INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The RhaS protein is the L-rhamnose-responsive transcription activator of the Escherichia coli L-rhamnose catabolic and transport operons rhaBAD and rhaT, respectively (12, 13, 52, 53, 57), and is a member of the AraC/XylS family of transcription activators (17, 18, 44, 53). Full activation of both the rhaBAD and rhaT promoters requires activation by CRP binding immediately upstream of RhaS (13, 57). RhaS alone is able to activate rhaBAD expression by about 1,000-fold (13). In the presence of RhaS, CRP activates rhaBAD an additional 30- to 50-fold; however, CRP is unable to activate to any significant extent in the absence of RhaS (13).

The AraC/XylS family of transcription activators is named for its most well-studied member, AraC. The AraC protein consists of two functionally separable domains (7). The N-terminal AraC domain is responsible for both dimerization and L-arabinose binding, while the C-terminal domain is responsible for both DNA binding and transcription activation. In RhaS, the C-terminal domain is also responsible for DNA binding (4), and it is likely that the N-terminal domain functions in dimerization and L-rhamnose binding. The AraC/XylS family consists of more than 130 proteins that are identified by a 99-amino-acid region of sequence similarity within the DNA-binding domain of AraC (17, 18, 44, 53). One subset of AraC/XylS family proteins regulates expression of genes involved in carbon metabolism. This group includes AraC, RhaS, RhaR, and MelR from E. coli and XylS from Pseudomonas putida, which are among the most well characterized of the AraC/XylS family proteins (4, 5, 8, 12, 13, 16, 19, 29, 30, 38, 39, 53-55). Another large and important subset of AraC/XylS family proteins are those that regulate expression of virulence factors in bacterial pathogens (18). A few examples of this large group include CfaD from enterotoxigenic E. coli, SprA from Salmonella enterica serovar Typhimurium, and UreR from a variety of enteric pathogens (9, 14, 28, 48).

While DNA binding has been well characterized in a number of AraC/XylS family members (4-6, 12, 43, 45, 54), transcription activation by AraC/XylS family proteins is less well understood. It has been shown that activation of several promoters dependent upon AraC/XylS family activators requires the C-terminal domain (CTD) of the  subunit of RNA polymerase (RNAP). The -CTD is the most well-characterized activation target and is required by a large number of activator proteins (reviewed in references 11 and 24). Perhaps the most direct evidence for an interaction between an AraC/XylS family activator and -CTD has been found with the Ada protein at the alkA promoter. In this case mobility shift assays showed that a substitution in -CTD eliminated the ability of purified  subunit to supershift the DNA-bound form of either Ada or ^meAda (34). Strong evidence also exists for an interaction between -CTD and the MarA, SoxS, and Rob proteins in cases where these activators bind to DNA upstream but not overlapping the 35 region of the promoter (25-27). Finally, at a truncated rhaBAD promoter where RhaS was the only activator, deletion of -CTD led to a 180-fold defect, and alanine substitutions identified eight residues in -CTD that were candidates for making contacts with RhaS (23).

There is also evidence that the mechanism of transcription activation by some AraC/XylS family proteins may involve interactions with the ⁷⁰ subunit of RNAP, usually in cases where the binding site for the activator overlaps the 35 region of the promoter. In fact, the very first substitution isolated in ⁷⁰ (originally named alt and with the substitution R596H) involved an interaction with AraC. This substitution increased the ability of AraC to activate transcription in the absence of CRP, such that cya mutant cells regained the ability to use arabinose as the sole carbon source (50, 56). The more recent finding that other ⁷⁰ substitutions at positions near R596, especially K593A, significantly reduce activation by AraC in the absence of CRP supports the hypothesis that wild-type AraC and ⁷⁰ make an interaction that contributes to transcription activation (36).

Biochemical evidence for an interaction between ⁷⁰ and Ada also exists. Ada differs from many other AraC/XylS family proteins in that it can activate transcription from either a site that overlaps the 35 region (at alkA) or from a site that is 5 to 7 bp upstream of the 35 region (at ada and aidB) (1, 15, 35, 47). The N-terminal half of Ada, which includes the AraC/XylS family domain, is capable of binding to DNA and activating transcription at the alkA promoter but is not sufficient for transcription activation at promoters where Ada binds upstream of the 35 region (1). At the alkA promoter, a set of positively charged amino acids in ⁷⁰ was important for activation by Ada (33). A heparin-resistant ternary complex could be formed between DNA, Ada, and RNAP containing wild-type ⁷⁰, but not with RNAP containing ⁷⁰ substitutions K593A, K597A, or R603A (33), indicating that these ⁷⁰ residues might be directly involved in an interaction with Ada.

