Identification of Genes Controlled by the Essential YycG/YycF Two-Component System of Staphylococcus aureus

Sarah Dubrac; Tarek Msadek

doi:10.1128/JB.186.4.1175-1181.2004

. 2004 Feb;186(4):1175–1181. doi: 10.1128/JB.186.4.1175-1181.2004

Identification of Genes Controlled by the Essential YycG/YycF Two-Component System of Staphylococcus aureus

Sarah Dubrac ¹, Tarek Msadek ^1,^*

PMCID: PMC344212 PMID: 14762013

Abstract

The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37°C and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5′-TGT(A/T)A(A/T/C)-N₅-TGT(A/T)A(A/T/C)-3′]. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.

The major human pathogen Staphylococcus aureus is responsible for a broad spectrum of infections, ranging from food poisoning and superficial skin abscesses to more serious diseases such as pneumonia, meningitis, endocarditis, septicemia, and toxic shock syndrome. The unique adaptive potential displayed by S. aureus to environmental conditions and stress has made it one of the main causes of nosocomial infections, emphasized by the rapid emergence of multiple-antibiotic-resistant strains over the past few decades. Until now, vancomycin had remained the weapon of last resort, but the recent appearance of the vanA vancomycin resistance gene cluster in S. aureus highlights the growing threat this bacterium poses to human health and the urgent need to develop novel therapeutic approaches (5). In many cases, virulence gene expression in pathogens is controlled by two-component histidine kinase-response regulator signal transduction systems (TCSs) (9), yet apart from the well-studied AgrC/AgrA system of S. aureus (30) and the CovS/CovR system of S. pyogenes (16), little is known about the function of TCSs in gram-positive pathogens. The high degree of conservation among TCSs, their ubiquitous nature, and the fact that several are essential for cell viability have made them an attractive target for novel classes of antimicrobial compounds (3, 37). The YycG/YycF TCS is specific to low-G+C gram-positive bacteria, where it is the most highly conserved TCS. It has been shown to be essential in Bacillus subtilis and Streptococcus pneumoniae, and a thermosensitive mutation of the system in S. aureus has been shown to be lethal at nonpermissive temperatures (11, 23, 27, 40, 42). Attempts to inactivate the yycFG genes in Streptococcus pyogenes and Listeria monocytogenes have failed, suggesting that the system is also essential in these bacteria (12, 19).

The yycG/yycF genes are expressed during exponential growth and rapidly shut off at entry into stationary phase, suggesting that this system is active, and also necessary, during the exponential growth phase (11). All of the YycG kinase orthologs are similar in domain organization and contain a PAS domain, as determined by using the SMART database (36). PAS domains are signaling modules essentially found in proteins involved in signal transduction. They have been involved in monitoring changes in light, redox potential, oxygen, small-ligand concentration, and more generally, intracellular energy status (39).

Previous studies on the YycG/YycF system have shown that it is involved in competence development and virulence in S. pneumoniae (10, 42). In S. aureus, the YycG/YycF system is thought to play a role in membrane permeability, and a temperature-sensitive mutant of S. aureus yycF exhibited attenuated virulence in an in vivo model (27). Furthermore, links were established among YycG/YycF, resistance to macrolide-lincosamide-streptogramin B (MLS_B) antibiotics, and a secreted antigen, SsaA (26). The YycG/YycF ortholog system in B. subtilis is composed of the YycG histidine kinase and the YycF response regulator (11). Although the exact role of the YycG/YycF system in B. subtilis remains to be established, it has been suggested to be involved in cell division by modulating the expression of the ftsAZ operon (13). During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence consisting of two hexanucleotide direct repeats separated by five nucleotides [5′-TGT(A/T)A(A/T/C)-N₅-TGT(A/T)A(A/T/C)-3′] (18). In addition to the ftsAZ cell division genes, many of the genes controlled by the YycG/YycF system in B. subtilis play a role in cell wall metabolism (tagA/tagD in teichoic acid biosynthesis; yocH, encoding a potential autolysin; and ykvT, encoding a potential spore cortex lytic enzyme) (18).

Since the YycF response regulators of B. subtilis and S. aureus are highly conserved (74% amino acid sequence identity), particularly in the recognition helix of the OmpR-type winged helix-turn-helix DNA-binding domains, it is highly likely that the two regulators recognize the same DNA sequence. With our previously defined consensus sequence, a detailed DNA motif analysis of the S. aureus genome was performed and a list of genes potentially regulated by the YycG/YycF system was established. These encode proteins involved in cell wall metabolism, membrane-bound transport systems, and pathogenicity, including two major antigenic proteins, SsaA and IsaA, and three proteins implicated in interactions between the bacteria and the host matrix. We have shown that YycF of S. aureus binds directly to the regulatory upstream regions of three of these genes, indicating that the regulator recognizes the same consensus established in B. subtilis. Furthermore, these results suggest that the YycG/YycF system regulates the expression of several virulence genes by direct interaction with their promoter regions. This is the first report of genes directly controlled by the YycG/YycF system of S. aureus, whose essential nature in a major pathogen makes it a prime target for the development of novel antibacterial compounds.

MATERIALS AND METHODS

Bacterial strains and growth media.

Escherichia coli K-12 strain TG1 (14) was used for cloning experiments, and E. coli strain BL21λDE3 (38) was used for protein overexpression and purification. E. coli strains were grown in Luria-Bertani medium and transformed by electroporation (35). S. aureus RN4220 (21) and its derivatives were grown in Trypticase soy broth (TSB) medium and transformed by electroporation, with selection on plates supplemented with chloramphenicol (10 μg · ml⁻¹).

Plasmids and plasmid constructions.

Standard procedures were used for DNA manipulation (35). Plasmid pDH88 was used for isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible gene expression (17). Plasmid pET28/16 (6), a derivative of plasmid pET28a (Novagen), was used for protein overexpression and purification.

To generate a strain in which the yyc operon was placed under the control of the inducible Pspac promoter, a 432-bp fragment generated by PCR with primers OSA34 and OSA35bis (Table 1), carrying the ribosome-binding site and 5′ portion of yycF, was digested with XbaI and BglII and cloned into pDH88 to generate pSD3-3. The circular plasmid was then transformed into S. aureus strain RN4220, and integration of the plasmid through a single crossover event generated strain RN4220Pspac-yycF, in which the yycF operon was placed under Pspac control.

