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Infection and Immunity, May 2002, p. 2434-2440, Vol. 70, No. 5
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.5.2434-2440.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

rgf Encodes a Novel Two-Component Signal Transduction System of Streptococcus agalactiae

Barbara Spellerberg,^1* Eva Rozdzinski,² Simone Martin,¹ Josefine Weber-Heynemann,¹ and Rudolf Lütticken¹

Institute of Medical Microbiology and National Reference Center for Streptococci, University Hospital Aachen, D-52057 Aachen,¹ Department of Medical Microbiology and Hygiene, University of Ulm, D-89081 Ulm, Germany²

Received 13 August 2001/ Returned for modification 8 October 2001/ Accepted 7 February 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The adhesion of gram-positive bacteria to extracellular matrix (ECM) proteins is regarded as an important determinant of pathogenicity. A comparison of the adhesion of Streptococcus agalactiae strain O90R to different ECM proteins showed that the most pronounced binding could be observed for immobilized fibrinogen. To investigate the genetic determinants of S. agalactiae fibrinogen binding, a pGhost9:ISS1 mutant library was screened for mutants displaying reduced agglutination of fibrinogen-coated latex beads. A putative two-component signal transduction system was identified and designated rgfBDAC. It comprises genes encoding a putative response regulator of 218 amino acids and a putative histidine kinase of 426 amino acids. Comparison of the deduced proteins with the GenBank database revealed a significant similarity to quorum-sensing systems of gram-positive pathogens. Transcription analysis of the rgf locus showed that the encoding genes are located on one transcript. To further characterize the influence of the putative histidine kinase encoded in the rgf locus on the adhesion of S. agalactiae to immobilized fibrinogen, a targeted mutant of rgfC was generated. In comparison to the wild-type strain this mutant demonstrated altered fibrinogen binding capacities depending on bacterial cell density. Transcription analysis of secreted and surface-localized S. agalactiae proteins in the wild type and the rgfC mutant strain revealed that mRNA levels of the C5a peptidase gene scpB were increased in the mutant strain while the transcription of the secreted CAMP factor gene cfb was unaffected by this mutation. Based on these results, we hypothesize that rgf regulates the expression of bacterial cell surface components.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Streptococcus agalactiae (group B streptococcus) is an important human pathogen causing neonatal pneumonia, sepsis, and meningitis and severe infections in immunocompromised adult patients. It has the ability to survive and multiply in various host compartments requiring the expression and coordinate regulation of multiple pathogenicity factors. Several group B streptococcal molecules important for pathogenicity and virulence have been identified and characterized. However, the regulation and control of these factors are poorly understood, and a typical two-component regulatory system that controls their expression has not yet been described.

In gram-positive bacteria, peptide-based signal transduction appears to be the preferred mechanism to sense and respond to population density and coordinate the expression of molecules that are important for pathogenicity. A comprehensive review of bacterial quorum sensing has been published recently (6). Peptide-controlled two-component signal transduction systems regulate the production of pathogenicity factors in Staphylococcus aureus (20), competence of streptococci (10), and bacteriocin production in lactic acid bacteria (7). The agrBDCA (accessory gene regulator) locus encodes the quorum-sensing system that controls the expression of S. aureus virulence factors. In S. aureus the transcription of secreted proteins, including several of the known S. aureus toxins, is upregulated and the transcription of cell surface-associated proteins such as protein A, coagulase, and extracellular matrix (ECM) adhesins is downregulated by agr. Signaling occurs through the peptide pheromone AgrD, and AgrB appears to be involved in the processing of AgrD (11). The typical response regulator and histidine kinase are encoded by agrA and agrC.

