition, in vitro study demonstrated that the GdpS proteins in both S. epidermidis and S. aureus cannot synthesize c-di-GMP. Therefore, the authors suggested that staphylococci may have only remnants of the c-di-GMP signaling pathway (23).

In this study, we investigated the function of the S. aureus NCTC8325 GdpS protein, which comprises the N-terminal sensor domain and the C-terminal GGDEF domain. We identified the transcriptional profiling affected by GdpS by using microarray analysis, and we carried out further study of its influence on the virulence of S. aureus, especially on spa expression. Our data indicated that GdpS affects spa transcription through SarS but not Agr, SarA, SarT, or Rot. In addition, consistent with the previous work on GdpS in S. epidermidis, the GdpS protein in S. aureus was observed to function similarly, depending on the N-terminal domain rather than the C-terminal GGDEF domain.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth media.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. Escherichia coli DH5α was grown in Luria-Bertani (LB) medium, while the plasmid-containing E. coli strains were grown in the same medium but with added antibiotics (ampicillin, 100 mg/liter; kanamycin, 50 mg/liter). Staphylococcus aureus and its derivative strains were grown in tryptic soy broth (TSB) (soybean-casein digest medium USP; Oxoid) medium, and when necessary, erythromycin (2.5 mg/liter) and chloramphenicol (15 mg/liter) were added. The media were solidified with 1.5% (wt/vol) agar if needed.

TABLE 1.
Bacterial strains and plasmids used in this study

DNA manipulation.

Genomic DNA of S. aureus NCTC8325 was prepared using a standard protocol for gram-positive bacteria (15). Plasmid DNA from E. coli was extracted using a plasmid purification kit (Promega) according to the manufacturer's instructions. Plasmid DNA from S. aureus was extracted using the same kit, except that the cells were incubated for at least 30 min at 37°C in the solution of lysostaphin (Sigma) before the extraction process. The Taq and Pfu DNA polymerases were obtained from Promega, and the Primer Star DNA polymerase was obtained from Takara. Restriction enzymes were obtained from New England BioLabs, and the incubation conditions were as recommended by the suppliers. Staphylococcus aureus was transformed by electroporation as described previously (28).

Construction of the S. aureus gdpS mutant strain.

To construct the deletion mutants, a 380-bp fragment that flanked the upstream region of the gdpS (SA OUHSC_00760) sequence and a 500-bp fragment that flanked the downstream region of the gdpS sequence were amplified by PCR with, respectively, the primers up-gdp-f-EcoRI, up-gdp-r-XbaI, down-gdp-f-XhoI, and down-gdp-r-SalI, using chromosomal DNA from S. aureus NCTC8325 as the template. A 1.5-kb erythromycin resistance gene was PCR amplified using primers Em-f-XbaI and Em-r-XhoI from pEC1 (6). The three fragments were mixed and digested by XbaI and XhoI, and then the digestions were purified and ligated. Using the ligation product as the template, 2.4-kb fragments were amplified by PCR with the primers up-gdp-f-EcoRI and down-gdp-r-SalI. The 2.4-kb fragments were then digested by EcoRI and SalI and cloned into the EcoRI/SalI sites of the shuttle plasmid pBT2 to create pBTgdpS. Allelic replacement of the native gdpS gene with the resulting plasmid in the genomic DNA of S. aureus NCTC8325 with Emr was carried out as described previously (6). Erythromycin-resistant and chloramphenicol-sensitive colonies were screened. PCR and sequencing were out to confirm that the desired gene inactivation had occurred by double-crossovercarried recombination. The sequences of all primers used in this study are listed in Table Table22.

TABLE 2.
Oligonucleotide primers used in this study

Complementation of the gdpS mutant.