TABLE 1.

Oligonucleotides used in this study

Name	Sequence
OSA34	5′-TCTAGATATTAATGATTTAAGAAAAGAGG-3′
OSA35bis	5′-AGATCTCCAGTGTCTTGTGCTGGTTGTGAG-3′
OSA54	5′-TTTTCTGATTAAAAGTCCTATATTATGCAC-3′
OSA55	5′-ATCCTGCAGTTGCGATAGTAGCTGTAGCGA-3′
OSA90	5′-GAGAAGCATATCACTATTAGAAAGTTG-3′
OSA91	5′-GCGCCATTGTAAATGTAGCGAAGCCC-3′
OSA92	5′-GGGTTGATTGGATTCTAAAGGGCACATATTTC-3′
OSA93	5′-GCGTAACCTGTTACACCTAATGCCACTGC-3′
TM314	5′-CCACCATGGCTAGAAAAGTTGTTGTAGTTG-3′
TM315	5′-CTCCTCGAGCTCATGTTGTTGGAGGAAATATCCA-3′
TM316	5′-GGTGGTCTCCCATGTCCAGAGGTAACTATACGC-3′
TM317	5′-CTCCTCGAGTTCATCCCAATCACCGTCTTCAATG-3′

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The YycG histidine kinase has two amino-terminal transmembrane domains, located between residues 14 and 34 and between residues 183 and 203, as determined with von Heijne's algorithm (7). YycF and ′YycG (isolated cytoplasmic histidine kinase domain, residues 220 to 609) were overproduced with plasmids pETyycF and pETyycG, respectively, constructed by cloning a PCR-generated NcoI/XhoI DNA fragment corresponding to the yycF coding sequence (715 bp, PCR TM314/TM315) or a PCR-generated BsaI/XhoI DNA fragment corresponding to the ′yycG coding sequence (1,189 bp, PCR TM316/TM317) between the NcoI and XhoI sites of plasmid pET28/16, replacing the stop codons with an XhoI restriction site. The BsaI enzyme cleaves after its restriction site, generating a DNA fragment whose cohesive end was defined to be compatible with NcoI. This allows the creation of translational fusions adding six histidine residues to the carboxy terminus of the corresponding protein, placing expression of the genes under the control of a T7 bacteriophage promoter.

Overproduction and purification of ′YycG and YycF.

Plasmids pETyycF and pETyycG were introduced into a BL21/λDE3 strain, in which the T7 RNA polymerase gene is under the control of the inducible lacUV5 promoter, which also carries the pREP4 plasmid, allowing coproduction of the GroESL chaperonin, in order to optimize recombinant protein solubility (2). The resulting strains were grown in 2 liters of Luria-Bertani medium at room temperature, expression was induced during the mid-exponential growth phase by addition of 1 mM IPTG, and incubation was pursued for 4 h. Cells were centrifuged at 10,800 × g for 30 min and resuspended in 1/50 of the culture volume of buffer I (50 mM NaH₂PO₄ [pH 8], 300 mM NaCl, 20 mM imidazole). Cells were disrupted by sonication, and cell debris was removed by two consecutive 30-min centrifugation steps at 17,200 × g. E. coli crude protein extracts were loaded onto a 0.2-ml Ni-nitrilotriacetic acid agarose (Qiagen) column equilibrated with buffer I. The column was washed with 10 volumes of buffer II (50 mM NaH₂PO₄ [pH 6], 300 mM NaCl, 30 mM imidazole), and the proteins were eluted with an imidazole gradient (30 to 500 mM). Fractions were pooled and dialyzed against buffer III (50 mM NaH₂PO₄ [pH 8], 300 mM NaCl, 50% glycerol) to remove imidazole and concentrate the protein solution approximately fourfold.

Protein phosphorylation assays.

For ′YycG autophosphorylation, 1.5 μg of protein was incubated for 1 min at room temperature in 10 μl of phosphorylation buffer (100 mM Tris HCl [pH 8], 200 mM KCl, 4 mM MgCl₂, 0.5 mM dithiothreitol, 0.1 mM EDTA, 3.5% glycerol, 2.5 μM ATP, 4 μCi of [γ-³²P]ATP). For phosphotransfer from ′YycG to YycF, equal amounts of both proteins were incubated together and the reaction was initiated by the addition of a radiolabeled ATP mixture as described above. Reactions were stopped by adding 2 μl of sodium dodecyl sulfate (SDS) loading buffer and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 12% acrylamide gels, followed by autoradiography.

Gel shift assay.

A 381-bp DNA fragment corresponding to the ssaA promoter region was generated by PCR with Pwo polymerase (Roche) and oligonucleotides OSA54 and OSA55 (Table 1). Labeling, DNA binding, and electrophoresis were performed as described previously (8).

DNase I footprinting.

DNA fragments corresponding to the promoter regions of ssaA (381 bp, OSA54/OSA55), isaA (311 bp, OSA92/OSA93), and lytM (430 bp, OSA90/OSA91) were generated by PCR with Pwo polymerase (Roche) and the indicated oligonucleotide pairs (for sequences, see Table 1). Labeling of DNA fragments was performed as described previously (8). YycF binding to DNA (5 × 10⁴ cpm per reaction mixture) was performed at room temperature in a buffer containing 25 mM NaH₂PO₄ (pH 8), 50 mM NaCl, 2 mM MgCl₂, 1 mM dithiothreitol, and 10% glycerol. DNase I treatment and electrophoresis were then performed as described previously (8).

Database comparisons and sequence analysis.

Computations were performed with the TopPredII (7) program and the SubtiList and AureoList relational databases (http://genolist.pasteur.fr) (29). Sequence comparisons with the GenBank database were accomplished with the National Center for Biotechnology Information BLAST2 (1) web server by using the default parameter values provided.

RESULTS

Growth and stress resistance of a conditional yycFG mutant.

