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Signal Transduction and Regulatory Mechanisms Involved in Control of the ^S (RpoS) Subunit of RNA Polymerase

Institut für Biologie, Mikrobiologie, Freie Universität Berlin, 14195 Berlin, Germany

SUMMARY

INTRODUCTION

THE PROBLEM OF MULTIPLE STRESS SIGNAL INTEGRATION

REGULATION OF rpoS TRANSCRIPTION

Promoters Contributing to rpoS Transcription

Trans-Acting Factors Controlling rpoS Transcription

cAMP-CRP and EIIA(Glc).

The GacS-GacA two-component and Las-Rhl quorum-sensing systems in pseudomonads.

BarA, a histidine sensor kinase in search of a response regulator.

PsrA in pseudomonads: a TetR-like regulator.

Role for Polyphosphate in rpoS Regulation

Small Molecules That Influence rpoS Transcription

ppGpp.

Is rpoS expression controlled by quorum sensing?

Homoserine lactone and homocysteine thiolactone.

Acetate and other weak acids.

Cellular NADH-to-NAD+ ratio.

REGULATION OF rpoS TRANSLATION

Role of rpoS mRNA Secondary Structure

trans-Acting Factors Involved in rpoS Translation

The RNA binding protein Hfq (HF-I).

HU: a nucleoid protein that also stimulates rpoS translation.

H-NS and StpA: histone-like proteins acting as RNA chaperones?

Role of small regulatory RNAs in rpoS translation: DsrA, OxyS, and RprA.

The LysR-like regulator LeuO: a repressor for dsrA expression.

DnaK and DksA: a link to heat shock and chaperones.

EIIA(Glc): a link to the carbon source and energy supply.

The cold shock domain proteins CspC and CspE.

A Small Molecule That Influences rpoS Translation: UDP-Glucose

rpoS Translational Control Network and Stress Signal Input

REGULATION OF {sigma}S PROTEOLYSIS

{sigma}S Degradation by the Complex ATP-Dependent ClpXP Protease

The Response Regulator RssB: a {sigma}S Recognition Factor with Phosphorylation-Modulated Affinity

The Turnover Element: the RssB Binding Site within {sigma}S

Initiation of {sigma}S Proteolysis: the RssB Cycle

Signal Integration in the Control of {sigma}S Proteolysis

Additional Factors with Uncharacterized Molecular Functions in {sigma}S Turnover

RssA.

The histone-like protein H-NS.

The LysR homolog LrhA.

The DnaK chaperone.

A Small Molecule That Affects {sigma}S Proteolysis: Acetyl Phosphate

REGULATION OF {sigma}S ACTIVITY

In Vivo Evidence for Regulation of {sigma}S Activity

The Response Regulator RssB Can Act Like an Anti-Sigma Factor for {sigma}S

CONCLUSIONS AND PERSPECTIVES

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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^S, or RpoS, is a sigma subunit of RNA polymerase in Escherichia coli that is induced and can partially replace the vegetative sigma factor ⁷⁰ (RpoD) under many stress conditions. As a consequence, transcription of numerous ^S-dependent genes is activated (for reviews, see references 75, 77, 79, 124, and 125). Consistent with the multiple functions of the ^S regulon, the rpoS gene was discovered independently and named accordingly by several groups (recently summarized in references 79 and 105). It was identified as a gene involved in near-UV resistance (nuv) (216); as a regulator for the katE-encoded catalase HPII (katF) (126, 186), exonuclease III (xthA) [186], and acidic phosphatase (appR) (211); and, finally, as a starvation-inducible gene encoding a central regulator for stationary phase-inducible genes (csi-2) (113). Only then was it recognized that all the previous studies had described alleles of the same gene (113, 212), which codes for a sigma factor (152, 156, 209). Because of its crucial role in stationary phase or under stress conditions, the name ^S or RpoS was proposed (113). In addition, the term ³⁸ is used sometimes (although the molecular mass of ^S deviates from 38 kDa in various species and even in some E. coli strains). The rpoS gene has also been identified in other enteric and related bacteria. At present, it seems that ^S occurs in the branch of the proteobacteria, i.e., in a group of gram-negative bacteria that includes many species with special importance for humans because of their pathogenic or beneficial potential. With minor variations, the general function of ^S in these bacteria appears to be similar to that in E. coli (summarized in reference 79).

In more recent studies, it was demonstrated that ^S and ^S-dependent genes not only are induced in stationary phase but actually respond to many different stress conditions (76, 82, 121, 144, 148). Therefore, ^S is now seen as the master regulator of the general stress response, which is triggered by many different stress signals, is often (though not always) accompanied by a reduction or cessation of growth, and provides the cells with the ability to survive the actual stress as well as additional stresses not yet encountered (cross-protection). This is in pronounced contrast to specific stress responses, which are triggered by a single stress signal and result in the induction of proteins that allow cells to cope with this specific stress situation only. While specific stress responses tend to eliminate the stress agent and/or to mediate repair of cellular damage that has already occurred, the general stress response renders cells broadly stress resistant in such a way that damage is avoided rather than needing to be repaired (for a recent review of different bacterial stress responses, see reference 203).