The gdpS gene and its promoter from S. aureus NCTC8325 were amplified by PCR with primers c-gdpS-f-EcoRI and c-gdpS-r-BamHI, while the gdpS promoter and its N-terminal 5TMR-LYT domain were amplified by PCR with primers c-lyt-f-EcoRI and c-gdpS-r-BamHI. The PCR products were, respectively, cloned into pLI50 to create plasmids pLIgdpS and pLIlyt. The method of site-directed mutagenesis by PCR was used to mutate the conserved GGEEF motif or the 5TMR-LYT domain of the GdpS protein. For example, to create the plasmid pLIgdpSM, the plasmid pLIgdpS was used as a template for the PCRs, and the primers for constructing a deletion mutation (GGEEF to EEF) were m-sagdpS-f and m-sagdpS-r. A DNA fragment (6,678 bp, containing the full length of pLIgdpS except the 6-bp nucleotide bases) was amplified by PCR with Primer Star DNA polymerase. The PCR products were digested with DpnI to remove the template plasmids, and subsequently, the digested products were phosphorylated, self-ligated, and transformed into E. coli DH5α. The positive clones with mutational plasmids were verified by DNA sequencing, yielding pLIgdpSM. The four plasmids were transformed by electroporation into S. aureus RN4220 and subsequently transferred to strain S. aureus NCTC8325 gdpS::ermB.

Biofilm and autolysis assays.

Semiquantitative measurements of biofilm formation under static conditions were done using Costar 3599 96-well plates (Corning), as described previously (12, 44). Triton X-100-stimulated autolysis was measured as described previously (43). The cells were grown in PYK medium (5.0 g of Bacto Peptone, 5.0 g of yeast extract, and 3.0 g of K2HPO4 per liter at pH 7.2) to mid-exponential phase at 30°C with constant shaking (200 rpm). After centrifugation, the cells were washed with cold double-distilled water, resuspended in 0.05 M Tris-HCl (pH 7.5) buffer containing 0.05% (wt/vol) Triton X-100 in spectrophotometer vials, and incubated at 30°C with constant shaking (200 rpm). The decrease in the optical density at 580 nm (OD580) was measured every 30 min.

Proteolytic activities and fibronectin binding assays.

Proteolytic activities were determined using the insoluble proteolytic substrate azocoll (Sigma) as previously described (17), with some modifications. The substrate (4 mg/ml) was suspended in 100 mM phosphate buffer (pH 7.0). Staphylococcus aureus strains were grown in TSB for 12 h, and every strain was grown to the same OD. About 400 to 500 μl of culture supernatant was added to 400 μl of the substrate suspension. The mixtures were incubated for 4 h at 37°C with constant shaking, and the reaction was stopped by removing the substrate by centrifugation. An aliquot was removed, and the absorbance was measured at 530 nm.

The fibronectin binding assay was essentially a modification of that used by Ahmed et al. (1). Costar 3590 96-well plates (Corning) were coated with 100 μl of 0.02% sodium carbonate (pH 9.6) containing fibronectin (10 mg/ml; Sigma) overnight at 4°C and then blocked with 100 μl of 2% bovine serum albumin solution for 1 h at 37°C. The wells were washed thrice with 100 μl of phosphate-buffered saline. Subsequently, 100 μl of bacteria (corresponding to 106, 107, or 108 cells) was added, in quadruplicate, to the appropriate wells and incubated for 2 h at 37°C. After that, the bacteria were fixed with 100 μl of 25% formaldehyde for 10 min. Subsequently, 100 μl of 0.5% crystal violet was added to each well and left for 1 min, and the absorbance was measured at 570 nm.

Total RNA isolation, cDNA generation, and microarray processing.

Total RNA was isolated using RNeasy Mini kits (Promega) according to the manufacturer's instructions. The cDNA was synthesized and labeled according to the manufacturer's suggestions for S. aureus antisense genome arrays (Affymetrix Inc., Santa Clara, CA). Further preparation, hybridization, and scanning were carried out by Biochip Company of Shanghai. Real-time reverse transcription-PCR (RT-PCR) was also performed as previously reported (51), using an Applied Biosystems 7000 real-time PCR system (Applied Biosystems). The 16S rRNA was used for normalizing all the reactions, and its transcript levels showed minimal variation between wild-type and mutant cells (data not shown). Microarray data were analyzed with the Affymetrix Microarray Suite software 5.1 (Affymetrix Inc.) and a four-comparison survival method (8).

