Molecular Pathogenesis Program, Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA
Correspondence
Richard P. Novick
novick{at}saturn.med.nyu.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our studies of the regulation of extracellular protein production, we have thus far concentrated on the agr system. The agr locus controls genes encoding most extracellular staphylococcal proteins, which constitute the agr regulon, and is conserved throughout the staphylococci. Regulation of the component genes is primarily at the level of transcription, though several of the genes are secondarily regulated at the translational level. Nearly all the currently available data on the regulation of extracellular protein genes are from studies on S. aureus; results with other staphylococci are consistent with these (Vuong et al., 2000). In general, during aerobic planktonic growth in vitro, genes encoding secreted proteins are up-regulated during the post-exponential phase, whereas genes encoding surface proteins are up-regulated very early in growth and down-regulated shortly thereafter. The intracellular effector of both types of agr-determined regulation is a regulatory RNA, RNAIII. However, several of the environmental factors acting as external inputs into this regulatory system inhibit the production of protein A (a surface protein), as well as of many secreted proteins, and act independently of agr, whereas others may interact with agr (Chan & Foster, 1998a
; Lindsay & Foster, 1999
). saeRS appears to be a key element in the regulatory cascade governing the staphylococcal virulon. saeRS was originally identified as a Tn551 insertion with an exoprotein-defective phenotype (Giraudo et al., 1994
) and subsequently shown to be a two-component signal transduction module with the transposon insertion in saeR, the putative response regulator gene (Giraudo et al., 1999
). In preliminary studies (Ross & Novick, 2001
; Novick, 2003
), we have observed that sae is a more complicated locus than originally envisioned (Giraudo et al., 1999
) and that it has a complex transcriptional pattern that is profoundly influenced by agr and by certain environmental stimuli. In this report, we present a detailed study of the agrsae interaction, the results of which suggest that sae may have a major role in the integration of cell density signalling with signalling through environmental stimuli and other regulatory elements.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Northern blot hybridization.
Culture samples representing equal numbers of bacteria were centrifuged, washed in 2 ml TE (10 mM Tris/HCl, 1 mM EDTA, pH 7·5) and resuspended in the same buffer. RNA was prepared by the method of Kornblum et al. (1988), separated by agarose gel electrophoresis (1 % agarose in standard Tris/borate buffer), electroblotted to nitrocellulose filters and hybridized to probes prepared by PCR using [32P]dATP (Amersham). Blots were scanned with a Molecular Dynamics phosphorimager and analysed using IMAGEQUANT software (NIH). Sequences of the primers used in this study are listed in Table 2
.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
The sae locus
It is clear from these blots that the sae locus is larger and more complex than originally envisioned by Nagel and coworkers (Giraudo et al., 1999). Analysis of the sae transcription pattern at two different time points, T=1 h (representing early-exponential phase) and T=5 h (representing post-exponential phase), using different probes is shown in Fig. 4
(b, c). Henceforth, the 2·1 kb sae transcript is referred to as A, the 2·6 kb sae transcript is referred to as B, the 3·1 kb sae transcript is referred to as C and the 0·5 kb sae transcript is referred to as D. These blots show that sae transcripts A, B and C all terminate at or near the end of saeS, and therefore have different 5' ends. The fourth transcript, D, is seen only with the upstream probe, P, and is therefore homologous to a region between the 5' ends of B and C. The directionality of this transcript is not known presently. One possibility is that B and C are transcribed from two different promoters and that D is transcribed independently from a third. Another possibility is that C is processed to give B+D. Transcript C includes two ORFs, of 146 and 157 codons, 5' to saeR, of which the latter is within B. Since the two upstream ORFs are within the sae operon, they are likely to be important for sae function and are here designated saeP and saeQ, respectively. There is a strong potential translational start within saeQ leading to a possible C-terminal protein, SaeQ'. Additionally, the potential secondary structure of the saeR transcript suggests that the translational start of SaeR is occluded, that the C-terminal end of the saeQ reading frame overlaps with the saeR start, and therefore that saeQ would have to be translated in order to permit translation of saeR.