Growth of a conditional Pspac-yycF mutant was monitored in TSB medium. An overnight culture of the mutant was inoculated with or without IPTG at an optical density at 600 nm (OD₆₀₀) equal to 0.005 and incubated at 37°C. After six generations without IPTG, growth was arrested, indicating that the system is essential at physiological temperatures (Fig. 1A). Interestingly, even after prolonged incubation in the absence of the inducer, the cells did not undergo lysis (Fig. 1A). The number of CFU was monitored in the same assay by plating the cultures on TSB medium plus IPTG. Although no cell lysis was observed in the absence of IPTG, the number of bacteria able to form colonies was drastically decreased, leading to 95% cell death 3 h after cessation of growth, suggesting that absence of yycFG expression is lethal for the cell (Fig. 1A).

FIG. 1. — IPTG-dependent growth of the conditional RN4220Pspac-*yycF* mutant strain. An overnight culture in TSB plus 1 mM IPTG was diluted to an OD₆₀₀ of 0.005 in TSB medium with (circles) or without (squares) 1 mM IPTG. (A) OD₆₀₀ values (solid symbols) and numbers of CFU per milliliter (open symbols) were measured in TSB medium. For measurement of the number of CFU per milliliter, cells were appropriately diluted in TSB liquid medium and plated on TSB plates with 1 mM IPTG. (B) OD₆₀₀ was measured with (circles) or without (squares) 1 mM IPTG. At the indicated times, 1 mM IPTG was added to the culture without IPTG. Each experiment was repeated three times, and the values obtained did not vary significantly.

Microscopic examination of the mutant cells cultivated in the absence of IPTG did not reveal any anomalous cell morphology as previously observed for a conditional mutant of B. subtilis (data not shown) (11, 13). In order to test the capacity of the surviving viable cells to resume a normal growth rate, IPTG was reintroduced 160 min after the growth arrest. After a lag phase, the surviving cells resumed exponential growth, indicating that, as on plates, the living cells recover the ability to divide once the YycG/YycF proteins are produced (Fig. 1B).

The S. aureus RN4220Pspac-yycF strain has a normal growth rate in TSB medium containing 1 mM IPTG, with a doubling time of 30 min. In order to identify stress conditions that require a high level of YycG/YycF in the cell, we determined the minimal inducer concentration that allows normal growth under laboratory culture conditions (rich medium at 37°C with aeration). We determined that the cells have the same doubling time when grown with 1 mM or 50 μM IPTG (data not shown).

We then compared the resistance of cells grown with 1 mM or 50 μM IPTG when exposed to alcohol stress (6% ethanol), salt stress (5% NaCl), high temperature (45°C), and acidic or basic stress (pH 4 or pH 10, respectively). Under these conditions, the cells grew equally well with minimal or maximal inducer concentrations, suggesting that the YycG/YycF system does not play an essential role in resistance to these stresses (data not shown).

Genome-based prediction of the YycG/YycF regulon.

The YycF regulator of S. aureus is very similar to its ortholog in B. subtilis. They have 75% amino acid sequence identity, and the winged helix-turn-helix DNA-binding domains have 30 identical amino acids out of 31 (Fig. 2). This very high level of identity between the two regulators suggested that they may recognize the same DNA sequences. We previously identified a consensus YycF recognition sequence in B. subtilis, consisting of two hexanucleotide direct repeats separated by five nucle-otides [5′-TGT(A/T)A(A/T/C)-N₅-TGT(A/T)A(A/T/C)-3′](18). We performed an in silico search for this motif in the intergenic regions of the complete S. aureus N315 genome with the Search Pattern function of the AureoList database (http://genolist.pasteur.fr/AureoList/) and have established a list of genes potentially regulated by YycG/YycF. Once matches located between convergent genes were eliminated, 34 potential binding sites on either strand within the 400-bp region upstream from the translation initiation codon were identified, corresponding to 31 genetic loci. Among these, only 12 genes encode putative proteins whose potential function can be deduced from sequence similarities (Table 2), whereas the remaining 19 are of unknown function (SA0100, SA0129, SA0364, SA0502, SA0570, SA0618, SA0620/SA0621, SA0674, SA0682, SA0710, SA0913, SA1898, SA1945, SA2097, SA2118, SA2305, SA2353, SA2439, and SA2481). Of the 12 genes potentially regulated by the YycG/YycF system listed in Table 2, 5 have been described as pathogenicity factors: 2 encode major antigens, SsaA and IsaA, and 3 others encode bacterial proteins implicated in interactions with the host matrix (a sialoprotein-binding protein, an elastin-binding protein, and a staphylokinase precursor). One gene, lytM, encodes a peptidoglycan hydrolase, a function shown to be regulated by the YycG/YycF system in B. subtilis (18, 33). We note that 3 of the 19 genes of unknown function preceded by a potential YycF binding site encode proteins with significant similarities to the SsaA antigen (SA0620, SA2097, and SA2353).

FIG. 2. — Amino acid sequence alignment of the *B. subtilis* and *S. aureus* YycF proteins. Conserved residues are shaded, and the DNA-binding winged helix-turn-helix domains are boxed.

TABLE 2.

Alignment of nucleotide sequences of putative YycF-regulated promoter regions

Gene	Sequence^a	Function
lytM*(SA0265)	`→TGTAATGACAA→TGTAAT...N₁₅₈...ATG`	Peptidoglycan hydrolase
prs (SA0458)	`→TGTAATATTAA→TGTAAT...N₁₀₃...ATG`	Ribose-phosphate pyrophosphokinase
sdrD (SA0520)	`→TGTTACAGAAA→TGTAAT...N₂₆₇...ATG`	Sialoprotein-binding protein
hu (SA1305)	`→TGTAATGCTTG→TGTTAA...N₇₄...ATG`	DNA-binding protein II
ebpS (SA1312)	`→TGTAAAATCAT→TGTAAT...N₃₈...ATG`	Elastin-binding protein
phoP (SA1516)	`→TGTAAAAAATG→TGTAAA...N₉₃...ATG`	Transcriptional regulatory protein
sak (SA1758)	`→TGTTAAATATT→TGTTAA...N₁₇₆...ATG`	Staphylokinase precursor
isaA* (SA2356)	`→TGTAAAGAAAG→TGTAAT...N₁₅₆...ATG`	Immunodominant antigen A
opp-2B (SA1214)	`←GTTACACTGCA←GTAACA...N₂₆₅...ATG`	Oligopeptide transporter membrane permease domain
ndhF (SA0411)	`←TTTACAAAATC←TTTACA...N₃₆₉...ATG`	NADH dehydrogenase
SA1221	`←TTTACATTTTC←TTAACA...N₁₂₉...ATG`	Thioredoxin reductase
ssaA* (SA2093)	`←ATTACAAAACG←TTAACA...N₁₃₆...ATG`	Staphylococcal secretory antigen
	`←ATTACAAATTT←GTAACA...N₂₆₅...ATG`

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The direct repeats of the potential YycF binding sites on either strand are shown in bold and indicated by arrows. Genes where direct binding by YycF was demonstrated are indicated by asterisks. The ssa gene promoter region contains two binding sites for YycF.