The adhesion of streptococci to ECM proteins appears to contribute to the colonization of mucosal surfaces and the internalization of pathogenic bacteria into host cells and interferes with the recognition and elimination of pathogenic bacteria by the host immune system. While the adhesion of S. agalactiae to fibronectin and fibrinogen has been described previously (14, 30) and a genetic locus mediating the binding to human laminin has been identified (28), our knowledge about the interaction of S. agalactiae with ECM proteins remains scarce. We compared the adhesion of S. agalactiae to several immobilized ECM proteins and found that the strongest binding was to immobilized fibrinogen. To identify genes involved in binding of S. agalactiae to fibrinogen, we screened a mutant library of strain O90R for mutants deficient in the agglutination of fibrinogen-coated latex beads. In the present study we report the identification and characterization of a genetic locus with four open reading frames that were designated rgfBDAC (for regulator of fibrinogen binding). The rgf locus encodes the typical histidine kinase and response regulator of a two-component regulatory system and displays similarities to quorum-sensing systems of gram-positive pathogens.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and media. The bacterial strains and plasmids used in this study are listed in Table 1. The S. agalactiae strain O90R (ATCC 12386), a strain derived from the serotype Ia strain O90, was used for nucleotide sequencing of the rgf locus, construction of the pGh9:ISS1 library, and targeted pGhost5 mutagenesis. Escherichia coli strain DH5 served as a host for recombinant pGhost5 plasmids. E. coli strain XL1-Blue MRF was used for screening of the lambda ZAP Express phage library. Streptococcal isolates were cultured on Columbia agar (Oxoid, Basingstoke, England) supplemented with 3% sheep blood, in Todd-Hewitt broth (THB) (Oxoid), or in THB supplemented with 0.5% yeast extract (THY) at 37°C. Mutant strains with chromosomally integrated pGhost5 or pGh9:ISS1 plasmids were cultured in medium containing erythromycin (5 mg/liter) at a temperature of 37°C. For the adhesion experiments, bacteria that were harvested in mid-logarithmic growth phase were grown to an optical density (OD) at 600 nm of 0.5, and stationary-growth-phase bacteria were harvested from an overnight culture with an OD of 1.1 ± 1.

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TABLE 1. Bacterial strains and plasmids

General molecular biology techniques. Standard recombinant DNA techniques were employed for nucleic acid preparation and analysis. PCR was carried out with Taq polymerase with 35 cycles of amplification steps of 1 min at 94°C, 1 min at 50 to 56°C, and 1 to 3 min at 72°C, depending on product size. Genomic streptococcal DNA was isolated as described previously (18). Plasmid DNA was isolated and purified using the Qiaprep Spin Miniprep kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR products were sequenced on an ABI 310 automated DNA sequencer using the ABI PRISM Big-Dye terminator cycle sequencing kit (PE Applied Biosystems, Weiterstadt, Germany). To confirm the presence of rgf in different serotypes, Southern blot analysis was performed after digestion of genomic DNA with EcoRI. Hybridization was carried out at 65°C overnight. The hybridization probe was generated from strain O90R by PCR using the primers 5'-CCT TTC AAT CAG TTC AAG TC-3' and 5'-GCT TCC TGT GAA AGT CC-3'. PCR products were labeled by adding digoxigenin-dUTP (DIG-dUTP) (Roche, Mannheim, Germany) at a final concentration of 5 µM. Hybridizing fragments were visualized with the chemiluminescent substrate CSPD (Serva, Heidelberg, Germany) as instructed by the manufacturer.

S. agalactiae strains were transformed according to the protocol of Ricci et al. (24). Nucleotide sequences adjacent to the pGh9:ISS1 insertion site in rgfB were obtained by screening of the S. agalactiae phage library in Lambda Zap Express as described previously (27).