|
sae autoregulation
Examination of the transcription pattern seen with the saeR : : Tn551 mutation (Fig. 5, left-hand side) shows that transcript A is present (indicated as A*), is longer owing to the inserted transposon and its presence is prolonged through the post-exponential phase, suggesting that it may be autorepressed by native SaeR. Furthermore, the upstream transcripts are absent (lower panel), suggesting that these may be autoinduced by SaeR. As expected, saeRS, cloned under Pbla control, restores the upstream transcripts in the saeR mutant (not shown). The right-hand part of Fig. 5
, in which RNA prepared from the agr-null, the wild-type and the saeR : : Tn551 strains was blotted with a combination of upstream probes P and Q (Fig. 4a
), demonstrates both the agr requirement and the saeR requirement for the expression of the upstream transcripts B, C and D. This result confirms the autoinduction of these transcripts and the cooperation between sae and agr for their activation.
|
Effects of environmental stimuli
It has been observed by several investigators that certain environmental stimuli profoundly affect the expression of certain exoprotein genes through B and SarA independently of agr (Chan & Foster, 1998a
, b
; Chan et al., 1998
; Cheung et al., 1999
; Cheung & Zhang, 2002
).
Accordingly, we analysed the transcription pattern of sae under the influence of certain environmental stimuli. Fig. 7(a) shows a series of Northern blots of whole-cell RNA samples prepared during growth in CYGP broth with the addition of 28 mM glucose, 1 M NaCl or subinhibitory clindamycin (SBCL). In cultures of this type grown with 28 mM glucose, the glucose is used up by the beginning of the stationary phase, by which time the pH has fallen to 55·5 (R. P. Novick & D. Jiang, unpublished data). As can be seen in the glucose panel, in which pH was monitored, the usual switch in transcription pattern occurs at T=2 h but as soon as the pH drops below 6, at T=4 h, sae transcription is turned off. We have observed independently that TSST-1 and other agr-regulated exoproteins, previously considered to be catabolite repressed (Hallis et al., 1991
; Iandolo & Shafer, 1977
; Coleman, 1983
), are produced in the presence of glucose but are not produced below pH 6 (B. Weinrick, H. F. Ross & R. P. Novick, unpublished data). This result suggests that sae may mediate the effects of a moderate decline in pH and that a considerable proportion of the apparent catabolite repression of exoprotein synthesis may actually be a pH effect. Similarly, as seen in the NaCl and SBCL blots, both 1 M NaCl and SBCL eliminate the temporal switch in sae transcription, consistent with their observed effects on overall exoprotein synthesis (Chan & Foster, 1998a
; Herbert et al., 2001
). Since the experiments were performed with an Agr+ strain, and since these stimuli do not affect agr activation, it is clear that the observed effects override the agr induction of the sae switch. This was confirmed for SBCL in an experiment with the Pbla : : RNAIII fusion in an agr-null background. As can be seen in Fig. 7(b)
, SBCL prevented the switch despite the induction of RNAIII. Note that, even without induction in this strain, transcript A is not detectable and there is a weak band representing transcript B. This seems inconsistent with results shown in Fig. 3
and is probably owing to a difference in growth conditions and the earlier time at which the samples shown in Fig. 3
were collected.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sae transcription pattern is complex and undergoes a critical change during the growth cycle. This change involves the disappearance of a 2·1 kb transcript (A) that is present from the outset and the appearance of three new ones, of 0·5 kb (D), 2·6 kb (B) and 3·1 kb (C), coincident with the onset of agr RNAIII synthesis. Partial confirmation of this has recently been reported by Giraudo et al. (2003)
. The new transcripts contain two additional reading frames, upstream of saeR, here designated saeP and saeQ, which are likely to be translated. The transcriptional switch is effected by sae, indicating that the locus is autoregulated. It is not affected by
B, but is blocked in agr and sarA mutants. Since SarA enhances RNAIII production, the effects of a sarA mutation on sae may be related to this. Whether other regulatory determinants also affect the sae switch is presently under investigation.
