Staphylococcus aureus ClpC Divergently Regulates Capsule via sae and codY in Strain Newman but Activates Capsule via codY in Strain UAMS-1 and in Strain Newman with Repaired saeS

Abstract

ClpC is an ATPase chaperone found in most Gram-positive low-GC bacteria. It has been recently reported that ClpC affected virulence gene expression in Staphylococcus aureus. Here we report that ClpC regulates transcription of the cap operon and accumulation of capsule, a major virulence factor for S. aureus. As virulence genes are regulated by a complex regulatory network in S. aureus, we have used capsule as a model to understand this regulation. By microarray analyses of strain Newman, we found that ClpC strongly activates transcription of the sae operon, whose products are known to negatively regulate capsule synthesis in this strain. Further studies indicated that ClpC repressed capsule production by activating the sae operon in strain Newman. Interestingly, the clpC gene cloned into a multiple-copy plasmid vector exhibited an activation phenotype, suggesting that ClpC overexpression has a net positive effect. In the absence of sae function, by either deletion or correction of a native mutation within saeS, we found that ClpC had a positive effect on capsule production. Indeed, in the UAMS-1 strain, which does not have the saeS mutation, ClpC functioned as an activator of capsule production. Our microarray analyses of strain Newman also revealed that CodY, a repressor of capsule production, was repressed by ClpC. Using genetic approaches, we showed that CodY functioned downstream of ClpC, leading to capsule activation both in Newman and in UAMS-1. Thus, ClpC functions in two opposite pathways in capsule regulation in strain Newman but functions as a positive activator in strain UAMS-1.

Staphylococcus aureus has a repertoire of virulence factors, including secreted toxins, enzymes, and cell wall-associated molecules, that allow it to infect and survive in a host. The ability of S. aureus to survive under various conditions relies on fine control of its virulence factors (23). Recent intense studies have shown that the virulence factors are regulated by an impressive number of regulators in a complex network involving transcriptional factors and two-component environmental sensing systems (4). The complexity of the regulatory network poses a challenge to understanding how virulence factors are controlled by various regulators in the context of pathogenesis.

Capsule is one of several virulence factors that contribute to S. aureus pathogenicity. This cell surface molecule is produced by most S. aureus strains, with type 5 or type 8 being the dominant serotype. The cap5 and cap8 genes, required for type 5 and type 8 capsule synthesis, respectively, are allelic and organized as an operon in which the polycistronic message is transcribed mainly by a promoter located at the beginning of the operon (21, 37). The nearly identical promoter regions of the cap5 and cap8 operons indicate that their mechanisms of regulation are similar. We have previously characterized the cap promoter in detail and have defined the control region within a short (60-bp) sequence that includes a cis-acting 10-bp inverted repeat (38). As the control region of the cap operon is relatively simple, it is surprising that 12 regulators have been reported to affect capsule production. The large number of regulators affecting capsule production suggests that these regulators form a regulatory network to fine-tune capsule production in response to various environmental conditions. Thus, studying capsule regulation would lead to further understanding of the complex regulatory network controlling virulence in S. aureus. Recently, we have shown that arlRS upregulates capsule production by activating mgrA and that sbcDC, which is repressed by LexA, downregulates capsule production by repressing arlRS and mgrA (8, 25). These studies therefore establish a connection between the SOS response and capsule gene regulation. More recently, Majerczyk et al. found that CodY repressed cap genes either directly or by repressing agr, thus linking the metabolic status sensed by CodY with the regulation of the capsule and other virulence genes (31). A further link between metabolism and capsule production was provided by the finding that tricarboxylic acid (TCA) cycle intermediates affected capsule production (41). In other studies, it has been reported that sigB activates capsule production through arlRS and spoVG (33, 44) and that the AI-2 autoinducer signaling system regulates capsule production through the KpdDE two-component system (52).

In a previous study, we identified several regulators that affect cap promoter activity by screening a transposon library (25). One of the genes we found was clpC. ClpC is an ATP-dependent Hsp100/Clp chaperone of the AAA+ superfamily involved in protein quality control by refolding or degrading misfolded proteins (20). ClpC is conserved in all low-GC Gram-positive bacteria. In Bacillus subtilis, ClpC can associate with ClpP protease and form a ClpCP proteolytic complex that has been shown to have a pleiotropic effect on cellular functions, including gene regulation (12, 20). In S. aureus, ClpC has been shown to be involved in metabolism, the oxidative stress response, survival, programmed cell death, and biofilm formation (5-7, 10, 11). In the present study, we found that ClpC affected capsule production through two independent pathways; the sae-dependent pathway led to negative control of capsule production, and the codY-dependent pathway led to positive control. Our data suggest that the sae locus, which has been shown to repress capsule genes (30, 46), plays a critical role in the divergent phenotypic effect.

