Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain David Tropel1* and Jan R van der Meer1,2 Process of Environmental Microbiology and Molecular Ecotoxicology, Swiss Federal Institute for Environmental Science and Technology (EAWAG), Dubendorf, Switzerland ă Department of Fundamental Microbiology, University of Lausanne, Switzerland Keywords Pseudomonas azelaica; r54-dependent transcriptional activators; XylR family Correspondence J R van der Meer, Department of Fundamental Microbiology, Batiment de Biologie, University of Lausanne, CH 1015 Lausanne, Switzerland Fax: +41 21 6925605 Tel: +41 21 6925630 E-mail: Janroelof.vandermeer@unil.ch *Present address M E Muller Institut-Biozentrum, ă Klingelbergstrasse 70, CH-4056 Basel, Switzerland (Received 25 November 2004, revised 27 January 2005, accepted 10 February 2005) doi:10.1111/j.1742-4658.2005.04607.x In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the r54-dependent PhbpC promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences Here, the HbpR–DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR ‘protomer’ in the oligomeric complex is impaired whilst leaving the other contacts intact An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints Wild-type HbpR was unable to activate transcription from the hbpC promoter when DA-HbpR was expressed in the same cell, suggesting the formation of (repressing) heterooligomers This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR The metabolism of 2-hydroxybiphenyl (2-HBP) in Pseudomonas azelaica HBP1 is initiated by enzymes encoded by the hbpCAD genes [1–4] The main regulator for hbpCAD expression is the HbpR protein, which, upon exposure to 2-HBP, activates transcription from promoters located upstream of hbpC (PhbpC) and hbpD (PhbpD) (Fig 1A) [4,5] HbpR is a member of the r54-dependent family of enhancer binding regulators (EBR), more specifically related to XylR and DmpR, regulators of xylene and phenol metabolism in P putida, respectively [4,6,7] EBRs control isomerization of the closed complex between r54RNA polymerase holoenzyme (RNAP) and the promoter DNA, to the open form [8–11] Isomerization requires ATP hydrolysis by the regulator, an activity that localizes in its strongly conserved central Abbreviations 2-HBP, 2-hydroxybiphenyl; CBP, calmodulin-binding protein; EBR, enhancer binding regulator; EMSA, electrophoretic mobility shift assay; RNAP, RNA polymerase holoenzyme; UAS, upstream activating sequence 1756 FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS D Tropel and J R van der Meer A B Fig (A) Genetic organization of the hbp genes in Pseudomonas azelaica strain HBP1 Open and grey bars (some as arrows) depict the orientation and the size of the genes; the solid line indicates noncoding DNA The two HbpR-regulatable promoters are indicated schematically with a small triangle and a ‘plus’; the location of the different upstream activation sites (UASs) (C-4 ⁄ C-3, C-2 ⁄ C-1 and D2 ⁄ D-1) is indicated by small black pillars HbpR is depicted schematically as a hexamer Enlarged is the operator region bound by HbpR within the hbpC promoter The arrows within the sequence point to two imperfect palindromic sequences (UASs C-1 and C-2) The grey boxes (R11, etc.) indicate the regions protected in DNaseI footprints by HbpR [19] and are used as such also in other figures Sequence numbers refer to the locations of the transcriptional start site of hbpC (B) Alignments of the sequences of three HbpR-binding sites within the hbp gene region [19] The lane ‘cons-1’ indicates the strictly conserved residues in the proximal UASs, ‘cons-2’ indicates those in the distal UASs, and ‘cons1+2’ indicates residues conserved in all six UASs (or C) domain [7,12,13] The ATPase activity of the C-domain is repressed by the regulator’s N-terminal or A-domain, but released in response to a specific signal [6] For regulators such as XylR, DmpR and HbpR, the signal is a chemical compound interacting directly with the A-domain [4,14,15] The complete signalling ⁄ derepression ⁄ activation pathway seems paralleled by cyclical changes in protomer configuration and the EBR–RNAP–DNA complex The regulatory protein apparently cycles between dimers in solution and oligomers on the DNA [16] Crystallization data of an NtrC1 single C-domain variant clearly showed a ringshaped heptameric configuration [17], but hexameric structures have also been observed [18] FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS HbpR-binding site Like all EBRs, HbpR exerts its control by binding to a specific operator sequence upstream of the promoter [19] The operator sequence for EBRs is usually named the ‘upstream activating sequence’ (UAS) and is characterized by two imperfect palindromes of 16 bp and a spacing of between 29 and 42 bp between their centres (Fig 1A) [20] The regions covered by XylR on the