Integration host factor suppresses promiscuous activation of the sigma ...

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7277-7281, August 1995 Microbiology

Integration host factor suppresses promiscuous activation of the a 54-dependent promoter Pu of Pseudomonas putida (enhancers/DNA bending/transcription)

J. PEREZ-MARTIN AND V. DE LORENZO* Centro de Investigaciones Biol6gicas, Consejo Superior de Investigaciones Cientificas, Velizquez 144, 28006 Madrid, Spain

Communicated by Howard A. Nash, National Institute of Mental Health, Bethesda, MD, April 24, 1995

In the presence of m-xylene, the Pu promoter ABSTRACT of the TOL plasmid of Pseudomonas putida is activated by the prokaryotic enhancer-binding protein XylR. The intervening DNA segment between the upstream activating sequences (UASs) and those for RNA polymerase binding contains an integration host factor (IHF) attachment site that is required for full transcriptional activity. In the absence of IHF, the Pu promoter can be cross-activated by other members of the o54-dependent family of regulatory proteins. Such illegitimate activation does not require the binding of the heterologous regulators to DNA and it is suppressed by bent DNA structures, either static or protein induced, between the promoter core elements (UAS and RNA polymerase recognition sequence). The role of IHF in some r54 promoters is, therefore, not only a structural aid for assembling a correct promoter geometry but also that of an active suppressor (restrictor) of promiscuous activation by heterologous regulators for increased promoter specificity.

plasmid-encoded XylR protein (10), of the o-54-dependent family of transcriptional regulators (2). Pu has a functional IHF-binding site within the intervening sequence, the occupation of which is required for full promoter activity (12, 13). In this work, we show that the absence of IHF results in availability of the Pu promoter to illegitimate activation by other regulators of the same family of proteins, in a fashion strictly dependent on promoter geometry. These data support the view that the role of the IHF-induced bend in Pu is not only a structural aid for assembling the right promoter geometry but also that of an active suppressor of cross-activation in vivo.

Bacterial promoters dependent on the alternative sigma factor 54 are one of the few cases of transcriptional activation at distance known in the prokaryotic world (1). These promoters include distant (>100 bp) enhancer-like upstream activating sequences (UASs), which are the target sites for a class of regulators belonging to the so-called NtrC family of regulators (2) or, more properly, to the o54-dependent group of activator proteins. Once attached to their cognate UAS in an active form, these regulators make the a-54-containing RNA polymerase (au54-RNAP), bound at specific sequences at -12/ -24, initiate transcription. Like their eukaryotic counterparts, some (but not all) prokaryotic enhancers of this type also activate transcription when they are placed in different orientations upstream and downstream of the promoter (3, 4). Since interactions between the activator bound to distant sites and ar54-RNAP require the looping-out of the intervening DNA (5), both the physical properties of the nucleotide sequence and the binding of proteins that alter DNA topology are predicted to have an effect on the final outcome of promoter activity (6). One class of oa54 promoters includes target sequences for the integration host factor (IHF) between the UAS and the binding site for the polymerase. IHF is a small protein (20 kDa) known to participate in a variety of cellular processes (7), which causes a sharp bend (- 140°) upon binding its target DNA sequence. It is currently believed that the role of IHF in (r54 promoters is that of providing a structural aid to improve contacts between the polymerase bound to the -12/-24 positions and the activator protein attached to the UAS, thereby enhancing promoter activity (8, 9). The Pu promoter of the TOL pWWO plasmid of Pseudomonas putida (Fig. 1) belongs to the o 54 class. When cells are exposed to m-xylene, this promoter becomes activated by the

