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Pseudomonas: Biology and Diversity |
Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain1
Author for correspondence: Víctor de Lorenzo. Tel: +34 91 585 4536. Fax: +34 91 585 4506. e-mail: vdlorenzo{at}cnb.uam.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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54-dependent activator XylR of the TOL plasmid pWW0 of Pseudomonas putida when bound to the upstream activating sequences (UAS) of the cognate Pu promoter was examined by transmission electron microscopy (TEM). To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylR
A, with or without ATP or its non-hydrolysable analogue ATP
S, and the resulting complexes were visualized by TEM. The different types of DNA–protein association were analysed and a statistical study of the frequency of the various forms was made. ATP appeared to establish an equilibrium between different molecular associations, as well as major changes in the physical shape of the DNA–protein complexes. The formation of higher nucleoprotein structures frequently bearing DNA bends became manifest. Such complexes often engaged otherwise separated UAS-containing plasmids, indicating that the ATP-driven multimer included XylR molecules recruited in trans. Whilst ATP caused the different types of XylR–DNA complex to occur at quite balanced frequencies, ATPS appeared to displace the distribution predominantly towards the higher order forms. These data are compatible with the notion that each time ATP is hydrolysed the transcriptional activation complex is disassembled. Keywords: Pseudomonas putida, TOL plasmid, Pu promoter, sigma 54, enhancers
Abbreviations: TEM, transmission electron microscopy; UAS, upstream activating sequences
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Expression of the upper operon is driven by the 54-dependent promoter Pu which, like other promoters of this type, is activated at a distance by the enhancer-binding and toluene-responsive protein XylR (Abril et al., 1991 ; Pérez-Martín & de Lorenzo, 1996b ). This process also requires the integration host factor (Abril et al., 1991 ; de Lorenzo et al., 1991 ), a binding sequence for which is present within the intervening region between the upstream activating sequences (UAS) bound to XylR and the -12/-24 sequence motifs for 54-RNA polymerase holoenzyme (Fig. 1a). XylR belongs to the group of activators that act in concert with 54 and are generically known as the NtrC family, named after the most studied member of the group (North et al., 1993 ). All these proteins seem to share a common mechanism of activation of their cognate promoters when bound to the UAS. Also like these proteins, XylR has a three-domain structure (Drummond et al., 1986 ; Inouye et al., 1988 ). The N-terminal or A domain is a signal receptor module that interacts directly with the aromatic effector, for example substrates of the TOL upper pathway (Delgado & Ramos, 1994 ). This event releases the intramolecular repression that the A domain exerts in the protein, thus generating an active form of the regulator. As a consequence, protein variants deleted of the A domain are constituive and can activate transcription in vivo (Pérez-Martín & de Lorenzo, 1995 ) and in vitro (Pérez-Martín & de Lorenzo, 1996c ) in the absence of the aromatic inducer. The A domain is connected to the central C module through a supposedly flexible linker or B domain. The central C domain is the most conserved region of this type of protein; it provides the ATPase activity that is required for transcriptional activation and presumably interacts with the 54 factor for the eventual formation of a transcriptionally competent open complex. Finally, the C-terminal domain of XylR contains a helix–turn–helix motif for binding the UAS of the Pu promoter (Inouye et al., 1988 ; Morett & Segovia, 1993 ).