Purification and phosphorylation of ′YycG and YycF.

In order to determine whether genes on this list are directly regulated by YycF, the ′YycG and YycF proteins were overproduced and purified. The yycF coding sequence was cloned into plasmid pET28/16, creating a translational fusion adding six histidine residues to the carboxy terminus of YycF and placing the gene under the control of an inducible T7 bacteriophage promoter (see Materials and Methods). In order to overproduce ′YycG, a DNA fragment encoding the isolated cytoplasmic histidine kinase domain was cloned into pET28/16, adding six histidine residues to the carboxy terminus of the protein (see Materials and Methods). His-tagged YycF and ′YycG were purified in a single step with an Ni-nitrilotriacetic acid agarose column (see Materials and Methods), and SDS-PAGE analysis revealed a purity greater than 90% (Fig. 3A, lanes 4 and 6, respectively). The purified ′YycG protein displayed the expected apparent molecular mass (approximately 44.8 kDa) (Fig. 3A, lane 6), whereas the recombinant YycF protein migrated slightly higher than the expected size (approximately 30 kDa instead of the deduced molecular mass of 28.2 kDa) (Fig. 3A, lane 4). Such anomalous electrophoretic migration has previously been reported for its ortholog in B. subtilis (18).

FIG. 3. — Purification and phosphorylation of ′YycG and YycF. (A) SDS-PAGE analysis of crude extracts from *E. coli* BL21/λDE3 carrying pET28/16 (lane 2), pETyycF (lane 3), or pETyycG (lane 5). Purified proteins (approximately 50 pmol) were loaded in lane 4 (YycF) and lane 6 (′YycG). Molecular size standards were loaded in lane 1. (B) In vitro phosphorylation of ′YycG and YycF. Lane 1, ′YycG incubated with [γ-³²P]ATP for 1 min at room temperature. ′YycG and YycF were incubated for various times following the addition of [γ-³²P]ATP. Lanes 2 to 7, 0.5, 1, 5, 10, 15, and 20 min, respectively.

Autophosphorylation of the ′YycG histidine kinase was demonstrated by incubating the protein with [γ-³²P]ATP and then subjecting it to SDS-PAGE and autoradiography, as shown in Fig. 3B, lane 1. When the purified YycF and ′YycG proteins were incubated together, autophosphorylation of ′YycG and phosphotransfer to YycF were observed upon addition of [γ-³²P]ATP, followed by SDS-PAGE and autoradiography (Fig. 3B, lanes 2 to 7). Phosphorylation of YycF by ′YycG reached equilibrium in approximately 10 min (Fig. 3B, lane 5).

YycF binds specifically to the ssaA promoter region.

As mentioned above, ssaA appears among the genes listed as potential members of the YycG/YycF regulon. In a previous report, it has been shown that the hypersensitivity to MLS_B antibiotics of a temperature-sensitive yycF mutant could be suppressed by ssaA overexpression, suggesting that ssaA expression could be regulated by YycF (26). To test this hypothesis, we have carried out assays of YycF binding to the ssaA promoter region. Gel mobility shift assays were performed with the purified YycF protein and a 381-bp radiolabeled DNA fragment corresponding to the ssaA promoter region and synthesized by PCR with oligonucleotides OSA54 and OSA55 (Table 1). The appearance of two different-sized protein-DNA complexes suggests that two YycF molecules can bind to the ssaA promoter region (Fig. 4A). DNase I footprinting assays were performed to precisely determine the location of the YycF binding site(s). As shown in Fig. 4B, YycF binds two distinct regions, extending from position −283 to position −255 and from −164 to −133 relative to the translation start site (Fig. 4C), in agreement with the two different protein-DNA complexes observed in the gel mobility shift experiments. These two regions each contain the direct repeat predicted to be a YycF target site (Table 2). These data constitute the first demonstration of direct binding of the YycF regulator on a promoter region in S. aureus. Furthermore, they provide a direct link between ssaA expression and the YycG/YycF system, since it seems that ssaA transcription is directly regulated by YycF.

FIG. 4. — Gel mobility shift assay and DNase I footprinting analysis of YycF binding to the *ssaA* promoter region. (A) DNA-binding reactions were performed with 0.1 pmol of a radiolabeled DNA fragment corresponding to the *ssaA* promoter region (−340 to +40 with respect to the translation initiation site) and purified YycF. Lanes: 1, no protein; 2, 26 pmol; 3, 34 pmol; 4, 53 pmol; 5, 68 pmol; 6, 106 pmol. (B) DNase I footprinting of YycF bound to the *ssaA* promoter region. Lanes contain approximately 0.5 pmol (5 × 10⁴ cpm per reaction mixture) of labeled nontemplate or template strand (NTS and TS, respectively) of *ssaA* (−340 to +40). Lanes: 1, no protein; 2, 140 pmol; 3, 212 pmol; 4, A+G Maxam and Gilbert reaction. Brackets indicate proximal and distal regions protected by YycF from DNase I cleavage (I and II, respectively). (C) Nucleotide sequences of the *ssaA* promoter region. Regions protected by YycF from DNase I cleavage are shown by bars, and the conserved repeats are indicated by arrows.

Binding of YycF in two additional promoter regions predicted to contain YycF binding sites.

