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TraP is a triply phosphorylated staphylococcal protein that has been hypothesized to be the mediator of a second Staphylococcus aureus quorum-sensing system, "SQS1," that controls expression of the agr system and therefore is essential for the organism's virulence. This hypothesis was based on the loss of agr expression and virulence by a traP mutant of strain 8325-4 and was supported by full complementation of both phenotypic defects by the cloned traP gene in strain NB8 (Y. Gov, I. Borovok, M. Korem, V. K. Singh, R. K. Jayaswal, B. J. Wilkinson, S. M. Rich, and N. Balaban, J. Biol. Chem. 279:14665-14672, 2004), in which the wild-type traP gene was expressed in trans in the 8325-4 traP mutant. We initiated a study of the mechanism by which TraP activates agr and found that the traP mutant strain used for this and other recently published studies has a second mutation, an adventitious stop codon in the middle of agrA, the agr response regulator. The traP mutation, once separated from the agrA defect by outcrossing, had no effect on agr expression or virulence, indicating that the agrA defect accounts fully for the lack of agr expression and for the loss of virulence attributed to the traP mutation. In addition, DNA sequencing showed that the agrA gene in strain NB8 (Gov et al., J. Biol. Chem., 2004), in contrast to that in the agr-defective 8325-4 traP mutant strain, had the wild-type sequence; further, the traP mutation in that strain, when outcrossed, also had no effect on agr expression.

In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.

Previous studies have shown that production of extracellular proteases in Staphylococcus aureus is stimulated by agr (RNAIII) and mgrA, and repressed by sarA. Protease expression is also repressed by rot, however this effect is generally observed only in agr mutants. Several other regulators (sarR, sarV, sarS, sae) that may impact protease expression have been described. As the interactions between all regulators that control protease gene expression are not fully understood, the present study was undertaken to elucidate the regulatory network governing aureolysin (aur) and staphylococcal serine protease (sspA) transcription. The regulation of both genes was studied as activation of the serine protease (SspA) zymogen requires aureolysin. For this purpose we have analyzed the effect of different combinations of regulatory mutations. The present study clearly shows that the positive effect of agr (RNAIII) on aur and sspA transcription requires rot, which is in accordance with the hypothesis that RNAIII acts by neutralizing Rot activity through binding [McNamara, P.J., Milligan-Monroe, K.C., Khalili, S., Proctor, R.A., 2000. Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus. J. Bacteriol. 182, 3197-3203]. Concomitantly, overexpression of rot in agr(+) strains or inactivation of rot in strains with low levels of RNAIII clearly affected aur and sspA transcription, indicating that the inhibiting effect of RNAIII on Rot could be titrated. Furthermore, our present data support that the only role of RNAIII in aur and sspA regulation is to counteract the repressive activity of Rot. Apart from an apparent direct positive effect of mgrA on sspA and aur transcription, these genes were mainly controlled through repression by sarA and rot, which seemed to occur via binding of SarA and Rot to the aur and sspA promoters, respectively. Maximum transcription of aur and sspA was obtained when both repressors were absent, either in a sarA mutant where Rot is neutralized by RNAIII during post-exponential phase, or in an agr sarA rot triple mutant. Interestingly, aur was much more sensitive to repression by sarA than by rot, whereas sspA was equally suppressed by sarA and rot. On the other hand, sspA was more sensitive to repression by rot than aur. Thus, SarA and Rot seemed to act independently in an additive way. Inactivation of sarR and sarS had no apparent effect on aur and sspA transcription, although overexpression of these regulators suppressed aur and sspA transcription, respectively, likely in a direct way as indicated by DNA binding experiments. In conclusion, our results indicate that aur and sspA transcription are coordinately regulated but can also be individually modulated by agr, sarA, rot, sarS, sarR, and mgrA. A provisional model for the regulation of aur and sspA transcription is presented.

It has been reported that high production of proteases and alpha-hemolysin in the prototype Staphylococcus aureus strain 8325-4 was associated with its sigmaB deficiency. Here we analyzed one fresh clinical isolate (KS26) and two ancient human isolates (Wood46 and V8) selected for high production of proteases and alpha-hemolysin. All three strains lacked yellow pigment and showed a low level of expression of sigB-dependent promoters, indicating sigmaB deficiency. Nucleotide sequencing of the sigB operon revealed that KS26 and Wood46 had stop codons in rsbU and sigB, respectively, while V8 had an insertion of an IS element in rsbU. Complementation experiments with sigB on a plasmid reduced expression of proteases and alpha-hemolysin dramatically, indicating that the high production of these exoproteins was associated with sigmaB deficiency. Although sigmaB-deficient strains show attenuated virulence in some animal models, our results indicate that such strains can cause infection in humans.

