SpdC, a novel virulence factor, controls histidine kinase activity in Staphylococcus aureus.

The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons.

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions.

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium.

Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni.

Expression of a cryptic secondary sigma factor gene unveils natural competence for DNA transformation in Staphylococcus aureus.

It has long been a question whether Staphylococcus aureus, a major human pathogen, is able to develop natural competence for transformation by DNA. We previously showed that a novel staphylococcal secondary sigma factor, SigH, was a likely key component for competence development, but the corresponding gene appeared to be cryptic as its expression could not be detected during growth under standard laboratory conditions. Here, we have uncovered two distinct mechanisms allowing activation of SigH production in a minor fraction of the bacterial cell population. The first is a chromosomal gene duplication rearrangement occurring spontaneously at a low frequency [≤10(-5)], generating expression of a new chimeric sigH gene. The second involves post-transcriptional regulation through an upstream inverted repeat sequence, effectively suppressing expression of the sigH gene. Importantly, we have demonstrated for the first time that S. aureus cells producing active SigH become competent for transformation by plasmid or chromosomal DNA, which requires the expression of SigH-controlled competence genes. Additionally, using DNA from the N315 MRSA strain, we successfully transferred the full length SCCmecII element through natural transformation to a methicillin-sensitive strain, conferring methicillin resistance to the resulting S. aureus transformants. Taken together, we propose a unique model for staphylococcal competence regulation by SigH that could help explain the acquisition of antibiotic resistance genes through horizontal gene transfer in this important pathogen.

The WalKR system controls major staphylococcal virulence genes and is involved in triggering the host inflammatory response.

The WalKR two-component system is essential for the viability of Staphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR in S. aureus through a phosphomimetic amino acid replacement (WalR(c), D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb, emp, fnbA, and fnbB), cytolysis (hlgACB, hla, and hlb), and innate immune defense evasion (scn, chp, and sbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalR(c) are strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively active walR(c) allele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.

Bacterial moving and shaking: the 11th blast meeting.

Since their inception 20 years ago, the biennial blast (Bacterial Locomotion and Signal Transduction) meetings instantly became the place to be for exchanging and sharing the latest developments in the field of bacterial motility and signalling. At the 11th edition, held last January in New Orleans, LA, researchers reported on the myriad of mechanisms involved in bacterial movement, sensing and adaptation, ranging from the molecular level to multicellular behaviour. New insights into bacterial signalling phenomena were gained, revealing previously unsuspected layers of complexity, particularly in mechanisms ensuring signal transduction fidelity and novel links to metabolic processes.

Bacitracin and nisin resistance in Staphylococcus aureus: a novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters.

Two-component systems (TCSs) are key regulatory pathways allowing bacteria to adapt their genetic expression to environmental changes. Bacitracin, a cyclic dodecylpeptide antibiotic, binds to undecaprenyl pyrophosphate, the lipid carrier for cell wall precursors, effectively inhibiting peptidoglycan biosynthesis. We have identified a novel and previously uncharacterized TCS in the major human pathogen Staphylococcus aureus that we show to be essential for bacitracin and nisin resistance: the BraS/BraR system (Bacitracin resistance associated; SA2417/SA2418). The braRS genes are located immediately upstream from genes encoding an ABC transporter, accordingly designated BraDE. We have shown that the BraSR/BraDE module is a key bacitracin and nisin resistance determinant in S. aureus. In the presence of low antibiotic concentrations, BraSR activate transcription of two operons encoding ABC transporters: braDE and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDE and vraDE promoter sequences, essential for their transcriptional activation by BraSR, suggesting it is the likely BraR binding site. We demonstrated that the two ABC transporters play distinct and original roles in antibiotic resistance: BraDE is involved in bacitracin sensing and signalling through BraSR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin and nisin resistance when produced on its own. We show that these processes require functional BraD and VraD nucleotide-binding domain proteins, and that the large extracellular loop of VraE confers its specificity in bacitracin resistance. This is the first example of a TCS associated with two ABC transporters playing separate roles in signal transduction and antibiotic resistance.

GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus.