Construction of S. agalactiae mutants. The vector pGhost9:ISS1 (17) was used for random chromosomal mutagenesis of S. agalactiae strain O90R as described previously (27). To identify the chromosomal insertion site, total genomic DNAs of the pGh9:ISS1 mutants were digested with EcoRI or HindIII, ligated, and transformed into E. coli EC 101 (16). Erythromycin-resistant E. coli clones harboring recombinant pGh9:ISS1 plasmids were selected on tryptic soy agar plates supplemented with erythromycin (150 mg/liter). Nucleotide sequencing of the streptococcal DNA adjacent to the pGh9:ISS1 insertion site was performed with primers annealing to pGh9:ISS1 vector sequences (5'-CGA GGT CGA CGG TAT CG-3' and 5'-TAG ACT TAT CAG GAA ACT TTG C-3'). The plasmid pGhost5 (3) was used for targeted insertion duplication mutagenesis of rgfC. For this purpose an internal 469-bp fragment of rgfC was amplified by PCR with the primers 5'-ATT GGA GAA TTC TCT TAT CTA TGG GCG-3' and 5'-GGC GGC GGA TCC CTA TCC ATC TTA GAG ATA GC-3' (the EcoRI and BamHI restriction sites are underlined) and subcloned into pGhost5. The resulting recombinant plasmid was transformed into strain O90R and integrated into the chromosome as described previously (17). Correct chromosomal integration of the plasmid was confirmed by PCR with primers annealing to vector sequences and genomic nucleotide sequence upstream or downstream of the duplication fragment and subsequent DNA sequencing of the resulting PCR products.

RNA preparation and analysis. Total RNA was prepared from S. agalactiae strains O90R, RGFB-K1, and RGFC-K2 grown to an OD of 0.2 to 0.8 in 10 ml of THB supplemented with 0.5% starch and 1% bovine serum. Cells were lysed mechanically with glass beads in a cell disrupter (Bio 101, Vista, Calif.). Purification of RNA was achieved with the RNeasy Mini kit (Qiagen) as instructed by the manufacturer. Northern blot analysis was carried out with 5 µg of RNA that was separated on a 1% agarose electrophoresis gel {containing 1.8% formaldehyde and 0.02 M MOPS [3-(N-morpholino)propanesulfonic acid]} and transferred to a Biodyne B nylon membrane (Pall, Portsmouth England). Overnight hybridization at 45°C was performed in DIG Easy HYB Solution (Roche) with DIG-dUTP-labeled PCR product generated with the primers 5'-CTA CAA GAG GTG AAC CAG AGC-3' and 5'-GCT ATC CTT TAA TGC TAA TGG AC-3'. Hybridizing fragments were visualized with the chemiluminescent substrate CSPD (Serva) as instructed by the manufacturer. To generate the scpB-specific probe that was used for Northern blot analysis, DIG-dUTP-labeled PCR products were generated with the primers 5'-CTC AAG CTC CTG CTA AGA CTG C-3' and 5'-GCT ACT ATC ATT ACC AGC TGA G-3'. Northern blot analysis of the cfb gene was performed as described previously (22).

For reverse transcription-PCR (RT-PCR) experiments, 100 ng of DNase-treated RNA was used as a template in an RT reaction with 2 pmol of primer annealing to rgfC (5'-TAC AAG TCC TAA TCT CAG AGC-3') or rgfA (5'-CCT TTC AAT CAG TTC AAG TC-3'). RT was carried out at 42°C for 50 min with 200 U of SuperscriptII reverse transcriptase (Gibco BRL, Karlsruhe, Germany). Subsequent PCRs were carried out with 5 µl of the RT reaction product as a template and the primer combination 5'-ACC TTT CGT TAT CAG GTA TCA GC-3' and 5'-TAC AAG TCC TAA TCT CAG AGC-3' for rgfC or 5'-TCA TAC TCG TCG TGC TCT GG-3' and 5'-GCT TCC TGT GAA AGT CC-3' for rgfA. To control for DNA contamination of RNA samples, 100 ng of DNase-treated RNA was used as the template in a PCR under the above-described conditions.

Adhesion of S. agalactiae to ECM components. Screening of the S. agalactiae pGh9:ISS1 library for adhesion-deficient mutants was performed with fibrinogen-coated latex beads. Latex beads (1 ml of 0.81-µm-diameter beads) (Difco Laboratories, Detroit, Mich.) were coated with human fibrinogen (Sigma, Deisenhofen, Germany) at a concentration of 50 µg/ml in 0.17 M glycine-NaOH (pH 8.2) supplemented with 0.01% ovalbumin and 0.01% Merthiolat as described previously (19). Single mutant strains were grown on erythromycin-supplemented blood agar plates, and bacterial cells from 1 CFU were mixed with 25 µl of fibrinogen-coated latex beads on a glass slide and inspected visually for a clumping reaction after 5 min. To confirm the agglutination deficiency, experiments were repeated twice.

