INTRODUCTION

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In natural environments, bacterial cells often face dramatic changes of environment, and they have evolved responses to adapt their physiology to such changes. To cope with adverse conditions, nonsporulating enterobacteria such as Escherichia coli or Salmonella enterica serovar Typhimurium can undergo a global programmed modification of their gene expression pattern, leading to better resistance to a number of chemical and physical stresses such as heat, oxidative agents, or hyperosmotic shock (16, 21, 22). Overall, these properties result in better survival of the cells. One key regulator of this genetic program is the product of the rpoS gene, which controls a large regulon expressed in response to starvation and during the transition to stationary phase (16). The osmC gene of E. coli is a member of this regulon and exhibits a complex regulatory pattern (4, 12, 15). osmC encodes an envelope protein of unknown function. It is transcribed from two overlapping promoters (Fig. 1A). The proximal promoter, osmCp₂, is mainly recognized by the ^s sigma factor and is responsible for growth phase regulation. It is also activated by the leucine responsive protein (LRP) and repressed by the nucleoid-associated protein H-NS. Transcription from the distal promoter, osmCp₁, occurs during exponential phase in a ^s-independent manner. It is repressed by both LRP and H-NS (4). Transcription from both promoters is stimulated by elevated osmolarity, and gel mobility shift experiments with crude extracts of E. coli have demonstrated that several proteins are able to bind to the osmC promoter region, suggesting that additional regulators are involved in the control of osmC expression (4, 15).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Sequence of the osmC promoter region and of its RcsB box. (A) The 10 and 35 regions of the two osmC promoters, osmCp1 and osmCp2, are underlined. Bent arrows show the osmCp1 transcription starts. The A-to-G substitutions in the 10 boxes that abolish either osmCp1 or osmCp2 activity (osmCp11 or osmCp21, respectively) are represented by G's in parentheses above the sequence. Lines under the sequence show the extents of the DNA fragments carrying osmCp1 that were used to locate the RcsB target site. The first three nucleotides at the 5' ends of these fragments are given. The sequence required for activation of the fts genes by RcsB is shown above the osmC sequence. The bases in that sequence whose mutations have a strong or mild effect on RcsB activity are indicated by asterisks or circles, respectively (5). (B) Alignment of the sequences of the RcsB and RcsAB boxes. The RcsB box is proposed from the comparison between the fts and osmC regulatory regions (W stands for A or T, K stands for T or G, M stands for C or A, R stands for A or G, Y stands for C or T, and S stands for C or G). Bases important for activation of the fts genes by RcsB are underlined. The symmetry of the motifs is highlighted by the vertical broken line. The sequence of the RcsAB box is from reference 35.

The two-component system rcsC-rcsB was initially identified as a regulator of the synthesis of the capsular polysaccharide in E. coli (14). It also regulates the synthesis of the exopolysaccharides of other enteric and plant-pathogenic bacteria (1, 8, 26, 34). By analogy with other two-component systems, the response regulator RcsB is thought to be activated through the transfer of a phosphate group from either its cognate sensor, RcsC (32), or another protein, RcsF (10). The rcsB-rcsC system has also been reported to be a positive regulator of cell division gene expression in E. coli (5, 11). The activation is direct and requires a specific sequence, the "RcsB box," centered at positions 44 and 43 from the transcription start (5). Besides these two fairly well characterized examples, RcsB has also been reported to induce the lytic growth of lambdoid prophages, probably by antagonizing cI repressor activity (27). In Salmonella enterica serovar Typhi, production of invasion proteins and flagella is repressed by RcsB in low-salt medium (2). A signal specifically recognized by the sensor RcsC has not yet been identified. In E. coli, the rcsC-rcsB regulation pathway is activated by desiccation and osmotic shock (24, 29), by the overproduction of the chaperon DnaJ-like transmembrane protein DjlA (6, 19), and by several mutations affecting the composition of the envelope (7; reviewed in reference 13).

This work reports the regulation of the E. coli osmC gene by RcsB and strengthens the notion that RcsBC is a major cellular regulatory system.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and bacteriophage. The bacterial strains used in this study, all derived from E. coli K-12, as well as the plasmids and bacteriophage used, are listed in Table 1.