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summarizes the series of events that, according to the model proposed by Pérez-Martín & de Lorenzo (1996a ), follow the release of the intramolecular repression caused by m-xylene binding to the A domain of XylR. Within this scheme, the activator bound to its cognate sequences at the Pu promoter cyclically changes its conformation upon ATP binding to build up a protein multimer that is competent to hydrolyse ATP and to channel the released energy into transcription initiation by the polymerase. This model is favoured by data (Pérez-Martín & de Lorenzo, 1996a ) from DNase I footprinting, from monitoring of the conformational states brought about by ATP binding to XylR, and from the behaviour of XylR variants apparently locked in either a non-multimerized state (G268N) or a multimerized state (R453H). The weakness of this model was the very circumstantial evidence for the proposition that the multimer becomes disassembled every time ATP is hydrolysed. This was largely based on the observation that mutant R453H (Fig. 1) seemed to keep the complex in a multimerized state but yet was unable to activate transcription. The behaviour of this mutant is, however, somewhat ambiguous. Whilst it footprinted the DNA and auto-crosslinked with a pattern similar to that of the wild-type protein in the presence of ATP (Pérez-Martín & de Lorenzo, 1996a ), an equivalent mutation found in NtrC (R358H) lacked any ATP-binding ability (North et al., 1996 ) and there was doubt over whether it polymerized spontaneously (Li et al., 1999 ). These observations encouraged us to re-examine the issue of the ATP-driven multimerization cycle proposed for XylR with a different technique, namely direct visualization of protein–DNA complexes by transmission electron microscopy (TEM). The work presented here analyses the effect of the ATP in the formation of different types of oligomeric complexes and their distribution under various conditions. The results are consistent with the reported transitions in the DNA–protein contacts observed by DNase I footprinting assays brought about by ATP (Pérez-Martín & de Lorenzo, 1996a ). Moreover, the data are compatible with the notion of an ATP-driven cycle as the basis for the transcriptional activation of Pu. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Its parental vector, pUC18, was used as the insertless control in the TEM experiments reported below. Metallo-affinity purification of the His-tagged XylRA protein was described in detail by Pérez-Martín & de Lorenzo (1996b ). High-purity plasmid DNA for TEM experiments was prepared by ultracentrifugation in isopycnic gradients of CsCl (Maniatis et al., 1982 ).
Preparation of samples for TEM of XylRA–DNA complexes.
Specimens for examination with the electron microscope were prepared by mixing the DNA under examination at a final concentration of 1 ng DNA µl-1 with increasing amounts of purified XylRA ranging from 5 nM to 300 nM in an assay buffer containing 20 mM Tris/HCl pH 7·5, 2 mM MgCl2, 1 mM EDTA and 40 mM KCl. Where indicated, 5 mM ATP or its non-hydrolysable structural analogue ATPS was added to the mixtures. The final volume of each reaction was 50 µl. The components were premixed on ice and incubated for 20 min at 30 °C. Reactions were then stopped by cooling, the mixtures were diluted twofold in the same buffer and 0·1% glutaraldehyde was added for fixation, followed by incubation for 30 min at 37 °C. The reaction was then stopped by adding NH4Cl to a final concentration of 10 mM. Fifty microlitres of each sample was layed on a piece of Parafilm for 15 min to let complexes become located homogeneously at the drop surface. The drops were then adsorbed to the inner face of an exfoliated mica piece for 20 s and dried. The samples underwent three 90 min washes with sterile water, followed by an additional washing step with absolute ethanol to dehydrate them. The alcohol was then removed to total dryness and the samples were rotary-shadowed with a platinum and carbon spray at a 3 ° angle using BALZERS 400T cryofracture equipment. The layers resulting from the shadowing were floated away from the mica and recovered on copper grids that were inspected in an electron microscope (JEOL JEM-1200 EXII). TEM images were captured at a magnification ofx30000.
Image analysis and statistical breakdown.