MATERIALS AND METHODS Strains, Plasmids, and General Procedures. Escherichia coli S90C strain and its himA (IHF-) derivative E. coli DPB102 have been described (12). E. coli MC4100 was made rpoNthrough P1 phage transduction of the ntrA208::TnlO marker from E. coli ET8045 (9) and, where required, was also made IHF- through transduction of the hip[A3::cat] marker from E. coli MC252 (14). Hybrid promoters Pu-AT4 and Pu-ST (9) are derivatives of the Pu promoter of the TOL plasmid, in which the naturally occurring IHF-binding site has been replaced either by a segment of DNA intrinsically bent by ,120° (Pu-AT4) or by an equivalent noncurved DNA sequence (Pu-ST). These hybrid promoters maintain the overall orientation of core elements on the DNA helix surface present in the wild-type Pu, but they are devoid of IHF sites (9). The Pu-4 promoter is a variant carrying a 4-bp insertion at position -40 of the Pu promoter sequence, while the Pu-6 promoter has a 6-bp insertion at -106 (15). Transcriptional fusions of the various promoters to lacZ were engineered on vector pRS551 (16), which prevents readthrough transcription from plasmid promoters into the reporter gene. pRS551 carrying fusions of lacZ to the Pu promoter (pRSPu) or its hybrid derivatives Pu-AT4 (pRSPu/AT4) and Pu-ST (pRSPu/S7) have been described (9). pRSPu-4 and pRSPu-6 were constructed by transferring the 0.3-kb EcoRI/BamHI inserts of pERD435 and pRED407NB (15), respectively, to the corresponding sites of pRS551. Where indicated, lacZ fusions were integrated into the chromosome of E. coli MC4100 and its rpoN- and IHFderivatives (see above) by recombination with ARS45 phage and further lysogenization of the strain of interest (16). Where required, the xylR gene was introduced into the chromosome of E. coli by using the specialized hybrid minitransposon mini-TnlO Ptt xylR/xylS (12). pTS174 (Cmr) is a xylR+ derivative of pACYC184 (17). pMC71A is a nifA+ derivative of pACYC184 (18). A plasmid expressing the central, activating domain of NifA (19) was designed as follows. A DNA fragment corresponding to positions 198-466 of the NifA amino acid sequence (the central domain of the regulator) was produced by subjecting pMC71A to a PCR with primers 5'-GGAAT-

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Abbreviations: UAS, upstream activating sequence; a54-RNAP, ,54containing RNA polymerase; IHF, integration host factor. *To whom reprint requests should be addressed.

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Microbiology: Perez-Martin and de Lorenzo CH3

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plasmid DNA of each strain and estimating the relative amount of each of the plasmids in agarose gels. Promoter activation by NifA or its truncated derivative (see above) was examined in cells cultured at 30°C and, where required, grown in the presence of 0.1 mM isopropyl f3-D-thiogalactopyranoside.

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FIG. 1. Regulation of thexyl genes in the TOL plasmid pWWO and organization of the aS4 promoters used in this work. As summarized above (not to scale), in the presence of m-xylene, the upper-operon promoterPu, of the a54 class, is activated by the cognate activator XylR (10). Other promoters and regulators present in the system are also indicated along with their aromatic inducers. Below the scheme of the operons, the Pu promoter region (and those of various Pu derivatives) used for construction of lacZ fusions is expanded, showing the location of relevant DNA sequences. These include UASs for XylR, the -12/-24 region recognized by (r54-RNAP (the nucleotide sequence of which is shown), and one IHF-binding site located within the intervening region. The Pu-AT4 promoter (9) is a Pu derivative in which the sequence -40 to -105 (i.e., spanning the former IHF site) has been substituted by a heterologous DNA segment that includes a 76-bp A+T tract endowing a static bend functionally equivalent to that caused by IHF binding (11). The Pu-ST promoter is equivalent to Pu-AT4 in its base composition and distances between the promoter elements, but the 76-bp A+T tract has been replaced by an unbent DNA sequence from pBR322 (9). Pu-4 and Pu-6 contain, respectively, 4- and 6-bp insertions at positions -40 and -106, which offsets by about a half-helix DNA turn the upstream region of the promoter with respect to the polymerase binding site.

TCGCCTGTACCCCCTTCGCGCGGT-3' and 5 '-CGGGATCCTCAGGCCAGCGCTTTCGGCGG-3'. The 0.8-kb amplification product was then inserted as an EcoRI/BamHI fragment in the lacIq/Ptrc-based expression vector pTrc99A (20). A 2.3-kb Sph I restriction fragment of the resulting plasmid containing the sequence of the NifA central domain (expressed through a heterologous translation initiation region downstream of the Ptrc promoter) and the lacIq gene was excised and inserted into the Sph I site of pACYC184, giving rise to plasmid pNifAAC. The latter was used as the source of the NifA central domain in vivo, as explained in Results. Quantification of Promoter Activity in Vivo. Transcriptional activity was monitored by assaying accumulation of ,B-galactosidase in cells carrying lacZ gene fusions to the promoter of interest (see above). For this purpose, cells were grown at 37°C to OD600 = 1.2 in LB liquid medium (21) supplemented with appropriate antibiotics and exposed for 4 h to saturating vapors of the XylR effector m-xylene. 3-Galactosidase assays were made on cells permeabilized with chloroform and SDS (21). The linearity of the assay with o-nitrophenyl 13-D-galactoside was verified in all cases. ,B-Galactosidase values were the averages of at least three independent measurements, each of which was from duplicate samples, with deviation not exceeding 15%. The maintenance of plasmid copy number under the different conditions used was monitored by miniprepping the

