INTRODUCTION

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The AraC family of transcription activators (sometimes also called the AraC/XylS family) was found to consist of more than 100 members in the last published homology search (performed in March 1997) (13). Activators in this family are found in a wide variety of bacterial species. Many members of this family regulate expression of genes involved in carbon metabolism, stress responses, or pathogenesis. Examples of proteins that regulate carbon metabolism (and the carbon source and species they are found in) are AraC (L-arabinose, Escherichia coli), RhaS and RhaR (L-rhamnose, E. coli), XylS (benzoates, Pseudomonas putida), and MelR (melibiose, E. coli) (2, 14, 33, 36). Some of the proteins that regulate stress response genes are SoxS (oxidative stress, E. coli), MarA (multiple antibiotic resistance, E. coli), Ada (alkylating agents, E. coli), and Rob (originally identified as binding to oriC but cross-activates SoxS- and MarA-regulated genes, E. coli) (6, 18-21, 30, 39). Finally, some AraC family proteins that regulate virulence factors in pathogens include CfaD (fimbriae, enterotoxigenic E. coli) and VirF (invasion proteins, Shigella dysenteriae) (11, 17, 31, 32).

The RhaS protein, one of the AraC family members, activates expression of the E. coli rhaBAD and rhaT operons (10, 38). rhaT encodes the L-rhamnose transport protein (35). rhaBAD encodes the enzymes required for catabolism of the sugar L-rhamnose (25, 28); hence RhaS binding and activation of rhaBAD occurs only in the presence of L-rhamnose (9, 10). In the absence of L-rhamnose, there is apparently insufficient active RhaS to saturate the rhaBAD promoter, resulting in a lag in rhaBAD mRNA accumulation after L-rhamnose addition (10). RhaR, another AraC family protein (12, 13, 29, 36), activates rhaSR expression (37). Although RhaS and RhaR are encoded in the same operon, there apparently is sufficient RhaR to saturate the rhaSR promoter in the absence of L-rhamnose, as there is no detectable lag in rhaSR mRNA accumulation after L-rhamnose induction (10). As a result, L-rhamnose induction of rhaBAD expression first requires induction of rhaSR expression, which leads to accumulation of RhaS and finally activation of rhaBAD expression.

Egan and Schleif (9) used DNase I footprinting and point mutations to locate the DNA-binding site for RhaS (rhaI) to a region spanning 32 to 81 relative to the rhaBAD transcription start site (Fig. 1). The symmetry of important single-base mutations in rhaI led to the prediction that the RhaS binding site is an inverted repeat of two 17-bp half-sites (rhaI₁ and rhaI₂) separated by 16 bp of uncontacted DNA. Based on the fact that AraC binds to DNA as a dimer (16) and the size of the RhaS binding site, it is predicted that RhaS binds to rhaI as a dimer, with each RhaS monomer contacting one half-site. When viewed from one face of the DNA helix, each rhaI half-site consists of two major grooves, separated by a minor groove. All of the base pairs found important for RhaS binding lie in the major grooves of rhaI (9), suggesting that RhaS makes all of its sequence-specific DNA contacts within the major grooves (Fig. 1). This is in agreement with the previous findings that AraC and RhaR also interact with their DNA binding sites within two major grooves of each half-site (3, 37). We expected that in order to contact two major grooves, each RhaS monomer would possess two DNA-contacting motifs.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   (a) Model for transcription activation at rhaBAD, showing the two activator proteins, cyclic AMP receptor protein (CRP) and RhaS, and RNA polymerase (RNAP) shown. The bent arrow represents the rhaBAD transcription start site. (b) DNA sequence of the RhaS binding site (rhaI). The two half-sites, rhaI1 and rhaI2, are inverted repeats, as indicated by the arrows above the sequence. Sequences within the half-sites are divided into two major grooves separated by a minor groove, with the outer and inner major grooves indicated. Down arrows indicate bases previously identified as important for interaction with RhaS (9); their positions relative to the rhaBAD transcription start site are indicated below. Bases overlapping the 35 region of the promoter were not analyzed in this previous study since their effects on RhaS binding and RNA polymerase binding could not easily be distinguished by the analysis used. Notice that all of the bases important in rhaI1 are identical in rhaI2.