The major function of the general stress response is thus preventive, which is clearly reflected in the ^S-dependent multiple stress resistance observed with starved or otherwise stressed cells (82, 113, 136) (for a recent review of the general stress response that also includes physiological aspects, see reference 79). Accordingly, the majority of the more than 70 ^S-dependent genes known so far confer resistance against oxidative stress, near-UV irradiation, potentially lethal heat shocks, hyperosmolarity, acidic pH, ethanol, and probably other stresses yet to be identified. Additional ^S-controlled gene products generate changes in the cell envelope and overall morphology (stressed E. coli cells tend to become smaller and ovoid). Metabolism is also affected by ^S-controlled genes, consistent with ^S being important under conditions where cells switch from a metabolism directed toward maximal growth to a maintenance metabolism. ^S also controls genes mediating programmed cell death in stationary phase, which may increase the chances for survival for a bacterial population under extreme stress by sacrificing a fraction of the population in order to provide nutrients for the remaining surviving cells (22). Finally, a number of virulence genes in pathogenic enteric bacteria have been found to be under ^S control, consistent with the notion that host organisms provide stressful environments for invading pathogens (recently summarized in reference 80). However, even though numerous ^S-dependent genes have been identified (see references 77, 79, and 125 for recent compilations), many more such genes will probably be found in the future. Moreover, the functions of the genes known so far are incompletely understood. Even after more than 10 years of intensive research on ^S, much remains to be learned about the physiology of the ^S-mediated response. The same is true for the regulatory interdependencies within the large regulatory network directed by ^S. By contrast, the basic mechanisms of regulation of ^S itself are now reasonably well understood and are the subject of this review (for recent minireviews on the same subject, see references 81 and 125). Since by far most of the relevant work has been done with E. coli and Salmonella, the systems referred to in this review are those described for these enteric bacteria if not otherwise mentioned. Whereas the physiological function of ^S is comparable in all species where it has been discovered to date, there are significant differences between enteric bacteria and pseudomonads in the regulation of ^S that are outlined specifically.

THE PROBLEM OF MULTIPLE STRESS SIGNAL INTEGRATION

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Complex and physiologically far-reaching bacterial responses often use a single master regulator at the interface of upstream signal processing and downstream regulatory mechanisms. In the general stress response of E. coli, ^S plays the role of this top-level master regulator. Other examples of such central regulators are ^B in the general stress response of various gram-positive bacteria (172) and the response regulator Spo0A in sporulation initiation of Bacillus subtilis (202). The master regulators serve as the decisive information processing units, which connect complex signaling networks with downstream regulatory cascades or networks that ultimately control the expression of numerous structural genes associated with a response. These regulatory networks exhibit a hierarchical and modular structure; i.e., they can be subdivided into lower-level smaller modules that are under the control of secondary regulators, which also allow specific signal input at such lower and more confined levels. A master regulator may also commit the cell to a certain complex developmental program, with specific temporal and spatial control being exerted by secondary regulalors.

Depending on the type of master regulator (sigma factor, two-component response regulator, etc.) as well as on whether its cellular level or its activity (or both) is the decisive parameter, signal transduction and integration upstream of the central regulator can be very different. In the general stress response of E. coli, the decisive parameter is the cellular level of ^S (however, as described below, there is now some initial evidence that ^S may also be subject to some sort of activity control). ^S levels increase in response to starvation for carbon, nitrogen, or phosphate sources as well as for amino acids. This leads to entry into stationary phase, i.e., a complete cessation of growth, but ^S can also be induced by a partial reduction of the growth rate (64, 89, 92, 113, 114, 157, 210). Additional inducing conditions are hyperosmolarity (148), nonoptimally high (144) or low (199) temperature, acidic pH (18, 121), high cell density (114), and probably other environmental stress situations.

How can so many different stress signals be integrated toward a single parameter, i.e., the intracellular ^S concentration? Do they generate a common intracellular signal? Initially it seemed that a reduction or cessation of growth might be such a signal. However, ^S is also induced in late exponential phase (provided that a certain cell density is reached) without any change of growth rate (114). In response to the classical heat shock procedure (i.e., a shift from 30 to 42°C), ^S is induced even though growth is accelerated (144). On the basis of more recent studies, it has become clear that the concept of a unifying intracellular ^S-inducing signal cannot be correct, simply because different environmental signals affect different levels and therefore completely different processes in the regulation of ^S.

Regulation of ^S occurs at nearly every theoretically possible level (Fig. 1). rpoS transcription is stimulated by controlled downshifts in growth rate in a chemostat (157, 210) as well as by continuous reduction in growth rate which results in an inversely correlated increase in rpoS transcription (5- to 10-fold) (113, 114). By contrast, abrupt cessation of growth, as for example, in response to sudden glucose starvation, only weakly increases rpoS transcription (less than twofold) (113, 114). rpoS translation, i.e., the rate of translation of already existing rpoS mRNA, is stimulated (i) by high osmolarity (hyperosmotic shift rapidly activates translation more than fivefold, but also continuous growth at high osmolarity has clear effects) (148), (ii) during growth at moderately low temperatures (e.g., at 20°C) (199), (iii) on reaching a certain cell density (approximately 1 x 10⁸ to 2 x 10⁸ cells ml^-1) during growth in minimal glucose medium (114), and (iv) in response to a pH downshift from pH 7 to pH 5 in rich medium (G. Kampmann and R. Hengge-Aronis, unpublished data).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Various levels of S regulation are differentially affected by various stress conditions. An increase of the cellular S level can be obtained either by stimulating S synthesis at the levels of rpoS transcription or rpoS mRNA translation or by inhibiting S proteolysis (which under nonstress conditions is extraordinarily rapid). The most rapid and strongest reaction can be achieved by a combination of these processes (as observed, e.g., on hyperosmotic or pH shifts). For further details, see the text.

In recent years, considerable progress has been made in identifying (i) cis-acting regulatory regions at the DNA, mRNA, or protein levels; (ii) trans-acting factors such as regulatory proteins or regulatory RNAs; and (iii) additional large or small molecules that modulate ^S regulation at these different levels. In general, the closer to the rpoS gene, its mRNA, or the ^S protein itself these factors act, the better understood is their molecular mechanism of action. By contrast, the upper parts of the corresponding signal transduction pathways have remained more elusive. It is, however, already clear that these pathways do not operate independently and in parallel until they finally converge to influence, e.g., rpoS mRNA secondary structure or the activity of a specific ^S recognition factor for proteolysis. Rather, these pathways are highly interconnected, such that specific stress conditions can influence the cellular ^S concentration by multiple mechanisms. Moreover, specific components of these pathways also control each other. Thus, ^S is controlled by a complex signal transduction network whose redundancy, additiveness, and internal feedback regulatory loops are crucial for its admirable signal-integrative power (and probably its nonlinear behavior) but at the same time have made life difficult for researchers trying to elucidate these pathways.