To investigate the adhesion of S. agalactiae to immobilized ECM components, adherence assays were performed in ECM-coated Terasaki plates as published previously (25). To investigate fibrinogen adhesion, 60-well Terasaki plates were incubated for 18 h with human fibrinogen (Sigma) reconstituted in Dulbecco's phosphate-buffered saline (DPBS). After washing in DPBS, fibrinogen-coated wells were treated for 1 h in 5% bovine serum albumin blocking solution at 37°C and washed again; 5 x 10⁶ fluorescein isothiocyanate (FITC)-labeled bacteria were added per well, and after 1 h of incubation, nonadhering bacterial cells were removed by washing (five times) in DPBS and plates were treated with 2.5% glutaraldehyde. Adherent bacteria were quantified in a fluorescence counter (Cytofluor II; Perseptive Biosystems Inc.).

Nucleotide sequence accession number. The nucleotide sequence of the coding regions for the S. agalactiae rgf locus has been submitted to the EMBL/GenBank/DDBJ nucleotide sequence data libraries and assigned accession no. AF390107.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adhesion of S. agalactiae to immobilized ECM proteins. To characterize the interaction of the S. agalactiae strain O90R to ECM proteins, laminin, fibronectin, fibrinogen, thrombospondin, vitronectin, and collagen types I and IV were immobilized on microtiter dishes and incubated for 1 h with fluorescently labeled bacteria. While no significant binding of S. agalactiae strain O90R could be detected for collagen types I and IV, binding of S. agalactiae to laminin, fibronectin, fibrinogen, and vitronectin could be observed. Among the different ECM proteins that were studied under these experimental conditions, the most pronounced binding of strain O90R was observed for fibrinogen (Fig. 1). The adhesion to immobilized human fibrinogen was about four times the amount observed for fibronectin, and further studies were focused on investigating the genetic basis of the S. agalactiae fibrinogen adhesion.

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FIG. 1. The adhesion of the S. agalactiae strain O90R to immobilized ECM components was tested in Terasaki plates coated with 100 µg of ECM proteins per ml as indicated. Bacteria were grown in THY, harvested in mid-logarithmic phase, and labeled with FITC. Bacteria (5 x 106) were added to each well and incubated for 60 min at 37°C. Following the removal of nonadherent bacteria, adhesion was quantified in a fluorescence counter. Error bars indicate standard deviations.

Identification and nucleotide sequence characterization of the rgf locus. To identify S. agalactiae genes involved in fibrinogen adhesion, an O90R pGh9:ISS1 mutant library was screened for mutants deficient in their ability to agglutinate fibrinogen-coated latex beads. Four different mutants were isolated in the screen. Analysis of the plasmid insertion sites revealed that in mutants 1, 2, and 3 open reading frames with similarities to a polysaccharide transporter, a tRNA synthase gene, and an amino acid transporter were affected by the mutation. Nucleotide sequence characterization of the pGh9:ISS1 insertion site in mutant 4 revealed that it carried an insertion in a genetic locus with characteristics of a two-component regulatory system, and further studies were focused on this mutant. A schematic representation of the locus is shown in Fig. 2. Genes coding for a typical response regulator and a typical histidine kinase and two additional putative genes could be identified. The first open reading frame is 816 nucleotides long and was designated rgfB. Comparison of the deduced protein with the GenBank database showed 66% identity and 81% similarity with a hypothetical protein of Streptococcus pyogenes (AE006621) that has been identified in the S. pyogenes genome project (8). However, a putative function has not been assigned to the S. pyogenes protein, and no putative protein domains could be identified in the deduced amino acid sequence of rgfB by searching the conserved domain database via the National Center for Biotechnology Information website.

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FIG. 2. Schematic representation of the rgf locus. Open reading frames and approximate sizes are indicated. Primer annealing sites for RT-PCR experiments and the subsequently generated PCR products are shown.