In addition to ssaA, four genes closely linked to S. aureus virulence have the YycF consensus sequence in their promoter regions: sdrD, encoding a sialoprotein-binding protein; ebpS, encoding an elastin-binding protein; sak, encoding a staphylokinase precursor; and isaA, encoding immunodominant antigen A (Table 2). Furthermore, as described in B. subtilis (18), it seems that the YycG/YycF system also regulates cell wall metabolism in S. aureus via regulation of lytM, which encodes a peptidoglycan hydrolase (33). DNase I footprinting assays were performed on radiolabeled DNA fragments corresponding to the lytM and isaA promoter regions, generated by PCR with oligonucleotides OSA92/OSA93 (isaA) and OSA90/OSA91 (lytM) (the oligonucleotide sequences are listed in Table 1). YycF bound specifically to these two promoter regions (Fig. 5A and B). The regions protected by YycF binding are located around the previously defined consensus sequence (Fig. 5C). The YycF regulator binds the isaA DNA fragment at two distinct regions (Fig. 5A and C). The region with higher apparent affinity for YycF (region I in Fig. 5A and C) contains the direct repeat proposed to be the YycF consensus recognition sequence. The second site (region II in Fig. 5A and C) contains only one-half of the direct repeat, which could explain the lower affinity observed for YycF binding.

FIG. 5. — DNase I footprinting assay of YycF bound to the *isaA* and *lytM* promoter regions. Lanes contain approximately 0.7 pmol (5 × 10⁴ cpm per reaction mixture) of labeled nontemplate strands of *isaA* (−250 to +59 with respect to the translation initiation codon) (A) or *lytM* (−321 to +60 with respect to the translation initiation codon) (B). Fragments were incubated with increasing amounts of purified YycF. (A) Lanes: 1, no protein; 2, 34 pmol; 3, 68 pmol; 4, 136 pmol; 5, 204 pmol; 6, A+G Maxam and Gilbert reaction. (B) Lanes: 1, no protein; 2, 136 pmol; 3, 272 pmol; 4, 408 pmol; 5, A+G Maxam and Gilbert reaction. The brackets indicate regions protected by YycF from DNase I cleavage. (C) Nucleotide sequence of the *isaA* and *lytM* promoter regions. Regions protected by YycF are indicated by bars, and the conserved direct repeats are indicated by arrows.

DISCUSSION

This report is the first description of genes directly regulated by the essential YycG/YycF TCS in S. aureus. Previous work has highlighted several phenotypes linked to a thermosensitive mutation of the yycF locus such as hypersensitivity to MLS_B antibiotics and to unsaturated long-chain fatty acids and partial suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations (27).

Although these characteristics led the authors to suggest that the YycG/YycF TCS could be implicated in regulation of bacterial cell wall or membrane composition, no genes regulated by this system had been identified. A recent report had focused on gene products able to complement the hypersensitivity to MLS_B antibiotics of the conditional yycF mutant, and it was suggested that the loss of function or expression of ssaA was responsible for this phenotype (26). Although we were not able to reproduce the MLS_B hypersensitivity phenotype with the RN4220Pspac-yycF mutant (data not shown), specific binding of the purified YycF protein to the ssaA promoter region supports the hypothesis of direct regulation of ssaA expression. The ssaA gene encodes a major antigenic protein, SsaA (staphylococcal secretory antigen), whose function has been investigated in S. epidermidis (22). As SsaA from S. epidermidis has 74% amino acid sequence identity with SsaA from S. aureus, we can postulate that these two proteins have similar functions in these two closely related bacteria. It has been reported that a high rate of anti-SsaA immunoglobulin G antibodies can be detected in sera of patients with S. epidermidis endocarditis, suggesting a role for SsaA in the pathogenesis of this disease (22). Interestingly, three genes of unknown function potentially belonging to the YycF regulon of S. aureus encode proteins with strong similarities to SsaA (SA0620, SA2097, and SA2353). We have also shown that another important antigenic protein, IsaA (immunodominant staphylococcal antigen), is probably regulated by the YycG/YycF system as well. Although the putative function of this protein is still unknown, the high rate of antibodies raised against IsaA and found during sepsis caused by methicillin-resistant S. aureus shows that this protein is expressed at a high rate during infection, which could suggest a putative role in colonizing host tissues (25). Although it has to be verified, three other genes encoding potential virulence factors implicated in interactions with the host extracellular matrix may also be regulated by YycG/YycF. The sdrD gene encodes a Ser-Asp-rich sialoprotein-binding protein, a function reported to be important in the pathogenicity of osteomyelitis (41). EbpS is implicated in interactions between S. aureus and elastin, one of the major protein components of the extracellular matrix (31, 32). Finally, Sak, the staphylokinase, may have an important physiological function in helping cells to disseminate in host tissue by activating plasminogen (28). A comparison of candidates for YycG/YycF regulation in B. subtilis and S. aureus reveals several similarities. The sizes of the regulons are quite similar since the B. subtilis YycF regulon comprises 14 genes, including 4 genes for which specific binding of YycF has been shown (18), whereas the S. aureus YycF regulon is composed of 12 genes, and specific binding was shown for 3 of these. YycF-dependent regulation of yocH, which encodes a peptidoglycan hydrolase, has been demonstrated in B. subtilis (18). In S. aureus, the present work has highlighted binding of YycF to the lytM promoter region. LytM is a glycylglycine endopeptidase (33, 34), indicating a likely role for the S. aureus YycG/YycF system in controlling cell wall metabolism, as shown for its ortholog in B. subtilis. The yycF operon is transcribed during exponential growth in B. subtilis, and its expression is rapidly shut down as cells enter stationary phase (11). The yocH gene follows the same expression profile, and the results suggest that the YycG/YycF system is only active during the exponential growth phase (18). Expression of the S. aureus yycF operon has not been studied, but a predicted σ^A promoter was suggested, 215 bp upstream from the yycF translation start site, in agreement with expression of the operon during exponential growth (27). This correlates with the lytM expression profile since LytM is essentially synthesized during exponential growth (34). The second common link between the B. subtilis and S. aureus YycF regulons is an oligopeptide ABC transporter. Potential YycF binding sequences are found in front of the opp (oligopeptide permease) operons of B. subtilis and S. aureus, as well as in front of the oppA gene of L. monocytogenes and the Streptococcus agalactiae gbs0144 gene, which encodes a protein similar to the binding protein of the Opp system. This suggests that YycF and their orthologs in closely related bacteria could control synthesis of the Opp system. The Opp system has a very pleiotropic role since it is implicated in the uptake of various oligopeptides that act as signals for the cell (for a review, see reference 24). In B. subtilis, the Opp system imports extracellular peptides that influence competence and sporulation (20). In L. monocytogenes, the Opp system is implicated in bacterial growth at low temperature and intracellular survival (4), whereas it controls extracellular protein production and virulence in Bacillus thuringiensis (15).