Genetic techniques. Standard procedures were used for transduction with phage P1vir (23). Strain CLG723 carries a (malP-lacZ) transcriptional fusion in which an intact lac operon is fused to the first gene of the malPQ operon (9). Strains carrying osmCp-lac fusions were constructed as follows. Diverse DNA fragments harboring osmCp were PCR amplified with oligonucleotides introducing EcoRI sites at both ends. The templates used in the PCRs were plasmid pCG321 (osmCp₁⁺ osmCp₂⁺) or derivatives of pCG321 carrying the mutations osmCp₁₁ (osmCp₁ osmCp₂⁺) or osmCp₂₁ (osmCp₁⁺ osmCp₂), respectively (4) (Table 1). The resulting EcoRI fragments were cloned into the unique EcoRI site of pOM41 (33). After transformation of the resulting plasmids into CLG723, osmCp was inserted in front of the (malP-lacZ) fusion by homologous recombination, as described previously (15).

-Galactosidase assay. To test the effect of overexpressed RcsB on the osmC promoters, cultures were grown in Luria-Bertani broth (LB) aerobically at 37°C. Overnight cultures were diluted 1,000-fold and grown for five generations, then diluted 40-fold in prewarmed medium with or without 500 µM isopropyl--D-thiogalactopyranoside (IPTG). Samples for the assay were collected after 2 h (optical density at 600 nm [OD₆₀₀], ~0.2). For osmotic shock assays, cultures were made in LB medium without NaCl (LB0) at 30°C. Overnight cultures were diluted 1,000-fold and grown for five generations, and then a NaCl solution was added to reach a final concentration of 0.5 M NaCl. In the control sample, the same volume of water was added. Specific -galactosidase activities are expressed in Miller units (23).

In vitro transcription. DNA templates for the transcription assays were generated by PCR and purified by exclusion chromatography (MicroSpin S-300 HR columns; Amersham Pharmacia Biotech). Single-run transcription assays were performed in 15 µl of buffer (50 mM Tris-HCl [pH 7.8], 50 mM KCl, 3 mM MgCl₂, 0.1 mM EDTA, 0.1 mM dithiothreitol, 25 µg of bovine serum album/ml) at 37°C. A total of 0.3 pmol of templates and 0.3 U of RNA polymerase (Roche Molecular Biochemicals) were used in each reaction. Templates were incubated with 10 µM RcsB protein in 7.5 µl of buffer for 5 min; then RNA polymerase was added. Five minutes later, 7.5 µl of a mixture containing 0.25 mM ATP, GTP, and CTP, 0.17 mM UTP, 5 µCi of [³²P]UTP, and heparin (0.6 µg/ml) was added. After a 5-min incubation, the reaction was stopped with 15 µl of sequence loading buffer. A 5-µl sample of the reaction product was loaded onto an 8% denaturing polyacrylamide gel. Signals were quantitated with a PhosphorImager.

Gel mobility shift assay. DNA templates were generated by PCR with 5'-end-labeled primers and purified from an agarose gel (Qiaex II Kit; Qiagen). Reactions were performed at 35°C in 10 µl of the transcription buffer described above in the presence of 10% glycerol. After the template was incubated with RcsB (10 µM) for 10 min, RNA polymerase (60 nM) was added for 10 min, followed by addition of poly(dI-dC) · poly(dI-dC) (Sigma) to achieve a final concentration of 0.2 µM. The reaction products were loaded into a native 4% polyacrylamide gel at 4°C.