REGULATION OF rpoS TRANSCRIPTION

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Soon after ^S had been recognized as a stationary-phase regulator that was itself induced in stationary phase (113), it became clear that much of its regulation was due to posttranscriptional mechanisms (114, 127, 135). Although ^S protein levels are very low in exponentially growing cells, relatively high levels of rpoS mRNA are present and do not seem to change in response to several stresses that actually result in strongly elevated ^S protein levels. Therefore, attention has focused on the control of rpoS translation and ^S proteolysis (see below) whereas rpoS transcription has remained insufficiently characterized. Nevertheless, transcriptional regulation of rpoS occurs, e.g., during the gradual decrease in growth rate when cells grow in rich medium and finally enter stationary phase. Under these conditions, rpoS transcription is activated approximately 5- to 10-fold (113, 114, 135, 153, 190, 208).

View larger version (33K): [in this window] [in a new window]   FIG. 2. Transcriptional control regions upstream of the rpoS gene. (A) The nlpD-rpoS operon is located at 61.76 min on the E. coli chromosome, where it is trancribed in counterclockwise direction. (B) The operon promoters (nlpDp1 and nlpDp2) contribute to basal expression of rpoS but are not regulated by growth rate or growth phase (115). (C) The major rpoS promoter (rpoSp) is located within the nlpD gene, is flanked by two putative cAMP-CRP binding sites (CRP box I and II), and is subject to stationary-phase induction when cells are grown on rich medium (112). Broken lines in panel A indicate the relative positions of the sequences shown in panels B and C.

Two putative cAMP-CRP binding sites are present upstream and downstream of rpoSp (Fig. 2), and the role of these potential binding sites is currently under investigation. Whereas the location of the upstream cAMP-CRP box is similar to that in the lac promoter and corresponds to a classical activator position at a class I promoter (31), the location of the second cAMP-CRP box downstream of the transcriptional start site may suggest an inhibitory action. In addition, cAMP-CRP may also have an indirect effect on rpoS expression, since cya or crp mutants exhibit a reduced growth rate, which in turn can affect rpoS transcription, as mentioned above.

Adenylate cyclase activity is positively modulated by the crr-encoded EIIA(Glc), which is the soluble part of the glucose-specific EII component of the phosphotransferase system for solute uptake. Consistent with this, a crr mutation results in elevated ^S levels during exponential phase, which reflects increased rpoS transcription as well as increased rpoS translation. The former can be suppressed by cAMP addition, indicating that EIIA(Glc) affects rpoS transcription through its modulation of adenylate cyclase activity (217). Phenotypically, the crr mutant thus seems to mimic the log-phase behavior of the cya mutant.

Polyamines stimulate adenylate cyclase expression at the level of translational initiation, and at the same time they lead to 2.3- and 4-fold increases of the cellular levels of ^S and ²⁸, respectively (235). This polyamine-induced upregulation of ^S may mimic the positive effect of cAMP-CRP on rpoS transcription during entry into stationary phase.

The GacS-GacA two-component and Las-Rhl quorum-sensing systems in pseudomonads. GacA is a two-component response regulator in various Pseudomonas species that has long been known to positively affect the production of secondary products such as antibiotics, toxins, and lytic exoenzymes during entry into stationary phase (117, 123, 178). The cognate histidine sensor kinase is the GacS (LemA) protein (85, 178). What is actually sensed by GacS has not been clarified. Homologs of GacA are present in Salmonella (SirA, which controls certain virulence genes [93]) and E. coli (YecB or UvrY [see below]). The GacS-GacA two-component system is at the top of a regulatory cascade that controls the LasI-LasR quorum-sensing system, which in turn regulates a second quorum-sensing system, RhlI-RhlR (110, 167, 176). These quorum-sensing systems are crucial for the control of virulence factors, exoenzymes, and stress-protective proteins as well as for the formation of biofilms (62, 74, 111, 159, 232); for summaries of quorum-sensing systems, see references 61 and 231).

Mutations in gacA or gacS also result in a more than 80% reduction of ^S levels and equally reduced expression of a transcriptional rpoS::lacZ fusion specifically during transition into stationary phase (227). Whether this regulation of rpoS by the Gac system is direct or indirect has not been demonstrated. There are also conflicting data about how rpoS is linked to the Las-Rhl cascade. An earlier study had indicated that rpoS is under positive control of both the Las and Rhl systems (110). More recently, however, rpoS was found not to be affected by rhl mutations; in contrast, rhlI is upregulated in rpoS mutants, indicating that ^S is a negative regulator of the Rhl system (228). This fits with the observation that pyocyanin and pyoverdin are overproduced in rpoS mutants (205), since these virulence-associated factors are under positive control of the Rhl system (27).

In contrast to the situation in Pseudomonas, the GacA homolog YecB (UvrY) in E. coli appears not to be involved in the control of rpoS expression. Even though YecB overproduction stimulated the expression of transcriptional and translational rpoS::lacZ fusions and resulted in a twofold-higher level of ^S specifically during transition into stationary phase, a yecB::cat knockout mutation did not alter the expression of these rpoS::lacZ fusion, nor were ^S levels affected (Kampmann and Hengge-Aronis, unpublished). While ^S plays similar physiological roles in E. coli and Pseudomonas (188, 205), differential control by YecB-GacA may reflect the different environmental conditions characteristic of the natural habitats of these bacteria.

BarA, a histidine sensor kinase in search of a response regulator. The E. coli homolog of GacS is a hybrid sensor kinase called BarA (40% identity and 59% overall similarity at the amino acid level to P. aeruginosa GacS). BarA was previously found as a multicopy suppressor of an envZ mutation; i.e., when present at high levels, BarA is able to cross-phosphorylate the response regulator OmpR and thereby activate porin synthesis (88, 154). Under the name AirS, BarA has also been identified as a virulence factor in uropathogenic E. coli (241). There is evidence that BarA plays a positive role in rpoS expression. A strain with a lacZ insertion in the chromosomal copy of barA, which was originally isolated as a hydrogen peroxide-sensitive mutant, exhibits reduced levels of ^S (150, 151). In the mutant, rpoS mRNA levels were reduced during exponential phase but were normal in stationary phase. Therefore, BarA was suggested as a positive regulator of rpoS transcription (150). By specifically affecting rpoS mRNA levels in exponential phase, this control may determine the range within which ^S levels can be modulated by posttranscriptional control mechanisms in response to various stress conditions.