MATERIALS AND METHODS

Bacterial strains, culture media, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table 1. Staphylococci were cultured in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI) or glucose-depleted TSB (TSB-0G). Antibiotics were added to culture medium as appropriate at final concentrations of 10 μg/ml chloramphenicol, 3 μg/ml tetracycline, 10 μg/ml erythromycin, 50 μg/ml spectinomycin, and 100 μg/ml penicillin. Escherichia coli strains DH5α and XL1-blue were used for plasmid construction and maintenance. E. coli was cultivated in Luria-Bertani broth or agar (Difco). Phage 52A, 80α, or φ11 was used for plasmid and chromosomal DNA transduction between S. aureus strains.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid	Relevant characteristic(s)	Reference or source
S. aureus
UAMS-1	Wild-type clinical isolate CP8 strain	14
Newman	Wild-type CP5 strain	T. Foster
CYL6841	Newman ΔclpC::cat	This study
CYL11015	Newman ΔsaeR	This study
CYL11624	Newman ΔsaeR (in-frame deletion)	This study
CYL11027	Newman ΔclpC::cat ΔsaeR	This study
CYL11592	Newman ΔsaeS	This study
CYL11771	Newman ΔsaeRS	This study
CYL11791	Newman ΔclpC::cat ΔsaeRS	This study
CYL11481	Newman saeS(P18L)	This study
CYL11233	Newman ΔcodY::ermC	This study
CYL11390	Newman ΔsaeR ΔcodY::ermC	This study
CYL11391	Newman ΔclpC::cat ΔsaeR ΔcodY::ermC	This study
CYL11315	UAMS-1 ΔhsdR	This study
CYL11461	UAMS-1 ΔclpC::cat	This study
CYL11491	UAMS-1 ΔsaeR	This study
CYL11569	UAMS-1 ΔsaeS	This study
CYL11772	UAMS-1 ΔsaeR ΔsaeS	This study
MS1	UAMS-1 ΔcodY::ermC	32
CYL11983	UAMS-1 ΔclpC::cat ΔcodY::ermC	This study
E. coli
DH5α	Host strain for plasmids	42
XL1-Blue	Host strain for plasmids	42
Plasmids
pLL28	Temperature-sensitive vector	19
pLL29	Single-copy integration vector	26
pLL31	E. coli-S. aureus shuttle vector	This study
pLL33	Translational blaZ fusion vector	25
pLL35	Transcriptional blaZ fusion vector	25
pLl47	E. coli-S. aureus shuttle vector
pLL97	E. coli-S. aureus shuttle vector	This study
pKOR1	Vector for allele replacement	2
pTL3736	pLL97 containing Pmspac-clpC	This study
pTL3737	pLL97 containing Pmspac-saeR	This study
pCL3817	pLL29 containing Pmspac-clpC	This study
pCL3899	pLL31 containing Pspac-clpC	This study
pCL11979	pLL31 containing Pspac-codY	This study
pTL3675	pLL33 containing PsaeP1-blaZ	This study
pTL3678	pLL33 containing PsaeP3-blaZ	This study
pTL3722	pLL35 containing PsaeP1-blaZ	This study
pTL3723	pLL35 containing PsaeP3-blaZ	This study
pCL3884	pLL47 containing Ppcn-lacI	This study

Plasmid and strain construction.

Allele replacement of the clpC gene in strain Newman was performed as described previously, by cloning the appropriate PCR fragments into pLL28 (28). As a result, a 1,464-bp internal fragment of the clpC gene was replaced with the cat gene from pC194. The mutation was backcrossed to strain Newman using phage 80α, and the resulting strain was named CYL6841. The pKOR1 system (2) was used as described previously (9) to construct mutant strains CYL11015, CYL11624, CYL11592, CYL11481, CYL11315, CYL11491, CYL11569, and CYL11772 (Table 1). The mutants were verified by PCR amplification. For strain CYL11481, the saeS(P18L) mutation was also verified by DNA sequencing of the amplified fragment containing the mutation. Double or triple mutants of strain Newman containing ΔclpC::cat (i.e., CYL11027, CYL11791, and CYL11391 in Table 1) were constructed by phage 80α transduction of the ΔclpC::cat allele from CYL6841. To construct strain UAMS-1 ΔclpC::cat (CYL11461), the ΔclpC::cat mutation was transduced from CYL6841 first to a restriction-deficient strain (CYL11315) and then to strain UAMS-1. Strains Newman ΔcodY::ermC (CYL11233) and UAMS-1 ΔclpC::cat ΔcodY::ermC (CYL11983) were constructed by phage 52A transduction of the ΔcodY::ermC allele from MS1 to strains Newman and CYL11461, respectively.

Plasmid pLL31 was constructed by replacing the pBS KS+ ori region of pLL2443 (19) with the pGB2 ori region from pLL29 (26). In addition, the cat gene was deleted. Plasmid pLL31 contains the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter originally from pSI-1 (51) for expressing promoterless genes. Plasmid pLL47 was constructed by replacing the tetK gene of pCL96 (25) with the ermC gene of pE194. Plasmid pLL97 was constructed from pLL47 by inserting the lacI-Pspac region from pCL15 in which the Pspac promoter was modified by the addition of lacO₃ (designated Pmspac). Although the dual lacO sites should render tighter control by LacI (36), we found that clpC or codY cloned under the control of Pmspac in pLL97 was still leaky. Cloning of the clpC, saeR, and codY genes under the control of the Pspac or Pmspac promoter in pLL29, pLL31, or pLL97 was achieved by ligating appropriate PCR fragments to the vector. Translational or transcriptional fusions of sae promoters to blaZ were achieved by cloning appropriate PCR fragments to the vector. The constructs were verified by DNA sequencing. The sequences of the PCR primers used for strain and plasmid construction in this study are available upon request.

RNA methods.

RNA was isolated as described previously (27). Cultures were grown overnight in TSB and diluted to a starting optical density at 600 nm (OD₆₆₀) of 0.05 in fresh medium with a flask-to-medium ratio of ∼20:1 and incubated for 4 h (OD₆₆₀ of 2.8 to 3.2) or 18 h (OD₆₆₀ of 7.5 to 8.1) with shaking at 225 rpm at 37°C.