	RESULTS

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RcsB activates the osmCp₁ promoter. The expression of osmC is affected by the growth rate and the osmolarity of the medium. The increased expression of the gene at the end of the exponential phase depends on the stationary-phase sigma factor ^s. Despite the identification of two other factors involved in osmC regulation, LRP and H-NS, neither of these factors is related to the osmolarity-dependent expression of the gene, suggesting that other regulators might control osmC expression (4, 15). With the aim of identifying these putative regulators, we screened an E. coli genomic library for genes able to affect osmC transcription. Chromosomal DNA was extracted from the wild-type E. coli strain MG1655, partially digested with Sau3A, and ligated to vector pJPB209 (25). The ligation products were used to transform strain CLG684, a lac derivative of strain MG1655 containing a chromosomal (osmCp₁-osmCp₂-lac) transcriptional fusion. Cells were plated on MacConkey lactose medium to screen for clones in which osmC expression was affected. Plasmids from those clones were purified, and their inserts were sequenced. This genetic screen yielded several genes able to stimulate transcription of osmC promoters (I. Toesca, C. Pérard, C. Gutierrez, and A. Conter, unpublished data). In particular, three different plasmids isolated in this experiment contained the transcription regulator gene rcsB. To confirm the activator role of RcsB in osmC expression, we introduced plasmid pFAB1, expressing RcsB from the lacUV5 promoter, into strain CLG684. After plating onto MacConkey lactose medium, CLG684/pFAB1 exhibited a darker red phenotype than the control strain CLG684/pJPB209. Strains CLG685 and CLG686 carry osmC-lacZ transcriptional fusions expressed under the control of only osmCp₂ or osmCp₁, respectively, owing to point mutations in the 10 boxes of the promoters (Fig. 1A) (12). To identify the target of RcsB, pFAB1 was introduced into CLG685 and CLG686. The -galactosidase assays for which results are presented in Table 2 indicated that the activity of the promoter osmCp₁ was activated by overexpression of RcsB during the exponential phase (Table 2, CLG686, 300 versus 35 Miller units). A similar stimulation was obtained in an rpoS background, demonstrating that it does not involve a shift in the utilization of sigma factors and is most probably due to activation of transcription of osmCp₁ by E⁷⁰. In contrast, expression of osmCp₂ during exponential phase was not stimulated by overexpression of RcsB (Table 2, CLG685, 7 versus 4 Miller units). Assays performed during the decelerating phase demonstrated that RcsB did not stimulate osmCp₂ at this stage of growth, either (data not shown). Therefore, we conclude that osmC is regulated by RcsB only through the distal promoter osmCp₁.

Osmotic activation of osmCp₁ does not require the rcs genes. Sledjeski and Gottesman (29) have reported that the expression of the cps genes is induced by an osmotic shock and that this induction is dependent on the rcsB and rcsC genes and partly dependent on the rcsA gene. The expression of osmC is also stimulated by increasing the osmolarity of the medium (12, 15). In order to test whether osmotic regulation of osmC was dependent on the rcs genes, we monitored expression of an osmCp₁-lacZ fusion in wild-type, rcsB, rcsA, or rcsC strains following an osmotic shock. In LB0, the basal expression level of the fusion was reduced to 50% that of the wild-type in the rcsB background (Fig. 2A). After an osmotic shock, the expression of the fusion transiently increased five- to sixfold in both the wild-type and rcsB backgrounds (Fig. 2A). In the presence of rcsA or rcsC mutations, we observed results identical to those of the wild-type and rcsB strains, respectively (data not shown). In contrast, as shown in Fig. 2B, expression of a cps-lacZ fusion was osmotically stimulated in a wild-type but not in an rcsB strain, in agreement with an earlier report (29). Data obtained with rcsA and rcsC mutations were also similar to those reported previously (data not shown). We therefore conclude that the osmotic regulation of osmCp₁ is independent of the rcs genes, in contrast to that of cps.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Induction of osmCp1 following an osmotic shock does not require RcsB. Bacterial cells of wild-type or rcsB strains were grown at 30°C in LB0, and -galactosidase specific activity was assayed at the times shown. At the time indicated by the arrow, NaCl was added to a final concentration of 0.5 M (+). The same volume of water was added to unshocked controls (). (A) Expression of an osmCp1-lacZ fusion; (B) expression of a cps-lacZ fusion.

The sequence required for RcsB activation is immediately adjacent to the osmCp₁ 35 box. To identify the sequence required for RcsB stimulation, we constructed a set of osmCp₁-lac fusion strains in which osmCp₁ was carried on various DNA fragments. These strains were transformed with pFAB1, and -galactosidase activities were monitored with or without induction of rcsB expression. The osmCp₁ fragments differed from each other by having different 5' ends upstream of the promoter (Fig. 1A). The results of this deletion analysis, shown in Table 2, indicated that 16 bp upstream of the osmCp₁ 35 box was sufficient for RcsB activity (C10E insert in CLG740). Stimulation was no longer observed with a deletion leaving only 6 bp upstream of the 35 box (C13E insert in CLG743 [Fig. 1A]), indicating that the sequences required for RcsB activity have been deleted in the construction.

