Bacteria possess a repertoire of distinct regulatory systems promoting survival in disparate environments. Under in vitro conditions it was demonstrated for the human pathogen Staphylococcus aureus that the expression of most virulence factors is coordinated by the global regulator agr. To monitor bacterial gene regulation in the host, we developed a method for direct transcript analysis from clinical specimens. Quantification of specific transcripts was performed by competitive reverse transcription-PCR, and results were normalized against the constitutively expressed gene for gyrase (gyr). Using sputum from cystic fibrosis (CF) patients infected with S. aureus we examined the transcription of the effector molecule RNAIII of agr, of spa (protein A), generally repressed by agr, and of hla (alpha-toxin), generally activated by agr. In the CF lung RNAIII was expressed poorly, indicating an inactive agr in vivo. Despite the low level of RNAIII expression, spa was detectable only in minute amounts and an irregular transcription of hla was observed in all sputum samples. After subculturing of patient strains agr-deficient isolates and isolates with unusual expression profiles, i.e., not consistent with those obtained from prototypic strains, were observed. In conclusion, the agr activity seems to be nonessential in CF, and from the described expression pattern of spa and hla, other regulatory circuits aside from agr are postulated in vivo.Over time, bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments (25). Our understanding of such adaptation processes during infection is limited by our ability to recreate host conditions in an experimental setting. Therefore, the sequential gene expression essential for host colonization, evasion of the immune system, tissue invasion, and maintenance in different organs remains to be determined for most bacterial infections. In recent years new approaches have been developed to identify bacterial genes which are induced during infection (15,23). While these methods are very useful for screening new candidate genes involved in pathogenesis, until now no method has been available to discern the transcription pattern of characterized virulence genes directly during infection.Staphylococcus aureus causes a variety of local and systemic infections in humans and is one of the most important community-acquired and nosocomially acquired pathogens. S. aureus infections are probably established via the coordinated synthesis of extracellular and cell-bound virulence factors (32). The expression of most virulence factors is controlled by the global regulator agr, which is thought to be a prime pathogenesis factor in S. aureus. The agr locus is composed of two divergent transcriptional units (RNAII and RNAIII). The RNA molecule RNAIII is the effector of the operon, which exhibits negative and positive regulatory functions (17, 29), activating extracellular proteins such as the hemolysins but repressing others, for exampl...

The global regulator sar in Staphylococcus aureus controls the synthesis of a variety of cell wall and extracellular proteins, many of which are putative virulence factors. The sar locus in strain RN6390 contains a 339-bp open reading frame (sarA) and an 860-bp upstream region. Transcriptional analyses of this locus revealed three different transcripts of 0.58, 0.84, and 1.15 kb (designated sarA, sarC, and sarB, respectively). All three transcripts seemed to be under temporal, growth cycle-dependent regulation, with sarA and sarB being most abundant in early log phase and the sarC concentration being highest toward the late stationary phase. Mapping of the 5 ends of the sar transcripts by primer extension and modified S1 nuclease protection assays demonstrated that transcription is initiated from three separate, widely spaced promoters. The 3 ends of all three sar transcripts are identical, and transcriptional termination occurs upstream of a typical prokaryotic poly(T) termination signal. Northern (RNA) analysis of sar mutant clones containing plasmids that comprised various promoters and the termination signal revealed that individual transcripts can be generated from each of the three promoters, thus suggesting possible activation as independent promoters. The multipromoter system, from which transcription is initiated, bears conserved features for recognition by homologous 70 transcription factors and also by those expressed in the general stress response. Downstream of the two distal promoters (P3 and P2) are two regions potentially encoding short peptides. It is conceivable that posttranslational cooperation between these short peptides and the sarA gene product occurs to modulate sar-related functions. Complementation studies of a sar mutant with a clone expressing all three sar transcripts showed that this clone was able to restore the sar wild-type phenotype to the sar mutant.Staphylococcus aureus is a major human pathogen (30). Infections caused by this organism range from abscesses, endocarditis, pneumonia, meningitis, and sepsis to severe toxemia including toxic shock syndrome. The recent increase in antibiotic resistance and a lack of potential vaccine candidates have highlighted the importance in finding new approaches to control this important pathogen.Infections caused by S. aureus are probably related to the organism's striking capability to react to changing environments during the infection process. In any stage of surface colonization, entry, and invasion, this highly coordinated response seems to be modulated by the expression of appropriate genes via signal transduction pathways. These modulators may interact with other regulatory elements which, in turn, control the transcription of a wide variety of unlinked genes, many of which are involved in pathogenesis (12, 30). Accordingly, an understanding of the genetic basis in the expression of virulence factors in S. aureus is a prerequisite to the identification of target sites for the development of novel antimicrobial agents based on the glo...