The homology to the GacS-GacA system, as well as recent biochemical data (166), suggests that BarA is a cognate sensor kinase for YecB. Therefore, it seems surprising that BarA but not YecB (see above) is involved in ^S control. However, it is possible that BarA acts on more than one response regulator with an unknown target response regulator being involved in rpoS control. BarA (GacS) belongs to the complex "built-in phosphorelay" sensor kinases in which sensor, transmitter, receiver, and histidine-containing phosphotransfer domains are combined in a single polypeptide chain. In view of their multiple interactions, phosphorelay components seem especially adequate for establishing such phosphotransfer networks.

PsrA in pseudomonads: a TetR-like regulator. A search for insertional mutations that downregulated the expression of a transcriptional rpoS::lacZ fusion in P. putida yielded a mutant defective in a gene termed psrA (for "Pseudomonas sigma regulator"), which encodes a TetR repressor-like regulatory protein. psrA is required for increased rpoS transcription during entry into stationary phase and is negatively autoregulated (104). Whether this control of rpoS is direct or indirect is currently unknown.

Polyphosphate-free ppk mutants are multiple stress sensitive and impaired in stationary phase survival (36, 175). Consistent with these phenotypes, ^S levels as well as the expression of a transcriptional rpoS::lacZ fusion are reduced in a strain that overproduces yeast exopolyphosphatase and is therefore depleted of polyphosphate (195). These findings indicate that polyphosphate somehow stimulates rpoS transcription and thereby contributes to stationary-phase induction of rpoS (which in rich media is partly due to increased rpoS transcription). However, polyphosphate fails to stimulate rpoS transcription in vitro and therefore may exert an indirect influence in vivo (195).

Such ppGpp-free mutants contain strongly reduced ^S levels. Glucose and phosphate starvation, but not amino acid limitation, still induce ^S in these mutants (albeit to lower levels than in the wild type). On the other hand, ^S accumulation can be triggered by artificially stimulating ppGpp accumulation (64).

ppGpp affects rpoS transcription, as demonstrated with transcriptional rpoS::lacZ fusions (112). However, ppGpp does not seem to specifically target the promoters involved in rpoS transcription, since a transcriptional rpoS::lacZ fusion construct, in which these natural promoters were deleted (and basal expression was due to vector-dependent transcriptional readthrough activity), exhibited similarly reduced expression as the promoter-carrying construct in a ppGpp-free genetic background. It was therefore proposed that in the case of rpoS, ppGpp may affect transcriptional elongation or transcript stability rather than transcriptional initiation (112). In the absence of ppGpp, starvation may result in an uncoupling of transcription and translation, which may lead to increased premature termination, as demonstrated for lacZ mRNA (52, 219, 220).

It is also unclear whether this ppGpp effect is direct or indirect. An increase in the cellular ppGpp content results in the accumulation of polyphosphate, which also stimulates rpoS transcription by an unknown mechanism (see above). It is therefore conceivable that ppGpp acts indirectly via polyphosphate. The finding that a polyphosphate-depleted strain is not impaired in ppGpp accumulation but contains strongly reduced ^S levels is consistent with such an indirect mode of action (195). Clearly more research is required to elucidate these relationships at the molecular level.

Is rpoS expression controlled by quorum sensing? Sometimes high cell density in a bacterial population turns out to be the inducing signal for "stationary phase-inducible" genes. The classical "quorum-sensing" system is the lux system in Vibrio fischeri, where a membrane-permeable acylated homoserine lactone (acylated HSL, the "autoinducer") is produced by LuxI and accumulates in the medium. Beyond a certain threshold concentration, the autoinducer binds to and activates LuxR, which stimulates the expression of the luxI and luxR genes themselves and the luciferase structural genes (60, 61, 187). Numerous bacterial species contain homologs of the LuxI-LuxR pair, which control a wide variety of output functions (recently summarized in reference 231). Other types of quorum-sensing systems use different kinds of inducing molecules; e.g., gram-positive species use small peptides in general. A hallmark of all these systems is their inducibility on addition of conditioned medium, i.e., spent supernatant obtained from a culture grown to relatively high cell density, which contains the inducing molecule in sufficient concentration.

With respect to rpoS induction by conditioned medium, conflicting data have been reported. Such induction (approximately fourfold) was observed for a transcriptional rpoS::lacZ fusion, and acetate was proposed as the inducing agent (190). With a different rpoS::lacZ operon fusion present in multiple copies, fourfold induction was also found with conditioned Luria broth medium (153), but when the same fusion was present in single copy in the chromosome, induction by spent medium was reduced to a mere 1.6-fold (197). In another study, a transcriptional rpoS::lacZ fusion was found to be completely unaffected by conditioned medium (63). With a set of single-copy transcriptional and translational rpoS::lacZ fusions (114), very little if any induction was obtained, no matter whether conditioned rich or minimal medium was used, and even spent medium freshly prepared in parallel with the induction experiments (to compensate for potential instability of a putative inducer) had little effect on rpoS expression levels (D. Traulsen and R. Hengge-Aronis, unpublished results). It therefore seems that quorum sensing mediated by some excreted medium component does not play a significant role in the regulation of rpoS in E. coli. Therefore, translational induction of rpoS beyond a certain cell density (114) may also be connected to some metabolic alterations rather than to quorum sensing mediated by an excreted substance (see below). The finding that rpoS itself is probably not or is only weakly controlled by quorum sensing does not preclude certain ^S-transcribed genes from being subject to such regulation, which may affect the promoters of these genes directly (11, 197, 206).

The E. coli genome sequence (23) does not reveal obvious homologs of genes encoding known acyl-HSL synthases of the LuxI and AinS families (17, 61, 66). There is, however, a LuxR-related protein, SdiA, which may respond to an unidentified acyl-HSL (63, 197). Expression of SdiA itself, as well as the activity of a known target promoter, ftsQp2, responds negatively to conditioned (E. coli) medium, which may mean that the potential of E. coli to respond to an acyl-HSL via SdiA (and thereby activate cell division genes) is downregulated in stationary phase.rpoS, however, does not seem to be under the control of SdiA (63).