Western blot analysis of Spa.

Western blot analysis was performed using a modified method as previously described (18). The cells were harvested at an OD600 of 1.5, and the cell wall-associated proteins were released from the bacterial cells by lysis after incubation with lysostaphin (24 U/ml) and were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto a polyvinylidene difluoride membrane (Hybond; GE). The membranes were incubated with anti-Spa rabbit IgG (Sigma), and the IgG bound to Spa was detected using horseradish peroxidase-conjugated sheep anti-rabbit antibodies (Pierce).

Microarray data accession number.

The microarray data have been submitted to the CIBEX database (http://cibex.nig.ac.jp) with the accession number CBX74.

RESULTS

Bioinformatic analysis of GdpS in S. aureus.

The available information on the S. aureus genome shows that gdpS (SAOUHSC_00760) is an independent open reading frame (Fig. (Fig.1A).1A). Upstream of gdpS, there exists an operon predicted to be involved in glutathione metabolism. A gene called llm is located downstream of gdpS, encoding a lipophilic protein that affects the lysis rate and methicillin (meticillin) resistance level (31). The gdpS encodes a protein containing two domains: the N-terminal 5TMR-LYT domain and the C-terminal conserved GGDEF domain (Fig. (Fig.1B).1B). The N terminus of GdpS contains several transmembrane regions, five of which form a 5TMR-LYT domain, which has been proposed to be the sensor of the LytS-YhcK-type histidine protein kinase. The histidine kinase LytS affects autolysis and is involved in the regulation of murein hydrolases (2, 7, 32). The C-terminal GGDEF domain of GdpS contains most of the motifs and residues involved in GTP binding. However, we observed several nonhomologous residues between the GdpS GGDEF domain and the invariable residues of the GGDEF domain consensus sequence, which is consistent with previous findings (23). The study of the GdpS protein in staphylococci has indicated that the GdpS proteins in both S. aureus and S. epidermidis, which share 82.0% similarity at the amino acid level, have only GTP-binding ability and not c-di-GMP synthetic activity (23).

FIG. 1.
Bioinformatics identification of gdpS (SAOUHSC_00760) in S. aureus NCTC8325. (A) Chromosomal organization of the gdpS gene and its surrounding region. Upstream of gdpS, there exists an operon predicted to be involved in glutathione metabolism. Downstream ...

Phenotypic assays of the gdpS mutant strain.

To ascertain whether GdpS affects the growth of S. aureus, we assessed the growth rates of the mutant strain and the parental strain, and our results showed no remarkable difference in the growth of the two strains when they were grown in LB or TSB medium (data not shown). Previous reports have suggested that the lytS mutant of S. aureus exhibits increased autolysis and a marked propensity to form aggregates in liquid culture (7). Our results showed that the gdpS mutant exhibited almost the same rate of autolysis as the parental strain (data not shown), suggesting that the N terminus of GdpS might not function similarly to the general LytR-LytS two-component system in S. aureus. We also performed biofilm assays. Although a previous study indicated that GdpS influenced biofilm formation in S. epidermidis when the cells were grown in brain heart infusion (BHI) medium supplemented with 4% NaCl (23), the gdpS mutant of S. aureus NCTC8325 showed the same biofilm formation capacity as the wild type when grown in TSB, BHI medium, or BHI medium supplemented with 4% NaCl (data not shown). In addition, we performed phenotypic assays to monitor the protease activity and fibronectin binding of both the gdpS mutant and the wild type. The total protease activity in the culture supernatant of the gdpS mutant increased by >60% relative to that for the wild type, as determined with azocoll, whereas the gdpS mutant demonstrated a level of adherence to fibronectin similar to that of the wild type (data not shown). To further investigate how gdpS deletion affects the cellular activities, we performed microarray analysis of these two strains.

Effect of GdpS on S. aureus gene transcription.