The focus of our work has been the mechanism of transcription activation by the RhaS protein. The binding site for RhaS overlaps the 35 region of both the rhaBAD and rhaT promoters by 4 bp, and hence it seemed likely that a target of transcription activation by RhaS might be ⁷⁰. To test this possibility, we first tested activation by RhaS in strains expressing a library of ⁷⁰ derivatives with single alanine substitutions in region 4.2 and at the very C-terminal end of ⁷⁰. We found that activation by RhaS was defective in the presence of several ⁷⁰ derivatives, most notably K593A and R599A, but only at truncated promoters that lacked the binding sites for the second activator, CRP. In an effort to identify RhaS amino acids that might contact these positively charged ⁷⁰ residues, we constructed alanine substitutions in nearly all of the negatively charged residues in the C-terminal domain of RhaS. A number of the RhaS derivatives were defective for activation in combination with wild-type ⁷⁰. Finally, we combined the RhaS and ⁷⁰ derivatives and found one combination, RhaS D241A plus ⁷⁰ R599A, which showed no greater defect than the individual derivatives at both the truncated rhaBAD and rhaT promoters. This phenotype suggests that the two substitutions may define an interaction between the RhaS and ⁷⁰ proteins that is important for transcription activation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Culture media and growth conditions. Cultures for -galactosidase assay were grown in 1× MOPS buffered medium (42); 1× MOPS consisted of 40 mM 3-(N-morpholino)propanesulfonic acid (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, and 0.002% thiamine. For other experiments (cloning, strain construction, Ter test, etc.), cells were grown in tryptone-yeast extract medium (37), with or without antibiotic, or TB maltose (0.8% Bacto-Tryptone, 0.5% NaCl, 0.2% maltose). Ampicillin was used at 125 or 200 µg/ml, as indicated.

General methods. Standard methods were used for restriction endonuclease digestion, ligation, transformation, and purification of plasmid DNA. Primers for automated DNA sequencing were IRD41 dye labeled (Table 1) and custom synthesized by LI-COR, Inc. (Lincoln, Nebr.). DNA sequences were verified by automated dideoxy sequencing on a LI-COR 4000L sequencer. Sequencing reactions were performed using the Thermo Sequenase fluorescence-labeled-primer cycle sequencing kit from Amersham Pharmacia Biotech (Piscataway, N.J.). All DNA sequences were confirmed on both strands.

Strains, plasmids, and phage. The E. coli strains,  phage, and plasmids used in this study are described in Table 2. All assays were performed using cultures of strains derived from ECL116 (2). In all cases, lacZ translational fusions were assayed as single-copy lysogens integrated into the E. coli chromosome at att. A library encoding wild-type ⁷⁰ and alanine substitution derivatives of ⁷⁰ were a gift from C. Gross and were carried on the plasmid pGEX2T (10, 36).

Construction of rhaT-lacZ fusions. The full-length rhaT promoter (including both the CRP and RhaS-binding sites) was amplified by PCR using primers 2097 and 2096 and whole cells of E. coli ECL116 as the source of template DNA. The truncated rhaT promoter (with only the RhaS-binding site) was amplified by PCR using primers 2096 and 2152 and whole cells of E. coli DH5 as the source of template DNA. The PCR products were digested at the EcoRI site in 2097 and 2152 and the BamHI site in 2096 and cloned between the EcoRI and BamHI sites of pRS414, yielding plasmids carrying full-length (pSE203) and truncated (pSE204) fusions, respectively. The DNA sequence of the promoter regions and fusion junctions were sequenced on both strands. The translational fusions thus constructed with full-length and truncated rhaT promoters were transferred to RS45 and RS74 (both imm²¹), respectively, by in vivo recombination (51) to generate recombinant phages SME107 and SME108 (Table 2). Strains SME2186 and SME2187 carrying a single-copy lysogen of the recombinant  phage were obtained by transducing ECL116 with phage carrying the full-length and the truncated fusions, respectively. Lysogens carrying the full-length fusion were identified as pinpoint blue colonies amid a white lawn on a nutrient agar plate containing X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) and L-rhamnose. Lysogens of the truncated fusion were selected by spreading the transduction mixture on a plate carrying a lawn of gt30 (imm²¹). In this case, lysogens were differentiated from -resistant cells by their sensitivity to the heteroimmune phage Ch6 (imm⁴³⁴) when cross-streaked. For both the full-length and the truncated fusions, single lysogens were identified by the Ter test (22) and confirmed by -galactosidase assay. P1 phage-mediated generalized transduction (40) was used to introduce an in-frame deletion of approximately two-thirds of rhaS (13) linked to Tn10 into SME2186 and SME2187 to generate SME2341 and SME2342, respectively. The presence of the rhaS deletion was confirmed by PCR analysis.