The gene is preceded by a typical ribosome binding site that is located 7 nucleotides upstream. A second putative start codon is located 15 nucleotides upstream of the start codon and could alternatively serve as the start codon; however, a typical ribosome binding site could not be found upstream of this ATG codon. The pGh9:ISS1 insertion site of the initially isolated mutant was located 343 nucleotides downstream of the ATG start codon of rgfB. The next open reading frame in this locus starts 113 nucleotides downstream of rgfB; it is 129 bp long and was designated rgfD. No significant similarities were found upon comparison of the deduced protein with the database. However, the first 20 amino acids of the deduced sequence are hydrophobic according to Kyte-Doolittle analysis (13) and could represent a signal sequence that is cleaved off during processing of the final peptide. The gene of the putative signal peptide is followed by another open reading frame of 657 nucleotides that was designated rgfA. The encoded amino acid sequence is 60% identical and 77% similar to a response regulator of Streptococcus pneumoniae (AJ006401) (15); while it has high similarities to a number of streptococcal response regulators, it also displays a 34% identity and 55% similarity to agrA of S. aureus. The gene rgfA is followed by the last open reading frame in this locus, which was designated rgfC and codes for a putative histidine kinase of 426 amino acids. Conserved residues in H, N, and G₂ blocks of histidine protein kinases (29) are present at amino acids 254 (H), 354 (N), and 409 to 411 (GxG) of the deduced amino acid sequence of rgfC. Again the highest identities (41%) and similarities (61%) of rgfC were observed for the histidine kinase of the previously mentioned S. pneumoniae two-component signal transduction system TCS 13 (15). Comparison of the deduced amino acid sequence with AgrC revealed 23% identity and 45% similarity.

Presence of rgf in different S. agalactiae serotypes. To determine the presence of rgf in selected S. agalactiae strains of different serotypes, a fragment of the rgf gene was amplified by PCR and used as a hybridization probe for Southern blot analysis of S. agalactiae strains of serotypes Ia, Ib, and II to VIII. Hybridization of the probe with chromosomal DNA could be detected as a single band in all of the serotypes tested (data not shown), confirming the presence of this novel two-component regulatory system in all of the known serotypes of S. agalactiae.

Transcription analysis of the rgf locus. The genetic organization of the rgf locus is compatible with polycistronic transcription. To investigate if the encoded genes are located on one transcript, Northern blot analysis and RT-PCR experiments were performed. Hybridization experiments with a probe to rgfB detected an mRNA transcript of more than 3 kb in strain O90R in the late logarithmic growth phase at ODs of 0.6 and 0.8 (Fig. 3). The size of this transcript is compatible with that of the total rgf locus, which comprises 3,129 nucleotides from the start codon of rgfB to the stop codon of rgfC. This transcript was absent in the rgfB mutant strain (Fig. 3) and could not be detected in Northern blots of early-log-phase cultures (OD of 0.2) of the wild-type strain. To investigate whether rgfDAC are also located on this transcript, RT-PCR experiments were performed, and these experiments revealed that these genes are cotranscribed with rgfB.

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FIG. 3. Transcription analysis of the rgf locus was performed by Northern blot analysis (A and B) and RT-PCR (C). (A) Strain O90R was harvested at an OD of 0.6. Lane M, RNA molecular size marker. (B) Strain O90R and the rgfB mutant strain RGFB-K1 were harvested at an OD of 0.8. In both panels A and B, 5 µg of total RNA was probed with an rgfB-specific hybridization probe. Nucleotide sizes in kilobases are indicated at the left. (C) Strain O90R was grown to an OD of 0.8, and 100 ng of total RNA was used as a template for reverse transcription. RT primer annealing sites and the subsequent PCRs are shown in Fig. 2. DNA served as a positive control, and 100 ng of RNA that has not been subjected to an RT reaction was used to control for DNA contamination of RNA samples.