In summary, our results show that the YycF regulator of S. aureus recognizes a tandemly repeated hexanucleotide sequence with a spacer of five nucleotides [5′-TGT(A/T)A(A/T/C)-N₅-TGT(A/T)A(A/T/C)-3′] and that it binds specifically to genes involved in virulence and cell wall metabolism. But the main questions concerning the essential nature of the YycG/YycF system remain unanswered since none of the 12 putative YycF-regulated genes in S. aureus have been shown to be essential. In B. subtilis, it was suggested that the essential nature of the YycG/YycF system could be due to the regulation of the essential tagD/tagA genes involved in teichoic acid biosynthesis (18), but we were unable to demonstrate specific binding of YycF to the tag operon promoter region of S. aureus (data not shown). Two hypotheses could explain the essentiality of the YycG/YycF system. Either it controls several functions that are essential not on their own but only in combination, or it regulates an essential function that was not picked up in our DNA motif analysis. As a complementary approach to identify new genes directly controlled by YycF in S. aureus, ChIP-to-chip experiments (chromatin immunoprecipitation followed by hybridization to DNA macroarrays) are now in progress.

Acknowledgments

We are grateful to G. Rapoport for helpful discussion and critical reading of the manuscript.

This work was supported by research funds from the Institut Pasteur, Centre National de Recherche Scientifique, and the European Commission (grant QLG2-CT-1999-01455).

REFERENCES

1.Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Amrein, K. E., B. Takacs, M. Stieger, J. Molnos, N. A. Flint, and P. Burn. 1995. Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc. Natl. Acad. Sci. USA 92:1048-1052. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Barrett, J. F., and J. A. Hoch. 1998. Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob. Agents Chemother. 42:1529-1536. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Borezee, E., E. Pellegrini, and P. Berche. 2000. OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect. Immun. 68:7069-7077. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Chang, S., D. M. Sievert, J. C. Hageman, M. L. Boulton, F. C. Tenover, F. P. Downes, S. Shah, J. T. Rudrik, G. R. Pupp, W. J. Brown, D. Cardo, and S. K. Fridkin. 2003. Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. N. Engl. J. Med. 348:1342-1347. [DOI] [PubMed] [Google Scholar]
6.Chastanet, A., J. Fert, and T. Msadek. 2003. Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other gram-positive bacteria. Mol. Microbiol. 47:1061-1073. [DOI] [PubMed] [Google Scholar]
7.Claros, M. G., and G. von Heijne. 1994. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10:685-686.7704669 [Google Scholar]
8.Derre, I., G. Rapoport, and T. Msadek. 1999. CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria. Mol. Microbiol. 31:117-131. [DOI] [PubMed] [Google Scholar]
9.Dziejman, M., and J. J. Mekalanos. 1995. Two-component signal transduction and its role in the expression of bacterial virulence factors, p. 305-317. In J. A. Hoch and T. J. Silhavy (ed.), Two-component signal transduction. ASM Press, Washington, D.C.
10.Echenique, J. R., and M. C. Trombe. 2001. Competence repression under oxygen limitation through the two-component MicAB signal-transducing system in Streptococcus pneumoniae and involvement of the PAS domain of MicB. J. Bacteriol. 183:4599-4608. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Fabret, C., and J. A. Hoch. 1998. A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J. Bacteriol. 180:6375-6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Federle, M. J., K. S. McIver, and J. R. Scott. 1999. A response regulator that represses transcription of several virulence operons in the group A Streptococcus. J. Bacteriol. 181:3649-3657. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Fukuchi, K., Y. Kasahara, K. Asai, K. Kobayashi, S. Moriya, and N. Ogasawara. 2000. The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis. Microbiology 146:1573-1583. [DOI] [PubMed] [Google Scholar]
14.Gibson, T. J. 1984. Studies on the Epstein-Barr virus genome. Ph.D. thesis. University of Cambridge, Cambridge, United Kingdom.
15.Gominet, M., L. Slamti, N. Gilois, M. Rose, and D. Lereclus. 2001. Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol. Microbiol. 40:963-975. [DOI] [PubMed] [Google Scholar]
16.Graham, M. R., L. M. Smoot, C. A. Migliaccio, K. Virtaneva, D. E. Sturdevant, S. F. Porcella, M. J. Federle, G. J. Adams, J. R. Scott, and J. M. Musser. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc. Natl. Acad. Sci. USA 99:13855-13860. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Henner, D. J. 1990. Inducible expression of regulatory genes in Bacillus subtilis. Methods Enzymol. 185:223-228. [DOI] [PubMed] [Google Scholar]
18.Howell, A., S. Dubrac, K. K. Andersen, D. Noone, J. Fert, T. Msadek, and K. Devine. 2003. Genes controlled by the essential YycG/YycF two-component system of Bacillus subtilis revealed through a novel hybrid regulator approach. Mol. Microbiol. 49:1639-1655. [DOI] [PubMed] [Google Scholar]
19.Kallipolitis, B. H., and H. Ingmer. 2001. Listeria monocytogenes response regulators important for stress tolerance and pathogenesis. FEMS Microbiol. Lett. 204:111-115. [DOI] [PubMed] [Google Scholar]
20.Koide, A., and J. A. Hoch. 1994. Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation. Mol. Microbiol. 13:417-426. [DOI] [PubMed] [Google Scholar]
21.Kreiswirth, B. N., S. Lofdahl, M. J. Betley, M. O'Reilly, P. M. Schlievert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709-712. [DOI] [PubMed] [Google Scholar]
22.Lang, S., M. A. Livesley, P. A. Lambert, W. A. Littler, and T. S. Elliott. 2000. Identification of a novel antigen from Staphylococcus epidermidis. FEMS Immunol. Med. Microbiol. 29:213-220. [DOI] [PubMed] [Google Scholar]
23.Lange, R., C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein. 1999. Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae. Gene 237:223-234. [DOI] [PubMed] [Google Scholar]
24.Lazazzera, B. A., and A. D. Grossman. 1998. The ins and outs of peptide signaling. Trends Microbiol. 6:288-294. [DOI] [PubMed] [Google Scholar]
25.Lorenz, U., K. Ohlsen, H. Karch, M. Hecker, A. Thiede, and J. Hacker. 2000. Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus. FEMS Immunol. Med. Microbiol. 29:145-153. [DOI] [PubMed] [Google Scholar]
26.Martin, P. K., Y. Bao, E. Boyer, K. M. Winterberg, L. McDowell, M. B. Schmid, and J. M. Buysse. 2002. Novel locus required for expression of high-level macrolide-lincosamide-streptogramin B resistance in Staphylococcus aureus. J. Bacteriol. 184:5810-5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Martin, P. K., T. Li, D. Sun, D. P. Biek, and M. B. Schmid. 1999. Role in cell permeability of an essential two-component system in Staphylococcus aureus. J. Bacteriol. 181:3666-3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Molkanen, T., J. Tyynela, J. Helin, N. Kalkkinen, and P. Kuusela. 2002. Enhanced activation of bound plasminogen on Staphylococcus aureus by staphylokinase. FEBS Lett. 517:72-78. [DOI] [PubMed] [Google Scholar]
29.Moszer, I., L. M. Jones, S. Moreira, C. Fabry, and A. Danchin. 2002. SubtiList: the reference database for the Bacillus subtilis genome. Nucleic Acids Res. 30:62-65. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Novick, R. P. 2003. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Microbiol. 48:1429-1449. [DOI] [PubMed] [Google Scholar]
31.Park, P. W., T. J. Broekelmann, B. R. Mecham, and R. P. Mecham. 1999. Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS). J. Biol. Chem. 274:2845-2850. [DOI] [PubMed] [Google Scholar]
32.Park, P. W., J. Rosenbloom, W. R. Abrams, and R. P. Mecham. 1996. Molecular cloning and expression of the gene for elastin-binding protein (ebpS) in Staphylococcus aureus. J. Biol. Chem. 271:15803-15809. [DOI] [PubMed] [Google Scholar]
33.Ramadurai, L., and R. K. Jayaswal. 1997. Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus. J. Bacteriol. 179:3625-3631. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Ramadurai, L., K. J. Lockwood, M. J. Nadakavukaren, and R. K. Jayaswal. 1999. Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus. Microbiology 145:801-808. [DOI] [PubMed] [Google Scholar]
35.Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
36.Schultz, J., F. Milpetz, P. Bork, and C. P. Ponting. 1998. SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. USA 95:5857-5864. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Stephenson, K., and J. A. Hoch. 2002. Virulence- and antibiotic resistance-associated two-component signal transduction systems of gram-positive pathogenic bacteria as targets for antimicrobial therapy. Pharmacol. Ther. 93:293-305. [DOI] [PubMed] [Google Scholar]
38.Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189:113-130. [DOI] [PubMed] [Google Scholar]
39.Taylor, B. L., and I. B. Zhulin. 1999. PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev. 63:479-506. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Throup, J. P., K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham. 2000. A genomic analysis of two-component signal transduction in Streptococcus pneumoniae. Mol. Microbiol. 35:566-576. [DOI] [PubMed] [Google Scholar]
41.Tung, H., B. Guss, U. Hellman, L. Persson, K. Rubin, and C. Ryden. 2000. A bone sialoprotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family. Biochem. J. 345(Pt. 3):611-619. [PMC free article] [PubMed] [Google Scholar]
42.Wagner, C., A. Saizieu Ad, H. J. Schonfeld, M. Kamber, R. Lange, C. J. Thompson, and M. G. Page. 2002. Genetic analysis and functional characterization of the Streptococcus pneumoniae vic operon. Infect. Immun. 70:6121-6128. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r1] 1.Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r2] 2.Amrein, K. E., B. Takacs, M. Stieger, J. Molnos, N. A. Flint, and P. Burn. 1995. Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc. Natl. Acad. Sci. USA 92:1048-1052. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r3] 3.Barrett, J. F., and J. A. Hoch. 1998. Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob. Agents Chemother. 42:1529-1536. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r4] 4.Borezee, E., E. Pellegrini, and P. Berche. 2000. OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect. Immun. 68:7069-7077. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r5] 5.Chang, S., D. M. Sievert, J. C. Hageman, M. L. Boulton, F. C. Tenover, F. P. Downes, S. Shah, J. T. Rudrik, G. R. Pupp, W. J. Brown, D. Cardo, and S. K. Fridkin. 2003. Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. N. Engl. J. Med. 348:1342-1347. [DOI] [PubMed] [Google Scholar]