To characterize the gene transcriptional profiling influenced by GdpS, DNA microarray assays were performed using the parental strain NCTC8325 and the gdpS deletion mutant strain. The cells were grown in TSB medium to an OD600 of 1.7. A twofold induction ratio was used as a cutoff limit to compare the transcriptional profiling in the wild type and the gdpS mutant strain. Microarray data indicated that 77 genes were induced and 47 genes were repressed in the gdpS mutant strain. Among these genes, several major classes were associated with metabolism, signal transduction, and virulence of S. aureus (Table (Table3).3). The transcript levels of a range of virulence factors were altered in the gdpS mutant strain, such as sdrC/sdrD (encoding Ser-Asp-rich fibrinogen-binding and bone sialoprotein-binding protein), sspA/sspB/sspC (encoding extracellular proteases), and particularly spa (encoding the IgG-binding protein A precursor), whose transcript level decreased eightfold in the gdpS mutant strain. Interestingly, the transcript level of sarS, which has been demonstrated to be involved in the positive regulation of spa transcription by directly binding to the spa promoter, decreased about threefold in the mutant strain compared with the parental strain, suggesting that GdpS might influence spa transcription via SarS. Furthermore, the transcript levels of a set of selected genes were verified with real-time RT-PCR measurements. Figure Figure22 shows that there was a positive correlation between the two techniques.

FIG. 2.
Correlation of microarray and real-time RT-PCR results (microarray versus real-time RT-PCR). The differences in the transcription of seven genes were log2 transformed and plotted against each other. The diagonal line indicates that the ratio from the ...
TABLE 3.
Main genes affected by GdpS

GdpS affects Spa at the transcription level via a SarS regulating pathway.

According to our DNA microarray data, the transcript level of spa decreased about eightfold in the gdpS mutant strain compared with the parental strain. spa has been suggested to be regulated by a range of factors, including Agr, SarA, SarS, SarT, and Rot. In the Agr system, RNAIII has been suggested to repress spa expression. As RNAIII is a small RNA whose expression information was not reflected in the microarray assay, we performed real-time RT-PCR to determine if the change in spa transcription was associated with RNAIII repression. The transcript levels of agrA, sarA, sarS, sarT, and rot in the wild type and the gdpS mutant were also compared using real-time RT-PCR analysis. Interestingly, the transcript levels of agr, sarA, sarT, and rot displayed no apparent alteration in gdpS mutant compared with the parental strain. In contrast, the expression of sarS was influenced by GdpS (Fig. (Fig.3).3). As mentioned above, the microarray data showed that the transcript level of sarS decreased in the gdpS mutant, and our real-time RT-PCR indicated that GdpS indeed repressed the transcription of sarS. Previous studies have shown that the expression of spa was lower in the sarS mutant than in the parental strain, S. aureus NCTC8325-4 (11, 35). To further evaluate whether GdpS affects Spa via SarS, we attempted to complement the gdpS mutant with a plasmid containing the full-length gdpS. As shown in Fig. Fig.3,3, the transcript levels of spa and sarS in the gdpS mutant were complemented almost to the same extent as those in the wild type by the full-length gdpS.

FIG. 3.
Comparative measurement of spa and its regulator gene transcripts by real-time RT-PCR in S. aureus NCTC8325 (wild type [WT]), SX3 (gdpS mutant), and SX4 (gdpS mutant with a plasmid encoding full-length GdpS). All the strains were grown in TSB medium to ...

To further demonstrate that the gdpS gene influences spa expression, we carried out Western blot analysis. The results showed that the expression level of Spa was higher in the wild type than in the gdpS mutant at an OD600 of 1.5 (Fig. (Fig.4B4B).

FIG. 4.
(A) The transcriptional regulation of spa and sarS expression by GdpS was compared using real-time RT-PCR in S. aureus NCTC8325 (wild type [WT]), SX3 (gdpS mutant), SX4 (gdpS mutant with a plasmid encoding full-length GdpS), SX5 (gdpS mutant with a plasmid ...