Fibrinogen binding of rgfC mutant and wild-type strains. To characterize the adhesion of the wild-type strain and the rgfC mutant strain to immobilized fibrinogen, fluorescently labeled bacteria were allowed to adhere to microtiter dishes coated with fibrinogen at concentrations ranging from 1 to 200 µg/ml. In both strains a dose dependence of the fibrinogen adherence could be observed, reaching a saturation level for microtiter dishes that were coated with fibrinogen at a concentration of 50 µg/ml (data not shown). At fibrinogen concentrations from 1 to 200 µg/ml, the mutant strain (RGFC-K2) harvested in mid-logarithmic growth phase (OD of 0.5) displayed a significantly reduced binding to immobilized fibrinogen compared to the parent strain O90R. In contrast to these findings, a significantly increased adherence to immobilized fibrinogen could be observed for the RGFC-K2 strain grown to stationary phase (Fig. 4). The adherence values for the RGFC-K2 strain harvested in stationary phase increased to 400% of the adherence values that were determined for the same strain harvested in mid-logarithmic growth phase. In the wild-type strain O90R the adherence to immobilized fibrinogen was not altered for bacteria harvested in different growth phases (Fig. 4).

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FIG. 4. Adhesion of the wild-type strain O90R and the rgfC mutant RGFC-K2 to immobilized fibrinogen. Bacteria were grown in THY and labeled with FITC, and 5 x 106 bacteria per well were allowed to adhere for 1 h at 37°C and subsequently quantified in a fluorescence counter. Values represent means ± standard deviations. For the comparison of adhesion during stationary- and logarithmic-phase growth, strains O90R and RGFC-K2 were grown overnight in THY or harvested at an OD at 600 nm of 0.5. Terasaki plates were coated with 10 µg of fibrinogen per ml.

Influence of rgfC on transcription levels of secreted and surface-localized S. agalactiae proteins. Due to the similarities of the deduced rgf proteins with components of known quorum-sensing systems, we hypothesized that rgf might be involved in the regulation of secreted and surface-localized S. agalactiae proteins. To test this hypothesis, the transcription of the C5a peptidase gene scpB and the CAMP factor gene cfb in the S. agalactiae wild-type strain O90R and the isogenic rgfC mutant was investigated. ScpB harbors the bacterial cell surface anchor motif LPTXG (26), while the CAMP factor is an extracellular protein that is secreted into the growth medium (2). Transcription of scpB and cfb was investigated for bacteria harvested at different growth phases (Fig. 5). While the transcription of cfb appeared to be unaffected by the rgfC mutation, increased transcription of scpB could be observed in rgfC mutants (Fig. 5).

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FIG. 5. Influence of rgfC mutation on transcription of S. agalactiae genes. Total RNAs of S. agalactiae wild-type strain O90R and rgfC mutant RGFC-K2 were harvested at the indicated optical densities (600 nm). Northern blot analysis was performed with labeled PCR products directed toward the C5a peptidase gene scpB (A) and the CAMP factor gene cfb (B). Molecular sizes in kilobases are indicated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In most gram-positive bacteria, pathogenicity is a multifactorial process depending on the coordinate expression of surface-associated and secreted molecules. In S. aureus this process is controlled by the quorum-sensing system agr. This peptide-based cell density-sensing system controls a number of different pathogenicity factors, including the S. aureus enterotoxins, the different hemolysins, and surface-associated factors that mediate the binding of S. aureus to ECM proteins (20) (32). In a screen for S. agalactiae mutants with altered agglutination properties with fibrinogen-coated latex beads, we identified a genetic locus with similarities to quorum-sensing systems of gram-positive pathogens. It contains four open reading frames (rgfBDAC) encoding a putative histidine kinase, RgfC, and a putative response regulator, RgfA. A corresponding peptide hormone may be encoded by rgfD. A close homologue of rgfB was identified in the S. pyogenes genome sequencing project; however, the function of both genes remains unknown. The hypothesis that rgfB encodes a factor that is required for processing of a putative signal peptide will be investigated in future experiments. Transcription analysis of rgfBDAC shows that these genes are cotranscribed in the S. agalactiae wild-type strain O90R (Fig. 3). Investigation of rgf transcription at different growth phases showed strong expression in the late exponential growth phase, resembling the transcription patterns of agr of S. aureus (21). The closest amino acid similarities of the rgf locus were observed for the TCS 13 two-component regulatory system of S. pneumoniae (15), which has been proposed to be involved in pheromone-peptide sensing. Despite the lack of direct experimental evidence that the rgf locus encodes a pheromone-peptide sensing system, the identification of a putative signal peptide and the considerable similarity to the peptide-controlled two-component regulatory systems of other gram-positive pathogens support the hypothesis that the rgf locus represents a quorum-sensing system.