Microarray profiling was performed essentially as described previously (3), using two RNA samples from each strain prepared independently. Data were analyzed using Gene-spring GX software (Agilent). Genes with at least a 2-fold change in expression, statistically significant as determined by Student's t test (P ≤ 0.05), and detectable above background levels under the included conditions based on Affymetrix algorithms were considered to be differentially expressed under the conditions indicated. Real-time quantitative reverse transcription (qRT)-PCR was performed essentially as described previously (24).

Other methods.

For capsule quantification, overnight cultures grown in TSB-0G were diluted in fresh medium to an OD₆₆₀ of 0.05 with a flask-to-medium ratio of ∼20:1. Cultures were further incubated for 4 h (OD₆₆₀ of 1.7 to 2.4) or 18 h (OD₆₆₀ of 4.0 to 5.6). Bacterial cells were harvested according to the OD₆₆₀ and processed as described before (28), with minor modifications—the lysostaphin and DNase I treatments were at 37°C for 30 min; the proteinase K digestion was at 50°C for 30 min and repeated once. The crude capsule preparations were serially diluted 3-fold and blotted to Immobilon-P membrane (Millipore) using a dot blot apparatus (Bio-Rad). Membranes were treated with anti-capsule antibody, followed by peroxidase-conjugated anti-rabbit antibody, and finally developed with Immobilon Western horseradish peroxidase chemiluminescent substrate according to the manufacturer's instructions (Millipore). Anti-type 5 capsule antibody was used for capsules from strains with the Newman background, whereas anti-type 8 capsule antibody was used for capsules from strains with the UAMS-1 background. Unless indicated specifically, all capsule assays were performed with cultures harvested at 4 h and 18 h, but only results from 4 h are shown since the results obtained at the two harvest time points are comparable. β-Lactamase (BlaZ) assays for the promoter fusions were done with cultures grown in TSB-0G as previously described (29).

RESULTS

Effect of ClpC on capsule production in strain Newman.

We previously found that a Tn551 insertion in the clpC gene (Fig. 1) resulted in higher activity of the cap promoter in a genetic screen in strain COL for genes affecting capsule expression, suggesting that ClpC represses capsule expression (25). To confirm those results and study the regulation of capsule production by ClpC in more detail, we constructed a clpC deletion mutation in strain Newman. As expected, the clpC mutation resulted in increased capsule production; however, we observed that the mutant strain occasionally produced the same amount of capsule as the wild-type strain or even less. Nonetheless, this inconsistency was eliminated when the strains were grown in TSB-0G. Using this modified medium, we showed that the clpC mutation resulted in increased capsule production, as well as cap mRNA (Fig. 1). It should be noted here that all of the experiments described here were conducted with this modified medium unless indicated otherwise. To further confirm that the mutation is due to the clpC mutation, we cloned the wild-type clpC gene under the control of the IPTG-inducible Pmspac promoter in a multiple-copy plasmid vector for complementation. Unexpectedly, we found an increase in capsule production in the complementation strain compared to that of the mutant or the wild-type strain with or without the addition of 0.5 mM IPTG (Fig. 2 A). To determine whether multiple copies of the clpC gene contributed to the unexpected complementation results, we cloned the Pmspac::clpC construct in a single-copy integration vector, pLL29 (26). The resulting plasmid was integrated at the phage φ11 attachment site on the chromosome. As shown in Fig. 2B, capsule production in the ΔclpC mutant carrying Pmspac-clpC in the chromosome was about half of that of the mutant alone. The fact that partial complementation of the mutation occurred only when clpC was supplied in a single-copy vector suggests that the repression of capsule production by ClpC occurs when ClpC is produced at a low level. This also suggests that clpC cloned under the control of the Pmspac promoter is still expressed at a higher level than clpC at its chromosomal locus. To further reduce clpC expression, we provided the lacI gene from pSI-1 (51) in plasmid LL47 (note that pLL29 does not contain the lacI gene) and repeated the complementation experiment in the presence of increasing IPTG concentrations. We found that complementation to the level of the wild type could only be achieved without IPTG and that an IPTG concentration as low as 0.25 mM resulted in partial complementation (Fig. 2C). Taken together, the results of the complementation tests suggest that ClpC has a negative effect on capsule production when it is produced at a low level but has a positive effect when it is overproduced in the strain Newman background.

FIG. 1.

Effect of clpC mutation on capsule from overnight cultures. RNAs from strain Newman and the ΔclpC::cat mutant CYL6841 were isolated and quantified by qRT-PCR using capD-specific primers. The n-fold change is expressed relative to the level of strain Newman. Data represent two independent experiments (left panel). Capsules were isolated from strain Newman and ΔclpC::cat mutant CYL6841, serially diluted, and analyzed by immunoblotting (right panel). The genetic organization of the clpC locus with the Tn551 insertion site of the original clpC mutant is shown at the top.

FIG. 2.