One DNA-binding motif in AraC has been predicted to be a helix-turn-helix (H-T-H) motif consisting of residues 196 to 215 (3). Ser208 and His212 of this motif have been shown to make base-specific contacts with DNA (3, 27). Asp256 of AraC has also been shown to be important for DNA sequence recognition and was predicted to be a part of a second H-T-H motif (H-T-H 2) (27). Recently Rhee et al. (30) determined the crystal structure of the first AraC family member, MarA. MarA is one of relatively few AraC family members that consists of a DNA-binding domain without any dimerization domain, and it activates transcription as a monomer (6, 23). The MarA structure includes two H-T-H DNA-binding motifs; the first (H-T-H 1) aligns with AraC Ser208 and His212, and the second (H-T-H 2) aligns with AraC Asp256.

In this study, we used a genetic analysis to identify RhaS amino acids that make specific contacts with rhaI. Our results provide further evidence that AraC family transcription activators utilize two H-T-H motifs to contact DNA. Further, this work is the most extensive functional analysis of the second AraC family H-T-H motif to date. Our data suggest that Arg202 and Arg206 of RhaS, which align with H-T-H 1 of MarA and AraC, make base-specific contacts with rhaI. Both of these amino acids contact the inner major grooves of the inverted repeat RhaS binding site (Fig. 1). Our results further show that residues within the 247 to 254 region of RhaS are crucial for contact with rhaI. We found that Asp250 and Asn252, which lie in this region, make specific contacts with the outer major grooves of rhaI and are part of H-T-H 2 of RhaS.

	MATERIALS AND METHODS

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Culture media. Cultures for -galactosidase assay were grown in 1× MOPS [3-(N-morpholino)propanesulfonic acid]-buffered medium (26) (40 mM MOPS, 4 mM Tricine, 0.01 mM FeSO₄, 9.5 mM NH₄Cl, 0.276 mM K₂SO₄, 0.5 µM CaCl₂, 0.528 mM MgCl₂, 50 mM NaCl, 3 × 10⁹ M Na₂Mo₄, 4 × 10⁷ M H₃BO₃, 3 × 10⁸ M CoCl₂, 10⁸ M CuSO₄, 8 × 10⁸ M MnCl₂, 10⁸ M ZnSO₄, 1.32 mM K₂HPO₄, 10 mM NaHCO₃, 0.2% Casamino Acids, 0.002% thiamine). Ampicillin and tetracycline were used at 125 and 12.5 µg/ml, respectively, when necessary. For other experiments (cloning, strain construction, Ter test, etc.), cells were grown in tryptone-yeast extract medium (22) with or without antibiotic or in TB-maltose (0.8% Bacto-Tryptone, 0.5% NaCl, 0.2% maltose).

DNA sequencing. IRD41 dye-labeled primers for DNA sequencing (Table 1) were custom made by LI-COR, Inc. (Lincoln, Neb.). DNA sequences were verified by automated dideoxy sequencing on a LI-COR 4000L sequencer. Sequencing reactions were performed with a Thermo Sequenase fluorescent-labelled primer cycle sequencing kit from Amersham Life Science.

Plasmids, phage, and strains. Wild-type rhaS was amplified by PCR using primers 1170 and 898 and plasmid pSE101 as the template. The PCR product was digested at the EcoRI site in 1170 and the HindIII site in 898 and cloned between the EcoRI and HindIII sites of Tet^r pALTER-1, resulting in pSE160. An Ap^r version of the clone (pSE159) was made by using Promega Altered Sites II in vitro mutagenesis system. The DNA sequence of the cloned rhaS gene in both pSE159 and pSE160 was verified by DNA sequencing on both strands.