The rgf locus was initially identified in a screen for S. agalactiae mutants that demonstrate altered binding to fibrinogen-coated latex beads. A novel two-component regulatory system of S. pyogenes which regulates the binding to ECM components has recently been described. However, the genetic structure of the fas two-component system encodes two histidine kinases upstream of a response regulator and differs considerably from the genetic structure of the rgf system (12). Beside the agr genes, the sar locus (4) and the sae locus (9) are involved in the control and regulation of virulence factors of S. aureus, and multiple regulators have been described for S. pyogenes (1, 12, 23). Even though a typical two-component regulatory system controlling virulence factors has not yet been characterized in S. agalactiae, the control of virulence-associated genes most likely requires the presence of multiple regulators as in other gram-positive pathogens.

Adherence of S. agalactiae to fibrinogen has been described previously (14), but an S. agalactiae adhesin interacting with fibrinogen has not yet been identified. Since the genetic background of fibrinogen binding is still unclear, we could investigate only the altered fibrinogen binding phenotype. Our initial screen isolated mutants that lost the ability to agglutinate fibrinogen-coated latex beads. This assay does not, however, allow a quantitative measurement of fibrinogen binding. To confirm the involvement of rgf in the regulation of fibrinogen binding, a targeted mutation of the gene rgfC was created by insertion duplication mutagenesis. Since the rgf genes are cotranscribed and targeted insertional mutagenesis of rgfB and rgfA results in downstream effects on rgfC, rgfC was chosen for targeted insertion mutagenesis and further investigation. In contrast to the wild-type strain O90R, which showed constant adherence values in different growth phases, the mutant strain displayed growth phase-dependent binding to immobilized fibrinogen. While binding to immobilized fibrinogen was significantly reduced for the rgfC mutant grown to the mid-logarithmic growth phase, bacteria harvested from an overnight culture showed increased binding to fibrinogen in comparison to the wild-type strain. These findings led to the hypothesis that the rgf system is cell density dependent and that fibrinogen binding molecules on the surface of S. agalactiae are controlled through rgf. To further substantiate the hypothesis that cell surface-located molecules are controlled by rgf, we investigated the transcription of a known cell surface-located virulence factor of S. agalactiae, the C5a peptidase, in the wild type and the rgfC mutant strain. The C5a peptidase (ScpB) interferes with the recruitment of granulocytes to sites of infection through the cleavage of the chemotactic complement component C5a (5, 31). It harbors the cell surface anchor motif LPTXG at its C terminus. Transcription of scpB was increased in the rgfC mutant strain (Fig. 5). The increased fibrinogen binding of the rgfC mutant in the late exponential growth phase, as well as the increased transcription of scpB in this mutant strain, shows similarities to the control of cell surface-associated molecules through agr of S. aureus. The agr system is responsible for a decreased expression of cell surface-associated molecules in the late exponential growth phase.

In summary, our screen for S. agalactiae mutants with altered fibrinogen binding properties led to the identification of a novel two-component regulatory system that was designated rgf. The locus comprises four genes, rgfBDAC, that are cotranscribed and appear to control the expression of cell surface-located molecules in a cell density-dependent manner.

 
The work of B.S. was supported by a grant from the DFG (Sp511/2-2).

 
* Corresponding author. Present address: Department of Medical Microbiology and Hygiene, University of Ulm, Robert Koch Str. 8, D-89081 Ulm, Germany. Phone (49)-731-50024615. Fax (49)-731-50024619. E-mail: barbara.spellerberg{at}medizin.uni-ulm.de.

Editor: E. I. Tuomanen

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Infection and Immunity, May 2002, p. 2434-2440, Vol. 70, No. 5
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.5.2434-2440.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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