Complementation of the clpC mutation in strain Newman by capsule assay. (A) Capsules were isolated with no IPTG induction from strain Newman(pLL97) and the ΔclpC::cat mutant CYL6841 carrying either pLL97 or pTL3736 (i.e., pLL97 containing the clpC gene under the control of Pmspac). (B) Capsules were isolated with no IPTG induction from strain Newman(pLL29) and ΔclpC::cat mutant CYL6841 carrying either pLL29 or pCL3817 (i.e., pLL29 containing clpC under the control of Pmspac). (C) Capsules were isolated with IPTG induction as indicated from the ΔclpC::cat mutant CYL6841 carrying pCL3817 and pCL3884 (i.e., pLL47 expressing the lacI gene). Strains Newman and CYL6841 carrying vectors pLL29 and pLL47 were used as controls.

Microarray analysis of clpC regulon.

Since ClpC is not a DNA-binding regulator, we envisioned that it might affect capsule production by interacting with a regulator(s) affecting capsule production. To understand how capsule production is regulated by ClpC, we performed a microarray analysis study to identify genes differentially regulated by ClpC. For these experiments, we first performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis using total cell lysates to determine the time course of the effect of the clpC deletion on the protein profile. We found that ClpC had a profound effect on the protein profile at 4 h of incubation time compared to that at 8 h and 18 h (data not shown). It has been shown that the expression of cap genes starts at mid-log phase, reaches a maximum at the beginning of stationary phase, and remains constant thereafter (29). For these reasons, we chose to perform transcriptional-profiling experiments using bacterial cultures harvested at 4 h and 18 h. As shown in Tables S1 and S2 in the supplemental material, we found that 195 genes were upregulated and 242 genes were downregulated by ClpC by at least 2-fold. Notably, many more genes were affected by ClpC at 4 h than at 18 h. Almost half of the genes affected are involved in metabolism. In addition, 64 virulence genes and 19 genes involved in transcriptional regulation were affected. As expected, all of the cap5 genes, except one, were increased in the mutant at 4 h. These results validate the notion that ClpC represses capsule expression. To verify the microarray analysis results, we performed qRT-PCR with selected genes, including one of the capsule genes, capD. The results (Fig. 3) matched well with those of the microarray analysis. It should be noted here that the microarray analysis experiments described in this section were conducted using cultures grown in regular TSB medium.

FIG. 3.

Confirmation of microarray analysis results by qRT-PCR. RNAs isolated at 4 h from strain Newman and the ΔclpC::cat mutant CYL6841 were analyzed by qRT-PCR using gene-specific primers. The n-fold change is expressed relative to the level of the clpC mutant on a log scale. Data represent the means with standard errors from at least two independent experiments.

ClpC represses capsule production by activating sae genes.

To understand the regulation of capsule production by ClpC, we focused on the regulatory genes affected by ClpC. Based on the microarray analysis data (Table 2 and 3), the sae genes were highly reduced in the clpC mutant (the n-fold changes in the four sae genes affected by ClpC ranged from 30.3- to 656.9-fold; Table 2), indicating that ClpC is an important positive regulator of sae. The effect of ClpC on sae was also confirmed by qRT-PCR (Fig. 3). The sae operon contains four genes; the saeRS genes comprise a two-component module, whereas the upstream saePQ genes have no known function. SaeRS have been previously shown to repress capsule production in strain Newman (30, 46), although previous global gene-profiling studies failed to identify the cap genes as the target genes affected by SaeRS (22, 40, 49). Thus, we hypothesized that SaeRS functioned downstream of ClpC in the repression of the cap genes. To test this hypothesis, we first assessed whether SaeRS represses capsule production. As shown in Fig. 4 A, the saeR mutant had increased capsule production 8.5-fold ± 0.7-fold, whereas the mRNA increased by 4.5-fold as measured by qRT-PCR. These results showed that SaeR acted as a repressor of capsule production in strain Newman. However, the mutations were complemented only partially with the wild-type saeR gene cloned under the control of the Pmspac promoter in the presence of 0.5 mM IPTG. This partial complementation is most likely the result of suboptimal expression of saeR by the artificial promoter, since it has been shown that the sae genes are highly expressed in strain Newman (30). On the other hand, sae has no effect on clpC transcription by qRT-PCR (not shown). Thus, ClpC is most likely an upstream activator of sae transcription.

TABLE 2.

Regulatory genes upregulated by clpC

ORFa	Changeb at:		Gene	Description
	4 h	18 h
SACOL0567	2.4		ctsR	Transcriptional regulator CtsR
SACOL0404	2.4			Transcriptional regulator, MarR family
SACOL2531	3.5			Transcriptional regulator, MarR family
SACOL1639	2.1		hrcA	Heat-inducible transcription repressor HrcA
SACOL0765	30.3	46.0	saeS	Sensor histidine kinase SaeS
SACOL0766	43.0	62.3	saeR	DNA-binding response regulator SaeR
SACOL0767	254.9	44.8	saeQ	Conserved hypothetical protein involved in sae regulation
SACOL0768	656.9	143.3	saeP	Conserved hypothetical protein involved in sae regulation
SACOL0246	2.9		lytR	Response regulator LytR
SACOL1002	2.0		spx	Conserved hypothetical protein

^aORF, open reading frame.

^bValues represent n-fold wild-type/mutant changes.

TABLE 3.

Regulatory genes downregulated by clpC (numbers represent fold changes of mutant/wild type)

ORFa	Changeb at:		Gene	Description
	4 h	18 h
SACOL1272	2.0		codY	Transcriptional regulator CodY
SACOL0179	3.9			Phosphosugar-binding transcriptional regulator, RpiR family
SACOL2308	2.5			Phosphosugar-binding transcriptional regulator, RpiR family
SACOL0518	2.4			Transcriptional regulator, GntR family
SACOL2384		2.09	sarZ	Staphylococcal accessory protein Z
SACOL2546	12.5			Perfringolysin O regulator protein, putative
SACOL2646	2.6			DNA-binding response regulator

^aORF, open reading frame.