XylR-Independent Activity of Pu in the Absence of IHF. The nucleotide sequence of the Pu promoter region contains a consensus IHF site that strongly binds the protein in vitro and is required for full promoter activity (12, 13). The lack of IHF, however, results in nontrivial behavior of the transcriptional activity of the promoter in vivo. As shown in Fig. 2, IHF absence not only prevents Pu from reaching its full potential activity, it also substantially increases the basal level of transcription (i.e., under uninduced conditions) with respect to the IHF+ counterpart. It should be noted that the lacZ fusions used to monitor Pu activity in vivo, whether in multicopy (Fig. 2) or in monocopy (Fig. 3), were placed downstream of strong transcriptional terminators. Therefore, the phenotype caused by the absence of IHF cannot be explained in terms of readthrough transcription from host or plasmid promoters. [= Uninduced

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FIG. 2. Characterization of XylR-dependent and -independent activity of Pu. E. coli S90C strain and its isogenic derivative E. coli DBP102 himA (IHF-) were cotransformed with the xylR+ plasmid pTS174 (or with the vector pACYC184 for xylR- strain) and each of the lacZ plasmids carrying fusions to the promoters indicated at the bottom. Cotransformants were grown in liquid medium and induced with saturating vapors of m-xylene (m-xyl) as described. Accumulation of ,B-galactosidase (3-Gal, Miller units) by cells after 4 h of induction is represented.

Microbiology: Pe'rez-Marti'n and de Lorenzo =Uninduced

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FIG. 3. Control of XylR-independent activity of Pu promoter by s54. Specialized A phages carrying lacZ fusions to Pu and the hybrid promoters Pu-AT4 and Pu-STwere used to lysogenize isogenic strains E. coli MC4100 xylR+hip+ (IHF+) rpoN+, E. coli MC4100 xylR+hip(IHF-) rpoN+, E. coli MC4100 xylR-hip- (IHF-) rpoN+, and E. coli MC4100 xylR-hip- (IHF-) rpoN- in order to have all the regulatory elements of the system in monocopy gene dosage. Cultures of each strain were exposed to saturating vapors of m-xylene (m-xyl) as described and 13-galactosidase (13-Gal) accumulation was recorded in Miller units after 4 h of induction. No activity above background was observed in the xylR-, IHF-, rpoN- strain (d).

To determine whether such relaxed behavior of Pu could have arisen from an adventitious cross-activation of the promoter by heterologous regulators able to contact n-54-RNAP holoenzyme (i.e., activators of the a54-dependent family), as has already been suggested (22), or whether it actually results from a residual activation by XylR even without its effector m-xylene, we compared the activities of a Pu-lacZ fusion in an isogenic collection of xylR+/xylR- and IHF+/IHF- host strains. The results of Fig. 2 show that, expectedly, the absence of XylR resulted in the loss of inducibility by m-xylene. However, while the basal activity of Pu is kept virtually negligible in an IHF+ background, it increased considerably in IHF- cells regardless of the presence or absence of the corresponding activator. Therefore, the significant basal levels of expression of Pu, in an IHF- background, are XylR independent. To rule out that the XylR-independent activity of Pu could be originated from an overlapping promoter structure other than the authentic (-54-dependent group, the same lacZ fusion was placed into the chromosome of isogenic rpoN+/rpoN- strains with or without IHF and XylR. The results of Fig. 3 show that Pu lost any significant activity when placed in a rpoN- strain, thus indicating that XylRindependent transcription still originated from the same o54_ promoter structure, which normally responds to XylR. Static and Protein-Induced DNA Bends Suppress CrossActivation of Pu by Noncognate Regulators. The observations discussed above indicated that the role of IHF binding to Pu could be more than a mere structural aid for assembling a correct promoter geometry. Besides this function, the results

Proc. Natl. Acad. Sci. USA 92 (1995)