Homoserine lactone and homocysteine thiolactone. Nonacylated HSL has been implicated in rpoS control in E. coli. It was reported that a thrA metL lysC mutant, which is deficient early in the branched pathway that leads to biosynthesis of lysine, methionine, threonine, and isoleucine, had reduced ^S levels, which apparently could be suppressed by exogenously adding HSL (at concentrations up to 1 mM). This suppression was weaker in the presence of multiple copies of RspA. Such overexpression also reduced stationary-phase expression of an rpoS::lacZ fusion (which apparently was a transcriptional fusion). Therefore, it was hypothesized that HSL is an inducer for rpoS expression and that RspA may be involved in the degradation of HSL (86). At the time this work was published, this seemed to be in line with quorum-sensing studies that demonstrated the role of acylated HSLs in gene regulation. However, it is now known that free HSL is not the precursor for acylated HSLs that serve as autoinducers in LuxI-LuxR-like systems (72, 165) but, rather, plays the role of an intracellular metabolite. Moreover, there is good evidence against quorum sensing affecting rpoS regulation (see above).

More recently it was found that HSL (up to 1 mM) added to wild-type E. coli did not induce rpoS (197), and the RspA overproduction effect on rpoS is now considered nonphysiological (67). Nevertheless, the idea that RspA may degrade HSL is consistent with the recent observation that RspA-overproducing strains indeed seem to have increased homoserine and decreased HSL levels (U. Sauer, personal communication). Unexpectedly, these strains actually show elevated ^S levels, which would not seem consistent with HSL being an inducer for rpoS (226).

Another recent study (67) implicates specifically the methionine biosynthesis pathway in ^S control. A metE mutant, which is deficient for conversion of homocysteine to methionine and therefore accumulates homocysteine thiolactone (HCTL), exhibits increased ^S levels. Moreover, an asd mutant, which is deficient earlier in methionine biosynthesis (and therefore also in homocysteine and HCTL formation), shows decreased ^S levels, and this phenotype could be suppressed by exogenous HCTL (1 mM) (67). During entry into stationary phase in minimal glucose medium, a 2.5-fold accumulation of HCTL was observed in wild-type cells (67). Under these conditions, however, there is little if any activation of rpoS transcription, but ^S accumulation is due to posttranscriptional control (114; also see below). Unfortunately, HCTL effects were demonstrated by assaying for ^S protein only, and so the level of control affected remains open to speculation (67).

In summary, these studies (67, 86) have demonstrated that amino acid biosynthetic pathways, in particular the branch that leads to methionine (with HCTL as the putative effector), can influence the expression of rpoS. It has not been clarified, however, whether this effect is at the level of rpoS transcription, and the underlying molecular mechanism remains unknown.

Acetate and other weak acids. In an early study that reported the induction of a transcriptional rpoS::lacZ fusion in spent culture supernatant, the fermentation product acetate (used at 40 mM) was found to activate rpoS expression (190). In another study, however, acetate did not have an effect on rpoS expression (153). The studies agreed that benzoate (10 or 25 mM) has an inducing effect, and it was concluded that this may be so in general for weak acids with a pKa of 4.8 to 4.9 (40 mM propionate was also found to be effective) (153, 190). However, a translational rpoS::lacZ fusion did not show this induction by weak acids (190). One has to take into account, however, that these studies were performed before the advent of limitless precise PCR cloning, and therefore reporter gene fusions had to be constructed in rather complicated ways and often remained incompletely characterized. To finally settle the issue of weak acids in rpoS control, these experiments would have to be repeated and expanded with the precisely characterized systems available today.

Recent genome-wide analyses have shown that addition of acetate to buffered medium results in the activation of various ^S-dependent genes and proteins, but unfortunately the cellular concentration and regulation of ^S itself were not studied under these conditions (7, 100). Similar conditions resulted in increased synthesis of ^S in Salmonella, but the underlying control mechanisms seemed at least in part posttranscriptional (39).

Cellular NADH-to-NAD⁺ ratio. Experiments with a transcriptional rpoS::luxAB fusion in a nuoG mutant background (which is defective in a subunit of NADH dehydrogenase) suggested that a high NADH-to-NAD⁺ ratio somehow downregulates rpoS transcription. Consistent with this proposal, rpoS transcription is low under oxygen-limited (microaerobic) growth conditions, where NADH levels should increase due to the scarcity of oxygen as an electron acceptor for respiration (194). The mechanistic basis of this effect is unclear.

REGULATION OF rpoS TRANSLATION

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Initial evidence for posttranscriptional regulation of rpoS was provided by clearly different patterns of expression of transcriptional and translational rpoS::lacZ fusions (114, 127, 135). Translational rpoS::lacZ fusions can actually reflect regulation of rpoS translation as well as of ^S proteolysis (114, 148, 192). The latter can be excluded by using translational fusions that contain fewer than 173 N-terminal codons of rpoS, since an essential proteolytic recognition element is located at and around K173 in ^S (20; also see below).

Translation of rpoS mRNA is stimulated by a shift to hyperosmolarity (114, 148), by low temperature (199), by a shift to acidic pH (pH 5; Kampmann and Hengge-Aronis, unpublished), or during late exponential phase when a growing culture reaches a certain cell density (114). After the onset of carbon starvation, i.e., on entry into stationary phase, rpoS translation is reduced again, and further increases in ^S levels are then due to inhibition of ^S degradation, as described below (114).

Even under conditions where ^S protein is hardly detectable, cells produce fair amounts of rpoS mRNA, which seems to remain constant under the translation-inducing conditions mentioned above (8, 147). It is generally believed that control of the rate of translation of already existing complete rpoS mRNA is based on an mRNA secondary structure in which the translational initiation region (TIR) is based paired and therefore not sufficiently accessible to ribosomes (under noninducing conditions). Certain stress signals are hypothesized to trigger changes in this mRNA secondary structure that allow more frequent translational initiation. However, the actual appearance of this rpoS mRNA structure is still largely a matter of speculation.