Pu and of HbpR on the PhbpC promoter in DNaseI footprinting experiments were slightly larger than the sequences of the palindromes [19,21], but the exact contribution of nucleotides in the binding site contacted by the proteins is not known The current hypothesis of hexameric EBR oligomers on their operator DNA is not congruent with the idea regarding the configuration of the binding site A binding site of two palindromes suggests that the regulator’s configuration would be formed by two dimers, a tetramer or any larger-order structure in which not all subunits contact the DNA Strangely enough, the distance between the two palindromes can be increased by one helical turn, but not by half a turn, without losing activation and binding affinity [19–22] In DNaseI footprinting analysis of HbpR on the hbpC operator, six distinct protected regions are visible [19] (Fig 1A), which would fit a hexameric EBR symmetry in which each protomer contacts the DNA The objectives of this work were to identify critical motifs in the binding site for HbpR and to examine the necessity of the sensing A-domain of HbpR for DNA binding The role of individual nucleotides and nucleotide motifs was characterized by site-directed mutagenesis and affinity binding by purified HbpR In several cases, DNaseI footprints were conducted to confirm the binding site contacts Additionally, we determined whether the A-domain of HbpR is important in forming the oligomeric structure and for DNA binding, by cloning an hbpR gene devoid of the A-domain, purifying this protein, and analyzing its DNA-binding characteristics and its activation capacity of the hbpC promoter in Escherichia coli Results Mutagenesis of the HbpR-binding sites Sequence alignment of the UASs found in the HbpR operators in the hbpC and hbpD promoters defined a set of strictly conserved nucleotides: GnnTTnAnn AnnTnnTnA (Fig 1B) When only the proximal or the distal UASs were compared, additional conserved nucleotides were found, which differed slightly between the proximal and the distal sites Some were located 1757 HbpR-binding site D Tropel and J R van der Meer Fig DNA sequence of the different mutants and the HbpR concentration required for 50% binding in electrophoretic mobility shift assays [expressed as nM calmodulin-binding protein (CBP)–HbpR fusion protein] Conserved residues in the proximal or in the distal upstream activation sites (UAS) are in bold (line ‘cons’, from Fig 1) The residues conserved among DmpR-, HbpR-, TouR- and XylR-binding sites are depicted by an asterix above the top sequence Only the mutated nucleotides are presented (for the upper strand), with the appropriate mutant names indicated on the right Arrows point at palindromic symmetry in the distal UASs Strictly conserved residues in both proximal and distal UASs (‘cons1+2’, Fig 1) are repeated at the bottom The maximum relative induction level of HbpR-mediated luciferase expression in Escherichia coli from the mutant promoters compared to the wild-type promoter was calculated on the basis of previous results [23] outside the UASs (Fig 1A) Five of the distally and seven of the proximally conserved nucleotides also occurred in palindromes in the XylR, DmpR, and TouR promoters (Fig 2) Instead of separately mutating every single nucleotide in the binding sites, it was decided to construct group-wise mutations with the idea that this would destabilize symmetrical structures more easily Some of these groups were based on sequence motifs occurring in the Pu promoter (e.g mutants 1–7, Fig 2) All mutant binding sites were then tested for affinity binding in electrophoretic mobility shift assays (EMSAs) with increasing concentrations of calmodulin binding protein (CBP)–HbpR (or His6–HbpR) (Fig 3A) (0–1200 nm), upon which the concentration at which 50% binding occurred was interpolated (Fig 3B) A first set of mutations consisted of changing the conserved ‘T’ and ‘A’ residues in the distal UAS (Fig 2) Mutants 12 and 15, with two mutations, could still be bound by HbpR but with about twofold less affinity, as determined by EMSA (Fig 2) More severely, HbpR lost about fourfold binding affinity for mutants 13 and 14, with four and six conserved residues mutated This suggests that the conserved 1758 residues indeed played an important role in contacting HbpR at critical positions That the rigidity of the binding region was not influenced by the change from four AT to four or six GC, was shown by mutants and (each with 3· GC and five mutations) Both mutants and were bound equally well by HbpR under the conditions of EMSA Changing, more or less randomly, other sets of bp (mutants 10, 11 and 16) did not influence the capacity to be bound by HbpR Strangely enough, however, mutations in residues, other than the conserved residues, resulted in the same loss of binding affinity as observed for