The GdpS effect on spa depends on the N-terminal domain.

The previous report focusing on GdpS in S. epidermidis demonstrated that GdpS functions independent of the GGDEF domain (23). To investigate whether the effect of GdpS on spa expression was also independent of the C-terminal GGDEF domain in S. aureus, we conducted several types of complementation experiments. Four kinds of plasmids encoding the sequences of the whole GdpS, the N-terminal domain, GdpS with a mutated GGDEF domain (GGEEF to EEF), and GdpS with a mutated N terminus were constructed and transformed into the gdpS mutant. The transcript levels of sarS and spa in the above-mentioned three strains were compared with those in the wild-type strain using real-time RT-PCR. The results showed that the transcript levels of spa in strains SX4 (gdpS mutant with a plasmid encoding full-length GdpS), SX5 (gdpS mutant with a plasmid encoding the N terminus of GdpS), and SX6 (gdpS mutant with a plasmid encoding GdpS with mutated GGDEF domain) were complemented to nearly the same extent as that in the wild-type strain (Fig. (Fig.4A).4A). However, in strain SX7 (gdpS mutant with a plasmid encoding GdpS with a mutated N terminus), the transcript levels of spa were still as low as that in the mutant strain. Taking all this into account, it can be concluded that the effect of GdpS on spa relies on the N-terminal domain rather than the C-terminal domain.

DISCUSSION

Existing experimental evidence suggests that GGDEF/EAL are generally soluble cytoplasmic domains located C terminal to the often multiple sensory and signal transduction domains. A significant fraction of the GGDEF and EAL domains is linked to the cytoplasmic sensory domains involved in the binding of small-molecule ligands or in protein-protein interaction, while another sizable fraction is linked to the N-terminal periplasmic or integral membrane sensory domains, whose ligand-binding specificity is unknown (25, 39, 47). The N terminus of the S. aureus NCTC8325 GdpS protein is a transmembrane domain proposed as a sensor of the LytS-YhcK-type histidine protein kinase. Bioinformatic analysis demonstrated that LytS and YhcK, which share a conserved membrane-spanning domain with five transmembrane helices, are both parts of the signaling complexes that might be involved in cell wall metabolism. However, in this study, we found that the rates of autolysis in the gdpS mutant and the wild-type strain displayed no apparent difference, and accordingly, the microarray data indicated that no factors associated with autolysis and murein hydrolases were affected by GdpS. Our study suggested that the specific LytS-YhcK sensor domain might function based on a novel mechanism. Interestingly, the GdpS protein in S. aureus was found to be related to the expression of some virulence factors, including proteases and protein A.

We carried out a more detailed study of protein A, as it is a major determinant of the virulence of S. aureus. Previous studies of spa regulation in S. aureus revealed that the regulatory elements Agr/RNAIII and SarA both play important roles in repressing spa transcription not only through direct regulation but also by interacting with SarS (48). Expression of sarS is strongly repressed by agr and sarA. In contrast, SarT and Rot were observed to influence the expression of spa only though positive regulation of sarS expression (41, 42). According to our study, the gdpS deletion affected spa transcription and also altered the transcription of its regulator gene, sarS. However, the gdpS gene exhibited no influence on agr, sarA, sarT, and rot. From these results, we can conclude that GdpS affects the expression of spa via SarS in an RNAIII-, SarA-, SarT-, and Rot-independent manner (Fig. (Fig.5),5), although the detailed mechanism remains to be further explored.

FIG. 5.
Proposed regulation of Spa by GdpS, SarS, RNAIII, SarA, SarT, and Rot. Previous work has shown that expression of spa is negatively controlled by Agr/RNAIII and SarA through both direct regulation and interaction with SarS. SarT and Rot upregulate the ...

As a regulator of virulence factor gene expression, SarS has been reported to negatively regulate serine protease and positively regulate the expression of several surface proteins (35). Both the microarray data and results of the phenotypic assays in this study showed that the protease activity was enhanced in the gdpS mutant, indicating that the expression of ssp might also be influenced by GdpS through SarS.