^bValues represent n-fold wild-type/mutant changes.

FIG. 4.

Effect of saeR mutation on capsule. (A) Cultures of strain Newman(pLL97) and ΔsaeR mutant CYL11015 carrying either pLL97 or pTL3737 (i.e., pLL97-saeR) were induced with 0.5 mM IPTG for 4 h. Capsules were isolated and analyzed by immunoblotting. Quantitation of capD by qRT-PCR from three independent experiments is shown to the right. The n-fold change (y axis) is expressed relative to the level of strain Newman. (B) Effect of SaeS(P18L) on capsule production in strain Newman. Capsules were isolated at 4 h from strain Newman, the saeS(P18L) mutant CYL11481, and the ΔsaeS mutant CYL11592 and subjected to immunoblotting. Quantitation of capD by qRT-PCR from two independent experiments is shown to the right. The n-fold change (y axis) is expressed relative to the level in strain Newman.

To further test the hypothesis, we sought a genetic approach by complementing a clpC saeR double mutant of strain Newman with either clpC or saeR cloned under the control of the Pmspac promoter. Our results showed that clpC cloned into a plasmid vector increased capsule production in the clpC mutant and in the clpC saeR double mutant (see Fig. S1 in the supplemental material), consistent with the results shown in Fig. 2. In single copy, we found that the cloned clpC gene did not affect capsule production in the clpC saeR mutant (see Fig. S2 in the supplemental material). Since clpC in single copy partially complemented the clpC mutant, the fact that the clpC gene did not affect capsule production in the double mutant suggests that ClpC functions upstream of SaeRS in capsule regulation. However, surprisingly, SaeR overexpression did not affect capsule production in the double mutant (see Fig. S1 in the supplemental material), though Pmspac-saeR in a multiple-copy plasmid partially complemented the saeR mutation (Fig. 4A; see Fig. S1 in the supplemental material). Thus, our genetic experiments did not fully support our contention that ClpC repression of capsule production is through the sae locus. Nonetheless, we argue in favor of this pathway based on the findings that ClpC had a very strong effect on the sae genes and SaeR repressed capsule production but had no effect on clpC expression. The inability of saeR to complement the clpC saeR double mutant was most likely due to the absence of ClpC in the double mutant, as we show below that ClpC was also required for sae operon autoactivation.

Restoration of a single amino acid variation of SaeS in strain Newman abolished sae repression of capsule production.

During the course of our study, it was revealed that among the strains whose genomes have been sequenced there was a single nucleotide variation in the saeS gene of strain Newman that resulted in an amino acid substitution at position 18 of SaeS, within the membrane-spanning domain of the protein (1, 13). This variation has been shown to be responsible for the unusually high expression of the sae genes in strain Newman, due to autoactivation via SaeR phosphorylation, relative to the sae genes in other strains (13, 30, 43). To test how the saeS variation in strain Newman affects capsule production, we changed the point mutation in the strain Newman chromosome such that the proline amino acid of SaeS^P18 was converted to leucine, thereby matching the SaeS^L18 amino acid sequence of other sequenced strains. This mutant Newman saeS(P18L) strain (CYL11481) produced a 7.2-fold higher capsule level than the wild-type Newman strain (Fig. 4B), suggesting that this single amino acid change alters the regulation of capsule production. To determine how strongly the SaeS(P18L) substitution affects capsule repression, we compared the saeS deletion mutant with the restored strain. As shown in Fig. 4B, the amount of capsule produced by strain Newman saeS(P18L) was only ∼2-fold less than that produced by the saeS deletion strain, suggesting that the single amino acid substitution in SaeS effectively reduces the SaeS regulatory function with respect to capsule repression in strain Newman. It should be noted here that the saeR and saeS double and single mutants produced indistinguishable amounts of capsule (data not shown), indicating that saeR and saeS function in unison with respect to capsule regulation.

The above results indicate that the saeS(P18L) substitution has about the same effect as the saeS deletion in strain Newman, suggesting that for strains such as UAMS-1, in which there is no mutation in saeS, the capsule would not be strongly affected by sae mutation under our growth conditions. To test this, we constructed saeR and saeS mutations in strain UAMS-1, a clinical strain originally isolated from an osteomyelitis patient. Deletion of saeR, saeS, or both genes had a negligible effect on capsule production (data not shown), suggesting that the polymorphism in the saeS gene is the cause of phenotypic variation between strains with respect to capsule repression by the sae locus.

ClpC activates capsule production by repressing CodY.

The clpC complementation results (Fig. 2A) showing clpC cloned into a multiple-copy plasmid augmented capsule production led us to hypothesize that ClpC has both positive and negative effects on capsule production. Since we showed above that ClpC repression of capsule production was most likely through sae, we predicted that ClpC would act as a positive regulator under sae-null conditions. To confirm this possibility, we measured the effect of the clpC mutation on capsule production in sae deletion mutants, in which the repression effect on capsule production via sae is eliminated. As shown in Fig. 5 A, deletion of clpC in an saeRS mutant strain resulted in reduced capsule production compared to that of the saeRS mutant, suggesting that clpC has a positive regulatory effect on capsule production when SaeRS function is blocked. To further test the sae-independent positive regulatory effect of ClpC, we again employed strain UAMS-1 since the capsule production in this strain is independent of sae. As shown in Fig. 5B, deletion of clpC reduced capsule production by ∼8.7-fold and complementation with multiple-copy clpC increased capsule production by 2.7-fold, compared to that of wild-type UAMS-1.