mutants 12, 13 and 15 Gradually changing the distal UAS to the sequence of the Pu promoter (mutants 1–4) very quickly resulted in a loss of HbpR-binding affinity, which culminated in a fourfold loss of binding affinity in mutant The fourfold loss of binding affinity did not translate to a fourfold decrease of HbpR-mediated expression of the luxAB reporter genes from the hbpC promoter with the same type mutation in E coli [23] In fact, the mutant promoter still allowed activation to about 72% of the maximum level observed for the wild-type promoter (Fig 2) Furthermore, mutations in the proximal binding site, which did not per se FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS D Tropel and J R van der Meer A HbpR-binding site Combining mutations in the distal and the proximal site very drastically reduced the binding affinity by HbpR and also by HbpR-mediated luciferase expression in E coli (mutants 72, 45, 46 and 47, Fig 2) DNaseI footprinting analysis B Fig Examples of several electrophoretic mobility shift assays (EMSAs) with mutated operator fragments and calmodulin-binding protein (CBP)–HbpR, and representation of the calculation of the half-binding concentration (A) EMSAs performed with DNA fragments containing the wild-type upstream activation sites (UASs) C-1 ⁄ C-2 (WT) sequence, or different mutants (as indicated at the top of each panel), in the presence of increasing concentrations of HbpR fusion protein (0–1200 nM) Assignment of the UAS mutant names corresponds to those given in Fig (B) Graphical representation of the relative densities of the free DNA measured at increasing concentrations of HbpR The percentage of free DNA was calculated from densitometric measurements of the radiolabeled bands as the density of the remaining unbound operator fragment at any HbpR concentration divided by that without HbpR (first lane of each panel) The dashed arrow points to the graphical interpolation used to determine the concentration at which 50% binding occurred A summary of all values is presented in Fig influence the conserved residues, changed the binding affinity for HbpR (mutants 5–7, Fig 2) Mutants 3, 8, and 11 disrupted the twofold symmetry in the distal UAS, but without direct effect on binding affinity Mutant was designed to maintain twofold symmetry, yet binding affinity was reduced twofold Finally, mutant 17 contained a set of four subsequent mutations, but outside the palindromes Still, binding affinity was reduced more than twofold FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS In order to reveal whether the differences in binding affinity were caused by changes in interaction patterns, DNaseI footprinting of CBP–HbpR was performed on mutant operator types 2, 4, 5, 7, 72 and 45 (Fig 4) Interestingly, this showed that a loss of binding affinity correlated to a deterioration of binding contacts in only those regions with new mutations, not throughout the whole operator region For example, HbpR binding to the mutant operator behaved like the wildtype operator as far as the UAS C-1 region was concerned (Fig 4) Binding to the UAS C-2 region (in which the mutations had been placed), was impaired both at the 3¢ end of the central regions R22 and R23, whereas the region R21 and the 5¢ part of R22 were again protected, as for the wild-type region This suggested a weaker interaction within this particular region, but retainment of the overall binding complex (although with weaker affinity, Fig 2) In contrast, the DNaseI footprint left by HbpR on the mutant type operator showed normal protection of UAS C-2, but almost no protection of the R13 region and the 3¢ end of R12 in UAS C-1 (Fig 4) The region R11 was again normally protected, showing that the mutated bp in operator type must have affected the binding of one particular set of contacts only, although still allowing overall binding The combination of mutation types and (operator type 45), on the other hand, not only totally abolished the binding affinity (Fig 2), but resulted in almost no protection on any of the binding regions, except at very high HbpR concentrations (1000 nm) Similarly, mutant operators 2, and 72 were analyzed for DNaseI footprints left by HbpR (Fig 4) As far as could be determined, the protection pattern of HbpR on mutant type was not affected, except that protection of the UAS C-2 region occurred at a higher HbpR concentration than for mutant type Mutant type 7, like mutant 5, was impaired in binding of the R13 region Both of these patterns contributed to the loss of binding contacts of mutant 72 Construction of a DA HbpR derivative In order to study whether the A-domain was important for DNA binding, several HbpR derivatives (devoid of parts of their A-domain) were constructed One of these, the DA-HbpR variant (in plasmid 1759 HbpR-binding site D Tropel and J R van der Meer Fig DNaseI