We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

The temporal expression of most virulence factors in Staphylococcus aureus is regulated by pleiotropic loci such as agr and sar. We have previously shown that the sar locus affects hemolysin production because it is required for agr transcription. To delineate the sar genetic determinant required for agr transcription, single copies of fragments from the sar locus, encompassing the individual sar transcripts (sarA, sarC, and sarB), were introduced into a sar mutant via the integration vector pCL84. Although a DNA fragment encompassing the sarA transcript plus a 189-bp upstream region was sufficient for agr expression, complementation analysis revealed that the sarB transcript was the most effective in augmenting agr transcription as determined by RNAII and RNAIII transcription and gel retardation assays with the P2 and P3 promoters of agr. As the region upstream of the sarA transcript encodes a 39-amino-acid open reading frame, ORF3, it is possible that posttranslational cooperation between the sarA gene product and ORF3 may be necessary for optimal agr expression. Deletion studies demonstrated that an intact sarA gene is essential for agr transcription. However, mutagenesis and in vitro translation studies revealed that unlike the agr locus, the required element is the SarA protein and not the RNA molecule. Taken together, these results indicate that the sarA-encoded protein, possibly in conjunction with peptides encoded in the upstream region, regulates hemolysin production by controlling agr P2 and P3 transcription.The control of extracellular protein production in bacteria is dependent upon global regulatory systems in which a single regulatory locus controls the expression of several unlinked target genes, often in a temporal manner. Regulation of virulence determinants in Staphylococcus aureus has been shown to be under the control of at least two genetic loci, agr and sar (6, 15). The agr locus encodes two divergent transcripts (RNAII and RNAIII). The promoter P2 initiates the formation of a 3-kb transcript designated RNAII that encodes four genes (agrA through agrD), while the promoter P3 initiates the formation of a 500-bp transcript called RNAIII that codes for the ␦-hemolysin (15). The agr operon mediates control of extracellular and surface proteins via the transcript RNAIII (26). However, in-frame deletions of any of the four genes in the RNAII coding region resulted in diminished transcription of RNAIII, thus indicating that RNAIII transcription is likely dependent on the activation of RNAII (23). We recently reported that the transcription of RNAII and RNAIII was partially dependent on the sar locus, which is encoded within a 1.2-kb DNA fragment that encompasses three overlapping transcripts (1, 13) (Fig. 1). These transcripts, designated sarA (0.58 kb), sarC (0.84 kb), and sarB (1.15 kb), have common 3Ј ends but three distinct promoters. A major 339-bp open reading frame (ORF), sarA, together with widely spaced upstream promoter sequences, is present in this locus (7, 13).Phenotypic analysis reve...

The expression of cell wall and extracellular proteins in Staphylococcus aureus is controlled by global regulatory systems, including sar and agr. We have previously shown that a transposon insertion into the 372-bp sarA gene within the sar locus resulted in decreased expression of several extracellular and cell wall proteins (A. L. Cheung and S. J. Projan, J. Bacteriol. 176:4168-4172, 1994). In this study, Northern (RNA blot) analysis with a 732-bp sarA probe indicated that two major transcripts (0.56 and 1.2 kb) were absent in the sar mutant compared with the parental strain RN6390. Additional transcriptional studies revealed that the sarA gene is encoded within the 0.56-kb transcript. Notably, a plasmid carrying the sarA gene together with a 1.2-kb upstream fragment (1.7 kb total) was able to reestablish the 1.2-kb transcript in the mutant. Although reconstitution of the parental phenotype by the sarA gene was incomplete, the introduction of a plasmid carrying the 1.7-kb fragment to the mutant restored the parental phenotype. Transcription of RNAII and RNAIII, which encode the structural and regulatory genes of agr, respectively, was diminished in the mutant but restored to wild-type levels by complementation with the 1.7-kb fragment. In gel shift assays, cell extracts of this clone were able to retard the mobility of a labeled RNAII promoter probe but not an RNAIII promoter element. These data suggest that sarA and the adjacent upstream DNA are essential to the expression of a DNA-binding protein(s) with specificity for the RNAII promoter, thereby controlling agr-related transcription.The emergence of antibiotic-resistant strains of Staphylococcus aureus has raised concern because of the organism's ability to cause a wide range of diseases. S. aureus has the capacity to synthesize a large number of extracellular and cell wall-associated proteins, some of which are involved in pathogenesis (9, 23). Many of the exoproteins (e.g., ␣-hemolysin and toxic shock syndrome toxin) are secreted during the postexponential growth phase (9), while certain surface proteins (e.g., protein A and fibrinogen and fibronectin-binding proteins) are repressed postexponentially (9, 12). Temporal expression of many of the proteins involved in virulence has been shown to be under the control of global regulatory systems, in which a common regulator directs the expression of multiple genes.In S. aureus, at least three global regulatory systems (agr, xpr, and sar) have been shown to play a role in the production of surface and extracellular proteins (6,12,21). Among these, the agr locus is the best characterized and has been shown to act at the transcriptional level to control the synthesis of both extracellular and cell wall proteins (12). Transposon-mediated inactivation of the agr locus leads to decreased production of exoproteins, while synthesis of some surface proteins is increased (19). The agr locus consists of at least five genes (agrA, B, C, and D and the ␦-hemolysin gene). Analysis of the agr sequence revealed features sugge...