FIG. 5.

Positive effect of clpC on capsule production in a sae-null background. (A) Capsules isolated at 4 h were compared among strain Newman, the ΔclpC::cat mutant CYL6841, the ΔsaeRS mutant CYL11771, and the ΔclpC::cat ΔsaeRS mutant CYL11791. (B) Effect of clpC on capsule production in UAMS-1. Capsules were isolated at 4 h from UAMS-1(pLL97) and the ΔclpC::cat mutant CYL11461 containing either pLL97 or pTL3736 (i.e., pLL97-clpC) and assayed by immunoblotting.

Recently, CodY has been found to repress capsule production (31). Our microarray analysis results (Table 3) and qRT-PCR data (Fig. 3) above showed that the codY gene was repressed by ClpC. These results suggest that ClpC could activate capsule production by repressing codY expression. To test this possibility, we chose to conduct genetic epistasis experiments in the absence of sae such that the ClpC repression of capsule production through sae could be avoided. To this end, we first showed that codY was not affected by saeR mutation by qRT-PCR (results not shown), suggesting that codY repression by ClpC is sae independent. We next assayed epistasis by analyzing capsule production in clpC and codY single and double mutants in the strain Newman saeR mutant background. As shown in Fig. 6 A, the clpC codY saeR triple mutation resulted in a phenotype similar to that of the codY saeR mutant but different from that of the clpC saeR mutant, indicating that codY is epistatic to clpC. Furthermore, the phenotype of the clpC codY saeR mutant could be complemented by Pspac-codY in a multiple-copy plasmid vector but could not be complemented by Pspac-clpC. Taken together, these results suggest that CodY acts downstream of ClpC in the capsule regulatory pathway. In the UAMS-1 background, where capsule production is largely unaffected by sae, the same results were obtained in similar epistasis assays (Fig. 6B), further confirming that CodY is involved in ClpC activation of capsule production in a sae-independent pathway.

FIG. 6.

Regulation of capsule production by clpC and codY. Each strain used in these assays carries pLL31, pCL3899 (i.e., pLL31-clpC), or pCL11979 (i.e., pLL31-codY). (A) Capsules were isolated without IPTG induction from the following strain Newman-derived strains with a saeR-null background containing the indicated plasmids: ΔsaeR mutant CYL11015, ΔclpC::cat ΔsaeR mutant CYL11027, ΔsaeR ΔcodY::ermC mutant CYL11390, and ΔclpC::cat ΔsaeR ΔcodY::ermC mutant CYL11391. (B) Capsules were isolated without IPTG induction from the following UAMS-1-derived strains containing the indicated plasmids: UAMS-1, ΔclpC::cat mutant CYL11461, ΔcodY::ermC mutant MS1, and ΔclpC::cat ΔcodY::ermC mutant CYL11983.

ClpC activates sae transcription by promoting sae autoregulation in strain Newman.

The four sae genes are transcribed by two promoters, P1 and P3, of which the P1 promoter has been shown to be highly autoactivated and the P3 promoter has been shown to be slightly autorepressed (1, 13, 35). To understand how ClpC activates the sae locus, we transcriptionally or translationally fused the two promoters with the blaZ reporter gene. We found that the P1 promoter, but not the P3 promoter, was strongly upregulated by ClpC (Fig. 7) by either a transcriptional or a translational fusion assay, indicating that ClpC affects sae at the P1 promoter, which drives the transcription of all four genes, primarily at the transcriptional level. Since P1 is the promoter for sae autoactivation, one possibility for how ClpC could activate the sae operon is by facilitating its autoregulation. If this were the case, one would also predict that ClpC and SaeR would affect sae transcription to the same extent. We therefore compared the saeP mRNA levels among clpC and saeR single and saeR clpC double mutants of strain Newman using qRT-PCR. We found that the saeP transcript was reduced drastically compared to that of the wild type and that there was no significant difference among the three strains (P values range from 0.3905 to 0.8748 by paired t test in Fig. 8). Thus, sae transcription, as measured by saeP mRNA, depends on not only SaeR (and therefore SaeS) but also on ClpC, suggesting that ClpC is required for autoactivation of the P1 promoter by SaeR to drive the transcription of the sae operon. Since SaeR phosphorylation by SaeS has been shown to be required for autoregulation (30, 47), one possibility is that ClpC could activate sae by promoting phosphorylation. This contention is consistent with the notions that ClpC is not a DNA-binding protein and that the most likely ClpC regulatory mechanism is by interacting with a regulator at the protein level.

FIG. 7.

Effect of clpC on sae promoters. BlaZ activities were assayed in strain Newman and the ΔclpC::cat mutant CYL6841 containing the transcriptional PsaeP1-blaZ fusion plasmid pTL3722 or the PsaeP3-blaZ fusion plasmid pTL3723 (A) and the translational PsaeP1-blaZ fusion plasmid pTL3675 or the PsaeP3-blaZ fusion plasmid pTL3678 (B). The BlaZ activities are expressed as the ratio of A₄₈₂ of the enzymatic reaction to the OD₆₆₀ of the culture. The error bars represent standard deviations of three independent experiments. The genetic organization of the sae locus is shown at the top.

