INTRODUCTION

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Rns, a transcriptional regulator belonging to the AraC family (16), is present in some strains of human disease-associated enterotoxigenic Escherichia coli (ETEC). Rns is required for the expression of CS1 and CS2 pili, as well as for its own expression (6, 15). To fully activate the CS1 pilin promoter, Pcoo, Rns uses two DNA binding sites upstream of the transcription start site, site I centered at bp 109.5 and site II centered at bp 37.5 (24). As is the case for nearly all other prokaryotic activators of ⁷⁰-dependent promoters (17), both sites are upstream of the promoter 10 hexamer. When bound at site II, which overlaps the 35 hexamer, Rns may make direct contacts with the  or  subunits of RNA polymerase, as has been shown for other activators (4, 19, 33). A potential Rns binding site has also been identified upstream of the CS2 pilin genes at the same position as site II (24), suggesting that the mechanism of transcriptional activation may be similar at both promoters.

Although Rns binding sites are located within the expected region at the CS1 and CS2 pilin promoters, the arrangement of Rns binding sites at Prns is unprecedented for an activator. Site 1 is centered 224.5 bp upstream of the transcription start site (25), well outside the promoter-proximal region where activators typically bind. Despite its distance from Prns, site 1 is required for Rns-dependent expression from this promoter because nucleotide substitutions within site 1 that interfere with Rns binding in vitro abolish Rns activation of Prns in vivo. Although other activators of ⁷⁰ promoters may also have promoter-distal upstream binding sites, these are invariably accompanied by promoter-proximal binding sites for the activator or for an auxiliary regulator (17). Unlike these other activators, there are no additional Rns binding sites between site 1 and the transcription start site of Prns, and, by itself, Rns facilitates the formation of an RNAP-open complex at Prns in vitro (25).

Rns has two additional binding sites near Prns; however, both of these sites are downstream of the transcription start site: site 2 is centered at bp +43.5 and site 3 is at bp +83.5. The locations of these sites suggest that Rns negatively regulates its own transcription because proteins that bind downstream of the 10 hexamer invariably act as repressors (17). However, nucleotide substitutions within site 3 abolished Rns-dependent expression from Prns, demonstrating that Rns, unlike nearly all other characterized prokaryotic activators, is not restricted to upstream binding sites (25). Thus, Rns is capable of activating transcription from an unprecedented variety of binding site locations, and this suggests there may be no intrinsic limitation to binding-site locations for activators.

Although Rns is unusual in the location of its binding sites, it probably binds to DNA in the same manner as other AraC family members do, because it shares with them a conserved secondary structure (16). Like most AraC proteins, Rns has two predicted helix-turn-helix (HTH) motifs within its carboxy terminus. For other AraC family members (3, 32) and presumably also for Rns, each motif participates in DNA binding by placing a recognition helix in the major groove of DNA. The recognition helices of Rns are identical or very similar to those of a group of regulators within the AraC family (Fig. 1), which suggests these regulators bind to DNA sequences similar to Rns binding sites. However, a direct comparison between the binding sites for these regulators is not possible because only those that are more distantly related to Rns, UreR (39) and VirF of Yersinia spp. (43), have binding sites that have been clearly defined experimentally. Attempts to characterize others biochemically (41, 42) have been hampered by their insolubility, a trait common to members of the AraC family (16).

View larger version (97K): [in this window] [in a new window]   FIG. 1.   Identity of Rns to other regulators within the AraC family. The carboxy termini of regulators with significant homology to Rns are shown. Amino acids identical to those of Rns are shaded. Numbering is relative to Rns, a 31-kDa protein. The predicted HTH motifs of Rns, which are thought to be involved in DNA binding, are overlined. MarA, for which there is structural information, is also shown with its HTH motifs underlined. Abbreviations: Ec, E. coli, Sf, S. flexneri; Pm, P. mirabilis; Yp, Y. pestis.

While little is known about their actual binding sites, the regulators with HTH motifs similar to that of Rns (Fig. 1) are required for the expression of genes known or thought to be required for virulence of bacterial pathogens. For example, like Rns, several of these activators are needed for the expression of pili. CfaR and CsvR activate the expression of colonization factor antigen I (CFA/I) and CS4 pili, respectively, in different human disease-associated ETEC strains (7, 44). AggR is required for the expression of aggregative adherence fimbriae I and II (AAF/I and AAF/II) in enteroaggregative E. coli (14, 27). FapR positively regulates the expression of 987P pili in porcine strains of ETEC (20). PerA positively regulates the expression of bundle-forming pili in enteropathogenic E. coli (EPEC) (41).