An average of 100–150 random plasmid molecules in the TEM images were inspected in each condition to produce enough samples for a statistically significant analysis. The fidelity of the statistical breakdown was monitored by examination of the error rate in the confidence interval corresponding to P=0·95 (error rate 0–4·9%) and P=0·99 (error rate 0–6·3%).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A of nonspecific DNA sequences adjacent to the UAS of Pu ) that XylR binding in vitro to the two sites present at the Pu enhancer (termed, respectively, distal and proximal) is not cooperative per se, i.e. full occupation of the proximal site occurs at much lower protein concentrations than the distal one. However, ATP addition causes a marked cooperativity in the binding of XylRA to the two sites of the Pu enhancer. This has been reported to alter the pattern of DNA–protein interactions between the activator and the target UAS as detected by DNase I footprinting. A close inspection of the footprinting data of Pérez-Martín & de Lorenzo (1996a ) reveals an interesting feature of the DNA–XylRA contacts. Addition of ATP caused XylRA not only to fully bind the two UAS, but also to significantly protect the region immediately upstream of the distal UAS. It should be noted that in these and in all other experiments presented below, we used an XylR variant that has been deleted of its N terminus (XylRA; Fig. 1) and is thus frozen in the constitutively active form equivalent to the protein after activation by m-xylene (Pérez-Martín & de Lorenzo, 1996c ). The extension of the DNase I protection, which engaged at least 44 additional bp upstream of the UAS, is not easy to explain. The enlargement of the footprint occurs towards the 5' end of the promoter sequence, and thus cannot simply be attributed to a nonspecific polymerization of the protein along the DNA by an excess of protein. A possible explanation could be that a protein multimer brought about by ATP binding could be wrapped by the DNA nucleated around the primary binding sites (the UAS). Since this is anticipated to cause a major change in the physical form of the DNA, we resorted to TEM of the protein–DNA complexes to examine the issue directly, and also to visualize possible intermediates of the XylR oligomerization cycle proposed for the activation of the Pu promoter (Pérez-Martín & de Lorenzo, 1996a ).
Typology of simple XylRA–DNA complexes visualized by TEM
To gain an insight into the structural changes undergone by the XylR–DNA complexes during activation of Pu, we prepared mixtures of pEZ9, a supercoiled plasmid bearing the Pu promoter as a 301 bp fragment (Fig. 1a), and the purified protein XylRA. As mentioned above, XylRA resembles the form of the protein that follows the release of intramolecular repression upon exposure to TOL inducers (Férnandez et al., 1995 ; Pérez-Martín & de Lorenzo, 1996c ). The complexes may thus reflect the type of DNA–protein associations that exist after that step in vivo. The mixtures of pEZ9 and XylRA were made at concentrations that are significant in ATPase (Pérez-Martín & de Lorenzo, 1996c ), footprinting (Pérez-Martín & de Lorenzo, 1996b ) and in vitro transcription assays (Pérez-Martín & de Lorenzo, 1996c ) and in a buffer whose composition was similar to that used for these assays. In all (100%) cases, the TEM images obtained from preparations of pEZ9 devoid of any protein corresponded to the form that in Fig. 2 is termed type I (single, naked plasmid contours devoid of any attached electron-dense spots). On this basis, we examined samples with increasing concentrations of XylRA without any other addition (Fig. 3). Even at protein concentrations as low as 10 nM, we started to detect a proportion of the species named type IIA in Fig. 2. These bear visible electron-dense, well-defined dots attached to the plasmid contour and are likely to reflect the simpler XylRA–UAS complexes detected with DNase I (Pérez-Martín & de Lorenzo, 1996a ). Both type I and type IIA forms remained well discernible at higher protein concentrations (Fig. 3), although the bulk of the plasmids in the samples remained in the unbound form. At 100 nM protein, another species (named type III in Fig. 2), although minor (<15%), became apparent as well. This form consisted of associations of two plasmids nucleated around a bigger electron-dense dot. Further increases in XylRA concentrations (up to 300 nM) resulted in large aggregates, which were considered artefactual and thus were disregarded for further studies (not shown). When the same experiments were made with plasmid pUC18 devoid of the Pu insert, every image observed corresponded to the type I form (not shown). This suggested that the electron-dense spots observed upon simple addition of XylRA to pEZ9 corresponded to genuine XylRA–Pu complexes. Since the type I form (naked plasmids) was predominant (60–100%) at both the protein concentrations used, we concluded that in the absence of any other addition, the interaction of XylRA with Pu is transient and that XylRA does not have per se an important affinity for the UAS. This matches previous results with DNase I footprint assays (Pérez-Martín & de Lorenzo, 1996a ) in which full occupation of the proximal site of the UAS (Fig. 1) was only detected at XylRA concentrations of 100 nM, whilst the occupation of the distal site required concentrations of the activator higher than 800 nM.