FIG. 8.

Effects of clpC and saeR on autoregulation of the sae operon. RNAs from the ΔclpC mutant CYL6841, the ΔsaeR mutant CYL11015, and the ΔclpC::cat ΔsaeR mutant CYL11027 were compared by qRT-PCR. The n-fold change is expressed relative to the level of strain Newman.

DISCUSSION

It is well documented that many, if not all, S. aureus virulence factors are regulated by multiple regulators (4). To understand the complex network of virulence gene regulation, we have focused on capsule as a model virulence factor. In this study, we attempted to delineate the role of ClpC in capsule gene regulation. ClpC is not a DNA-binding regulator; instead, it has a chaperonic activity that is capable of binding to misfolded proteins (20). ClpC, therefore, is unlikely to regulate capsule gene expression by direct binding to a DNA control region. We envision that ClpC exerts its regulatory functions by affecting the activities of other regulators, perhaps by direct protein interaction or by proteolytic cleavage when it associates with the ClpP protease. In B subtilis, ClpCP has been shown to be involved in several important regulatory pathways (12, 20). By transcriptional profiling, we found that the sae and codY genes could be the downstream regulators. Our data suggest that, in strain Newman, ClpC strongly activates the sae operon, presumably by facilitating sae autoactivation, which, in turn, represses capsule production. In S. aureus strains Newman and UAMS-1, ClpC represses the codY gene, which represses the cap operon either through direct binding or through agr, thereby promoting capsule production. We also found that the mutation within saeS of strain Newman was responsible for the difference in regulation between the two strains. Based on these data, we propose a model, as depicted in Fig. 9, in which ClpC regulates capsule production through two independent pathways with opposite effects on capsule production.

FIG. 9.

Proposed regulatory pathways for cap operon regulation by clpC. Arrows indicate positive regulation, and blocked arrows indicate negative regulation.

We initially focused on the sae genes since all four sae genes in the operon were strongly upregulated by clpC. However, the sae locus has been shown to repress capsule production only in strain Newman (30, 46). During the course of our study, it has been made clear that a saeS missense mutation in strain Newman contributes to the difference in sae regulatory function (13, 30, 43). In this study, we confirmed that the mutation had a strong effect on capsule repression since restoring the mutation effectively abolished the sae repression of capsule production with an increase in capsule production of ∼8-fold in strain Newman (Fig. 4B). Interestingly, this restoration also abolished the ClpC effect on sae activation. One question, then, is how ClpC, which has no DNA-binding motif, activates sae in strain Newman but not in other strains. Since we showed that ClpC activation of sae required SaeS^P18 but not SaeS^L18, and since it has been shown that sae autoactivation requires phosphorylated SaeR, and SaeS^P18 constitutively phosphorylates SaeR (30, 47), we speculate that ClpC, as a molecular chaperone, may facilitate the folding of strain Newman SaeS^P18, but not UAMS-1 SaeS^L18, to promote phosphorylation, thereby activating sae gene transcription under our experimental condition. Alternatively, it is possible that ClpC forms a proteolytic complex with ClpP to degrade a specific inhibitor of SaeS^P18.

The biological functions of S. aureus ClpC have not been well studied, but several recent studies have indicated that ClpC plays an important role in maintaining cell viability in senescence but only a minor role in the early phase of cell growth (6). Indeed, a study by Chatterjee et al. using microarray analysis and proteomics (7) showed that ClpC affected many more genes (or proteins) at 72 h than at 8 h (135 at 72 h versus 49 at 8 h by microarray analysis). However, our microarray analyses showed that ClpC had a profound effect at log phase (390 genes at 4 h) but much less of an effect at stationary phase (88 genes at 18 h) (see Tables S1 and S2 in the supplemental material). By comparing their results to our microarray analysis results, we found one common feature in that ClpC had a major effect on various genes involved in cellular metabolism. Despite this common theme, we found that there were very few common genes between the two studies, indicating major differences in the genes regulated by ClpC at these time points (only 13 genes are in common). The numbers of genes affected at different time points suggest that ClpC is very active in log phase, minimally active in stationary phase, and then active again at late stationary phase (death phase). However, the differences could be due, in part, to strain differences between the studies especially because we used strain Newman with the saeS^P18 mutation. Nonetheless, the difference in genes affected at different time points was striking.

Since ClpC has a strong effect on sae in strain Newman, one would expect that genes controlled by sae would also be controlled by ClpC in strain Newman. Several microarray analysis studies on saeRS using various strains have been reported (22, 40, 49). Differences in sae-regulated genes identified in these studies are expected due to differences in strains, microarray analysis technology, or growth conditions. In one of the studies, Rogasch et al. (40) used strain Newman to examine the sae effect at log phase and stationary phase, similar to our experiments. Among the 33 genes found to be affected by an sae mutation in their study, we found that 18 were also affected by the clpC mutation in our study, suggesting that these genes are regulated by ClpC through the sae locus. Most of these genes encode surface or secreted proteins and toxins. Interestingly, these genes were also identified by sae profiling studies using a different strain (22, 49). However, it should be noted here that the cap genes were not detected in the microarray analysis study of Rogasch et al., even though the same strain was used in their study and ours.

