INTRODUCTION

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In the enteric bacteria, including Escherichia coli and Salmonella typhimurium, the rpoS gene encodes an accessory sigma (specificity) factor for RNA polymerase (also called ^S or ³⁸ [36, 49]). RpoS is required for the transcription of many genes expressed during the onset of stationary phase. RpoS-dependent adaptations to nutrient limitation and starvation identified so far in E. coli include not only shifts in metabolic pathways but also resistance mechanisms protective against life-threatening stresses, such as high osmolarity, low pH, heat shock, elevated H₂O₂, and UV light (reviewed in references 19 to 21 and 29). RpoS is also a virulence factor for S. typhimurium (15) and other enteric bacteria.

RpoS abundance can be increased in exponential-phase cells by a variety of induction treatments, including osmotic challenge (22, 27, 34 [reviewed in reference 21]). High levels of RpoS are also seen in cells grown to stationary phase in rich medium (reviewed in reference 19). It has been demonstrated that RpoS abundance is increased by provoking an increase in the level of the alarmone ppGpp (18), and this may explain why so many treatments which induce RpoS also decrease the growth rate, at least transiently. For osmotic challenge and stationary phase, control of RpoS occurs both at the level of synthesis and by regulated proteolysis. Genetic analysis of RpoS regulation revealed a requirement for the energy-dependent ClpXP protease (41), which promotes RpoS turnover with the help of other factors (4, 32, 38). Both clpXP mutants and hns mutants lacking the abundant DNA-binding protein H-NS have an increased level of RpoS during the exponential phase (2, 53).

Comparative studies with rpoS-lac protein and operon fusions have shown that control of RpoS synthesis occurs mainly at a posttranscriptional level (27). Host factor I (HF-I) is an RNA-binding protein that was discovered through its role in the replication of Q, an RNA bacteriophage that infects E. coli (9, 16, 17, 23, 24, 42). The function of HF-I in uninfected cells has been unknown, but hfq mutants are quite pleiotropic (35, 51, 52). In recent work, it has been shown that S. typhimurium and E. coli hfq mutants lacking HF-I have substantially reduced expression of rpoS (7, 33). The defect in rpoS expression is posttranscriptional.

Some additional insight has been gained by analysis of mutations that restore expression of rpoS in hfq mutants (8; unpublished work cited in reference 33). Most of these mutations disrupt a predicted secondary structure that would sequester the rpoS ribosome binding site (RBS) (8). One attractive model is that control of rpoS synthesis at the translational level involves regulation of ribosome access by this inhibitory RNA secondary structure. The RNA-binding protein HF-I may be directly involved in relieving this inhibition, for example, as an RNA chaperone which promotes equilibration between different RNA secondary structures. However, other models of HF-I action are possible.

In this work, we tested two simple predictions of the translational control model for rpoS. Two different promoters were substituted for the native rpoS promoters: translational regulation was retained in these constructs and was still dependent on HF-I function. However, deletion analysis showed a requirement for sequences well upstream of the proposed secondary structure, suggesting that interaction of HF-I with this structure is probably not sufficient for regulation.

	MATERIALS AND METHODS

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Bacterial strains and construction. The strains used in this study are derived from the wild-type S. typhimurium strain LT-2 (originally obtained from J. Roth). S. typhimurium does not carry the lac operon. The LT-2 derivatives used here carry various lac fusions to the rpoS gene of E. coli and the hfq-1::Mud-Cam insertion (7), where indicated, but no other mutations with respect to the parental LT-2 strain. Note that some strains of LT-2 have been shown to carry a mutation in rpoS (a change of ATGTTG in the rpoS initiation codon [3]) as well as a mutation inactivating mviA (the homolog of the E. coli gene rssB or sprE, which controls turnover of RpoS [4]). The LT-2 strain used here carries a mutation mapping in the mviA region that stabilizes RpoS (data not shown). The lac fusions used in this study do not encode the segment of the RpoS protein required for turnover (41), and the hybrid RpoS-LacZ protein produced is stable during exponential growth in minimal glucose medium (7).