A previous study based on the screening of an S. aureus S30 transposon mutant library showed that the mutation of the S. aureus gdpS gene impaired the biofilm formation capacity (49). However, our physiological data revealed that the gdpS mutant displayed no difference in biofilm formation capacity compared to the wild type, which may possibly be due to some characteristic physiological differences between the strains. S. aureus strain S30 exhibited a stable and strong biofilm-forming phenotype on a variety of substrates and under various culture conditions (49), whereas the wild-type strain S. aureus NCTC8325 failed to exhibit strong biofilm formation under our experimental conditions.

An earlier study of GdpS demonstrated that in S. epidermidis GdpS inactivation impaired the biofilm formation capacity in a c-di-GMP-independent manner and suggested that the GdpS proteins in staphylococci are only the remnants of the c-di-GMP signaling pathway as found in Archaea (23). Although GdpS is the only GGDEF domain-containing protein in S. aureus NCTC8325, our data further suggest that it might not function in c-di-GMP signaling.

Acknowledgments

We thank the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for providing the strains.

This work was supported by the One Hundred Talent Project of the Chinese Academy of Sciences, the Ministry of Science and Technology of China (2004CB518904), and the National Natural Science Foundation of China (30721002).

Notes

Editor: A. Camilli

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 April 2009.

REFERENCES

1. Ahmed, S., S. Meghji, R. J. Williams, B. Henderson, J. H. Brock, and S. P. Nair. 2001. Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria. Infect. Immun. 692872-2877. [PMC free article] [PubMed]
2. Anantharaman, V., and L. Aravind. 2003. Application of comparative genomics in the identification and analysis of novel families of membrane-associated receptors in bacteria. BMC Genomics 434. [PMC free article] [PubMed]
3. Archer, G. L., and M. W. Climo. 2001. Staphylococcus aureus bacteremia—consider the source. N. Engl. J. Med. 34455-56. [PubMed]
4. Bronner, S., H. Monteil, and G. Prevost. 2004. Regulation of virulence determinants in Staphylococcus aureus: complexity and applications. FEMS Microbiol. Rev. 28183-200. [PubMed]
5. Brouillette, E., M. Hyodo, Y. Hayakawa, D. K. Karaolis, and F. Malouin. 2005. 3′,5′-Cyclic diguanylic acid reduces the virulence of biofilm-forming Staphylococcus aureus strains in a mouse model of mastitis infection. Antimicrob. Agents Chemother. 493109-3113. [PMC free article] [PubMed]
6. Bruckner, R. 1997. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol. Lett. 1511-8. [PubMed]
7. Brunskill, E. W., and K. W. Bayles. 1996. Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus. J. Bacteriol. 178611-618. [PMC free article] [PubMed]
8. Chen, Y. W., P. Zhao, R. Borup, and E. P. Hoffman. 2000. Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology. J. Cell Biol. 1511321-1336. [PMC free article] [PubMed]
9. Cheung, A. L., K. Eberhardt, and J. H. Heinrichs. 1997. Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus. Infect. Immun. 652243-2249. [PMC free article] [PubMed]
10. Cheung, A. L., J. M. Koomey, C. A. Butler, S. J. Projan, and V. A. Fischetti. 1992. Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc. Natl. Acad. Sci. USA 896462-6466. [PMC free article] [PubMed]
11. Cheung, A. L., K. Schmidt, B. Bateman, and A. C. Manna. 2001. SarS, a SarA homolog repressible by Agr, is an activator of protein A synthesis in Staphylococcus aureus. Infect. Immun. 692448-2455. [PMC free article] [PubMed]