[r6] 6.Chastanet, A., J. Fert, and T. Msadek. 2003. Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other gram-positive bacteria. Mol. Microbiol. 47:1061-1073. [DOI] [PubMed] [Google Scholar]

[r7] 7.Claros, M. G., and G. von Heijne. 1994. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10:685-686.7704669 [Google Scholar]

[r8] 8.Derre, I., G. Rapoport, and T. Msadek. 1999. CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria. Mol. Microbiol. 31:117-131. [DOI] [PubMed] [Google Scholar]

[r9] 9.Dziejman, M., and J. J. Mekalanos. 1995. Two-component signal transduction and its role in the expression of bacterial virulence factors, p. 305-317. In J. A. Hoch and T. J. Silhavy (ed.), Two-component signal transduction. ASM Press, Washington, D.C.

[r10] 10.Echenique, J. R., and M. C. Trombe. 2001. Competence repression under oxygen limitation through the two-component MicAB signal-transducing system in Streptococcus pneumoniae and involvement of the PAS domain of MicB. J. Bacteriol. 183:4599-4608. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.Fabret, C., and J. A. Hoch. 1998. A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J. Bacteriol. 180:6375-6383. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Federle, M. J., K. S. McIver, and J. R. Scott. 1999. A response regulator that represses transcription of several virulence operons in the group A Streptococcus. J. Bacteriol. 181:3649-3657. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r13] 13.Fukuchi, K., Y. Kasahara, K. Asai, K. Kobayashi, S. Moriya, and N. Ogasawara. 2000. The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis. Microbiology 146:1573-1583. [DOI] [PubMed] [Google Scholar]

[r14] 14.Gibson, T. J. 1984. Studies on the Epstein-Barr virus genome. Ph.D. thesis. University of Cambridge, Cambridge, United Kingdom.