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A–DNA complexesA in a transcriptionally competent regulator. To examine how the nucleotide affected the formation and appearance of DNA–protein complexes, we prepared samples for TEM in the same fashion as above, but adding to the mixtures 5 mM ATP. The resulting images were more diverse than their counterparts without ATP (Fig. 4). Besides the type I and type IIA forms already seen, we also observed the interesting shape that we refer to as type IIB. Similar to type IIA, this new structure shows a plasmid contour bearing one electron-dense dot, but in this case it acts as the apex of a sharply bent DNA segment. Another novel structure became visible when the samples were added with ATP, named type IIC (Fig. 2). In this case, one electron-dense dot indistinguishable from those seen in types IIA and IIB appeared to contact simultaneously otherwise separated DNA sequences within the same plasmid. The three type II forms involving single plasmids with single dots in different arrangements coexisted in all samples with XylRA and ATP. In no case was a significant reduction in the size of the plasmid contour detected. However, the sum of all type II forms was still a relatively minor proportion of the total images at the lower concentrations of the protein (10 nM; Fig. 4). At the higher concentration (100 nM XylRA), however, the frequency of these forms equalled that of the naked plasmids. In addition, multi-plasmid complexes became the most abundant form in samples with 100 nM of the protein (Fig. 4). Similar to the situation mentioned above for the samples without ATP, control plasmid pUC18 produced no forms other than type I even at the higher protein concentrations assayed. This again suggested that the changes in appearance and distribution of the complexes upon ATP addition reflected significant variations undergone by the system upon ATP addition.
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A–DNA associations in the presence of ATP, as compared to those in its absence (see above) revealed two informative features. First, that the type IIB form (single plasmids sharply bent at an apex with an electron-dense dot) appeared exclusively in the presence of ATP. Second, that intra- and inter-molecular plasmid associations (types IIC and III), became predominant only at the higher concentrations of the protein and in the presence of ATP (Fig. 4). In other words, it appeared that ATP addition caused XylRA to adopt a form which bears non-saturated DNA-binding sites and is thus able to interact with otherwise distant DNA sequences in the same plasmid (type IIC) or even in trans with a different plasmid (type III). Furthermore, the frequency of type IIC and III forms (Fig. 4) in the samples with 100 nM XylRA plus ATP as compared to the same samples without the nucleotide (Fig. 3) suggested that ATP causes the protein to oligomerize into a form able to bind two DNA sequences separated in cis or in trans. Whilst in the case of the inter-molecular associations (type III), the protein oligomer probably engages plasmids specifically anchored through the UAS, it is possible that in the type IIC complexes (intra-molecular associations), the lack of specificity is compensated by the physical proximity of the bound DNA.
Another interesting aspect of the distribution of XylRA–DNA forms in the samples with 100 nM protein and ATP (Fig. 4) is the coexistence of all visual forms (types I, II and III) at quite balanced frequencies. This is not the case in the absence of ATP, where the predominant forms are the naked plasmids and the simple associations. We believe that this diversity of forms reflects the population of different protein–DNA species that arise during the hydrolysis of ATP so that the frozen images represent distinct stages of the process. In fact, that higher complexes subsist along with naked plasmid molecules suggests a cycle of XylR binding, oligomerization into a form able to bind distant DNA molecules, and then dissassembly of the complex.