Theoretical predictions generated with the MFOLD computer program (using complete or partial rpoS mRNA sequences) indicate that approximately 340 nucleotides at the 5' end of rpoS mRNA fold into an very stable and complex cruciform-type structure (Traulsen and Hengge-Aronis, unpublished). Further downstream, the putative structures are somewhat less stable and the TIR has the potential to fold into two energetically almost equivalent principal structures. One is characterized by a large hairpin that includes the Shine-Dalgarno sequence. There is genetic evidence against this structure playing a role in rpoS translational control (S. Bouché and R. Hengge-Aronis, unpublished results). In the second putative structure, the region around the Shine-Dalgarno sequence is partially base paired to an "internal antisense" region located further upstream, with a relatively long and probably internally structured intervening sequence. There are, however, several theoretical possibilities for the exact location of the "internal upstream antisense" region (Traulsen and Hengge-Aronis, unpublished). Several variations of this second theoretical structure have been published (30, 38, 120, 131), and it is generally believed that this structure may come close to the in vivo reality under noninducing conditions. The only preliminary experimental evidence that such an "internal upstream antisense" structure is in principle correct is provided by two different complementary double point mutations, which showed wild-type expression levels (although one double mutant altered the regulatory pattern, in particular Hfq dependency [see below]) (30, 38). However, the exact details of the in vivo rpoS mRNA secondary structure still await experimental clarification.

In fact, the problem of the correct in vivo rpoS mRNA secondary structure is more complex than, e.g., in the related case of rpoH mRNA, which encodes the heat shock sigma factor ³².rpoH mRNA folds into a translationally incompetent secondary structure also involving an internal antisense element, which opens up upon heat shock, resulting in a directly temperature-triggered translational induction of rpoH (summarized in reference 236). This process does not involve any regulatory proteins (143), and the experimentally demonstrated rpoH mRNA secondary structure is the one theoretically predicted (142). In rpoS mRNA, however, several proteins and small regulatory RNAs are positively or negatively involved in translational control, and at least some of these can directly bind to rpoS mRNA in vitro (see below). Therefore, theoretical calculations or in vitro structural probing based on rpoS mRNA alone is likely to yield incorrect or at least incomplete results. It seems that the only way of settling the issue of the correct rpoS mRNA secondary structure and its dynamics may be in vivo structural probing. Wild-type strains under different conditions as well as various mutants with cis- or trans-regulatory defects in rpoS translation will have to be tested in such experiments. However, in view of the technical difficulties of such an endeavor, especially with a large mRNA with complex and semistable secondary structure, it is not surprising that such data have yet to be reported for rpoS mRNA.

The molecular function of Hfq in rpoS translation is still relatively speculative. Epistasis experiments, where hfq mutations were combined to other mutations or overproduction constructs that affect rpoS translation indicated that Hfq is probably directly involved in translation initiation; i.e., it acts close to or at the level of rpoS mRNA (130, 146, 200, 217, 238). rpoS mRNA coimmunoprecipitates with Hfq in cellular extracts (238). Hfq also binds with high affinity to several sites in a large 5' fragment of rpoS mRNA synthesized in vitro, which is predicted to fold into the same secondary structure as the wild-type mRNA (Traulsen and Hengge-Aronis, unpublished). A 5' deletion analysis of rpoS mRNA indicated that regions relatively far upstream of the TIR are important for translational stimulation by Hfq (38). Thus, Hfq binds rpoS mRNA, just as it is able to bind Qß RNA. However, Hfq does not show similarity to RNA helicases. This makes an active processive unfolding activity unlikely. Alternatively, by binding to a few crucial positions of rpoS mRNA, Hfq may affect the equilibrium between possible alternative secondary structures that are differentially productive for translational initiation. Thus, Hfq may stabilize a semistable rpoS mRNA secondary structure, which can easily open up when some additional stimulating factor is induced or activated (e.g., HU protein or DsrA-RNA, [Fig. 3; see below]). Yet another possibility is that Hfq does not necessarily affect rpoS mRNA secondary structure (although this would not be excluded) but acts like a "platform" bound to rpoS mRNA that recruits additional factors involved in rpoS translational control. The finding that single potentially base-pair-disrupting point mutations in the TIR or in the region likely to be base paired to the TIR result in increased rpoS translation and reduced Hfq dependence (30) appears consistent with both of these putative mechanisms of Hfq action.

View larger version (18K): [in this window] [in a new window]   FIG. 3. The rpoS translational control network. rpoS mRNA is thought to occur in at least two different conformations, one being a more closed structure with the translation initiation region base paired to an upstream internal antisense element, and the other being a more open and translationally competent structure. The translation-stimulating factors Hfq, HU, and DsrA RNA can bind to rpoS mRNA (indicated by broken heavy lines) and together probably drive it into the translationally competent structure. The other components shown are likely to act more indirectly (for further details, see the text).

If so, stress signal input into rpoS translational control would not necessarily be via a control of the activity or the level of Hfq itself. In fact, hfq mutants show overall reduced activity of translational rpoS::lacZ fusions or rates of ^S synthesis (as measured in pulse-labeling experiments), but regulation by stress signals, e.g., by hyperosmotic shift, is not abolished (146). This suggests that stress signals affect the cellular concentrations or activities of the specifically translation-activating or inhibiting components (e.g., DsrA and OxyS) that can join the rpoS mRNA-Hfq complex. These components indeed exhibit pronounced regulation (see below).

HU: a nucleoid protein that also stimulates rpoS translation. Protein HU is a major protein component of the bacterial nucleoid. It affects overall nucleoid structure and topology but also participates in specific gene regulation, DNA recombination, and DNA repair (155). In addition, HU is required for optimal survival during prolonged starvation (35). In members of the Enterobacteriaceae and Vibrionaceae, two homologous subunits (HU and HUß [encoded by hupA and hupB, respectively]) contribute to the formation of active HU protein (158). During growth, the HU2 homodimer is abundant, whereas during late exponential phase, HUß is induced and HUß heterodimers are formed in E. coli (35). An HU-deficient hupAB double mutant exhibits strongly reduced ^S levels because of reduced rpoS translation (12).