Rns-related regulators may also regulate the expression of other types of virulence factors, either directly or indirectly through regulatory cascades. In addition to activation of BFP pili, PerA may activate the expression of virulence-associated genes within the 36.5-kb locus of enterocyte attachment and effacement (LEE) (21) indirectly through the activator Ler (22). PerA also positively regulates the expression of a polycistronic mRNA encoding Tir, CesT, and EaeA (22), but it is not clear whether this occurs through Ler or through another regulator yet to be identified (22, 37). VirF from Shigella flexneri, the causative agent of bacillary dysentery, positively regulates the expression of genes needed for S. flexneri to invade, replicate within, and spread between epithelial cells of the colon. VirF regulates some virulence genes directly, as in the case of virG, and regulates others indirectly by activating VirB, a regulator unrelated to the AraC family (12). UreR, which has been isolated from uropathogenic strains of E. coli and Proteus mirabilis, directly activates the expression of genes encoding the structural subunits of urease in the presence of urea (9). Although UreR is the only Rns-related activator that has been shown to require an inducer, it positively regulates its own expression, as do Rns and PerA (5, 10, 15).

The similarities of other activators within the AraC family to Rns suggest the possibility that, like Rns, some may be able to activate transcription from binding sites outside the upstream promoter-proximal region. In this work, we found that Rns can substitute for several related regulators and that all but one of these is also able to substitute for Rns at Pcoo and Prns. We also found that site 3, downstream of the transcription start site, is required by each of these regulators to activate Prns. Since the identity of Rns to some of these activators is limited to their HTH motifs, this motif is an indicator of the ability of AraC family members to substitute for each other. In this study we also demonstrate that regulators interchangeable with Rns recognize the same binding sites as Rns and that a prototypical binding site for the group can be defined from the known binding sites for Rns. This can then be used to predict the location of the binding sites for Rns-related virulence regulators and may facilitate the identification of new virulence genes by sequence analysis. Some of these predicted binding sites are downstream of transcription start sites and even within genes. Thus, transcriptional activation from downstream binding sites is not limited to a single activator or a single promoter. Rather, Rns represents a new class of transcriptional regulators that play a pivotal role in the virulence of bacterial pathogens and whose activity is not restricted to promoter-proximal upstream binding sites.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Plasmids and strains. Plasmids, strains, and reporter phage are summarized in Table 1. Reporter phage were constructed by cloning the promoter region of interest upstream of the promoterless lacZYA operon carried by plasmids pRS550, pRS551, or pRS415 (36). The derivative plasmids were then recombined with a resident prophage, RS45, by homologous recombination as described previously (36).

Enzymatic assays. Lysogens of E. coli strain DH5 (45) were used for analysis of the PureR and PureD promoters, and lysogens of strain MC4100 (8) were used for analysis of all other promoters. All strains were grown with aeration at 37°C in Luria Bertani (LB) medium with 100 µg of ampicillin per ml. For induction, strains carrying plasmids with regulators expressed from the promoter ParaBAD (18) were grown to log phase with 0.2% glucose, washed, and diluted into LB medium with 0.1% arabinose. For strains carrying plasmids with regulators expressed from Ptac (40), isopropyl--D-thiogalactopyranoside (IPTG) was added at a final concentration of 30 µM to log-phase cells. Aliquots of cells in log phase were assayed for -galactosidase activity (23) before and 2 h after induction with IPTG or arabinose. UreR-dependent expression of -galactosidase was assayed on MacConkey agar containing 300 mM urea. For regulators that were not under the control of inducible promoters, the strains were assayed in log phase.

Sequence analysis. A binding-site consensus and scoring index (28) was defined by aligning the five known Rns binding sites as previously described (25). This scoring index combines the redundancy index (RI) and the Berg vonHippel function (BvH) and is represented graphically by the Rns binding-site sequence logo (34). The RI ranks the importance of each position in the consensus in terms of the conservation at that position within the alignment (35). The BvH relates the occurrence of a query nucleotide to that of the consensus base (2). Each potential binding site is assigned an overall index ranking that is the cumulative sum of BvH times RI at each position. Potential binding sites with index rankings as good as or better than those of known Rns binding sites were defined as having high similarity to Rns binding sites. The index rankings of other sites were used to define them as having moderate or low similarity to known binding sites by assigning arbitrary cutoff values for each category.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Substitution for other regulators by Rns. The ability of Rns to substitute for other regulators was assayed using single-copy promoter-lacZ reporter fusions integrated into the chromosome of E. coli K-12. Each reporter strain was transformed with a plasmid carrying rns expressed from an arabinose-inducible promoter, a plasmid carrying the promoter's cognate regulator, or, as a negative control, the relevant vector plasmid carrying neither regulator. For the purposes of this study, these assays were sufficiently sensitive to determine which activators can substitute for one another, but they should not be interpreted as a comparison of the activation efficiency of different regulators because the regulators may be expressed at different levels.