Our initial complementation experiment in the strain Newman background showed that clpC cloned into a multiple-copy plasmid resulted in capsule overproduction in the clpC mutant. However, when clpC was cloned into a single-copy integrative vector in the presence of lacI, a condition that should reduce the expression of clpC, we observed that the increased capsule production was restored to the wild-type level. These results led us to uncover the dual regulatory actions of ClpC with respect to capsule production in which sae is involved in capsule repression by ClpC. Our genetic studies in the absence of sae (either with the strain Newman Δsae mutant or with strain UAMS-1) further demonstrated that CodY participated in the pathway of capsule activation by ClpC. Since the codY and sae genes do not affect each other's expression, ClpC therefore regulates capsule production in two independent but divergent pathways. However, one obvious question is how ClpC switches to activation of capsule production when it is in high copy but acts as a repressor when it is in chromosome-equivalent copy in strain Newman. The answer to this quandary may rest on the result that ClpC strongly activated sae genes but moderately repressed the codY gene, as we showed by microarray analysis and qRT-PCR in this study. Based on these data, we propose that in the wild-type Newman strain, ClpC in a small amount is capable of activating sae effectively but repressing codY only moderately, resulting in an overall capsule repression phenotype. As the ClpC amount increases (as in a multiple-copy vector), sae activation no longer occurs (because an additional amount of ClpC does not further activate sae) but repression of codY continues, resulting in an overall capsule activation phenotype in a ClpC-overproducing strain. Since we showed that there was little effect on sae by ClpC in UAMS-1 with SaeS^L18, which represents most, if not all, other S. aureus strains, one may question the biological significance of this regulatory pathway found in strain Newman. However, several studies have shown that sae is a key regulatory locus for virulence in animal models (13, 15-17, 22, 49). Strains other than Newman were used in some of these studies, suggesting that sae with saeS^L18 is induced in vivo. It is possible that SaeS^L18 senses an effector in vivo and this results in a conformation that can be recognized by ClpC for autoactivation. Thus, a role of ClpC activation of sae in vivo cannot be ruled out.

CodY is a conserved global transcriptional regulator in low-GC Gram-positive bacteria. In B. subtilis, in which it is studied the most, CodY is involved in adaptation to metabolic stress (45). Recently, it has also been found to regulate virulence genes in S. aureus, including capsule genes (31, 32, 39, 48). CodY represses the cap genes by direct binding to the promoter region, as well as by repressing the agr locus, an activator of the cap genes (29). Our results showed that ClpC activated cap by repressing codY. As codY has also been shown to be repressed by ClpP (34), it is possible that CodY is subject to proteolytic regulation by ClpCP, although there is no direct evidence in this study to suggest this mechanism. CodY, however, may not be the only regulator involved in the activation of the cap genes by ClpC. In our complementation experiments, we found that the strain Newman ΔcodY mutant showed increased capsule production when complemented with a plasmid vector containing the clpC gene (data not shown), suggesting that ClpC may promote capsule production via additional pathways that are codY independent. One potential pathway by which ClpC could activate capsule production is by the TCA cycle intermediates, since it has been shown that ClpC affects citB, which encodes the TCA cycle enzyme aconitase, and that capsule biosynthesis is dependent on TCA cycle intermediates (5, 41).

Two microarray analysis studies of CodY have been reported (31, 39). By comparing the genes affected by ClpC with those affected by CodY reported earlier (31), we found 57 genes that were activated by ClpC and repressed by CodY. Most of these genes are involved in amino acid metabolism, suggesting that these genes are activated by ClpC through CodY in a sae-independent manner. In addition, 17 genes, including 7 cap genes within the 16-gene cap operon, were repressed by both ClpC and CodY. Since the cap genes are regulated by ClpC through both sae- and codY-dependent pathways, it is possible that other genes could also be regulated by ClpC in a manner analogous to that of the cap genes.

Using a genetic approach, we were unable to clearly demonstrate that SaeRS functions downstream of ClpC in the repression of capsule production. Because ClpC and SaeRS both negatively affected capsule production, a simple genetic epistasis assay comparing the phenotypes of single and double mutants cannot be used in this situation. We therefore resorted to complementation of the clpC sae double mutant with each gene. The rationale is that the upstream regulator requires the presence of a downstream regulator for complementation. Using a single-copy approach, we were able to show that the clpC gene could not complement the double mutant as expected (see Fig. S2 in the supplemental material). However, we were unable to show that the sae genes (using either saeR or saeS) can complement the double mutant (see Fig. S1 in the supplemental material) even in a codY-null background (data not shown). We attributed this to the finding that ClpC was required for sae autoactivation. Therefore, complementation by sae in the absence of clpC is unlikely, as ClpC is unavailable for autoactivation despite the induction of saeR or saeS with IPTG. Nonetheless, our finding that the sae operon was highly activated by ClpC but that clpC expression was not affected by sae strongly suggests that ClpC functions upstream of sae.

It should be noted here that in strain Newman, the effect of clpC mutation on capsule production is not always consistent when measured in regular TSB medium. It is likely that batch variation of the medium caused this inconsistency. However, when glucose was excluded from the medium, consistent results were obtained. Glucose is readily consumed by S. aureus, resulting in a lower pH, which has been shown to affect sae expression and thus genes regulated by the sae locus (13, 18, 35, 50). It is therefore most likely that the effect of glucose on clpC regulation of capsule production is mediated through the sae locus. Our observation that there was no medium effect in UAMS-1 (data not shown), in which mutations in clpC had little effect on sae expression, further supports this contention.