12. Christensen, G. D., W. A. Simpson, J. J. Younger, L. M. Baddour, F. F. Barrett, D. M. Melton, and E. H. Beachey. 1985. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 22996-1006. [PMC free article] [PubMed]
13. D'Argenio, D. A., and S. I. Miller. 2004. Cyclic di-GMP as a bacterial second messenger. Microbiology 1502497-2502. [PubMed]
14. Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 1316-34. [PMC free article] [PubMed]
15. Flamm, R. K., D. J. Hinrichs, and M. F. Thomashow. 1984. Introduction of pAMβ1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44157-161. [PMC free article] [PubMed]
16. Forsgren, A., and J. Sjoquist. 1966. “Protein A” from S. aureus. I. Pseudo-immune reaction with human gamma-globulin. J. Immunol. 97822-827. [PubMed]
17. Fournier, B., and D. C. Hooper. 2000. A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus. J. Bacteriol. 1823955-3964. [PMC free article] [PubMed]
18. Fournier, B., A. Klier, and G. Rapoport. 2001. The two-component system ArlS-ArlR is a regulator of virulence gene expression in Staphylococcus aureus. Mol. Microbiol. 41247-261. [PubMed]
19. Galperin, M. Y., A. N. Nikolskaya, and E. V. Koonin. 2001. Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol. Lett. 20311-21. [PubMed]
20. Gao, J., and G. C. Stewart. 2004. Regulatory elements of the Staphylococcus aureus protein A (Spa) promoter. J. Bacteriol. 1863738-3748. [PMC free article] [PubMed]
21. Guss, B., M. Uhlen, B. Nilsson, M. Lindberg, J. Sjoquist, and J. Sjodahl. 1984. Region X, the cell-wall-attachment part of staphylococcal protein A. Eur. J. Biochem. 138413-420. [PubMed]
22. Heinrichs, J. H., M. G. Bayer, and A. L. Cheung. 1996. Characterization of the sar locus and its interaction with agr in Staphylococcus aureus. J. Bacteriol. 178418-423. [PMC free article] [PubMed]
23. Holland, L. M., S. T. O'Donnell, D. A. Ryjenkov, L. Gomelsky, S. R. Slater, P. D. Fey, M. Gomelsky, and J. P. O'Gara. 2008. A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP. J. Bacteriol. 1905178-5189. [PMC free article] [PubMed]
24. Huntzinger, E., S. Boisset, C. Saveanu, Y. Benito, T. Geissmann, A. Namane, G. Lina, J. Etienne, B. Ehresmann, C. Ehresmann, A. Jacquier, F. Vandenesch, and P. Romby. 2005. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J. 24824-835. [PMC free article] [PubMed]
25. Hurley, J. H. 2003. GAF domains: cyclic nucleotides come full circle. Sci. STKE 2003PE1. [PubMed]
26. Jenal, U., and J. Malone. 2006. Mechanisms of cyclic-di-GMP signaling in bacteria. Annu. Rev. Genet. 40385-407. [PubMed]
27. Karaolis, D. K., M. H. Rashid, R. Chythanya, W. Luo, M. Hyodo, and Y. Hayakawa. 2005. c-di-GMP (3′-5′-cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation. Antimicrob. Agents Chemother. 491029-1038. [PMC free article] [PubMed]
28. Kraemer, G. R., and J. J. Iandolo. 1990. High-frequency transformation of Staphylococcus aureus by electroporation. Curr. Microbiol. 21373-376.
29. Langone, J. J. 1982. Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci. Adv. Immunol. 32157-252. [PubMed]
30. Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339520-532. [PubMed]
31. Maki, H., T. Yamaguchi, and K. Murakami. 1994. Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus. J. Bacteriol. 1764993-5000. [PMC free article] [PubMed]
32. Mascher, T., J. D. Helmann, and G. Unden. 2006. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol. Mol. Biol. Rev. 70910-938. [PMC free article] [PubMed]
33. Morfeldt, E., L. Janzon, S. Arvidson, and S. Lofdahl. 1988. Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus. Mol. Gen. Genet. 211435-440. [PubMed]