footprinting analysis of the binding of calmodulin-binding protein (CBP)–HbpR complex to the upstream activation sites (UASs) of C-1 ⁄ C-2 mutants (top strand only) The 229 bp 32P-end-labelled fragments containing UASs C-1 and C-2 with mutation type 4, type 5, type 45, type 7, type or type 72 were incubated with increasing concentrations of HbpR (0–400 nM) White boxes on the side of each footprint correspond to the numbered regions in Fig Grey boxes illustrate weakly protected regions When the protected region is shorter than that of the C-1 ⁄ C-2 sequence of wild-type UASs, the boxes are hatched pHB171), was created by starting the hbpR reading frame in the Q-linker, but adding a short affinity tag at the new N terminus to facilitate recovery of the protein from cell lysates after overproduction The tag chosen was again the CBP, which had already been used for purification of HbpR itself and has been shown previously not to disturb HbpR-mediated transcription activation [19] SDS ⁄ PAGE of the soluble cell extracts of E coli BL21 (pHB171) showed a protein of 43 kDa (gel not shown), which corresponds to the molecular mass of DA-HbpR (39 kDa) plus that of CBP (4 kDa) This protein band was not detected in extracts of E coli BL21 (pCAL-n-) We concluded that the fusion protein was correctly expressed in E coli DNA-binding characteristics of the DA-HbpR protein EMSA performed on the wild-type HbpR-binding site and increasing amounts of DA-HbpR or HbpR in fusion with CBP showed that whereas 200 nm HbpR was sufficient to bind all the operator DNA (Fig 5A), even 1200 nm DA-HbpR bound hardly any operator DNA No proteinỈDNA complex was observed (as for HbpR), although a small fraction of the labelled DNA remained in the well at higher concentrations of DA-HbpR On DNaseI footprint analysis, however, a progressive protection was seen, of the six regions identified previously (R11–R23), with increasing amounts of both HbpR and DA-HbpR (Fig 5B) The 1760 protection pattern of the two proteins proved that the derivative devoid of the A-domain still correctly interacted with the six regions, although protection at R11 and R12 took place at higher DA-HbpR concentrations than for HbpR Hence, removing the A-domain of HbpR did not impair regulator interactions with the operator DNA per se, but affected the protein–DNA complex stability in EMSA Activation of the hbpC promoter by CBP–DA-HbpR Activity of the CBP–DA-HbpR protein was verified in vivo by means of plasmid pHB172, which bears the sequence encoding the truncated CBP-tagged protein and a PhbpC::luxAB fusion in E coli E coli expressing the full-length CBP–HbpR (plasmid pHB164, in which hbpR is expressed from exactly the same promoter as in pHB172, Fig S1), showed induction of luciferase from the hbpC promoter at increasing concentrations of 2-HBP, with a maximum induction factor of about 10-fold (Fig 5C) Surprisingly, however, E coli (pHB172) displayed a luciferase activity of about 500-fold lower, albeit with a slight increase at 2-HBP concentrations of 1, 2, 10 and 20 lm (Fig 5C) As we can assume that the expression level of CBP–DAHbpR was not different from that of CBP–HbpR, because both genes were expressed from exactly the same promoter in E coli, this meant that CBP–DAHbpR did not activate gene expression We then tested FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS D Tropel and J R van der Meer HbpR-binding site A C B D Fig Transcriptional and DNA-binding activities of HbpR and the HbpR derivative deleted of its A-domain (DA-HbpR) (A) Electrophoretic mobility shift assays (EMSAs) of a DNA fragment containing upstream activation sites (UASs) C-1 ⁄ C-2 incubated with calmodulin-binding protein (CBP)–HbpR (lanes 2–5) or CBP–DA-HbpR (lanes 7–10) Lanes and 6, no CBP fusion protein added; lanes and 7, 200 nM CBP fusion protein; lanes and 8, 400 nM CBP fusion protein; lanes and 9, 800 nM CBP fusion protein; lanes and 10, 1200 nM CBP fusion protein (B) DNaseI footprinting analysis of CBP–HbpR and CBP–DA-HbpR binding to the UASs C-1 ⁄ C-2 region A 229 bp 32P-end-labelled fragment containing the UAS C-1 ⁄ C-2 was incubated with increasing concentrations of fusion protein (0–400 nM) The boxes indicate the regions protected from DNaseI digestion upon the addition of CBP–HbpR and CBP–DA-HbpR (numbering corresponding to Fig 6) (C) Transcription activation from PhbpC in the presence of different concentrations of 2-hydroxybiphenyl (2-HBP) in Escherichia coli containing plasmid pHB164 bearing cbp–hbpR (grey bars) or in pHB172 bearing cbp–DA-hbpR (black bars) (D) Transcription activation from PhbpC in the presence of different concentrations of 2-HBP in E coli carrying pHYBP124 (expressing wild-type HbpR) plus one of the following plasmids: pHYBP103 (white bars, luxAB genes under control of the hbpC promoter); pHB164 (grey bars); or pHB172 (black bars) Relevant plasmid constructs are shown at the top of the diagrams Note the log scale of the light emission values Instrument background value:  20–50 units The values represent arithmetic averages from light emissions measured, after an induction time of 120 min, on triplicate induction assays of two independently grown cultures whether 2-HBP-dependent activation could be restored by complementation with a wild-type HbpR expressed from another plasmid in the same cell (pHYBP124, Fig 5D) The combinations pHYBP124, pHB164 (wild-type HbpR plus CBP–HbpR) and pHYBP124, pHYBP103 (wild-type HbpR plus plasmid with the luxAB genes under control of the hbpC promoter) were equally inducible with 2-HBP and to an induction factor of approximately tenfold, as observed with pHB164 alone Even more surprinsingly, however, the FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS combination of wild-type HbpR (pHYBP124) and CBP–DA-HbpR (pHB172) in the same cell resulted in no light emission from the cultures (Fig 5D) Several other HbpR variants were constructed with differently sized deletions in the A-domain, which were tested in combination with pHYBP103 in the same cell However, none was capable of 2-HBP-dependent luciferase induction (data not shown) A fusion of the DA-hbpR gene with the gene fragment for the A-domain of XylR was also constructed, which was 1761 HbpR-binding site again incapable of activating the hbpC promoter from pHYBP103 in the presence of 2-HBP or m-xylene, and displayed a low basal level of luciferase activity (not shown) Thus – contrary to similarly truncated XylR, TouR and DmpR derivatives [24–26] – DA-HbpR is not a constitutively active protein, but rather inactive D Tropel and J R van der Meer A Discussion From all the mutations created in UAS C-1 ⁄ C-2, we can conclude that the HbpR-binding site (and, for that matter, also similar binding sites for other EBRs) must have an intrinsic resilience against punctual changes Impairment of a few interactions did not disturb the overall protein–DNA complex until a point at which insufficient interactions remained to stabilize the complex Most mutations, in fact, reduced the binding affinity by two- to fourfold, which still permitted the correct binding of CBP–HbpR and allowed HbpRmediated activation of a reporter gene fused to the mutant operator ⁄ promoter [23] As expected, changing conserved nucleotide motifs in the binding sites (like in mutants 13 and 14) reduced the affinity of HbpR binding and thus one would tend to stress their importance for HbpR contacting the DNA However, of surprise was that changes in nonconserved basepairs (like mutants 2–4) could also cause the same decrease in HbpR binding This means that there must be additional global structures or motifs that are important for achieving optimal HbpR binding Mutations outside the directly conserved nucleotides (like mutant 17) were also found to be important in the binding process Only when mutations in both palindromic regions were combined did a complete loss of binding contacts occur (mutants 45, 46 and 47) DNaseI footprint analysis allowed a further refinement to be made to establish the loss of binding affinity in some of the mutants Although not all mutants were screened with HbpR in DNaseI footprint analysis, it was found that mutations causing a decrease in binding affinity resulted from a loss of interactions in one or two particular regions, but not from an overall decrease in binding contacts This would be consistent with a model in which the binding contacts in the (six) different regions (R11–R23, Fig 6) are the result of six different HbpR protomers contacting the DNA If mutations cause an unfavourable local structure, or result in incorrect nucleotide bp to which HbpR cannot bind, it is easy to envision that only this region would become more prone to DNaseI attack Within the limits of the resolution of the DNaseI mapping, it was also observed that loss of protection always extended in two regions (like R12 and R13), except in 1762 B * * * * * * * Fig Model for the upstream activation sites (UASs) C-2 ⁄ C-1 operator DNA wrapping around a hypothetical HbpR hexamer (A) The DNA is bent around six HbpR protomers (labelled A–F) Nonprotected and hypersensitive regions in previous HbpR DNaseI footprints are depicted as lightning arrows [19] (B) Nucleotide sequence corresponding to the C-2 ⁄ C-1 region of UASs, with protected nucleotides encircled in grey and the nonprotected nucleotides in white Positions of the HbpR monomers are boxed (only one of two possible positionings is shown) Every monomer ineracts approximately within one helical turn, leaving a few unprotected residues (probably those located on the other side of the DNA helix) mutant As there were unprotected residues in the HbpR-binding site (showing up as cleaved products) at