Production of protein A (SpA) in Staphylococcus aureus is controlled by several global regulators including agr (RNAIII), sarA, sarS, sarT, rot and mgrA, which appear to form a regulatory network. SarS, which is an activator of spa, seems to be a key regulator in this network. Previous studies have shown that expression of sarS is upregulated in agr, sarA and mgrA mutants, resulting in increased spa expression. In an agr mutant, upregulation of sarS and spa required rot and sarT. In this study regulatory proteins binding to spa and sarS promoter fragments coupled to magnetic beads were identified by MALDI-TOF-MS. Expression of sarS and spa in mutants deficient for one, two or three of the identified regulators and in mutants over-expressing selected regulators was analyzed by Northern and Western blotting. Binding and transcription analysis indicated that SarA represses sarS by direct binding to the promoter, and that stimulation of sarS by sarT was rot-dependent. We also found that MgrA bound to the sarS promoter and was required for expression of both sarS and spa in an agr mutant. However, mgrA and rot were not required for stimulation of sarS in the absence of SarA, suggesting that these regulators, together with SarT, counteract the repressive activity of SarA. Our data indicate that SarS competes with SarA for binding to the spa promoter. This is consistent with the requirement for sarS in spa transcription even in the absence of the repressor, SarA. We also demonstrated binding of MgrA and Rot to the spa promoter, and that rot could stimulate spa transcription even in a sarS mutant. A provisional model for spa regulation involving agr, sarA, sarS, sarT, rot and mgrA is presented.

The prototype Staphylococcus aureus strain 8325-4 produces high levels of hemolysins and proteases. Recently it has been shown that this property depends on a deficiency of sigma factor B (SigB) activity controlling the activation of regulatory genes such as agr and sarA. SigB deficiency is in turn due to a mutation in the rsbU gene, which is required for posttranslational activation of SigB. The rsbU defect of strain 8325-4 has recently been repaired, and we used this strain (SH1000), along with its isogenic sigB-negative mutant, to investigate the contributions of RsbU and SigB in a murine model of septic arthritis. Intravenous inoculation with the rsbU-repaired isogenic strain SH1000 resulted in significantly more severe arthritis, weight decrease, and mortality compared to those of the parental strain 8325-4 (rsbU-negative) or the isogenic sigB-negative mutant (MJH502). SH1000 also persisted more in kidneys and joints of infected mice. Our data strongly suggest that RsbU and SigB regulate important virulence factors, thereby contributing significantly to the outcome of staphylococcal infection.

The virulence determinants of Staphylococcus aureus are expressed in a growth phase-dependent manner governed by the autoinducible quorum-sensing system agr. Activation of the agr system results in a rapid increase in the regulator RNAIII and occurs in response to accumulation of AIP. In order to activate the agr system, a basal transcription of agr components must be assumed. This basal transcription of agr components seems to be stimulated by sarA. To better understand how SarA would affect activation of the agr system by modulating the basal agr activity, a mathematical model for autoactivation of the agr system was set up. The model predicted that the agr system is hysteretic, meaning that the agr system is activated at a specific concentration of autoinducing peptide (AIP), whereas it is inactivated at a specific lower concentration of AIP. According to the model, changing the basal agr activity only had a marginal effect on steady-state levels of RNAIII but changed the sensitivity of the cells to AIP. This was supported by Northern blot analysis of RNAIII in S. aureus mutants with different levels of SarA expression. Since natural antagonistic AIPs have been demonstrated, the effect of adding inhibitors to the system was analyzed., (Copyright (c) 2004 S. Karger AG, Basel.)

Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 strains appeared to be protease negative. The major protease genes were present in all protease-positive (n = 5) and protease-negative (n = 12) strains analyzed. Northern blotting showed that transcription of the protease genes was suppressed due to increased sigma factor B (SigB)-dependent expression of the protease repressor SarA. Other SigB-dependent traits such as pigmentation and expression of asp 23 were also increased in protease-negative compared to protease-positive strains. Inactivation of sarA in three protease-negative strains resulted in increased transcription of all protease genes and increased protease production, while overexpression of sarA in a strain producing protease at high levels repressed protease production. Our results suggest that the protease genes are conserved among clinical S. aureus strains and that the level of SigB-dependent expression of the protease repressor sarA determines the level of protease production in each strain.