Media and growth conditions. Bacteria were grown at 37°C in Luria-Bertani (LB) medium (43) or in minimal MOPS (morpholinepropanesulfonic acid) medium (37), modified as described in reference 6, with 0.2% glucose as the carbon and energy source. For experiments employing continuous growth in high-osmolarity medium, a modified version of the basal medium of reference 25 was used. The modified medium contained 7 g of nutrient broth and 1 g of yeast extract (Difco) per liter of NCE minimal salts medium (per reference 5, but without MgSO₄). This medium also contained 0.2% glycerol, and where indicated, sucrose was added at 15%. Plates were prepared with nutrient agar (Difco) with 5 g of NaCl per liter. Antibiotics were added to the following final concentrations: sodium ampicillin, 100 µg/ml; chloramphenicol, 20 µg/ml; kanamycin sulfate, 50 µg/ml; and tetracycline hydrochloride, 20 µg/ml.

Construction of lac fusions. The system we used has been described previously (14). Fusions were made in the pRS plasmid series of Simons et al. (44). Full-length rpoS-lac fusions have been described previously (7). Construct A was constructed by digesting pMMkatF2 (36) with ClaI and EagI, filling in the ends with the Klenow fragment of DNA polymerase I, and cloning the 1.6-kb fragment into pRS552 that had been digested with EcoRI and BamHI and filled in. To make construct B, an rpoS-lac operon fusion, the same 1.6-kb ClaI-EagI fragment of pMMkatF2 was filled in and inserted into the SmaI site of pRS415. The fragment was subsequently excised by digestion at the flanking EcoRI and BamHI sites and inserted into pTE583 (7). These parental lac protein and operon fusions to rpoS extend from the ClaI site upstream of nlpD to the EagI site at codon 73 of the rpoS gene. The operon fusion version includes an RNase III site directly upstream of the lacZ RBS (28).

-Galactosidase assays. Cells were centrifuged and resuspended in Z-buffer (100 mM NaPO₄ [pH 7.0], 10 mM KCl, 1 mM MgSO₄) and then permeabilized by treatment with sodium dodecyl sulfate and chloroform (7). Assays were performed in Z-buffer containing 50 mM -mercaptoethanol by a kinetic method using a plate reader (Molecular Dynamics). Activities (change in optical density at 420 nm [OD₄₂₀] per min) are normalized to actual cell density (OD₆₅₀) and were always compared to those of appropriate controls assayed at the same time. The results shown are from a single experiment; each experiment was repeated several times with similar results.

	RESULTS

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Deletion analysis of sequences required for translational control of RpoS. If HF-I or stimuli such as osmotic challenge regulate RpoS synthesis at the translational level, then we expect that the native rpoS promoters can be replaced by another promoter without loss of regulation. A more restrictive translational control model (discussed above) predicts that HF-I interacts only with the segment of mRNA containing a putative secondary structure that inhibits translation. The ultimate result of this interaction is increased availability of the rpoS RBS for binding to ribosomes. According to this model, transcript sequences upstream of the secondary structure should not be required for HF-I regulation of RpoS.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   rpoS-lac fusions. The top line shows a restriction map of the nlpD and rpoS genes of E. coli, including the restriction sites used to make lac fusions in this study. The positions of two promoters that serve rpoS are also shown by bent arrows. Each line below the top one represents a different fusion; these are referred to in the text as rpoS-lac fusions and are labeled (A to P). The extent of the nlpD-rpoS sequence present in each fusion is shown, together with the identity of the promoter substitution where appropriate: Ptac or Plac UV5. The nature of the lac fusion (protein [pr] or operon [op]) is also indicated. ATG start codons (solid squares) and their cognate stop codons (arrowheads) are shown. One-letter abbreviations are used for restriction sites (the full name of each site is on the top line). Additional sequence and construction details are given in Materials and Methods. The lac region is not drawn to scale. Expression of -galactosidase was measured in overnight LB cultures of S. typhimurium strains bearing each fusion in single copy in the bacterial chromosome and was compared to the activity seen in an otherwise isogenic hfq mutant. Values are in arbitrary units, normalized to that of construct A in a wild-type background (set as 100). The ratio of the enzyme activity in the wild type divided by that in the hfq mutant is also given. N.D., not determined.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Model of the antisense-RBS structure near the rpoS start codon. The structure presented here is that suggested on the basis of genetic analysis (8), and the previous numbering scheme is retained in this figure. Arrows point to the nucleotides altered in the compensatory mutations which support the structure (C126G and G206C, as well as a second pair not shown). The U at nt 111 (also marked with an arrow and denoted 2) corresponds to the first rpoS-specific nucleotide in constructs J and M; material upstream of this point is deleted in these fusions. Not shown are the additional 111 nt retained by 1 (constructs I and L of Fig. 1) or the additional 400 nt extending to the 5' end of the PrpoS transcript. Two stems which pair the nucleotides connecting the antisense and RBS elements have also been added to this model. S.D., Shine-Dalgarno sequence.