about every helical turn, this suggests that HbpR monomers must be contacting two sites within one helical turn, leaving one or a few residues exposed (Fig 6) As the pattern of unprotected nucleotide bp occurs at every helical turn in DNaseI footprints (across a region of  50 bp), as mutants were constructed which decreased binding in two adjacent regions, and because mutants outside the conserved palindrome (mutant 17) also decreased HbpR-binding affinity, we propose a hypothetical model in which a disc-shaped hexameric HbpR complex contacts five helices, which is the simplest model for using to explain our results (Fig 6B) An important presumption in this model is, of course, the crystal structure of the NtrC1 C-domain [17], the model for PspF [18] and previous findings that EBRs oligomerize on the DNA [7,16,27] This model can also explain HbpR binding to the hbpD promoter, which has a larger spacing between the palindromic sequences For this promoter, the DNA loop would extend further outwards between monomers C and D, leading to weaker binding but still sufficient for activation In fact, this has been observed previously [19,20] The alternative explanation of a higher FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS D Tropel and J R van der Meer order structure, in which only four subunits would fix the target DNA, seems less probable on the basis of our results, although, for NtrC from Salmonella typhimurium, an octamer structure binding two small sequences of DNA, each containing a pair of UASs, was proposed [28] However, it is clear that this model will need further confirmation via structural information on the HbpR–DNA complex If EBRs form such strong oligomers, can something general be said about whether the oligomers form spontaneously in the absence of their operator DNA, or if the oligomerization is dependent on the DNA? For DmpR it has been established that oligomers form in the absence of the operator DNA, but need the presence of chemical inducers and ATP [27] HbpR does not seem to follow this rule, as EMSAs performed with purified CBP– HbpR showed the same protein–DNA complex in both the absence and presence of ATP or 2-HBP [19] This suggests that HbpR can self-associate on its operator DNA in vitro without effector recognition or ATP binding The HbpR protein even seems to be able to form an oligomeric complex capable of protecting the operator DNA in the absence of its sensing A-domain, which can be concluded from our results with a purified DA-HbpR preparation (Fig 5B) Hence, our findings suggest that the N-terminal domain of HbpR stabilizes the oligomeric form in vitro, but an A-deleted derivative may still assemble as an oligomeric form and contact the operator DNA correctly, similarly to that observed previously for DA-XylR, DA-DmpR and DA-TouR [21,25,27] Contrary to XylR, DmpR and TouR derivatives deleted of their A-domain, which activated transcription in the absence of inducer [25,26,29], a similar HbpR derivative (DA-HbpR) was unable to activate transcription from the hbpC promoter (Fig 5C) As measurable light emission occurred in those cultures ( 2000–6000 units) we have to assume that the hbpC promoter is still transcribed by RNA polymerase at a low basal rate In contrast, no light output at all was observed in culture expressing both wild-type HbpR and CBP–DA-HbpR, and rendered the hbpC promoter completely inactive (Fig 5D, 20–50 units is instrument background) From the control experiments we conclude that both proteins are expressed in E coli and that wild-type HbpR alone efficiently activates the hbpC promoter in trans Previous results have shown that even in the absence of any HbpR-binding sites and HbpR, luciferase expression takes place from the hbpC promoter in E coli to a level of  5000 light units [20] and, thus, we find the hbpC promoter to be repressed in strains expressing both hbpR and cbp– DA-hbpR As both CBP–HbpR and CBP–DA-HbpR FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS HbpR-binding site complexes alone attach to the UASs and allow expression from the hbpC promoter, this must mean that when both are expressed in the same cell there is no formation of homogenous HbpR oligomers which compete as such with homogenous CBP–DA-HbpR complexes on the binding site (in which case some light emission would occur), but rather formation of a hetero-oligomeric complex of HbpR and CBP–DAHbpR This hetero-oligomeric complex somehow acts as a very strong repressor on expression from the hbpC promoter, perhaps because it has a very high affinity for the UAS C-1 ⁄ C-2 binding sites, or can no longer dissociate in individual dimers as part of the normal activation cycle [16] We recently postulated the presence of active heterocomplexes between HbpR and XylR, when expressed in the same cell [23] In contrast, but in agreement with the