Data have been presented indicating that Staphylococcus aureus cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that in sarA mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation of sarA mutant cells in the presence of the global protease inhibitor alpha(2)-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarA mutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either ssp or aur in the prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A.

Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

The pathogenicity of Staphylococcus aureus depends on the combined action of more than 40 different extracellular toxins, enzymes and cell surface proteins. A global regulator agr controls the production of many of these virulence factors by a regulating RNA molecule, RNAIII. Most of the virulence genes regulated by RNAIII are also regulated by SarA and a family of homologous proteins. The Sar proteins appear to repress transcription of individual virulence genes or sets of genes. As some Sar proteins also repress one or more sar homologous genes an increased production of a single Sar protein can result in decreased expression of some virulence genes, and an increased expression of others. Results are presented suggesting that RNAIII might function as an antirepressor, binding one or more of the Sar proteins.

The global regulators agr (accessory gene regulator) and sarA (staphylococcal accessory regulator) have been reported to be both activators and repressors of virulence gene expression in Staphylococcus aureus. How the effector of the agr system, RNAIII, interacts with target gene promoters is unknown. SarA, on the other hand, is a DNA-binding protein, which binds to conserved DNA motifs immediately upstream of both positively and negatively regulated promoters. Here, we searched for additional regulators that could explain the differential effects of RNAIII and SarA. Four differently regulated genes (hla, alpha-toxin; hld, RNAIII; spa, protein A; ssp, serine protease) were analysed for binding of potential regulatory proteins to the corresponding promoter DNA fragments, linked to magnetic beads. One protein (29 kDa), with affinity for all four promoters, showed a high degree of similarity to SarA and was named SarH1 (Sar homologue 1). Expression of sarH1 was strongly repressed by sarA and agr. Analysis of hla, hld, ssp and spa mRNAs in sarH1, sarA and agr mutants, and in sarA/sarH1 and agr/sarH1 double mutants, revealed that sarH1 has a strong repressive effect on hla and an activating effect on spa transcription. SDS-PAGE analysis of secreted proteins from the different mutants showed that the production of several other exoproteins was affected by sarH1. In conclusion, we show that both the agr-dependent suppression of protein A production and the sarA-dependent stimulation of alpha-toxin production is mediated via a new regulator, SarH1, which belongs to a family of Sar homologues.

Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including hla (alpha-toxin), saeB (enterotoxin B), tst (toxic shock syndrome toxin 1), and ssp (serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) and fnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5' half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient S. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5' and 3' halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.

The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA. RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally. Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity. During this period an mRNA fragment, lacking an intact 5' end, accumulates. This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway. During this lag, the levels of bulk protein are maintained. Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization. Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions.

The expression of many virulence genes in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, which is encoded by the agr locus. Transcription RNAIII requires the activity of the agrA, B, C and D genes, which code for components of a quorum sensing signal transduction system. In this report we describe the overexpression and purification of the response regulator, AgrA. Monoclonal antibodies were produced and used to detect AgrA in the cytosolic fraction of S. aureus cells. Purified AgrA did not bind to the RNAIII promoter region in a DNA mobility shift experiment. This confirms previous results obtained with protein extracts from agr+ and agr- cells.

Many of the genes coding for extracellular toxins, enzymes and cell-surface proteins in Staphylococcus aureus are regulated by a 510 nt RNA molecule, RNAIII. Expression of the RNAIII gene is positively controlled by the closely linked agr operon, which encodes a multicomponent signal-transduction system, and by an unlinked operon called sar. We have analysed the 120 bp region that separates the RNAIII promoter, P3, from the divergent agr promoter, P2. By transcription analysis, it was shown that P3 can function in trans of the agr operon. A stretch of 53 bp upstream of P3, containing an interrupted repeat of 7 bp, was found to be required for the agr-dependent expression of RNAIII. A single cytoplasmic protein was shown to bind to at least two sites within this regulatory region. The protein, which was absent in extracts from a sarA mutant, was identified as the sarA product by N-terminal amino acid sequencing. A DNA fragment from the P2 region, encompassing an almost identical repeated DNA motif, competed for the same protein. No interaction between the regulatory DNA sequence and any agr-dependent products could be demonstrated. The results of this study suggest that P3 and P2 are controlled by a mechanism involving the binding of the SarA protein to multiple sites within the regulatory regions immediately upstream of each promoter, and the as yet unknown activity of AgrA.