Recombination of rhaS alleles into the chromosome. Chromosomal replacements by mutant rhaS alleles were constructed using an E. coli strain carrying bacteriophage  recombination functions resulting in increased homologous recombination frequencies (41). SME2394 was constructed by P1 transduction of the  bet exo operon under the control of the lac promoter from KM22 into SME 2393 with selection for kanamycin resistance (41). The presence of P_lac-bet exo was confirmed by PCR with oligos 2161 and 2162. Alleles of rhaS to be recombined were amplified by PCR using oligos 898 and 1170 with the corresponding rhaS clone in pALTER-1 (pSE193-196 and pSE199) as a template. Then, 100 µl of CaCl₂-treated SME2394 competent cells were transformed with approximately 500 ng of unpurified PCR product. The transformation mixtures were plated onto nutrient agar plates containing X-Gal, IPTG (isopropyl--D-thiogalactopyranoside; 1 mM), and L-rhamnose and incubated at 37°C for 72 to 96 h. Tiny blue colonies picked from amid the white lawn were patched onto nutrient agar plates containing X-Gal and L-rhamnose with and without ampicillin. Blue, ampicillin-resistant colonies had been transformed with plasmid DNA that had served as template in the PCR reaction, while blue, ampicillin-sensitive colonies had been transformed with the PCR-generated DNA fragments and had undergone the desired chromosomal replacement. Performing PCR on blue, ampicillin-sensitive colonies with oligo 898 downstream and oligo 744 upstream identified replacements of the in-frame rhaS deletion in SME2394 with full-length rhaS alleles. The presence of the mutant rhaS allele was tested by the same diagnostic PCR used to identify the mutations after the initial site-directed mutagenesis. For DNA sequencing, the rhaS alleles were amplified by PCR using oligos 880 and 744 with whole cells as the source of template DNA. The PCR products were purified using QIAquick PCR purification kit (Qiagen, Inc.). Both strands of the PCR products were sequenced using the IRD44-labeled oligos listed in Table 1. Once confirmed, the wild-type and mutant rhaS alleles were introduced into SME1851 and SME2187 by phage P1-mediated generalized transduction (40) using selection for the linked Tn10. Finally, recA::kan was moved into each of the strains by P1 transduction with selection for kanamycin resistance.