RcsB stimulates the osmCp₁ promoter in vitro. The presence of the putative RcsB box in the osmCp₁ regulatory region suggested that RcsB directly activates osmCp₁. This hypothesis was tested by an in vitro transcription assay. Linear PCR-generated templates were incubated with RNA polymerase alone or with both RNA polymerase and purified RcsB. The RcsB protein used is a mutant form in which the conserved aspartate residue at position 56 was replaced by a glutamate residue. This mutation, probably by mimicking the phosphorylation state, makes the protein 3.6- to 6-fold more active in vivo on the expression on the cps genes (16; I. Burzala, F. Carballes, J.-P. Bouche, and K. Cam, unpublished data). As shown in Fig. 3, all osmCp₁-containing templates generated a transcript of a size in agreement with the previous determination of the in vivo transcription start (12). When purified RcsB was added to the reaction, osmCp₁ transcripts from templates C9E and C10E were five- and fourfold more abundant, respectively (Fig. 3). These templates carry an intact RcsB box. In contrast, the amount of osmCp₁ transcripts from the template in which part of the RcsB box was deleted did not increase with the addition of RcsB in the reaction (Fig. 3, C13E template). These results indicated that RcsB directly activates the osmCp₁ promoter. The activation factor, however, was much lower than in vivo, where it reached 80-fold with the C10E fusion. This difference indicates that the in vitro assay is not optimal, possibly because RcsB activity in vivo may require cofactors.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   RcsB activates transcription from osmCp1 in vitro. The transcription reaction was performed with (+) or without () 10 µM RcsB. The same downstream primer used to synthesize the templates was also used to generate a sequence ladder. The 10 sequence of the osmCp1 promoter and the transcription starts are shown. The origin of the transcript marked by a star is unknown. Note that the sequence carries the A-to-G mutation abolishing osmCp2 activity.

RcsB stimulates the binding of RNA polymerase to the osmCp₁ promoter in vitro In order to understand the mechanism of osmCp₁ promoter stimulation by RcsB, the abilities of RcsB and RNA polymerase to bind to the osmCp₁ promoter separately or together were tested in a gel retardation assay. As shown in Fig. 4, RcsB alone was unable to retard a fragment containing osmCp₁ and the RcsB box (C1E template). In contrast, 6% of the fragments were found in a retarded complex with RNA polymerase alone. When RcsB was added to the reaction mixture containing RNA polymerase, a retarded complex migrating at the same position as that in the reaction with RNA polymerase alone was observed. However in this case, 60% of the probe was found in the complex. Therefore, addition of RcsB stimulates the binding of RNA polymerase to the template. With a template in which part of the RcsB box was deleted (C13E), no stimulation of the binding of RNA polymerase to the promoter by rcsB was observed, indicating that the stimulation effect was sequence specific. Therefore RcsB stimulates the osmCp₁ promoter by increasing the affinity of RNA polymerase for the promoter.

View larger version (110K): [in this window] [in a new window]   FIG. 4.   Stimulation by RcsB of the binding of RNA polymerase to osmCp1 in a gel shift assay. Binding reactions were performed with or without 5 nM RNA polymerase and with or without 10 µM RcsB. The retarded complex discussed in the text is indicated by an arrow. The signal marked by a star is inconsistently also observed in reactions without RcsB.

	DISCUSSION

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Previous work had shown that two overlapping promoters direct osmC transcription in E. coli (12, 15). We show here that the transcriptional regulator RcsB of the two-component system RcsB-RcsC is involved in osmC regulation, through stimulatory action on the distal promoter, osmCp₁. Using mutations that allow the expression of each of the promoters to be monitored individually, we had shown that under standard laboratory conditions the proximal promoter, osmCp₂, is responsible for growth phase regulation of osmC, via its control by the stress-specific sigma factor ^s (4, 12). Under such conditions, osmCp₂ appeared to be 10-fold more active than osmCp₁, suggesting that the main function of osmCp₁ was to ensure a low level of expression during exponential phase. However, the data presented in this report show that under appropriate conditions, i.e., when stimulated by RcsB, osmCp₁ is at least as efficient as osmCp₂ and contributes significantly to the expression of osmC under stress conditions during exponential phase.