In this study, we extend earlier observations on the influence of growth rate on mRNA stability and rRNA processing in Escherichia coli during continuous culture, to the effect of anaerobiosis. During slow anaerobic growth (generation time 700 min) both ompA and bla mRNA had a prolonged half-life compared to that during slow aerobic growth and the processing of 9S RNA was even more profoundly retarded, which indicated a general slowing of mRNA turnover. The latter was confirmed by a nearly fourfold increase in the functional half-life of bulk mRNA. In spite of this difference in stability, steady state levels of RNA, as judged by those of the ompA and 9S transcripts, were the same in aerobic and anaerobic cells at a given growth rate. Furthermore, we found that RNA synthesis during anaerobiosis was a fraction of that observed during slow aerobic growth and it is proposed that this offsets the general increase in mRNA stability. Our data therefore suggest that a constant level of RNA is maintained by matching the rate of decay to the level of RNA synthesis.

Staph.aureus endophthalmitis was induced in both eyes of 32 rabbits. Endophthalmitis developed in all the 64 eyes within 24-36 h. All vitreous cultures were positive while only 21 simultaneous aqueous cultures were positive. Thus, the importance of vitreous cultures in diagnosing bacterial endophthalmitis is emphasised.

A Tn551 insertional mutation in the accessory gene regulator (agr) locus of the Staphylococcus aureus chromosome resulted in the decreased production of at least seven extracellular toxins and enzymes and a simultaneous increase in the production of protein A and coagulase (Recsei et al. 1986). Adjacent to this locus we have now identified another gene, hld, transcribed into a 0.5 kb RNA which codes for the staphylococcal delta-lysin. The expression of hld was totally repressed in a strain carrying the agr insertional mutation. Hybridization with strand-specific probes and primer extension analysis revealed that hld and agr are transcribed in opposite directions, starting 188 nucleotides apart. The hld gene is mainly expressed during the post-exponential growth phase and is totally repressed during early exponential growth. Determination of hld mRNA half-life in different growth phases indicated that this regulation is at the level of transcription.

Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60 000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000, 46 000 and 41 000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71 000, 60 000, 46 000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.

Insertion of the erythromycin resistance transposon Tn551 into a single site of the Staphylococcus aureus chromosome resulted in decreased production of alpha-toxin, serine and metallo-proteinases and several other extracellular proteins and a simultaneous increase in the production of protein A. The site of insertion, designated exp, was separate from the structural gene for alpha-toxin and protein A. Hybridization analysis showed that the effect of the insertional mutation on the expression of the alpha-toxin and protein A was at the level of transcription. The chromosomal DNA flanking the transposon and the corresponding DNA of the wild-type strain was cloned in Escherichia coli. Northern blot hybridization experiments revealed that the exp locus codes for a major RNA of approximately 3.5 kb. This RNA was not found in the insertional mutant nor in a spontaneous exp mutant. A map of the exp locus constructed by Northern blot and restriction enzyme analysis showed that the insertional mutation was located in the middle of the coding sequence of the 3.5 kb RNA. The insertional mutant was reverted to wild type by inserting a recombinant plasmid containing most of the coding sequence of the 3.5 kb RNA.

The membrane-bound ribosome protein (MBRP)-complex of Staphylococcus aureus was studied using antibodies to its individual components. The four polypeptides of the complex were firmly held together, and none were present in large excess. The membrane-bound fraction of the MBRP-complex was accessible to trypsin only after removal of the membrane-bound ribosomes; it also remained associated with the membrane-bound ribosomes even after solubilization of the membranes with Triton X-100. Furthermore, the amount of MBRP-complex in the membrane was proportional to the rate of exoprotein synthesis. These results strongly suggest a role for the MBRP-complex in protein secretion.

The formation of bacteriolytic enzymes of Staphylococcus aureus, with special reference to strain M18, was investigated under a variety of conditions. The bacteriolytic activity was tested by using whole cells of Micrococcus lysodeikticus as a substrate. Complex media were required for production, and a Casein Hydrolysate-Yeast Extract medium (CCY(I)) was superior to Brain Heart Infusion and Trypticase Soy Broth. The optimal pH level for production was 7.0. Effective oxygenation and exchange of the beta-glycerophosphate of the CCY(I) medium for glucose increased the rates of growth and autolysis and the rate of appearance of extracellular bacteriolytic enzymes. However, the extracellular lytic activity decreased more rapidly at the end of the growth period than under the standard culture conditions. The appearance of inhibitor(s), probably derived from autolysis, might be responsible for this rapid decrease. The highest yields were obtained in a continuous process in which the activity was almost twice that of batch cultures grown under the same conditions. The bacteriolytic activity produced in continuous culture had a considerably increased stability in the purification process. The advantage of producing unstable bacterial proteins in continuous culture under controlled growth conditions is discussed.