Secondary structure in rpoS transcripts from deletion constructs. The lack of response to HF-I by the deletion constructs, as described above, could have a simple explanation by analogy to the behavior of RNA II, the primer for ColE1 plasmid replication. RNA II folds into substantially different structures in its downstream half, depending on either the presence of upstream sequences or their sequestration in a complex with the antisense RNA, RNA I (29a). Thus, if the inhibitory structure did not form in an rpoS transcript which lacks important upstream sequences, then a failure to respond to HF-I would be understandable but not enlightening. As a test of this possibility, we prepared a set of rpoS-lac fusions from construct L to use the method of compensatory mutations as previously described (8). These fusions carry either of two mutations, C126G (SD2) or G206C (SD3), as well as the double mutant together with a wild-type control. Expression of rpoS-lac in wild-type and hfq mutant derivatives of these fusion strains is reported in Table 1. Since the elevated -galactosidase activity seen in the single mutants is restored nearly to wild-type levels in the double mutant (measured in an hfq mutant background), we conclude that at least the identified RNA secondary structure forms in transcripts from this construct. None of the constructs is significantly stimulated by the presence of HF-I, and the lack of response to HF-I cannot be ascribed to loss of this structural element.

Osmotic challenge. We extended the analysis to include osmotic challenge because it has been demonstrated that HF-I is required for osmotic control of rpoS translation in E. coli (33). Several deletion constructs were tested for their response to challenge with high salt. The first experiment employed an osmotic challenge in which cultures growing in minimal MOPS glucose medium were treated with 0.3 M NaCl. The wild-type rpoS-lac protein fusion (construct A) showed a 3.5- to 4-fold increase in lac expression over 40 min and then reached a plateau by 60 min (Fig. 3A). The operon fusion (construct B) was not induced by this treatment (Fig. 3F). When an hfq mutation was present, the response of construct A to the osmotic challenge was partially defective (Fig. 3D). The kinetics of the response were changed, with an 2-fold increase at 40 min, but lac expression continued to increase up to at least 90 min.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Induction of rpoS-lac after osmotic challenge. Cultures of S. typhimurium strains carrying a lac fusion (as identified in Fig. 1 and the text) and an hfq mutation where indicated were grown in minimal MOPS glucose medium to an OD600 of 0.4 and then challenged with 0.3 M NaCl. The activity of -galactosidase was determined at 20, 40, 60, and 90 min after challenge. Solid squares, cultures receiving NaCl; open squares, control cultures. Data are reported as a percentage of the initial value for each fusion. [pr], lac protein fusion; [op], operon fusion.

Continuous growth at high osmolarity. In addition to changing the osmotic strength of the medium, the osmotic challenge method also changes the bacterial growth rate (at least transiently). Therefore, we tested the effect of continuous growth in high-osmolarity medium. These experiments (modeled on those described in reference 38) were carried out in a phosphate-buffered rich medium including glycerol; sucrose was the solute used to vary the osmolarity. Assays for -galactosidase were performed with cells sampled at three densities: OD₆₀₀ of 0.15, OD₆₀₀ of 0.6, and after growth overnight to stationary phase. Similar to the observations with E. coli (38), cells grown at high osmolarity showed an increase in rpoS-lac expression in exponential phase (OD₆₀₀ of 0.15 or 0.6), but not in stationary phase (data for OD₆₀₀ of 0.6 are shown in Table 2).

	DISCUSSION

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Previous work with S. typhimurium and E. coli has shown that the rate of synthesis of RpoS protein is reduced four- to sixfold in hfq mutants which lack HF-I (7, 33). Comparison of rpoS-lac protein and operon fusions suggests that the defect in hfq mutants lies at a posttranscriptional step, but it is not established whether HF-I specifically increases the rate of translation initiation or affects mRNA stability instead. The lack of an HF-I requirement for expression of rpoS-lac operon fusions is not incompatible with a role in mRNA stabilization, since the lacZ RBS of operon fusions could be insulated from such effects (28). There is as yet no in vitro system showing HF-I dependence of RpoS expression, so it is not certain that HF-I acts directly.