In vitro, HU binds with high affinity to a small rpoS mRNA fragment (150 nucleotides covering the TIR and the upstream antisense region probably base paired to the TIR) (12) as well as to a larger fragment (covering more than 700 nucleotides starting from the original mRNA 5' end) that also binds Hfq (Traulsen and Hengge-Aronis, unpublished). As a DNA binding protein, HU has a strong preference for nicked or cruciform DNA (94). Thus, HU may preferentially recognize secondary-structure elements, such as pronounced bends or kinks, which also occur in RNA secondary structure. HU may directly alter the rpoS mRNA secondary structure, but it is unknown how this effects relates to that of Hfq or of other components that affect rpoS translation (Fig. 3).

Since the induction of HUß (which is under the negative control of the nucleoid protein FIS [34]) correlates with stimulation of rpoS translation during late exponential growth phase, and specifically since the HUß heterodimer is required for stationary phase survival (35), it is tempting to speculate that the heterodimer is the form of HU involved in rpoS translation. However, this hypothesis has yet to be tested experimentally. Nevertheless, phylogenetically, the occurrence of an HUß heterodimer correlates with the occurrence of ^S (with the exception of the Pseudomonas group, but regulation of ^S is significantly different in several aspects in this group).

H-NS and StpA: histone-like proteins acting as RNA chaperones? H-NS is an abundant histone-like protein with functions in nucleoid organization as well as in gene regulation, where in nearly all cases it acts as a repressor or silencer that can form large nucleoprotein complexes. StpA is a closely related paralog of H-NS with similar properties (although it seems more efficient as an RNA chaperone). Just as with HU and HUß, homo- as well as heterooligomers are formed by H-NS and StpA (for reviews, see references 9, 43, and 230).

H-NS-deficient mutants exhibit strongly increased ^S levels, which in exponential phase are already similar to those reached by the wild-type only in stationary phase or under other stress conditions (15, 234). In these hns mutants, the rate of rpoS translation is enhanced and proteolysis of ^S is strongly reduced or even abolished (234). The slow growth and genetic instability typical of hns mutants are at least partially connected to these abnormally high ^S levels, since they can be suppressed by mutations in rpoS (15).

Mechanistically, it is still unclear how H-NS downregulates rpoS translation, but there are a number of possibilities for direct or indirect influences (Fig. 3). Since H-NS can bind to RNA (although high-affinity specific binding has not yet been demonstrated [40, 48]), it may directly interact with rpoS mRNA and affect its secondary structure, perhaps in a transient way as an RNA chaperone. H-NS may also counteract the effects of positive regulators of rpoS translation such as Hfq and/or HU. These possibilities are not mutually exclusive, since the positively acting factors and H-NS may have opposite effects on the equilibrium between two rpoS mRNA conformations that can be translated with different efficiencies (Fig. 3). Consistent with H-NS counteracting Hfq, H-NS deficiency has no effect on rpoS translation in a hfq mutant background (146). H-NS and HU in general seem to play antagonistic roles, e.g., in determining DNA supercoiling (44) or in the expression of certain genes such as ompF (41, 164). Thus, it is also possible that H-NS inhibits rpoS translation by affecting the cellular level of HU or by directly counteracting the stimulatory effect of HU on rpoS translation.

Another candidate for promoting rpoS translation is the H-NS homolog StpA. Several studies have shown that StpA levels are significantly lower than H-NS levels (201, 239), although one report gives approximately equal numbers of H-NS and StpA molecules per cell (10). It seems clear, however, that H-NS and StpA regulate each other negatively at the level of transcription. Therefore, an hns mutant should have an increased cellular concentration of StpA (201, 239). Moreover, StpA is upregulated after a hyperosmotic shift (58). Thus, increased StpA levels appear to correlate with increased rpoS translation. Since StpA can act as a RNA chaperone (40), it was tempting to speculate that it may stimulate rpoS translation. However, high-log-phase levels of ^S in an hns mutant were not suppressed by introducing an stpA mutation, and also osmotic induction of rpoS translation was normal in a stpA mutant (Bouché and Hengge-Aronis, unpublished). Therefore, under these conditions, StpA does not seem to play a role in rpoS translation. This, however, does not exclude a potential involvement of StpA under different conditions or in other genetic backgrounds.

Role of small regulatory RNAs in rpoS translation: DsrA, OxyS, and RprA. Several small regulatory RNAs with important fine-tuning functions in complex regulatory circuits have been identified in E. coli (summarized in reference 1), and three very recently published studies suggest that small regulatory RNAs in E. coli are much more common and significant than previously thought (6, 179, 224). rpoS translation seems to be an especially prominent target for such regulation, with three regulatory RNAs having been found so far. While DsrA and RprA promote rpoS translation, OxyS has an inhibitory function.

DsrA was originally identified as a multicopy suppressor of H-NS-mediated silencing of the rcsA gene in E. coli (198) and was then found to be essential for increased rpoS translation at low temperature (199). DsrA is a stable 87-nucleotide RNA that folds into a three-stem-loop structure (119, 131). A region covering most of stem-loop 1 and the following single-stranded part of DsrA is complementary to an upstream "antisense element" in rpoS mRNA that is assumed to base pair with the TIR region, suggesting that DsrA functions by an "anti-antisense" mechanism that disrupts intramolecular basepairing in rpoS mRNA (119, 120, 131). DsrA plays only a minor role for rpoS translation in cells grown at 37 or 42°C yet becomes the major stimulating factor at 30°C and especially at 20°C (199). The basis of low-temperature translational induction of ^S is the clearly enhanced transcription of dsrA as well as a sixfold-increased stability of DsrA at low temperature (177). DsrA and Hfq were recently reported to interact specifically, and Hfq was suggested to stabilize DsrA as well as to alter its secondary structure in a way that promotes association with rpoS mRNA (200). Whether the formation of such a binary complex facilitates DsrA action on rpoS mRNA or whether Hfq already bound to rpoS mRNA (as described above) recruits DsrA, the result is likely to be a ternary complex (see also above). Hfq may affect the secondary structure of both RNAs such that they optimally interact, and with all partners involved interacting with each other, the complex is probably relatively stable. As a result, the formation of an "open" conformation at the TIR of rpoS mRNA that allows ribosome entry would be facilitated (Fig. 3).