-Galactosidase assay. Starter cultures of strains to be assayed were grown in tryptone-yeast extract broth containing appropriate antibiotic for approximately 7 h at 37°C; 40 µl of starter culture was then inoculated into 2.5 ml of overnight medium and grown for approximately 17 h. Overnight medium consisted of 1× MOPS medium containing 0.04% glycerol as the carbon source and appropriate antibiotic. A 100-µl volume of the overnight culture was inoculated into 10 ml of growth medium. Growth medium consisted of 1× MOPS medium containing 0.4% glycerol as the carbon source with 0.2% L-rhamnose added as inducer in all cases and appropriate antibiotic. Cultures were grown at 37°C with vigorous shaking in baffled, 125-ml Erhlenmeyer flasks to an A₆₀₀ of approximately 0.4. Growth medium cultures were then centrifuged, and the cells were resuspended in Z buffer (24). -Galactosidase activity was determined as described by Miller (24) except that incubation with substrate o-nitrophenyl--D-galactopyranoside was at room temperature. Specific activities were averaged from at least three independent assays, with two replicates in each assay.

Mutagenesis of rhaS. Oligonucleotide primers used for random and site-directed mutagenesis (Table 1) were synthesized by Oligos Etc. Inc. A 45-nucleotide oligonucleotide with degenerate sequence (5) over the region to be mutagenized was used to generate random mutations in the rhaS region encoding amino acids 246 to 255 (Table 1). The oligonucleotide was designed to introduce an average of 1.5 mutations within the 30-bp region and was used in a site-directed mutagenesis procedure (Promega Altered Sites II in vitro mutagenesis system) to create random mutations.

	RESULTS

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First RhaS DNA-contacting motif. AraC amino acids Ser208 and His212, which correspond to amino acids 2 and 6 of the DNA recognition helix of an H-T-H motif, have been shown to make specific contacts with base pairs within the AraC binding site (araI) (3, 27). These AraC amino acids align with RhaS Arg202 and Arg206. Hence, we created alanine substitutions at Arg202 and Arg206 of RhaS. Amino acid 1 of the recognition helix often is involved in making specific DNA contacts, and so we also made an alanine substitution at Leu201. Further, it has been recently shown that Gln45 of MarA makes specific contact with DNA (30). Since MarA Gln45 aligns with RhaS His205, we also created an alanine substitution at His205 of RhaS.

Random mutagenesis of the second possible RhaS DNA-contacting motif. As described above, RhaS is expected to have two DNA-binding motifs. It seemed likely that the RhaS H-T-H involving amino acids 201 to 206 would contact the inner major grooves of rhaI (Fig. 2a), based on the expectation that the promoter proximal RhaS and AraC monomers would bind to DNA in the same relative orientation (and possibly make conserved contacts with RNA polymerase). Egan and Schleif (9) previously observed that the outer major grooves of rhaI are nearly identical to the corresponding major grooves of the site for RhaR binding (Fig. 2a). This suggests that RhaS and RhaR may share a conserved DNA-binding motif. We found a 10-residue region which was highly conserved in RhaS (residues 246 to 255) and RhaR (residues 282 to 291) (Fig. 2b). Eight of the ten amino acids in this region are identical whereas the other two are similara degree of sequence similarity that is unusually high, even for the very similar RhaS and RhaR proteins. Therefore, we hypothesized that residues 246 to 255 might be part of the second DNA-binding motif of RhaS.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   (a) Comparison of the DNA-binding sites (bs) recognized by RhaS, RhaR, and AraC. Horizontal arrows indicate the half-sites and their relative orientations. Notice that the downstream half-sites are all in the same relative orientation whereas the upstream half-sites are not. Bracketed bases are conserved in the RhaS and RhaR DNA-binding sites. The outer and inner major grooves of the RhaS binding site are indicated. The 35 regions of the promoters are shown. Vertical arrows indicate amino acid-base pair contacts between AraC H-T-H 1 and its DNA-binding site. (b) Sequence of amino acids 246 to 255 of RhaS, showing high similarity with the aligned region of RhaR (amino acids 282 to 291). Solid lines indicate identical amino acids; broken lines represent similar amino acids.