Suppressor mutations that decrease the in vivo dependence of rpoS-lac expression on HF-I function were found to map to a region encompassing 100 bp near the rpoS ATG codon, and genetic analysis of compensatory mutations suggests that an RNA secondary structure formed in this region limits rpoS expression (i.e., sequestration of the rpoS RBS by an intramolecular antisense RNA [8]). Perhaps the simplest model would be that HF-I binds to a site(s) in or near this region and disrupts the antisense pairing to allow ribosomes access to the rpoS mRNA. But although suppressor mutations may be suggestive of a potential mechanism, they do not necessarily recapitulate the role of HF-I for wild-type rpoS.

In this work, we tested whether such potential interactions of HF-I and the rpoS RBS region are sufficient for HF-I function by making promoter substitutions and by deletion of upstream segments from rpoS-lac transcripts expressed from the P_tac and P_{lac UV5} promoters. The results indicate that nonnative promoters still allow correct regulation by HF-I during growth into stationary phase and after osmotic challenge. However, in contrast to the prediction of the simple model, some sequences required for HF-I regulation of rpoS lie >100 nt upstream of the rpoS transcript antisense element. Reapplication of the method of compensatory mutations indicates that correct folding of the antisense-RBS structure is likely to be preserved in these deletion variants. Thus, HF-I must do more than simply melt this duplex.

We favor the idea that HF-I acts in the rpoS system very similarly to its function in the replication of E. coli RNA phage Q. There, HF-I is specifically required for copying of Q plus strands (1, 17, 47). Electron microscopy of HF-I protein bound to Q RNA reveals doubly looped structures that indicate specific and simultaneous interaction with two widely separated internal sites (independently bound by the replicase) which are then brought together with the RNA 3' end (30). Such interactions are consistent with the multimeric nature of HF-I protein (17, 24) and build on Senear and Steitz's demonstration of site-specific RNA binding activity by HF-I directed at RNA targets from phages R17 and Q (42). Mutations that disrupt the terminal RNA duplex of phage Q overcome the requirement for HF-I protein in phage replication (40), which suggests that HF-I might facilitate melting of the phage RNA 3' end to allow initiation of replication. This terminal stem of 5 bp is not by any means the most stable element in the highly structured phage RNA, which suggests a specific role for HF-I in replication initiation.

Thus, we could imagine that HF-I binds to a specific upstream site in the rpoS mRNA and from that position interacts with downstream elements to melt the antisense-RBS duplex. This type of model could also accommodate recent genetic studies showing that the DsrA RNA, a small untranslated RNA of E. coli (45), acts directly to increase rpoS expression by pairing with and sequestering the upstream rpoS antisense element (18a, 46). DsrA-rpoS antisense RNA pairing requires HF-I, and this suggests that HF-I might act like the Rop (Rom) protein, a facilitator of the RNA-antisense RNA pairing that controls ColE1 plasmid copy number (50). Or perhaps, as suggested previously, HF-I is actually a chaperone for RNA and promotes structural rearrangements (35).

It seems less likely but still possible that HF-I has its primary effect on rpoS mRNA stability. RNase E cleavage sites are rich in A and U residues (reviewed in reference 10); these are also favored by HF-I. Another possibility is that bound HF-I might direct rpoS mRNA down an alternative folding pathway by preventing the formation of particular duplexes which are targets for RNase E. Effects on mRNA turnover have been suggested to explain the autoregulation of hfq gene expression in E. coli (52). Consistent with this, we have found that HF-I is much more promiscuous in its RNA binding activity than predicted from earlier studies (unpublished data). However, changes in mRNA turnover may also be a secondary consequence of changes in the rate of initiation of translation (11), so only a demonstration that rpoS mRNA turnover is not affected by HF-I would be definitive. Finally, any model must explain why the benefits of the postulated mRNA stabilization are not evident for an mRNA having an unfettered, strong RBS or for a variety of other rpoS single mutants that alter the antisense RNA element and thereby have become completely independent of HF-I (unpublished data).

In summary, current evidence in this system still allows a number of possible models including: (i) a looping interaction between HF-I complexed to rpoS mRNA at far upstream sites and at sites closer to the AUG codon; (ii) the existence of additional proteins, acting together with HF-I or indirectly under its control, which might bind to upstream sequences; (iii) a subtle influence of upstream sequences on the ultimate folding pattern of rpoS mRNA in the region near the AUG initiation codon; and (iv) a requirement for other components, such as small regulatory RNAs, including DsrA RNA, to observe tight complex formation between HF-I and the rpoS mRNA.