ATPS fixes the XylRA–DNA complexes in the higher order forms
Since ATP hydrolysis is a dynamic process, the hypothesis of a protein–DNA oligomerization cycle driven by the nucleotide makes some predictions that can be examined by TEM. In particular, that the addition of a non-hydrolysable ATP analogue (such as ATPS) to the XylRA–DNA samples should eliminate the diversity of forms and displace all molecular species towards the complex formed just following ATP binding but prior to ATP hydrolysis. This is facilitated by the observation (Pérez-Martín & de Lorenzo, 1996a ) that the affinity of ATPS for XylRA is comparable to that of its non-hydrolysable counterpart. To ensure the visualization of events occurring on individual plasmids (rather than involving two or more), we ran the corresponding experiments with ATPS using a low range of concentrations of XylRA (5 and 10 nM) within which some effects started to be detected with ATP (Fig. 4). The analysis of the resulting images (Fig. 5) clearly revealed that the predominant forms (
90%) of the molecules even at the lowest protein concentration were those in which individual plasmids were bound by one single electron-dense dot (type IIA). Only at higher protein concentrations did multi-plasmid associations start to increase in frequency, becoming fully dominant at 20 nM (not shown). Interestingly, the type I form (naked plasmids) entirely disappeared from the visual field (
1%) at any protein concentration in the range 5–50 nM in the presence of ATPS, thereby suggesting that every available DNA target was engaged in stable interactions with the protein. Taken together, these results suggested that ATP binding (but not its hydrolysis) both increases the affinity of XylRA for DNA and gives rise to the protein species able to bind simultaneously two or more distant DNA sequences. Finally, the loss of diversity of complexes at the lower protein concentrations in the presence of ATPS (Fig. 5) as compared to the distribution of forms with ATP (Fig. 4) is consistent with the notion that ATP hydrolysis drives a protein multimerization/disassembly cycle that is interrupted at the early multimer stage if the nucleotide is not hydrolysed (see below). Inspection of the various complexes formed in the presence of ATPS also revealed the virtual absence of type IIB forms, in which the DNA seems to be sharply bent in association with the protein. This suggested that production of such species may be dependent on ATP hydrolysis.
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A with the UAS of Pu by using DNase I (Pérez-Martín & de Lorenzo, 1996a ) and hydroxyl radical footprinting (Pérez-Martín & de Lorenzo, 1996b ), and on the various studies on the archetype of 54-dependent enhancer-binding proteins, NtrC (Rippe et al., 1998 ). Although our former work was done with linear DNA and the work presented here largely involves supercoiled DNA, we did not find any incompatibility in the outcome of the respective experiments. From the early DNase I footprint analyses (Pérez-Martín & de Lorenzo, 1996b ), it was clear that in the absence of ATP, the binding of XylRA was somewhat weak and non-cooperative. This has its TEM counterpart in the abundance of naked plasmids in samples treated with XylRA concentrations of 10–100 nM in the absence of the nucleotide (Fig. 3). ATP changes the protein–DNA interaction pattern (Pérez-Martín & de Lorenzo, 1996a ) in two ways. On one hand, it increases dramatically the affinity and the cooperativity of the protein for the UAS, but on the other hand, it extends the protection from DNase I towards the 5' region of the promoter. These changes could correspond to the transition between type II forms A and B (Fig. 2), in which the protein bound to the DNA appears to nucleate a sharp DNA bend. A simple explanation for this (Révet et al., 1995 ) could be that XylRA dimers bound to adjacent UAS may interact with each other upon ATP addition and thus bend the intervening DNA sequence. This is however unlikely, since such inter-UAS DNA segments are very short (Fig. 1a) and the extension in the protection from DNase I (Pérez-Martín & de Lorenzo, 1996a ) cannot be explained. A more likely explanation is that ATP addition may initiate a nucleoprotein structure around which adjacent DNA sequences may wrap, so that the sharp DNA bends of the type IIB forms are in fact looped structures. This might be possible if ATP causes formation of an XylRA multimer with non-saturated DNA-binding domains which can hold simultaneously two separated DNA sequences. Depending on the concentration of protein, such non-saturated domains may then either bind adjacent sequences in the same plasmid or engage sequences from two plasmids (Fig. 6). This could explain why the effect of adding ATP does not become truly significant until a certain concentration of XylRA (e.g. 100 nM in Fig. 4) has been added to the samples.