-Galactosidase assay. Strains to be assayed for -galactosidase activity were grown and assayed as previously described (4). Briefly, they were first grown in TY broth containing ampicillin and then transferred to 1× MOPS minimal medium with ampicillin (200 µg/ml) and limiting carbon source (0.04% glycerol) for overnight growth. The overnight culture was diluted 1:100 into 1× MOPS medium containing 0.4% glycerol as the carbon source; 0.2% L-rhamnose was added as the inducer along with ampicillin (200 µg/ml), and the culture was grown to an A₆₀₀ of approximately 0.4. Assays were performed as described by Miller (40) except that in assays of the truncated rhaT fusion [(rhaT-lacZ)84], cultures were concentrated 20-fold (⁷⁰ derivatives) or 114-fold (RhaS derivatives and RhaS-⁷⁰ derivative combinations) upon addition of Z buffer. This is much greater than the 2.5-fold concentration in the standard assay. These assays were allowed to incubate for up to 3 days. Assays of RhaS derivatives and combinations of RhaS and ⁷⁰ derivatives at the truncated rhaT fusion were performed in a total volume of 0.1 ml rather than in the standard 1 ml so that very large culture volumes did not need to be grown. Under these conditions, the vast majority of the optical-density-at-420-nm readings were greater than 0.1, while the very lowest readings were greater than 0.05. Specific activities were averaged from at least three independent assays, with two replicates in each assay.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Sigma⁷⁰ substitutions at rhaBAD. Lonetto et al. (36) constructed a library of 17 single alanine substitutions near the C-terminal end of the ⁷⁰ subunit of RNA polymerase. They found that ⁷⁰ residues in this region were required for activation of a variety of promoters in which an activator protein binds to a site that overlaps the 35 region of the promoter. To determine whether contacts with ⁷⁰ were important for activation by RhaS, we first tested the library of alanine substitutions in ⁷⁰ at the rhaBAD and rhaT promoters. The strains that we assayed had a gene encoding wild-type ⁷⁰ in the chromosome and carried the gene encoding the ⁷⁰ derivatives on a plasmid. Lonetto et al. (36) showed that in the absence of IPTG induction the ⁷⁰ derivatives were produced from these plasmids at a level that is only slightly higher than that of wild-type ⁷⁰; hence, only about 50% of the RNAP is expected to contain non-wild-type ⁷⁰. The strains also carried a wild-type rha locus at the normal chromosomal location and a single-copy  specialized transducing phage carrying a translational fusion of the rhaBAD or rhaT promoter with lacZ. The promoter fusions were either full length and included the binding sites for both the CRP and RhaS activators or truncated and included only the binding site for RhaS (Fig. 1). Deletion of the CRP-binding site from the fusions was preferable to deletion of the crp gene has been shown to decrease rhaBAD expression both due to the direct loss of CRP activation and to decreased rhaS expression from the CRP-dependent rhaSR promoter.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (a) Schematic representation of the rhaBAD and rhaT promoter regions. RNA polymerase and the two activator proteins CRP and RhaS are shown bound to DNA in their respective positions. (b) DNA sequences of the rhaBAD and rhaT promoter regions, extending from the 35 regions to the most upstream endpoint of the promoter fusions used in this work. The positions of the RhaS-binding sites are shown by everted arrows, and the positions of the CRP-binding sites are shown by inverted arrows. The 35 regions of each promoter are marked, and the upstream endpoints of promoter fusions with lacZ are identified by a "."

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Alanine substitutions in the 70 subunit of E. coli RNA polymerase analyzed at a full-length fusion of the rhaBAD promoter with lacZ [(rhaB-lacZ)110] (A) and at a truncated fusion of the rhaBAD promoter with lacZ [(rhaB-lacZ)84] (B). In each case, a strain carrying the indicated translational fusion as a single-copy  lysogen was transformed with a plasmid encoding either wild-type 70 or a derivative with a single alanine substitution at the positions indicated. -Galactosidase activity was measured from cultures grown in minimal medium with glycerol, L-rhamnose, and ampicillin. The x axis represents the 70 derivative. The y axis represents the -galactosidase specific activity for each 70 derivative as a percentage of the activity for wild-type 70. The wild-type activity in panel A was 402 Miller units, while the wild-type activity in panel B was 9 Miller units. Both were set to 100%.

⁷⁰ substitutions at rhaT. To develop a more general picture of the role of the C-terminal end of ⁷⁰ in activation by RhaS, we tested the ⁷⁰ library at rhaT promoter fusions. As shown in Fig. 1, the location of the RhaS and CRP-binding sites relative to the core promoter at rhaT is the same as that at rhaBAD. Assays of -galactosidase activity were modified as described in Materials and Methods to allow accurate measurement of the low activities expressed from the truncated rhaT fusion. At the full-length rhaT fusion that included both the RhaS and CRP-binding sites [(rhaT-lacZ)133], L595A was slightly defective, but none of the other substitutions were defective (Fig. 3A). When tested at a truncated rhaT promoter where only the RhaS-binding site was present [(rhaT-lacZ)84], five of the ⁷⁰ substitutions gave a value that was less than 80% of the wild-type activation (Fig. 3B). The largest defects were found with K593A and R599A, the same two substitutions that were defective at (rhaB-lacZ)84, while smaller defects were also seen with L595A, L598A, and R608A. One of the ⁷⁰ substitutions, S604A, activated to more than 170% of the level of the wild type. This finding is similar to the increased activation observed with two other ⁷⁰ substitutions at the araBAD promoter, although the magnitudes of the effects were greater at araBAD (36).