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of Figs. 2 and 3 also suggested that the increased activity of Pu in the absence of IHF could be related to the bending of the DNA sequence between promoter core elements. Since IHF plays a role in many cellular processes (7) and its absence may lead to a pleiotropic phenotype, we sought to separate its outcome on promoter geometry from any other potential effect. For this, we used hybrid promoters Pu-AT4 and Pu-ST. In Pu-A T4, the IHF site has been substituted by a curved DNA sequence, which functionally replaces the DNA-bending effect of IHF in Pu. In Pu-ST, the curved sequence of Pu-AT4 has been exchanged by a nonbent DNA segment of the same size and base composition (9). The activity of each of the corresponding lacZ fusions was assayed again in IHF+ and IHFgenetic backgrounds as well as in rpoN+ and rpoN- cells. The results of Figs. 2 and 3 indicate that the relaxation of Pu regulation in the absence of IHF was reversed to a wild-typelike tight control when the IHF site was exchanged by a statically curved DNA sequence (as in the case with Pu-AT4). On the contrary, insertion of a noncurved DNA segment at the intervening region between the UAS and -12/-24 abolished any significant responsiveness to m-xylene/XylR and kept a high basal level of activity regardless of the presence or absence of IHF. These observations support the notion that the increased basal activity of Pu when IHF is not available is a phenomenon related to the presence or absence of a bent DNA segment (static or IHF induced) between the core promoter elements. In the presence of strong curvatures such as that caused by IHF binding to wild-type Pu (- 140°) or that present in the Pu-AT4 construct (-120°), the basal activity of the promoter remains low. On the contrary, the absence of such sharp curvatures seems to tolerate their illegitimate activation by other proteins that act in concert with ,54. Promiscuous Activation of Pu Depends on the Overall Configuration of the DNA Region. The observed illegitimate activation of Pu seems to rely on the local structure of the DNA of the region. To directly test this concept, we examined the activity of mutant promoters Pu-4 and Pu-6 in IHF+ and IHFgenetic backgrounds (Fig. 4). Pu-4 consists of a Pu sequence that has been inserted at -40 (i.e., between the IHF site and the u54-RNAP binding site; Fig. 1) with 4 additional base pairs. This mutation offsets entirely the orientation of the DNA bend caused by IHF binding with respect to the polymerase, thus Promoter activity, P-Gal x 0

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and Pu-6 are indicated with arrowheads.

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Proc. Natl. Acad. Sci. USA 92

eliminating any effect in the wild-type promoter of the local DNA geometry on the presentation of the enzyme to potential regulators. On the contrary, the Pu-6 mutant, containing a 6-bp insertion at position -106 (i.e., between the UAS and the IHF site), simply rotates the UAS by a half-helix turn, while maintaining the overall positioning of the cr54-RNAP with respect to DNA sequences upstream of the IHF site. As shown in Fig. 4, while Pu-4 displayed a substantial activity regardless of IHF, Pu-6 was considerably more active in the absence than in the presence of the protein. This confirms that the specificity-enhancing effect caused by IHF on Pu depends entirely on the maintenance of a locally bent DNA conformation supported by IHF binding. Promiscuous Activation of Pu Does Not Require Heterologous Activators to Bind DNA. To test directly the concept that o'54 promoters devoid of bent DNA sequences can be activated by heterologous proteins of the o54-dependent type of regulators, the effect of the NifA protein of Klebsiella pneumoniae on the activity of Pu, Pu-AT4, and Pu-ST promoters was studied. NifA is a convenient regulator for this purpose, since it is active in E. coli, it does not require any inducer (23), and it stimulates transcription even in the absence of DNA binding (23, 24). In one round of experiments, we produced wild-type NifA in IHF+/IHF- strains harboring lacZ fusions to Pu, Pu-AT4, or Pu-ST. The results shown in Fig. 5 indicate that NifA could activate Pu-ST regardless of the presence or absence of IHF, while Pu was activated only when IHF was missing. Pu-AT4 remained unresponsive to NifA under all circumstances assayed. Similar experiments were repeated with a truncated NifA protein (instead of wild-type NifA), which maintains its central activating domain but is unable to bind DNA (19). The similarity of the results obtained rule out the presence of cryptic UASs for NifA at the corresponding promoter region and they show that Pu activation in the absence of IHF does not require binding of the heterologous regulator to DNA sequences at the promoter region.

