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Research has been conducted on Staphylococcus aureus gene regulation. Results demonstrate that repressor of toxins, Rot, is a global regulator which regulates virulence gene transcription.

Soft-tissue infection is commonly found in patients bitten by Latin American Bothrops snakes. Staphylococcus aureus, which is not present in the mouth of the snake, is frequently isolated from these infections. The effects of B. asper venom on infection with S. aureus were analyzed in a model of infection in envenomated mouse gastrocnemius muscle. Inoculation of 50 colony-forming units (cfu) of S. aureus was enough to cause infection in envenomated muscle, compared with >5x104 cfu without venom. This effect was also achieved by injection of venom myotoxin III (an A(2) phospholipase). A sarA mutant strain in which production of extracellular toxins and enzymes is up-regulated and binding of fibronectin, fibrinogen, and other host proteins is down-regulated was much less virulent than the corresponding parental strain, indicating that the ability of S. aureus to mask itself with host molecules might be more important than the effects of secreted toxins and enzymes in this kind of infection.

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.

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.

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 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.

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.

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.

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.