34. Navarre, W. W., and O. Schneewind. 1999. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63174-229. [PMC free article] [PubMed]
35. Oscarsson, J., C. Harlos, and S. Arvidson. 2005. Regulatory role of proteins binding to the spa (protein A) and sarS (staphylococcal accessory regulator) promoter regions in Staphylococcus aureus NTCC 8325-4. Int. J. Med. Microbiol. 295253-266. [PubMed]
36. Palmqvista, N., T. Fosterb, A. Tarkowskia, and E. Josefsson. 2002. Protein A is a virulence factor in Staphylococcus aureus arthritis and septic death. Microb. Pathog. 33239-249. [PubMed]
37. Patel, A. H., J. Kornblum, B. Kreiswirth, R. Novick, and T. J. Foster. 1992. Regulation of the protein A-encoding gene in Staphylococcus aureus. Gene 11425-34. [PubMed]
38. Peterson, P. K., J. Verhoef, L. D. Sabath, and P. G. Quie. 1977. Effect of protein A on staphylococcal opsonization. Infect. Immun. 15760-764. [PMC free article] [PubMed]
39. Romling, U., M. Gomelsky, and M. Y. Galperin. 2005. C-di-GMP: the dawning of a novel bacterial signalling system. Mol. Microbiol. 57629-639. [PubMed]
40. Ross, P., H. Weinhouse, Y. Aloni, D. Michaeli, P. Weinberger-Ohana, R. Mayer, S. Braun, E. de Vroom, G. A. van der Marel, J. H. van Boom, and M. Benziman. 1987. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325279-281. [PubMed]
41. Said-Salim, B., P. M. Dunman, F. M. McAleese, D. Macapagal, E. Murphy, P. J. McNamara, S. Arvidson, T. J. Foster, S. J. Projan, and B. N. Kreiswirth. 2003. Global regulation of Staphylococcus aureus genes by Rot. J. Bacteriol. 185610-619. [PMC free article] [PubMed]
42. Schmidt, K. A., A. C. Manna, and A. L. Cheung. 2003. SarT influences SarS expression in Staphylococcus aureus. Infect. Immun. 715139-5148. [PMC free article] [PubMed]
43. Schrader-Fischer, G., and B. Berger-Bachi. 2001. The AbcA transporter of Staphylococcus aureus affects cell autolysis. Antimicrob. Agents Chemother. 45407-412. [PMC free article] [PubMed]
44. Shanks, R. M., N. P. Donegan, M. L. Graber, S. E. Buckingham, M. E. Zegans, A. L. Cheung, and G. A. O'Toole. 2005. Heparin stimulates Staphylococcus aureus biofilm formation. Infect. Immun. 734596-4606. [PMC free article] [PubMed]
45. Sjoquist, J., J. Movitz, I. B. Johansson, and H. Hjelm. 1972. Localization of protein A in the bacteria. Eur. J. Biochem. 30190-194. [PubMed]
46. Tamayo, R., J. T. Pratt, and A. Camilli. 2007. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu. Rev. Microbiol. 61131-148. [PMC free article] [PubMed]
47. Taylor, B. L., and I. B. Zhulin. 1999. PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev. 63479-506. [PMC free article] [PubMed]
48. Tegmark, K., A. Karlsson, and S. Arvidson. 2000. Identification and characterization of SarH1, a new global regulator of virulence gene expression in Staphylococcus aureus. Mol. Microbiol. 37398-409. [PubMed]
49. Tu Quoc, P. H., P. Genevaux, M. Pajunen, H. Savilahti, C. Georgopoulos, J. Schrenzel, and W. L. Kelley. 2007. Isolation and characterization of biofilm formation-defective mutants of Staphylococcus aureus. Infect. Immun. 751079-1088. [PMC free article] [PubMed]
50. Vandenesch, F., J. Kornblum, and R. P. Novick. 1991. A temporal signal, independent of agr, is required for hla but not spa transcription in Staphylococcus aureus. J. Bacteriol. 1736313-6320. [PMC free article] [PubMed]
51. Wang, L., J. Li, J. C. March, J. J. Valdes, and W. E. Bentley. 2005. luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic expression profiling. J. Bacteriol. 1878350-8360. [PMC free article] [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...