The sae transcription pattern suggests that the sae locus is more complex than originally envisioned and suggests that the upstream ORFs P and Q, or the transcripts B, C and D may play a role in the regulation by sae of at least some of the exoprotein genes. Other genes may be regulated by the sae two-component system, consisting of SaeR and S, alone. The potentially different roles of the sae upstream and downstream regions in the regulation of different exoprotein genes may explain the different classes of exoproteins in the exoprotein profiles in Fig. 1, and may also explain the paradox of sae appearing to be both epistatic to and downstream of RNAIII.
Activation of the sae system can be envisioned as starting (in vitro) with the activation of SaeS by an unknown ligand, possibly external, followed by the activation of SaeR, presumably by phosphorylation, though de-phosphorylation is certainly possible. Somewhat later, in mid-exponential phase in vitro, there is a critical regulatory transition in which agrRNAIII, in conjunction with activated SaeR (or, much less likely, SaeS), and possibly other regulatory elements, induces the three upstream transcripts, leading to production of SaeP and SaeQ. Concomitantly with induction of the upstream promoters, SaeR down-regulates (autorepresses) transcript A. Since transcripts B and C read through saeRS, the continuing transcription of SaeR is ensured. Translation of saeQ, however, is likely to be required for translation of saeR, and would therefore be an important feature of the sae autoregulation mechanism. Thus, the key regulatory transition must be a function of the upstream transcripts, presumably through SaeP and SaeQ, though possibly through an RNA-mediated effect. The alternative possibility that SaeR is the sole effector of sae-mediated regulation and that the upstream transcripts/products affect the production or activity of SaeR has not been ruled out.
Remarkably, the switch in sae transcription is blocked by diverse environmental signals, including 1 M NaCl, pH below 6 and SBCL. These stimuli have been shown to act downstream of agr and not through it; it is not presently known, however, whether they act directly or through other regulatory genes. Thus, sae may be a key intracellular coordinator of the agr quorum-sensing system with a variety of environmental signals that are well known to have profound effects on exoprotein synthesis (Chan & Foster, 1998a; Lindsay & Foster, 1999
).
It is concluded that, although the overall regulatory network governing the staphylococcal virulon seems to involve reciprocal interactions among various regulatory determinants (Novick, 2003), a central linear pathway in which saeRS is directly downstream from agr is beginning to take shape. At this stage, critical unknowns are the putative ligand for SaeS, and the mechanisms by which RNAIII induces and environmental stimuli block the mid-exponential regulatory transition responsible for activation of the sae system. Future studies will address these mechanisms and will also investigate the questions of translational and post-translational regulation of sae, the activity of SaeR and the role of its phosphorylation, and the regulatory role(s) of the upstream sae region.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chan, P. F. & Foster, S. J. (1998a). The role of environmental factors in the regulation of virulence-determinant expression in Staphylococcus aureus 8325-4. Microbiology 144, 24692479.[Abstract]
Chan, P. F. & Foster, S. J. (1998b). Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J Bacteriol 180, 62326241.
Chan, P. F., Foster, S. J., Ingham, E. & Clements, M. O. (1998). The Staphylococcus aureus alternative sigma factor B controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J Bacteriol 180, 60826089.
Cheung, A. L. & Zhang, G. (2002). Global regulation of virulence determinants in Staphylococcus aureus by the SarA protein family. Front Biosci 7, d18251842.[Medline]
Cheung, A. L., Coomey, J. M., Butler, C. A., Projan, S. J. & Fischetti, V. A. (1992). Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proc Natl Acad Sci U S A 89, 64626466.[Abstract]
Cheung, A. L., Chien, Y.-T. & Bayer, A. S. (1999). Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus. Infect Immun 67, 13311337.
Coleman, G. (1983). The effect of glucose on the differential rates of extracellular protein and -toxin formation by Staphylococcus aureus (Wood 46). Arch Microbiol 134, 208211.[Medline]
Garvis, S., Mei, J. M., Ruiz-Albert, J. & Holden, D. W. (2002). Staphylococcus aureus svrA: a gene required for virulence and expression of the agr locus. Microbiology 148, 32353243.