hypothesis that activation of HbpR must proceed differently from that of XylR, a fusion protein between DA-HbpR and the A-domain of XylR was found to be incapable of activating the hbpC promoter (not shown) Hence, we conclude that the A-domain of HbpR is needed to activate the ATPase activity of the C-domain, rather than to repress it (as for NtrC1, DctD, XylR, TouR and DmpR) [17,25,26,29,30] To date, such behaviour has only been observed for NtrC from S typhimurium [31] The repression mediated by the A-domain is correlated to the presence of a structured Q-linker between the A-domain and the C-domain [13,16, 17,32] Whereas EBRs such as NtrCST and HbpR not contain a structured linker, DctD, XylR, TouR and DmpR [13] In summary therefore, HbpR is the first characterized member of the XylR family to contain an A-domain that must control regulator activity in a positive manner Experimental procedures Strains and medium E coli DH5a [33] was used as host strain in routine cloning experiments and for luciferase expression studies E coli BL21 (DE3) pLysS (Stratagene) was used for overexpression and purification of HbpR E coli strains were grown at 25°, 30° or 37 °C on Luria–Bertani (LB) medium [33] When required, the medium was supplemented with ampicillin (100 lgỈmL)1) or chloramphenicol (25 lgỈmL)1) Recombinant DNA techniques DNA sequencing, plasmid DNA isolations, ligations, transformations and other DNA manipulations were carried out according to well-established procedures [33] Restriction 1763 HbpR-binding site endonucleases and other DNA-modifying enzymes were obtained from Amersham International plc, Roche Bio-chemicals and New England Biolabs Inc., and used according to the specifications of the manufacturer All PCR-generated binding-site mutations were verified by double-stranded template sequencing using a modified dideoxy-chain termination method [34] and primers that were labeled with the fluorescent dye IRD-800 at the 5¢-end, as described previously [35] Mutations of UASs C-1 ⁄ C-2 Promoter mutations were introduced by PCR using Pfu polymerase (Promega Corp.) and mutagenic overlapping primers, as described previously [23] Adequately sized PCR products were gel purified and cloned in pCR-Script from Stratagene, and sequenced on both strands to verify the accuracy of the introduced mutations The resulting mutations are shown in Fig For mutations constructed in both UASs, either pHB178 (containing mutant type 4) or pHB181 (containing mutant type 7) were used as template instead of pHYBP134 [4] with the wild-type hbpC promoter A list with all pHB plasmid names is available from the authors upon request Cloning of the DA-hbpR expression vector Plasmid pCAL-n- (Stratagene) was used for the production and purification of the HbpR protein deleted of its first 217 amino acids residues (DA-HbpR) in E coli The first 513 nucleotides of DA-hbpR were amplified by using the PCR on plasmid pHBP130 with primers hbpR5 (5¢-CGGCGGATCC.ATG.CAC.CCT.ATTCCCGATGAT), which introduces an ATG start codon in fusion with G651 of the hbpR open reading frame (Fig S1) and hbpR6 (5¢-CGGCGTAAAGATCCTCTCGGAAG) The PCR product was cloned in pGEM-T-easy (Promega), which, after transformation, resulted in plasmid pHB167 The complete DA-hbpR gene was then assembled as follows: a 1.090 kb HindIII ⁄ SalI fragment from pHB151 containing the remaining sequence of hbpR was used to replace a 325 bp fragment of pHB167 cut with HindIII and SalI (yielding pHB170) The 1.278 kb BamHI ⁄ SalI fragment from pHB170 containing DA-hbpR was cloned in plasmid pCAL-n- cut with BamHI and SalI (yielding pHB171) Plasmid pHB171 produced a DA-HbpR protein with an N-terminal CBP and a 12 bp thrombin tag (Fig S1) In vivo DA-HbpR activation To determine the in vivo activity of the CBP–DA-HbpR fusion protein, we measured activation of the luxAB genes, which were transcriptionally fused to the hbpC promoter, as described previously [4] Hereto, the CBP–DA-HbpR gene fusion of pHB171 was completed with the 2.75 kb 1764 D Tropel and J R van der Meer BamHI fragment of plasmid pHYBP109 (containing the native hbpRC intergenic region fused to the luxAB genes) This BamHI fragment was inserted at the single BglII site of plasmid pHB171 After transformation, plasmids in which cbp–DA-hbpR was expressed from the native hbpR promoter were selected (yielding pHB172, Fig S1) Plasmid pHB164, containing the same luciferase reporter system under control of the cbp–hbpR gene, served as a positive control for 2-HBP-dependent luciferase activation, as hbpR expression is driven from the same promoter as for pHB172 Competition assays between HbpR and CBP– DA-HbpR were carried out by cotransforming two plasmids in E coli DH5a and examining 2-HBP-dependent luciferase induction from the hbpC promoter All strains contained the wild-type hbpR gene cloned in vector pACYC184 (pHYBP124) [5], cotransformed either with pHB164 or with pHB172 As a control we used the same E coli DH5a (pHYBP124) cotransformed with pHYBP103, which contains the hbpC promoter fused to the luxAB genes [4] To ensure that the plasmid pHB172 did not change during its culture in E coli, we repurified the plasmid after induction of the culture with 2-HBP, immediately transformed it to E coli (pHYBP124) and re-examined 2-HBP induction Luciferase activity was measured in a culture with an attenuance (D) of 0.4, after 120 of induction at 30 °C with different 2-HBP concentrations (1, 2, 20, 20 and 200 lm), as previously described [19] Cultures were grown on LB containing the necessary antibiotics, harvested by centrifugation (5 min, 1575 g, at room temperature) and resuspended in Mops medium [5.5 gỈL)1 of Mops free acid, 5.1 gỈL)1 of Mops sodium salt, 0.5 gỈL)1 of NaCl, gỈL)1 of NH4Cl, 0.06 gỈL)1 of Na2HPO4Ỉ2H2O, 0.05 gỈL)1 of KH2PO4, mm MgSO4, 0.1 mm CaCl2, 0.2% (w ⁄ v) glucose, pH 7] Purification of HbpR fusion protein, EMSAs and DNaseI footprinting Purification of fusion proteins of the CBP to HbpR (CBP– HbpR) and to DA-HbpR (CBP–DA-HbpR) were carried out as previously described [19] Additionally, the hbpR gene was cloned in vector pET15b (Novagen, VWR International Life Science, Lucerne, Switzerland) in order to produce a His6tagged HbpR protein Purification of the His6–HbpR fusion protein was performed on Ni-nitrilotriacetic acid columns, according to the instructions of the supplier (Qiagen AG) pCR-Script and pGEM-T-Easy-derived plasmids containing the native PhbpC or modified PhbpC promoters were used in EMSA and DNase I footprinting Hereto, the fragment containing the PhbpC promoter insert was amplified and labelled in the PCR by using primers hbpCC and the [32P]ATP[cP] phosphorylated primer hbpC6 [19] EMSAs were conducted with the labelled PhbpC fragments, and different concentrations of CBP–HbpR, His6–HbpR or CBP–DA-HbpR, as described previously [19] FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS D Tropel and J R van der Meer Synthetic oligonucleotides and chemicals Primers labeled at the 5¢ end with the fluorescent dye, IRD800, were purchased from MWG-Biotech GmbH (Ebersberg, Germany) All other primers were from Microsynth GmbH (Balgach, Switzerland) All other chemicals were of the highest grade commercially available Acknowledgements The authors would like to thank Mrs Alexandra Baumeyer for her help in the luciferase reporter assays References Kohler H-PE, Kohler-Staub D & Focht DD (1988) Degradation of 2-hydroxybiphenyl and 2,2¢-dihydroxybiphenyl by Pseudomonas sp strain HBP1 Appl Environ Microbiol 54, 2683–2688 Kohler H-PE, Schmid A & van der Maarel M (1993) Metabolism of 2,2¢-dihydroxybiphenyl by Pseudomonas sp strain HBP1: production and consumption of 2,2¢,3trihydroxybiphenyl J Bacteriol 175, 1621–1628 Schmid A (1997) Der metabolismus von 2-hydroxybiphenyl-verbindungen in Pseudomonas azelaica HBP1 PhD Thesis, Universitat Stuttgart, Stuttgart ă Jaspers MC, Suske WA, Schmid A, Goslings DA, Kohler HP & van der 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5463–5467 35 Ravatn R, Studer S, Zehnder AJB & van der Meer JR (1998) Int-B13, an unusual site-specific recombinase of the bacteriophage P4 integrase family, is responsible for chromosomal insertion of the 105-kilobase clc element of Pseudomonas sp strain B13 J Bacteriol 180, 5505– 5514 Supplementary material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4607/EJB4607sm.htm Fig S1 Genetic constructions of the cbp–hbpR and cbp–DA-hbpR plasmids Inserts were assembled as described in the main text Relevant restriction sites and the direction of transcription for the hbpR and the luxAB genes are indicated (A) (B) Detail of the region of the calmodulin-binding protein (CBP) fusions The original hbp sequence is shown in italics The sequence derived from the pCAL-n- plasmids is shown in upper case The positions of BamHI, BglII and NdeI sites correspond to those in (A) Boxes indicate schematically the positions of the CBP, thrombin and FLAGsites M001 corresponds to the first native Met codon of HbpR M217 corresponds to the newly introduced Met codon for DA-HbpR FEBS Journal 272 (2005) 1756–1766 ª 2005 FEBS ... motifs in the binding site for HbpR and to examine the necessity of the sensing A -domain of HbpR for DNA binding The role of individual nucleotides and nucleotide motifs was characterized by site- directed. .. of the hbpC promoter in Escherichia coli Results Mutagenesis of the HbpR- binding sites Sequence alignment of the UASs found in the HbpR operators in the hbpC and hbpD promoters defined a set of. .. (UAS) and is characterized by two imperfect palindromes of 16 bp and a spacing of between 29 and 42 bp between their centres (Fig 1A) [20] The regions covered by XylR on the Pu and of HbpR on the