Substitution for Rns by other virulence regulators. To determine whether regulators homologous to Rns can substitute for Rns, we assayed their ability to increase the expression of -galactosidase from Pcoo-lacZ and Prns-lacZ reporters integrated into the chromosome of E. coli K-12. Each regulator was provided in trans from a plasmid. Some regulators were expressed from their native promoters (AggR, UreR, and CfaR), while others were under the control of inducible promoters. In each case, a strain without the gene for the regulator served as a negative control and a strain with rns expressed from an arabinose-inducible promoter served as a positive control. When the expression of VirF was induced, the expression of -galactosidase from both Pcoo and Prns increased (Table 3). Although this result is expected from the ability of Rns to substitute for VirF (Table 2), it disagrees with the previous conclusions of Porter et al. (29). Presumably their assay, which measured CS1 pilin expression from a Western blot of CooA, was too insensitive to detect activation. AggR and CfaR, which were expressed constitutively from their native promoters, also resulted in significantly higher -galactosidase expression from both Pcoo and Prns than in negative control strains (Table 3). These results show that CfaR, AggR, and VirF are able to substitute for Rns at both Pcoo and Prns.

Rns and related activators utilize the same binding site downstream of the transcription start site of Prns. The homology of the DNA binding domains of Rns, CfaR, AggR, VirF, and CsvR (Fig. 1) and their interchangeability imply that these regulators recognize similar DNA binding sites. In this study we have also shown that CfaR, AggR, and VirF can substitute for Rns to activate expression from Prns. This suggests that these other regulators are able to function as transcriptional activators when bound downstream of a promoter, because it has been shown that Rns requires binding site 3, downstream of the transcription start site, to activate Prns. However, because it is extremely rare for activators to utilize downstream binding sites, we sought to confirm this prediction experimentally by assaying the effect of a mutation in Rns binding site 3 on the ability of these regulators to activate Prns.

Positions of predicted binding sites in promoters regulated by Rns and related activators. Although VirF, CfaR, and AggR recognize the same binding sites as Rns does and utilize a downstream binding site to activate Prns, we wanted to determine if this promoter is a singular example or if other promoters regulated by these activators have downstream binding sites. However, Rns is the only activator within this group for which binding sites have been defined. Therefore we used the five experimentally determined Rns binding sites to define a binding site scoring index for this group of activators which is represented by the Rns binding-site logo (Fig. 2A). This scoring index was then used to search for potential binding sites, and these were ranked high, moderate, and low based on their similarity to the group of known Rns binding sites.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Rns binding-site logo and locations of predicted binding sites. (A) The consensus of the five known Rns binding sites is represented by the binding-site logo, where the height of each nucleotide is proportional to its frequency and the information content of that position. (B and C) Triangles represent predicted binding sites with similarity to the known Rns binding sites near the gene encoding activators homologous to Rns (B) or virulence genes regulated by these activators. (C) Right- and left-pointing triangles indicate binding sites located on the coding and noncoding strands, respectively. The known Rns binding sites are marked by asterisks, and dark, light, and no triangle shading indicates high, moderate, and low similarity of the predicted binding sites to the known binding sites, respectively. Numbering is relative to the beginning of the adjacent open reading frame. Transcriptional start sites that have been reported are shown as wavy arrows. The cognate regulator is shown to the left of each virulence gene, and the system encoded by these genes is shown to the right. Only a limited region of virF upstream sequence was available from GenBank. The nucleotide sequence of the CsvR-regulated locus, CS4, has not been reported.

Conclusions and evolutionary considerations. As an activator, Rns presents an unprecedented variability in the arrangement of the binding sites with which it interacts. However, in this study we have shown that Rns is not unique VirF, AggR, and CfaR also can activate expression from both Pcoo, with its more typical arrangement of binding sites, and Prns. Since the identity of VirF and AggR to Rns is limited to the HTH motifs, this suggests that identity within this region may by itself be a good indicator of which regulators within the AraC family can substitute for one another. Moreover, each of these regulatory proteins requires binding site 3, downstream of the transcription start site, to activate Prns. Thus, Rns is the prototype for a group of activators within the AraC family whose activity is not restricted to upstream binding sites. Although the number of regulators that function as activators when bound downstream of a promoter is still very small (25, 31, 38), they may be more common than previously thought because the AraC family has over 150 members (16), many of which have not been characterized.