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Alanine substitutions in the 70 subunit of E. coli RNA polymerase analyzed at a full-length fusion of the rhaT promoter with lacZ [(rhaT-lacZ)133] (A) and at a truncated fusion of the rhaT promoter with lacZ [(rhaT-lacZ)84] (B). In each case, a strain carrying the indicated translational fusion as a single-copy  lysogen was transformed with a plasmid encoding either wild-type 70 or a derivative with a single alanine substitution at the positions indicated. -Galactosidase activity was measured in cultures grown in minimal medium with glycerol, L-rhamnose, and ampicillin. The x axis represents the 70 derivative; the y axis represents the -galactosidase specific activity for each 70 derivative as a percentage of the activity for wild-type 70. The wild-type activity in panel A was 2.5 Miller units; in panel B it was 0.079 Miller units. Both were set to 100%.

Substitution of negatively charged amino acids in RhaS. We noticed that the two ⁷⁰ residues that were defective at rhaBAD were positively charged amino acids (K593A and R599A). While additional substitutions were also found at rhaT, the same two positively charged amino acids were defective at this promoter as well. It has been proposed that ⁷⁰ residues identified using this library define interactions with the activator protein that overlaps the 35 region of that promoter (32, 33, 36). It has previously been shown that an overexpressed DNA-binding domain of AraC could weakly activate transcription (7), indicating that this domain of AraC is capable of transcription activation. As this is the conserved domain of AraC/XylS family proteins, it is likely that many other family members utilized this same domain for transcription activation. We hypothesized that if the ⁷⁰ residues that were defective at rhaBAD indeed defined an interaction with RhaS, then the RhaS amino acids involved in this interaction would be among the 12 negatively charged amino acids located within the DNA-binding domain of RhaS (Fig. 4A and B). We previously determined that one of these, D250, was involved in base-specific DNA contacts at rhaBAD (4). We therefore constructed alanine substitutions by site-directed mutagenesis at 10 of the remaining 11 positions. Based on the position of residue 191 on the crystal structure of MarA (46) (Fig. 4B), it seemed very unlikely to contact ⁷⁰, so when technical difficulties were encountered in this construction it was not further pursued.

View larger version (67K): [in this window] [in a new window]   FIG. 4.   Model of the C-terminal domain of RhaS bound to DNA based on the crystal structure of a MarA-DNA complex (44). (A) "Front" view of RhaS C-terminal domain (white) in a space-filling model with the negatively charged residues highlighted and numbered. DNA is shown in a stick model and is colored cyan. RhaS residues (in red) were defective at both the rhaBAD and the rhaT promoters, while residues in orange were either not defective, were defective at only one promoter, or were not tested (D250 and D191). In this view the N-terminal subdomain of RhaS is on the left and the C-terminal subdomain is on the right. The approximate position of the 35 region of the promoter is shown as a gray bar. (B) Same as panel A, except rotated around the vertical axis by approximately 180° to give the "back" view (i.e., the N-terminal subdomain is on the right, and the C-terminal subdomain is on the left). (C) A model of the C-terminal region of 70 (residues 550 to 613, orange, based on the DNA-binding domain of NarL) has been added to the RhaS C-terminal domain model. RhaS is in the same view as in panel A, but only the RhaS residue 241 is highlighted in red. The 70 residue 599 is highlighted in violet. (D) Same as panel C, but rotated by somewhat less than 90° around the vertical axis. The modeling of 70 onto the MarA-DNA complex was performed using the program Insight II, and panels A through D were drawn using RasMol version 2.6 for the Macintosh.

RhaS substitutions at rhaBAD. We first tested the substitutions in negatively charged amino acids of RhaS at the rhaBAD promoter. At (rhaB-lacZ)110, two of the RhaS substitutions (E181A and D274A) showed slight defects of about 75% of the level of wild-type activation (Fig. 5A). At the truncated rhaBAD promoter fusion, (rhaB-lacZ)84, six of the alanine substitutions in RhaS were defective (Fig. 5B). E181A showed the greatest defect at 28% of the level of wild-type activation, while the other five defective substitutions activated to 56 to 68% of the wild-type level. It is also interesting to notice that the substitution at E236 resulted in a level of 279% of the wild-type activation at this truncated promoter but was not significantly different than wild-type at the full-length (rhaB-lacZ)110 fusion. This is similar to the increased activation by two of the ⁷⁰ substitutions (E591A and R596A) when tested at araBAD in the absence of CRP (36). E261A also resulted in greater than wild-type activation of (rhaB-lacZ)84, in this case to 166% of the wild-type level.