Alanine substitutions in the second RhaS H-T-H motif. We made individual alanine substitutions at most of the amino acid positions in the 246-255 region. Alanine substitutions were not constructed at Ser249 or Ser251 since random Gly substitutions at these positions had very little effect on RhaS function. We did not construct an alanine substitution at Gly247 since an alanine substitution at this position was isolated in the random mutagenesis described above. While we were in the process of creating and analyzing the alanine substitutions, publication of the MarA crystal structure showed that Arg96 makes specific contacts with DNA (30). Since, MarA Arg96 aligns with RhaS Thr256 (13), we also made an alanine substitution at this position.

Base-specific contacts by RhaS. To test whether base-specific contacts are made by any of the amino acids thought to be important in DNA binding, we used a genetic loss-of-contact approach (7, 8). The rationale behind the genetic loss-of-contact approach is that a protein with a single amino acid substitution will lose the ability to discriminate between the wild-type and mutant base pairs only at the position or positions in the DNA that are normally contacted by the substituted amino acid. As a result, any DNA positions normally contacted by the substituted amino acid can be mutated with relatively little effect, while mutations at other DNA positions will have large effects. This analysis will determine whether some of the amino acids that we have identified are directly involved in DNA binding and also identify the base pair positions contacted by such base-specific amino acids.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Alanine substitutions in RhaS H-T-H 1 and 2 analyzed at mutant rhaB-lacZ fusions. The x axis (labeled at the bottom) represents either the wild-type rhaBAD promoter or the position of point mutations in rhaI found to be important for RhaS binding (Fig. 1). Locations of the point mutations in either the inner or outer major grooves of rhaI are indicated. The y axis (labeled on the left) represents the percent -galactosidase specific activity for each RhaS alanine mutant at mutant rhaB-lacZ compared with the same mutant protein at the wild-type rhaB-lacZ fusion. The first bar in each graph represents the RhaS alanine mutant protein assayed at the wild-type rhaB-lacZ promoter and is set to 100%. Error bars are shown.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this work, we set out to determine the amino acids in the RhaS protein that contact rhaI, as well as the specific bases in rhaI that are contacted by those amino acids. Each of the rhaI half-sites is 17 bp long (Fig. 1), and therefore the face of the DNA helix likely to be contacted by RhaS will consist of two major grooves of DNA (9). This suggests that the RhaS protein contains two distinct DNA-contacting motifs in each monomer.

H-T-H 1 of RhaS is a DNA-binding motif. We constructed alanine substitutions at four amino acid positions within the recognition helix of a RhaS H-T-H DNA-binding motif proposed based on alignment with AraC (3). We found that mutations in the first (Leu201), second (Arg202), fifth (His205), and sixth (Arg206) amino acids of the recognition helix resulted in defects in RhaS activation from the wild-type rhaBAD promoter (Table 3). This result is consistent with the hypothesis that these amino acids may be involved in sequence-specific DNA contacts by RhaS. As discussed below, our results indicate that this motif contacts the inner major grooves of the rhaI inverted repeat.

H-T-H 2 of RhaS is also a DNA-binding motif. At the time we began these experiments, the second DNA-contacting motif had not been identified in any AraC family proteins. We hypothesized that the RhaS and RhaR proteins might use a conserved DNA-contacting motif to recognize the conserved outer major grooves of their binding sites (Fig. 2). We identified amino acids 246 to 255 of RhaS as a region having unusually high sequence similarity with RhaR and proposed that this region might be a part of the second DNA-contacting motif. We found that random substitutions at positions Gly247, Phe248, Asn252, and His253 resulted in a defect in the ability of RhaS to activate expression from the wild-type rhaBAD promoter (Table 4). Ala mutants at all of the amino acids from residues 247 to 254, except for 249 and 251 (which were tested as random substitutions only), were found to be at least twofold defective for activation from the rhaBAD promoter, and most were more than 10-fold defective (Table 5). We propose that this region is part of a second DNA-binding motif of RhaS that, based on alignment with the crystal structure of MarA (30), is likely an H-T-H motif.