Giraudo, A. T., Raspanti, C. G., Calzolari, A. & Nagel, R. (1994). Characterization of a Tn551-mutant of Staphylococcus aureus defective in the production of several exoproteins. Can J Microbiol 40, 677681.[Medline]
Giraudo, A. T., Rampone, H., Calzolari, A. & Nagel, R. (1996). Phenotypic characterization and virulence of a sae- agr- mutant of Staphylococcus aureus. Can J Microbiol 42, 120123.[Medline]
Giraudo, A. T., Cheung, A. L. & Nagel, R. (1997). The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level. Arch Microbiol 168, 5358.[CrossRef][Medline]
Giraudo, A. T., Calzolari, A., Cataldi, A. A., Bogni, C. & Nagel, R. (1999). The sae locus of Staphylococcus aureus encodes a two-component regulatory system. FEMS Microbiol Lett 177, 1522.[CrossRef][Medline]
Giraudo, A. T., Mansilla, C., Chan, A., Raspanti, C. & Nagel, R. (2003). Studies on the expression of regulatory locus sae in Staphylococcus aureus. Curr Microbiol 46, 246250.[Medline]
Hallis, B. A., Thurston, C. F. & Mason, J. R. (1991). Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP. FEMS Microbiol Lett 64, 247251.[Medline]
Herbert, S., Barry, P. & Novick, R. P. (2001). Subinhibitory clindamycin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus. Infect Immun 69, 29963003.
Iandolo, J. J. & Shafer, W. M. (1977). Regulation of staphylococcal enterotoxin B. Infect Immun 16, 610616.[Medline]
Kornblum, J. S., Projan, S. J., Moghazeh, S. L. & Novick, R. P. (1988). A rapid method to quantitate non-labeled RNA species in bacterial cells. Gene 63, 7585.[CrossRef][Medline]
Kullik, I., Giachino, P. & Fuchs, T. (1998). Deletion of the alternative sigma factor B in Staphylococcus aureus reveals its function as a global regulator of virulence genes. J Bacteriol 180, 48144820.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lindsay, J. A. & Foster, S. J. (1999). Interactive regulatory pathways control virulence determinant production and stability in response to environmental conditions in Staphylococcus aureus. Mol Gen Genet 262, 323331.[CrossRef][Medline]
Nicholas, R. O., Li, T., McDevitt, D., Marra, A., Sucoloski, S., Demarsh, P. L. & Gentry, D. R. (1999). Isolation and characterization of a sigB deletion mutant of Staphylococcus aureus. Infect Immun 67, 36673669.
Novick, R. P. (1991). Genetic systems in staphylococci. Methods Enzymol 204, 587636.[Medline]
Novick, R. P. (2003). Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 48, 14291449.[CrossRef][Medline]
Novick, R. P. & Richmond, M. H. (1965). Nature and interactions of the genetic elements governing penicillinase synthesis in Staphylococcus aureus. J Bacteriol 90, 467480.
Novick, R. P., Ross, H. F., Projan, S. J., Kornblum, J., Kreiswirth, B. & Moghazeh, S. (1993). Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12, 39673975.[Abstract]
Ross, H. F. & Novick, R. P. (2001). sae is a key intermediary in the activation by agr of the staphylococcal virulon. In CellCell Communication. Edited by S. Winans & B. Bassler. Snowbird, UT: American Society for Microbiology.
Vandenesch, F., Kornblum, J. & Novick, R. P. (1991). A temporal signal, independent of agr, is required for hla but not for spa transcription in Staphylococcus aureus. J Bacteriol 173, 63136320.[Medline]
Vojtov, N., Ross, H. F. & Novick, R. P. (2002). Global repression of exotoxin synthesis by staphylococcal superantigens. Proc Natl Acad Sci U S A 99, 1010210107.
Vuong, C., Gotz, F. & Otto, M. (2000). Construction and characterization of an agr deletion mutant of Staphylococcus epidermidis. Infect Immun 68, 10481053.
Yarwood, J. M., McCormick, J. K. & Schlievert, P. M. (2001). Identification of a novel two-component regulatory system that acts in global regulation of virulence factors of Staphylococcus aureus. J Bacteriol 183, 11131123.
Received 16 June 2003;
revised 10 July 2003;
accepted 11 July 2003.