View larger version (56K): [in this window] [in a new window]   FIG. 5.   Alanine substitutions in RhaS analyzed at a full-length fusion of the rhaBAD promoter with lacZ [(rhaB-lacZ)110] (A) and at a truncated fusion of the rhaBAD promoter with lacZ [(rhaB-lacZ)84] (B). In each case, a strain carrying the indicated translational fusion as a single-copy  lysogen was transformed with a plasmid encoding either wild-type RhaS or a derivative with a single alanine substitution at the positions indicated. -Galactosidase activity was measured from cultures grown in minimal medium with glycerol, L-rhamnose, and ampicillin. The x axis represents the RhaS derivative. The y axis represents the -galactosidase specific activity for each RhaS derivative as a percentage of the activity for wild-type RhaS. The wild-type activity in panel A was 453 Miller units for all of the assays except for E261A, where the wild-type activity was 204 Miller units, and in panel B it was 9.4 Miller units for all of the assays except for D241A, where the wild-type activity was 9.3 Miller units, and E261A, where wild-type activity was 3.9 Miller units. The activity in the case of E236A in panel B (marked with an asterisk) was 279%, but is drawn off the scale to avoid compression of the other values. The wild-type activity was set to 100%.

RhaS substitutions at rhaT. The same substitutions of negatively charged residues of RhaS were also tested for activation of rhaT. At the full-length rhaT promoter fusion [(rhaT-lacZ)133], four of the substituted RhaS proteins were slightly defective for activation (Fig. 6A). Each of the substitutions at positions E181, D182, D241, and D274 activated to 62 to 70% of the wild-type RhaS. Three of those four substitutions (E181, D182, and D241) were also defective at the truncated rhaT fusion that lacked the CRP-binding site [(rhaT-lacZ)84] (Fig. 6B). The defects of these substitutions at the truncated promoter were much more severe and resulted in only about 10% of the wild-type activation. Interestingly, the substitution at D274 was not defective at the truncated promoter. Two additional substitutions were somewhat defective at the truncated promoter but not at the full-length promoter (D186A and E187A).

View larger version (62K): [in this window] [in a new window]   FIG. 6.   Alanine substitutions in RhaS analyzed at a full-length fusion of the rhaT promoter with lacZ [(rhaT-lacZ)133] (A) and a truncated fusion of the rhaT promoter with lacZ [(rhaT-lacZ)84] (B). In each case, a strain carrying the appropriate translational fusion as a single-copy  lysogen was transformed with a plasmid encoding either wild-type RhaS or a derivative with a single alanine substitution at the positions indicated. -Galactosidase activity was measured from cultures grown in minimal medium with glycerol, L-rhamnose, and ampicillin. The x axis represents the RhaS derivative. The y axis represents the -galactosidase specific activity for each RhaS derivative as a percentage of the activity for wild-type RhaS. The wild-type activity in panel A was 1.38 Miller units for all of the assays except for with E261A, where the wild-type activity was 0.34 Miller units, and in panel B was 0.048 Miller units for all of the assays except for with E261A, where the wild-type activity was 0.027 Miller units. The wild-type activity was set to 100%.

Combination of RhaS and ⁷⁰ substitutions. We next wished to combine the RhaS and ⁷⁰ substitutions to test for evidence of interactions between combinations of alleles. We recombined the defective rhaS alleles into the normal chromosomal rhaS locus using the gene replacement strategy of Murphy (41). In this procedure an E. coli strain carries phage  recombination genes and, as a result, is capable of high-frequency replacement of chromosomal genes with alleles carried on PCR-generated DNA fragments. In the original description of this method, the recombined alleles could be identified by positive selection (for example lacZ::kan). Using a rhaB-lacZ fusion strain background, we were able to identify replacements of an in-frame deletion of rhaS with our partially functional rhaS alleles by screening for tiny blue colonies amid a lawn and so did not require positive selection (see Materials and Methods).