The GraSR two-component system (TCS) controls cationic antimicrobial peptide (CAMP) resistance in Staphylococcus aureus through the synthesis of enzymes that increase bacterial cell surface positive charges, by d-alanylation of teichoic acids and lysylination of phosphatidylglycerol, leading to electrostatic repulsion of CAMPs. The GraS histidine kinase belongs to the "intramembrane-sensing kinases" subfamily, with a structure featuring a short amino-terminal sensing domain, and two transmembrane helices separated only by a short loop, thought to be buried in the cytoplasmic membrane. The GraSR TCS is in fact a multicomponent system, requiring at least one accessory protein, GraX, in order to function, which, as we show here, acts by signaling through the GraS kinase. The graXRS genes are located immediately upstream from genes encoding an ABC transporter, vraFG, whose expression is controlled by GraSR. We demonstrated that the VraFG transporter does not act as a detoxification module, as it cannot confer resistance when produced on its own, but instead plays an essential role by sensing the presence of CAMPs and signaling through GraS to activate GraR-dependent transcription. A bacterial two-hybrid approach, designed to identify interactions between the GraXSR and VraFG proteins, was carried out in order to understand how they act in detecting and signaling the presence of CAMPs. We identified many interactions between these protein pairs, notably between the GraS kinase and both GraX and the VraG permease, indicating the existence of an original five-component system involved in CAMP sensing and signal transduction to promote S. aureus resistance.

The pleiotropic CymR regulator of Staphylococcus aureus plays an important role in virulence and stress response.

We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a DeltacymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the DeltacymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the DeltacymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host.

Natural transformation allows transfer of SCCmec-mediated methicillin resistance in Staphylococcus aureus biofilms.

SCCmec is a large mobile genetic element that includes the mecA gene and confers resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA). There is evidence that SCCmec disseminates among staphylococci, but the transfer mechanisms are unclear. Here, we show that two-component systems mediate the upregulation of natural competence genes in S. aureus under biofilm growth conditions, and this enhances the efficiency of natural transformation. We observe SCCmec transfer via natural transformation from MRSA, and from methicillin-resistant coagulase-negative staphylococci, to methicillin-sensitive S. aureus. The process requires the SCCmec recombinase genes ccrAB, and the stability of the transferred SCCmec varies depending on SCCmec types and recipients. Our results suggest that natural transformation plays a role in the transfer of SCCmec and possibly other mobile genetic elements in S. aureus biofilms.

Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.

CymR, the master regulator of cysteine metabolism in Staphylococcus aureus, controls host sulphur source utilization and plays a role in biofilm formation.

We have characterized the master regulator of cysteine metabolism, CymR, in Staphylococcus aureus. CymR repressed the transcription of genes involved in pathways leading to cysteine formation. Eight direct DNA targets were identified using gel-shift or footprinting experiments. Comparative transcriptome analysis and in vitro studies indicated that CysM, the OAS-thiol-lyase, was also implicated in this regulatory system. OAS, the direct precursor of cysteine, prevents CymR-dependent binding to DNA. This study has allowed us to predict sulphur metabolism functions for previously uncharacterized S. aureus genes. We show that S. aureus is able to grow on homocysteine as the sole sulphur source suggesting efficient MccA and MccB-dependent conversion of this compound into cysteine. We propose that SA1850 is a new thiosulphate transporter and that TcyP and TcyABC are l-cystine transporters. CymR directly controls the use of sulphur sources of human origin such as taurine and homocysteine. The cymR mutant also displayed a reduced capacity to form biofilms, indicating that CymR is involved in controlling this process in S. aureus via an ica-independent mechanism. These data indicate that fine-tuning of sulphur metabolism plays an important part in the physiology of this major pathogen and its adaptation to environmental conditions and survival in the host.

The Lactobacillus plantarum ftsH gene is a novel member of the CtsR stress response regulon.

FtsH proteins have dual chaperone-protease activities and are involved in protein quality control under stress conditions. Although the functional role of FtsH proteins has been clearly established, the regulatory mechanisms controlling ftsH expression in gram-positive bacteria remain largely unknown. Here we show that ftsH of Lactobacillus plantarum WCFS1 is transiently induced at the transcriptional level upon a temperature upshift. In addition, disruption of ftsH negatively affected the growth of L. plantarum at high temperatures. Sequence analysis and mapping of the ftsH transcriptional start site revealed a potential operator sequence for the CtsR repressor, partially overlapping the -35 sequence of the ftsH promoter. In order to verify whether CtsR is able to recognize and bind the ftsH promoter, CtsR proteins of Bacillus subtilis and L. plantarum were overproduced, purified, and used in DNA binding assays. CtsR from both species bound specifically to the ftsH promoter, generating a single protein-DNA complex, suggesting that CtsR may control the expression of L. plantarum ftsH. In order to confirm this hypothesis, a DeltactsR mutant strain of L. plantarum was generated. Expression of ftsH in the DeltactsR mutant strain was strongly upregulated, indicating that ftsH of L. plantarum is negatively controlled by CtsR. This is the first example of an ftsH gene controlled by the CtsR repressor, and the first of the low-G+C gram-positive bacteria where the regulatory mechanism has been identified.