Besides rpoS mRNA, DsrA has at least one other target, hns mRNA. While DsrA was initially thought to act like a conventional antisense RNA interfering with hns translation initiation (120), it now seems likely that a region corresponding to unfolded stem-loop 2 of DsrA forms a coaxial stack with two regions in hns mRNA. Negative regulation of hns expression is a consequence of more efficient degradation of hns mRNA within this complex (118). DsrA is predicted to form similar complexes with argR and ilvIH mRNAs, but an involvement of DsrA in the regulation of these genes has not yet been demonstrated (118).

Multifunctionality exerted by different regions may be common in small regulatory RNAs, since it has also been observed for OxyS. OxyS is a 109-nucleotide regulatory RNA that folds into a similar secondary structure to that of DsrA. As a member of the OxyR regulon, OxyS is induced by oxidative stress (hydrogen peroxide) and acts as a pleiotropic regulator (2). Small regions located in loops 1 and 3 of OxyS control translation of fhlA (which encodes a transcriptional activator) by forming a "kissing complex" with two sites of fhlA mRNA, one of which contains the Shine-Dalgarno sequence (3, 5). By contrast, the rather long A-rich single-stranded region between stem-loops 2 and 3 of OxyS is involved in negative regulation of rpoS translation, although this part of OxyS does not show significant sequence complementarity to rpoS mRNA. Coimmunoprecipitation experiments indicate that OxyS binds to Hfq protein. Thus, OxyS may sequester Hfq or form a translationally incompetent ternary complex with Hfq and rpoS mRNA (238) (Fig. 3). OxyS-mediated translational repression of rpoS may be a fine-tuning mechanism to avoid redundant overinduction of oxidative-stress protective genes (katG, gorA, and dps) that are under dual positive control of OxyR/⁷⁰ and ^S. It may also prevent the uneconomical induction of the large multifunctional ^S regulon under conditions where the cell has to cope with oxidative stress only, i.e., a situation that can be managed by the stress-specific OxyR-mediated response alone.

The third small regulatory RNA involved in rpoS translational control, RprA, was found as a multicopy suppressor for a dsrA mutation (130). In the dsrA mutant background, an rprA null mutation also reduces hyperosmotic stimulation of rpoS translation. However, in the presence of DsrA, neither RprA overproduction nor its knockout seems to affect rpoS expression. Thus, RprA clearly has the potential to stimulate rpoS translation, but the physiological conditions under which this becomes relevant are unknown. The rprA promoter is under positive control of RcsB, a response regulator that activates capsule synthesis (unpublished evidence mentioned in reference 130). RprA exhibits some sequence complementarity to the upstream "antisense" element that basepairs with the TIR of rpoS mRNA and may thus act similarly to DsrA (M. Majdalani and S. Gottesman, personal communication.

The LysR-like regulator LeuO: a repressor for dsrA expression. LeuO is a LysR-like regulator (189), which is strongly repressed by H-NS in growing E. coli cells (101). Overproduction of LeuO (either from a multicopy plasmid or in a mutant that carries a Tn 10 transposon immediately upstream of leuO with p_out of the transposase gene reading into leuO) reduces rpoS translation, especially at low temperature. This effect is entirely dependent on the presence of DsrA, and LeuO was shown to repress dsrA transcription (101). This regulation is direct since LeuO binding sites have recently been identified in the dsrA promoter region (177). A leuO knockout mutation, however, does not affect increased rpoS translation during late exponential phase or in response to high osmolarity or low temperature. This is not entirely surprising, since under these conditions, leuO expression is repressed or even "silenced" by H-NS (101). However, during entry into stationary phase, leuO is induced in a ppGpp-dependent manner (50). This ppGpp-mediated activation may be indirect, since leuO expression is subject to a "promoter relay" activation mechanism that involves the surrounding ilvIH and leuABCD operons (33, 51). As a consequence, LeuO probably downregulates DsrA in stationary phase. While this may alter the expression of other targets of DsrA, the ^S level is not affected (E. Klauck and R. Hengge-Aronis, unpublished results), probably because other ^S-inducing mechanisms compensate for the reduced levels of DsrA. When all these results are taken together, the physiological role of LeuO is far from clear. However, a hint may come from the "cryptic" bgl operon, where LeuO can antagonize H-NS-mediated (and under certain conditions also ^S-dependent) "silencing" (160, 218). Interestingly, the bgl operon becomes expressed in a mammalian host (97). It is thus conceivable that LeuO plays an important regulatory role in a host environment.

DnaK and DksA: a link to heat shock and chaperones. The heat shock chaperone DnaK, as well as a protein termed DksA (originally identified as a DnaK suppressor [95]), has been implicated in rpoS translation. A dnaK mutant exhibits a stationary-phase-specific multiple-stress-sensitive phenotype very similar to that observed for rpoS mutants (180, 181). This correlates with reduced ^S levels in starving dnaK mutant cells (144, 181). Part of this effect is due to reduced rpoS translation, since it can also be seen with RpoS::LacZ hybrid proteins that are not subject to proteolysis. The mechanism behind this effect remains unknown, but the overproduction of the heat shock sigma factor ³² in the dnaK mutant does not play a role, since a suppressor mutation that reduces the ³² level and/or activity does not suppress the dnaK effect on ^S (144).

DksA is a putative zinc binding protein with similarity to the transcriptional activator TraR (59) and other prokaryotic and eukaryotic regulators (103). The basis of dnaK suppression by multiple copies of dksA is still unclear, but it was suggested that production of some stress response factors might be involved (16). This is consistent with the more recent finding that dksA mutations in Salmonella exhibit reduced ^S induction in stationary phase and after a shift to acidic pH. Work with rpoS::lacZ translational fusions indicated that DksA affects rpoS translation by some not yet characterized mechanism (225). In P. aeruginosa, overexpression of DksA inhibits the expression of rhlI, rhlAB, and lasB (26). This would be in line with a repressing effect of ^S on the rhl system (228). However, additional data suggest th