View larger version (44K): [in this window] [in a new window]   FIG. 7.   Combinations of RhaS and 70 alanine substitutions at (rhaB-lacZ)84. aRhaS substitutions were tested in combination with either 70 K593A (A) or 70 R599A (B) at the (rhaB-lacZ)84 fusion. The -galactosidase specific activity for each combination is represented as a percentage of the activity found for the combination of wild-type RhaS and wild-type 70, which was 9.1 Miller units and was set to 100% for both graphs.

View larger version (51K): [in this window] [in a new window]   FIG. 8.   Combinations of RhaS and 70 alanine substitutions at (rhaT-lacZ)84. aRhaS substitutions were tested in combination with either 70 K593A (A) or 70 R599A (B) at the (rhaT-lacZ)84 fusion. The -galactosidase specific activity for each combination is represented as the percentage of the activity found for the combination of wild-type RhaS and wild-type 70, which was 0.18 Miller units and was set to 100% for both graphs.

Modeling of RhaS interaction with ⁷⁰. There are currently structures available for the DNA-binding domain of two AraC/XylS family proteins, MarA and Rob (31, 46). The C-terminal domain of RhaS shares 24% identity and 46% similarity with MarA and 31% identity and 45% similarity with Rob. According to Kwon et al., the main chain atoms of the conserved portions of the MarA and Rob structures are extremely similar, with a root mean square deviation of 0.9 Å (31), suggesting that modeling of RhaS residues onto either structure would give nearly the same result. The only major difference between the MarA and Rob structures is that MarA makes base-specific contacts with DNA using both of its helix-turn-helix motifs, while Rob only makes base-specific contacts with its N-terminal helix-turn-helix motif (31, 46). As we have evidence that both helix-turn-helix motifs of RhaS make base-specific contacts with DNA (4), RhaS was modeled based on the structure of the MarA-DNA complex (Brookhaven Data Bank file 1BLO) (Fig. 4) (46).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Activation by RhaS requires amino acids near the C-terminal end of ⁷⁰. Two residues near the C-terminal end of ⁷⁰, K593A and R599A, were found to be important for activation at lacZ fusions with both the truncated rhaBAD and rhaT promoters (Fig. 2B and Fig. 3B). Other work has shown that none of these ⁷⁰ substitutions are generally defective for transcription (32, 33, 36). These truncated promoters have binding sites for only one activator protein, RhaS. Hence, residues K593 and R599 in ⁷⁰ are apparently required for transcription activation by RhaS and might be involved in direct contacts with RhaS.

Negatively charged residues in RhaS important for activation. We reasoned that ⁷⁰ K593A and R599A might define interactions with RhaS and, if so, that the partner residues in RhaS would probably be negatively charged. Upon substitution of most of the Asp and Glu residues in the C-terminal domain of RhaS to Ala, we found that E181A, D182A, D186A, and D241A were defective at both the truncated rhaBAD and rhaT promoters (Fig. 5B and Fig. 6B). When modeled on the structure of MarA (46), the positions of these residues of RhaS suggest a possible face of RhaS that could interact with ⁷⁰ (Fig. 4A). RhaS E181, however, aligns with a residue on MarA, where alanine substitution resulted in a severe defect both at promoters where MarA binds overlapping the 35 region and at promoters where MarA binds further upstream (21), suggesting that this residue may have a role other than interaction with ⁷⁰. We cannot rule out that some of these RhaS residues are defective due to DNA-binding defects. We would argue, however, that the evidence for residue D241, in particular when in combination with substitutions in ⁷⁰ (Fig. 7 and 8 and see below), argues that the defect caused by at least this substitution is not due to a DNA-binding defect.

Genetic evidence for contacts between RhaS and ⁷⁰. If residues within two proteins are involved in direct protein-protein contacts with each other, than one would expect that substitution of either one or both of the residues might have the same phenotype (in this case, the same defect in transcription activation). It is also possible, however, that one or both of the residues will have secondary effects on protein folding or stability and therefore would have a larger overall effect on transcription activation. In this case, substitution of both of the residues involved in a contact would be expected to have the same defect as that of the single residue with the greater defect. On the other hand, the combination of two substitutions that do not define a direct protein-protein contact would be expected to have a defect that was greater than either of the individual substitutions. We have used this reasoning to analyze the combination of substitutions in ⁷⁰ and RhaS to determine whether any of the residues might define a protein-protein contact that might contribute to transcription activation.