[r15] 15.Gominet, M., L. Slamti, N. Gilois, M. Rose, and D. Lereclus. 2001. Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol. Microbiol. 40:963-975. [DOI] [PubMed] [Google Scholar]

[r16] 16.Graham, M. R., L. M. Smoot, C. A. Migliaccio, K. Virtaneva, D. E. Sturdevant, S. F. Porcella, M. J. Federle, G. J. Adams, J. R. Scott, and J. M. Musser. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc. Natl. Acad. Sci. USA 99:13855-13860. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r17] 17.Henner, D. J. 1990. Inducible expression of regulatory genes in Bacillus subtilis. Methods Enzymol. 185:223-228. [DOI] [PubMed] [Google Scholar]

[r18] 18.Howell, A., S. Dubrac, K. K. Andersen, D. Noone, J. Fert, T. Msadek, and K. Devine. 2003. Genes controlled by the essential YycG/YycF two-component system of Bacillus subtilis revealed through a novel hybrid regulator approach. Mol. Microbiol. 49:1639-1655. [DOI] [PubMed] [Google Scholar]

[r19] 19.Kallipolitis, B. H., and H. Ingmer. 2001. Listeria monocytogenes response regulators important for stress tolerance and pathogenesis. FEMS Microbiol. Lett. 204:111-115. [DOI] [PubMed] [Google Scholar]

[r20] 20.Koide, A., and J. A. Hoch. 1994. Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation. Mol. Microbiol. 13:417-426. [DOI] [PubMed] [Google Scholar]

[r21] 21.Kreiswirth, B. N., S. Lofdahl, M. J. Betley, M. O'Reilly, P. M. Schlievert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709-712. [DOI] [PubMed] [Google Scholar]

[r22] 22.Lang, S., M. A. Livesley, P. A. Lambert, W. A. Littler, and T. S. Elliott. 2000. Identification of a novel antigen from Staphylococcus epidermidis. FEMS Immunol. Med. Microbiol. 29:213-220. [DOI] [PubMed] [Google Scholar]

[r23] 23.Lange, R., C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein. 1999. Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae. Gene 237:223-234. [DOI] [PubMed] [Google Scholar]

[r24] 24.Lazazzera, B. A., and A. D. Grossman. 1998. The ins and outs of peptide signaling. Trends Microbiol. 6:288-294. [DOI] [PubMed] [Google Scholar]

[r25] 25.Lorenz, U., K. Ohlsen, H. Karch, M. Hecker, A. Thiede, and J. Hacker. 2000. Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus. FEMS Immunol. Med. Microbiol. 29:145-153. [DOI] [PubMed] [Google Scholar]

[r26] 26.Martin, P. K., Y. Bao, E. Boyer, K. M. Winterberg, L. McDowell, M. B. Schmid, and J. M. Buysse. 2002. Novel locus required for expression of high-level macrolide-lincosamide-streptogramin B resistance in Staphylococcus aureus. J. Bacteriol. 184:5810-5813. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r27] 27.Martin, P. K., T. Li, D. Sun, D. P. Biek, and M. B. Schmid. 1999. Role in cell permeability of an essential two-component system in Staphylococcus aureus. J. Bacteriol. 181:3666-3673. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r28] 28.Molkanen, T., J. Tyynela, J. Helin, N. Kalkkinen, and P. Kuusela. 2002. Enhanced activation of bound plasminogen on Staphylococcus aureus by staphylokinase. FEBS Lett. 517:72-78. [DOI] [PubMed] [Google Scholar]

[r29] 29.Moszer, I., L. M. Jones, S. Moreira, C. Fabry, and A. Danchin. 2002. SubtiList: the reference database for the Bacillus subtilis genome. Nucleic Acids Res. 30:62-65. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r30] 30.Novick, R. P. 2003. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Microbiol. 48:1429-1449. [DOI] [PubMed] [Google Scholar]

[r31] 31.Park, P. W., T. J. Broekelmann, B. R. Mecham, and R. P. Mecham. 1999. Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS). J. Biol. Chem. 274:2845-2850. [DOI] [PubMed] [Google Scholar]

[r32] 32.Park, P. W., J. Rosenbloom, W. R. Abrams, and R. P. Mecham. 1996. Molecular cloning and expression of the gene for elastin-binding protein (ebpS) in Staphylococcus aureus. J. Biol. Chem. 271:15803-15809. [DOI] [PubMed] [Google Scholar]

[r33] 33.Ramadurai, L., and R. K. Jayaswal. 1997. Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus. J. Bacteriol. 179:3625-3631. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r34] 34.Ramadurai, L., K. J. Lockwood, M. J. Nadakavukaren, and R. K. Jayaswal. 1999. Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus. Microbiology 145:801-808. [DOI] [PubMed] [Google Scholar]

[r35] 35.Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[r36] 36.Schultz, J., F. Milpetz, P. Bork, and C. P. Ponting. 1998. SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. USA 95:5857-5864. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r37] 37.Stephenson, K., and J. A. Hoch. 2002. Virulence- and antibiotic resistance-associated two-component signal transduction systems of gram-positive pathogenic bacteria as targets for antimicrobial therapy. Pharmacol. Ther. 93:293-305. [DOI] [PubMed] [Google Scholar]

[r38] 38.Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189:113-130. [DOI] [PubMed] [Google Scholar]

[r39] 39.Taylor, B. L., and I. B. Zhulin. 1999. PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev. 63:479-506. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r40] 40.Throup, J. P., K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham. 2000. A genomic analysis of two-component signal transduction in Streptococcus pneumoniae. Mol. Microbiol. 35:566-576. [DOI] [PubMed] [Google Scholar]

[r41] 41.Tung, H., B. Guss, U. Hellman, L. Persson, K. Rubin, and C. Ryden. 2000. A bone sialoprotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family. Biochem. J. 345(Pt. 3):611-619. [PMC free article] [PubMed] [Google Scholar]

[r42] 42.Wagner, C., A. Saizieu Ad, H. J. Schonfeld, M. Kamber, R. Lange, C. J. Thompson, and M. G. Page. 2002. Genetic analysis and functional characterization of the Streptococcus pneumoniae vic operon. Infect. Immun. 70:6121-6128. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Identification of Genes Controlled by the Essential YycG/YycF Two-Component System of Staphylococcus aureus

Sarah Dubrac

Tarek Msadek

Abstract