DISCUSSION In IHF- strains, the Pu promoter displays a 3- to 4-fold higher basal activity than in their IHF+ counterparts. This effect is detectable whether in multicopy or in monocopy gene dosage (Figs. 2 and 3) and seems to be due to the illegitimate activation of the polymerase bound to Pu promoter by heterologous a 54-dependent regulators. These may gain access to the enzyme bound to the promoter when the local DNA conformation is not maintained by bent DNA. This hypothesis is based on the following evidence: (i) The high basal transcription from Pu in the absence of IHF or an equivalent curved DNA is still dependent on o54 (Fig. 3) and, therefore, it necessarily requires an activator of the NtrC family (1). (ii) Basal transcription is independent of XylR (Figs. 2 and 3). (iii)

FI] Control (nifA -)

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Although Pu has no binding sites for NifA, it is possible to activate it in IHF- strains by overexpressing the heterologous regulator (Fig. 5). Although we have not addressed the nature of the regulator involved in the "illegitimate" activation of Pu, we believe that it may be not just one protein but rather the effect of several regulators of the family. In fact, various u54-dependent activators become constitutive when their N-terminal domain is deleted. These include NifA (24), DctD (25), and XylR itself (26). Since the interdomain region that connects the N-terminal receiving domain to the rest of the protein (the Q-linker) is fairly sensitive to proteases (27), it might be possible that cells always contain a certain pool of partially degraded regulators still capable of activation of promoter-bound o54-RNAP. In this respect, we have tried unsuccessfully to link the basal activity of Pu with activators of the same family of proteins such as NtrC. Although NtrC has been previously reported to stimulate in vivo the activity of Pu when overexpressed (28), we have not found any significant influence of ntrC mutations on the basal level of Pu in IHFhosts, regardless of the nitrogen status of the cells (data not shown). It is currently believed that the role of IHF in ao54 promoters is to facilitate contacts between the activator protein attached to the UAS and short-lived, prebound a-54-RNAP (9, 22, 29). However, although IHF sustains a loop that brings the two proteins in close proximity (thus increasing transcription efficiency) such effect may, at the same time, restrict the flexibility of the region. An IHF-induced bend can inhibit the function of an activator bound to a site that is not correctly placed with respect to the bend (29). The phenotypes endowed by Pu-AT4, containing a functional substitution of the former IHF-binding site of Pu by a sharp DNA curvature (-120°), are particularly revealing of the role of IHF in these systems. As shown in Figs. 2 and 3, Pu-AT4 maintained a low basal level of activity in the absence of IHF and was unresponsive to cross-activation by NifA. This suggests that the rigid conformation that DNA may adopt upon IHF binding increases activation specificity by preventing access to u54-RNAP of activators other than that properly bound and positioned in the UAS. In fact, the experiments carried out with NifAAA (Fig. 5) show that illegitimate activation of the Pu promoter can occur in the absence of any DNA binding by the heterologous activator. In this respect, the effect caused by bent DNA differs substantially from the inhibition of transcription observed when the activator protein is bound to a site that is not correctly placed with respect to the bend (29). It seems, therefore, that the role of J1F in this system is not only as coactivator (6, 30) but also as a suppressor of cross-regulation. We propose the term restrictor to describe operatively this specificity-enhancing function assigned to protein-induced or static bends in o-54-dependent promoters, although the concept may have a more general value (Fig. 6).

E nifAAC +

FIG. 5. Cross-activation of Pu by NifA. E. coli S9OC strain and its isogenic derivative E. coli DBP102 himA (IHF-) were cotransformed with pACYC184 (control, devoid of nifA), pMC71A (nifA+), or pNifAAC (expressing the activating central domain of NifA, unable to bind DNA), and each of the lacZ plasmids carrying fusions to the promoters is indicated above each panel. Cotransformants were grown overnight at 30°C in liquid medium (with 0.1 mM isopropyl 3-Dthiogalactopyranoside in the case of cells carrying pNifAAC). Accumulation of ,3-galactosidase ((3-Gal, Miller units) by cells is represented.

Microbiology: Pe'rez-Marti'n and de Lorenzo c

PO

FIG. 6. Restrictor effect of curved DNA on promoter activity. Spatial distribution of the DNA sequence surrounding the binding site for the RNAP (RNApol) affects availability of the enzyme to potential activators. Curved DNA (either static or produced by DNA-bending protein R) seems to sustain a conformation of the o54-promoter region, which prevents access of the RNAP to noncognate regulators. This effect is independent of the occupation of the UAS by the native activator (Act). On the contrary, the absence of protein-induced or intrinsic DNA curvature at the promoter region may expose activating surfaces of the polymerase to alien regulators. The result of this effect is the selection in vivo of a certain window of activity of the promoter from the whole potential available in the system.