Both promoters, osmCp₁ and osmCp₂, are stimulated by elevated osmolarity (4, 12, 15). This study has shown that activation of osmCp₁ by an osmotic shock does not require the rcs genes. The osmotic induction of osmCp₂ is also most probably independent of the rcs genes, because this promoter is not affected by overexpression of RcsB. Sledjeski and Gottesman (29) have reported that transcription from the cps promoter is induced by an osmotic shock in an RcsBC-dependent manner, and our data are perfectly consistent with these observations (Fig. 2B). The results obtained with osmCp₁ are therefore somewhat surprising, and further studies will be needed to understand why the rcs genes do not contribute to osmotic stimulation of osmCp₁.

The RcsB-RcsC system has been reported to regulate positively the expression of the genes directing exopolysaccharide synthesis in several bacterial species, as well as that of the cell division genes ftsA and ftsZ in E. coli (1, 5, 8, 14, 20, 34-36). Regulation of the exopolysaccharide synthesis genes by RcsB requires a cofactor, RcsA. The two proteins bind as a heterodimer to a specific sequence, the RcsAB box, located between 100 and 70 bp upstream of the putative 35 box of the promoters (13, 20, 35). Activation by RcsB of the cell division fts genes, in contrast, does not require RcsA (5, 10). We observed that an rcsA mutation has no effect on transcription from osmCp₁. Furthermore, overproduction of RcsA does not activate osmCp₁ (M. Davalos, A. Conter, C. Gutierrez, and K. Cam, unpublished data), indicating that RcsA is not involved in the activation of osmCp₁ by RcsB. When the sequences of the regions of the fts and osmC promoters required for RcsB activity are aligned, the following RcsB box, located next to the 35 box, emerges as a possible consensus: KMRGAWTMWYCTGS (where W stands for A or T, K stands for G or T, M stands for A or C, R stands for A or G, Y stands for C or T, and S stands for C or G). The respective positions of the bases important for activation by RcsB (underlined) revealed a symmetrical organization centered between the T and M bases, suggesting that RcsB binds to its target as a homodimer (5). Comparison of the RcsB box to the proposed RcsAB box, required for activation of the exopolysaccharide synthesis genes by RcsA-RcsB, showed that the left parts of the motifs are conserved, whereas the three bases at the ends of the right parts of the motifs differed (CTA instead of TGS [Fig. 1B]). These observations suggest an orientation for the binding of the RcsA-RcsB heterodimer to the RcsAB box, with the RcsB and RcsA monomers binding to the left and right parts of the motif, respectively.

Binding of the RcsA-RcsB heterodimer alone to the RcsAB box has been demonstrated (20). In contrast, binding of RcsB alone to the RcsB box has been observed neither at osmC nor at the fts cell division gene targets (Fig. 4) (I. Burzala, F. Carballès, J.-P. Bouché, and K. Cam, unpublished data). However, RcsB was able to potentiate the binding of RNA polymerase to the osmCp₁ promoter region, indicating that RcsB stimulates transcription by increasing the recruitment of RNA polymerase to the promoter. The Vibrio fischeri transcriptional activator LuxR is another member of the subfamily of bacterial regulatory proteins that includes RcsB (13). It has been reported that LuxR alone, like RcsB, is unable to bind to its target DNA but that it binds synergistically with RNA polymerase to the lux promoter (30). The binding properties of RcsB are likely to be similar to those of LuxR, but so far the formation of a three-component RcsB-RNA polymerase-promoter complex on the osmCp₁ and fts promoters remains to be demonstrated.

Finally, although four regulators involved in the control of osmC expression have now been identified (LRP, H-NS, ^s, and RcsB), none could explain the osmotic regulation of this gene. This suggests that there remains at least one additional regulator acting on the same promoter region. The participation of so many factors in the regulation of osmC illustrates the notion of cooperation of global regulators in the fine-tuning of stress-inducible genes (17). It also raises the question of the relationship between these factors. The complex osmC promoter region appears to be a good system in which to investigate these questions.