Characterization of a serine/threonine kinase involved in virulence of Staphylococcus aureus.

Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus. Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Deltastk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.

Comparative study of two plasticins: specificity, interfacial behavior, and bactericidal activity.

A comparative study was designed to evaluate the staphylococcidal efficiency of two sequence-related plasticins from the dermaseptin superfamily we screened previously. Their bactericidal activities against Staphylococcus aureus as well as their chemotactic potential were investigated. The impact of the GraS/GraR two-component system involved in regulating resistance to cationic antimicrobial peptides (CAMPs) was evaluated. Membrane disturbing activity was quantified by membrane depolarization assays using the diS-C3 probe and by membrane integrity assays measuring beta-galactosidase activity with recombinant strain ST1065 reflecting compromised membranes and cytoplasmic leakage. Interactions of plasticins with membrane models composed of either zwitterionic lipids mimicking the S. aureus membrane of CAMP-resistant strains or anionic lipids mimicking the negative charge-depleted membrane of CAMP-sensitive strains were analyzed by jointed Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and differential scanning calorimetry (DSC) to yield detailed information about the macroscopic interfacial organization, in situ conformation, orientation of the peptides at the lipid-solvent interface, and lipid-phase disturbance. We clearly found evidence of distinct interfacial behaviors of plasticins we linked to the distribution of charges along the peptides and structural interconversion properties at the membrane interface. Our results also suggest that amidation might play a key role in GraS/GraR-mediated CAMP sensing at the bacterial surface.

A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway.

The WalK/WalR (aka YycG/YycF) two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and specific to low G+C Gram-positive bacteria, including a number of important pathogens. An unusual feature is that this system is essential for viability in most of these bacteria. Recent studies have revealed conserved functions for this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism, that we have accordingly renamed WalK/WalR. Here we review the cellular role of the WalK/WalR TCS in different bacterial species, focusing on the function of genes in its regulon, as well as variations in walRK operon structure and the composition of its regulon. We also discuss the nature of its essentiality and the potential type of signal being sensed. The WalK histidine kinase of B. subtilis has been shown to localize to the divisome and we suggest that the WalKR system acts as an information conduit between extracytoplasmic cellular structures and intracellular processes required for their synthesis, playing a vital role in effectively co-ordinating peptidoglycan plasticity with the cell division process.

The Pta-AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis.

DegU is considered to be an orphan response regulator in Listeria monocytogenes since the gene encoding the cognate histidine kinase DegS is absent from the genome. We have previously shown that DegU is involved in motility, chemotaxis and biofilm formation and contributes to L. monocytogenes virulence. Here, we have investigated the role of DegU phosphorylation in Listeria and shown that DegS of Bacillus subtilis can phosphorylate DegU of L. monocytogenes in vitro. We introduced the B. subtilis degS gene into L. monocytogenes, and showed that this leads to highly increased expression of motility and chemotaxis genes, in a DegU-dependent fashion. We inactivated the predicted phosphorylation site of DegU by replacing aspartate residue 55 with asparagine and showed that this modified protein (DegU(D55N)) is no longer phosphorylated by DegS in vitro. We show that although the unphosphorylated form of DegU retains much of its activity in vivo, expression of motility and chemotaxis genes is lowered in the degU(D55N) mutant. We also show that the small-molecular-weight metabolite acetyl phosphate is an efficient phosphodonor for DegU in vitro and our evidence suggests this is also true in vivo. Indeed, a L. monocytogenesDeltaptaDeltaackA mutant that can no longer synthesize acetyl phosphate was found to be strongly affected in chemotaxis and motility gene expression and biofilm formation. Our findings suggest that phosphorylation by acetyl phosphate could play an important role in modulating DegU activity in vivo, linking its phosphorylation state to the metabolic status of L. monocytogenes.

Tearing down the wall: peptidoglycan metabolism and the WalK/WalR (YycG/YycF) essential two-component system.