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. It is assumed that, similar to NtrC (Rippe et al., 1998 ), the predominant form of XylRA in solution is a dimer. In the absence of ATP, such a dimer has a low affinity for the UAS, with which it interacts non-cooperatively. In the case of NtrC, it has been shown that although protein dimers bind cooperatively to the UAS of the glnA promoter in the absence of ATP (Porter et al., 1993 ), its phosphorylation increases such cooperativity (Porter et al., 1993 ). In the case of XylRA, ATP addition causes a conformational change that increases the affinity and cooperativity of the protein–DNA interaction. In addition, it triggers the formation of an oligomer whose buildup requires recruitment of further XylRA proteins from solution and whose frame bears non-saturated DNA-binding domains. This is also compatible with the data available for NtrC. In this case, scanning force microscopy suggests that transcriptional activation of glnAp2 may depend on the formation of an NtrC oligomer larger than a tetramer (Rippe et al., 1997 ; Wyman et al., 1997 ). Furthermore, analytical ultracentrifugation studies indicate that NtrC phosphorylation causes the formation of an octameric complex within the UAS of glnAp2 (Rippe et al., 1998 ) with an ability to bind simultaneously two target DNA sequences. In fact, our TEM observations with XylRA are fully compatible with the hydrodynamic model proposed for NtrC (Rippe et al., 1998 ), suggesting that during activation two dimers are directly bound to the enhancer but are attached to two additional dimers through protein–protein interactions. The resulting octamer thus has the ability to bind DNA through two independent surfaces. Although we do not know what are the actual protein species resulting from adding ATP to XylRA, it is reasonable to believe that the the type IIB, IIC and III forms involve multimers, perhaps octamers, which can bind DNA through two separate surfaces.
A final piece of information from the TEM images presented in this work is the accumulation of just one type of DNA–protein complex in the presence of ATPS (Fig. 5), as compared to the diversity of forms with ATP (Fig. 4). At the lowest protein concentration assayed (5 nM), 99% of the plasmids were found forming single complexes with XylRA, whereas more plasmids were engaged in the complexes at higher protein concentrations. The total lack of naked plasmids in these samples as compared to their presence in specimens treated with regular ATP, even at the highest protein concentrations (100 nM), suggests that ATP hydrolysis causes the full dissassembly of the multimer and the return to the first step of what appears to be a cycle (Fig. 6).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Assinder, S. J. & Williams, P. A. (1990). The TOL plasmids: determinants of the catabolism of toluene and xylenes. Adv Microb Physiol 31, 1-69.[Medline]
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Pérez-Martín, J. & de Lorenzo, V. (1995). The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor. Proc Natl Acad Sci U S A 92, 9392-9396.
Pérez-Martín, J. & de Lorenzo, V. (1996a). ATP binding to the 54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA. Cell 86, 331-339.[Medline]
Pérez-Martín, J. & de Lorenzo, V. (1996b). Physical and functional analysis of the prokaryotic enhancer of the 54-promoters of the TOL plasmid of Pseudomonas putida. J Mol Biol 258, 562-574.[Medline]
Pérez-Martín, J. & de Lorenzo, V. (1996c). In vitro activities of an N-terminal truncated form of XylR, a 54-dependent transcriptional activator of Pseudomonas putida. J Mol Biol 258, 575-587.[Medline]
Porter, S., North, A. K., Wedel, A. B. & Kustu, S. (1993). Oligomerization of NtrC at the glnA enhancer is required for transcriptional activation. Genes Dev 7, 2258-2273.
Révet, B., Brahms, S. & Brahms, G. (1995). Binding of the transcription activator NRI (NTRC) to a supercoiled DNA segment imitates association with the natural enhancer: an electron microscopic investigation. Proc Natl Acad Sci U S A 92, 7535-7539.
Rippe, K., Guthold, M., Hippel, P. H. & Bustamante, C. (1997). Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase 54 holoenzyme by scanning force microscopy. J Mol Biol 270, 125-138.[Medline]
Rippe, K., Mücke, N. & Schulz, A. (1998). Association states of the transcription activator protein NtrC from E. coli determinated by analytical ultracentrifugation. J Mol Biol 278, 915-933.[Medline]
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Received 11 February 2000;
revised 17 April 2000;
accepted 15 May 2000.
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