Identification of DNA contacts by RhaS. Further analysis of the mutants that we constructed in the two H-T-H motifs was performed by using a genetic loss-of-contact approach (outlined in Results) (7, 8). A genetic approach was chosen for initial characterization of these mutants since it is more informative than comparison of binding affinities of the mutants to wild-type and much easier than in vitro missing - contact probing (3, 4). In addition, a genetic approach was particularly useful in this case since the RhaS protein is severely insoluble (9).

Comparison with MarA structure. Both of the amino acids in H-T-H 1 of RhaS that make sequence-specific DNA contacts align with MarA residues also predicted to make specific DNA contacts. Arg202 of RhaS aligns with MarA Trp42. One of the base pairs contacted by each of these amino acids is a G-C base pair in the analogous position in the two binding sites (56 at marRAB and 67 at rhaBAD), while each of these residues also makes additional, different contacts (Fig. 4). Arg206 of RhaS aligns with Arg46 of MarA. In this case, the identical amino acid contacts the identical base pair in the analogous position in the two binding sites (G-C at 56 in marRAB and 67 in rhaBAD), and each amino acid also contacts a C-G base pair (55 at marRAB and 65 at rhaBAD). MarA has a third amino acid in H-T-H 1 that contacts DNA (Gln45). MarA Gln45 aligns with RhaS His205, which was found to be important for DNA binding by RhaS but not specific for any base positions. It seems likely that only Arg202 and Arg206 of RhaS H-T-H 1 make DNA contacts since two amino acid partners have been identified for each of the two important base pairs in the inner major grooves.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Summary of specific amino acid-base pair contacts by RhaS and comparison with MarA DNA contacts. DNA half-sites are divided into two major grooves and an intervening minor groove. Vertical and angled black lines represent amino acid-base pair contacts. The broken bar indicates a less conclusive DNA contact by RhaS. The side of the DNA sequence on which each amino acid is represented is arbitrary and is not meant to represent the base within the pair that is contacted. For MarA, the base(s) within each pair that is contacted is known (30); however, this information is not represented for simplicity and because the corresponding information is not known for RhaS. Relative positions of H-T-H 1 and 2 are shown below the two DNA sequences.

Have all of the base-specific contacts been identified? Curiously, at least two (or three if 77 is included) important base pairs in the outer major grooves of rhaI do not yet have identified amino acid partners. We have tested all of the amino acids ranging from the last amino acid of the first helix, through the turn, up to the sixth amino acid of the recognition helix, without finding any contacts with these base pairs. It is possible that additional amino acids of RhaS make DNA contacts; however, these amino acids are not located in the most common positions for H-T-H contacts. Alternatively, it is possible that amino acids that we tested make DNA contacts that were not detected by our analysis for some reason.

Nonspecific amino acid contacts. Our analysis identified six amino acids in RhaS that were defective for transcription activation at the rhaBAD promoter but that did not show evidence of base-specific DNA contacts. It is possible, although certainly not probable, that some of these amino acids are involved in transcription activation rather than DNA binding by RhaS. It is also possible that some of these amino acids make contacts with the phosphate backbone of the DNA, and therefore are directly involved in DNA binding, but do not make base-specific contacts. Eleven different amino acids in MarA make only phosphate backbone contacts with the DNA (30). Finally, it is possible that some of these amino acid substitutions alter the conformation of RhaS and therefore indirectly affect DNA binding. Inspection on the MarA structure of the amino acids that aligns with each of these nonspecific RhaS amino acids (by using RasMol version 2.6) indicates that all of them except Phe254 are significantly surface exposed and near the DNA backbone. Substitutions at Phe254 most likely alter the conformation of RhaS and thereby indirectly reduce DNA binding. Each of the other nonspecific amino acids could possibly make phosphate backbone contacts.