In order to survive, bacteria have developed avariety of highly sophisticated and sensitive signal transduction pathways with which they adapt their genetic expression to meet the challenges of their ever-changing surroundings. These mechanisms enable bacterial cells to communicate with their environment, their hosts and each other, allowing them adopt specific responses, or develop specialised structures such as biofilms or spores to ensure survival, colonization of their ecological niches and dissemination. As highlighted in this book, the so-called two-component systems (TCSs) are one of the most widespread and efficient strategies used for this purpose, where signal acquisition involves autophosphorylation of a sensor histidine kinase and transduction takes place when the kinase phosphorylates its cognate response regulator protein, leading in turn to specific alteration ofgene expression. In their simplest form, TCSs elegantly combine sensing, transducing and transcription activation modules within two proteins, effectively coupling external signals to genetic adaptation. The high degree of conservation among TCS phosphotransfer domains, their ubiquitous nature and the fact that several are essential for cell viability has made them an attractive target for novel classes of antimicrobial compounds. The WalK/WalR (aka YycG/YycF) two-component system, originally identified in Bacillus subtilis, is very highly conserved and specific to low G + C Gram-positive bacteria, including several pathogens such as Staphylococcus aureus. While this system is essential for cell viability, both the nature of its regulon and its physiological role had remained mostly uncharacterized. A number of recent studies have now unveiled a conserved function for this system in different bacteria, defining this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR. This review will focus on the cellular function of the WalK/WalR TCS in different bacterial species and the attractive target it constitutes for novel classes of antimicrobial compounds.

CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1.

Oenococcus oeni is a lactic acid bacterium responsible for malolactic fermentation of wine. While many stress response mechanisms implemented by O. oeni during wine adaptation have been described, little is known about their regulation. CtsR is the only regulator of stress response genes identified to date in O. oeni. Extensively characterized in Bacillus subtilis, the CtsR repressor is active as a dimer at 37°C and degraded at higher temperatures by a proteolytic mechanism involving two adapter proteins, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not encode orthologs of these adapter proteins and the regulation of CtsR activity remains unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of CtsR activity was studied using a heterologous expression system to demonstrate that O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni CtsR acts as a temperature sensor and is inactivated at growth temperatures above 33°C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni.

Inhibition of Rho Activity Increases Expression of SaeRS-Dependent Virulence Factor Genes in Staphylococcus aureus, Showing a Link between Transcription Termination, Antibiotic Action, and Virulence.

Staphylococcus aureus causes various diseases ranging from skin and soft tissue infections to life-threatening infections. Adaptation to the different host niches is controlled by a complex network of transcriptional regulators. Global profiling of condition-dependent transcription revealed adaptation of S. aureus HG001 at the levels of transcription initiation and termination. In particular, deletion of the gene encoding the Rho transcription termination factor triggered a remarkable overall increase in antisense transcription and gene expression changes attributable to indirect regulatory effects. The goal of the present study was a detailed comparative analysis of S. aureus HG001 and its isogenic rho deletion mutant. Proteome analysis revealed significant differences in cellular and extracellular protein profiles, most notably increased amounts of the proteins belonging to the SaeR regulon in the Rho-deficient strain. The SaeRS two-component system acts as a major regulator of virulence gene expression in staphylococci. Higher levels of SaeRS-dependent virulence factors such as adhesins, toxins, and immune evasion proteins in the rho mutant resulted in higher virulence in a murine bacteremia model, which was alleviated in a rho complemented strain. Inhibition of Rho activity by bicyclomycin, a specific inhibitor of Rho activity, also induced the expression of SaeRS-dependent genes, at both the mRNA and protein levels, to the same extent as observed in the rho mutant. Taken together, these findings indicate that activation of the Sae system in the absence of Rho is directly linked to Rho's transcription termination activity and establish a new link between antibiotic action and virulence gene expression in S. aureusIMPORTANCE The major human pathogen Staphylococcus aureus is a widespread commensal bacterium but also the most common cause of nosocomial infections. It adapts to the different host niches through a complex gene regulatory network. We show here that the Rho transcription termination factor, which represses pervasive antisense transcription in various bacteria, including S. aureus, plays a role in controlling SaeRS-dependent virulence gene expression. A Rho-deficient strain produces larger amounts of secreted virulence factors in vitro and shows increased virulence in mice. We also show that treatment of S. aureus with the antibiotic bicyclomycin, which inhibits Rho activity and is effective against Gram-negative bacteria, induces the same changes in the proteome as observed in the Rho-deficient strain. Our results reveal for the first time a link between transcription termination and virulence regulation in S. aureus, which implies a novel mechanism by which an antibiotic can modulate the expression of virulence factors.