The mechanism accounting for the restrictor effect of IHF Pu is clearly related to the binding of the protein to its cognate site at the promoter. This notion is also supported by results from other or4-dependent systems. For instance, the NifA-responsive ni/H promoter of K pneumoniae, which contains an IHF-binding site, could not be activated in vitro by the central domain of the regulator in the presence of IHF (19). Similarly, IHF inhibited transcriptional activation in vitro by NifA of the nifH promoter of Azotobacter vinelandii on a DNA template lacking the UAS but containing the IHF-binding site (31). Since the restrictor effect of IHF in Pu can be mimicked by statically curved DNA, it seems possible that the operative occlusion of the promoter by IHF is ultimately caused by DNA bending. Curved DNA could encourage a contact between DNA and the back side of RNAP (as has been suggested to CRP-induced DNA bending; ref. 32) that prevents extraneous protein-protein interactions. Alternatively, the DNA bending may sustain a local DNA geometry around the enzyme that hinders its presentation to nonspecific regulators from solution. Our genetic results do not ascertain whether, besides DNA bending, the restrictor effect originated by IHF involves protein-protein interactions with RNAP, while that caused by statically curved DNA could be assisted by specific recognition of upstream DNA by the enzyme. However, our data are consistent with the notion that DNA bending is, in both cases, the major cause of the observed effects. Although Giladi et al. (33) suggested that IHF could interact directly with the a subunit of RNAP during the activation of the phage A pL promoter, there seems not to be (unlike other activators like CAP) direct proof of such protein-protein interaction. Furthermore, the effect of IHF on pL is that of a positive regulator and it would be unlikely that the same protein could also behave as a repressor through direct contacts with the 0-54RNAP (that the geometry of the promoter would disfavor in any case; refs. 1 and 9). It is, however, possible that the occlusion effect caused by IHF or statically bent sequences is exacerbated in vivo through recruitment by the curved DNA of components of the bacterial nucleoid such as H-NS (34). Whether or not a similar type of masking plays a role in the silencing of E. coli promoters (35) deserves further study. From analysis of the various o-54-dependent systems known so far, a certain relationship between affinity of the polymerase for the promoter, presence of IHF-binding sites, and restrictor effects can be drawn. The requirement for IHF protein can be relieved in vitro by mutations around the on

Proc. Natl. Acad. Sci. USA 92 (1995)

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o-54-RNAP binding site that increase the affinity of the enzyme for the -12/-24 sequences (22). This is believed to make the binding of the enzyme to promoter longer lived and thus increase the probability of contacts with the activator. Although one role of IHF is increasing such contacts, the same effect may have different consequences in vivo. A polymerase too stably bound to the promoter could be more susceptible to promiscuous activation by regulators not bound to DNA. But even promoters with lower affinity for the polymerase (such as PnifH) can be activated by a form of NifA unable to bind DNA (19, 24) and, therefore, they could also become cross-activated by heterologous regulators. Santero et al. (22) suggested that IHF-induced bends in promoters that weakly bind r-54-RNAP ensure high fidelity and high efficiency of activation by the cognate activator while disfavoring the action of heterologous activators. In fact, a promoter with little affinity for o-54-RNAP along with an IHF-binding site should remain silent in the absence of induction due to the mutual specificity-enhancing effect of the two elements. All the data included in this work support this view. We are indebted to J. L. Ramos for the gift of strains. I. Cases is also gratefully acknowledged for inspiring discussions. This work was supported by Grant BI092-1018-CO2-01 of the Spanish Comisi6n Interministerial de Ciencia y Tecnologia and by Contract BI02-Cr920084 of the Biotech Program of the European Union. 1. Kustu, S., North, A. K. & Weiss, D. S. (1991) Trends Biochem. Sci. 16,

397-402. 2. North, A. K., Klose, K. E., Stedman, M. & Kustu, S. (1993)J. Bacteriol. 175, 4267-4273. 3. Reitzer, L. J. & Magasanik, B. (1986) Cell 45, 785-792. 4. Ninfa, A. J., Reitzer, L. J. & Magasanik, B. (1987) Cell 50, 1039-1046. 5. Su, W., Porter, S., Kustu, S. & Echols, H. (1990) Proc. Natl. Acad. Sci. USA 87, 5504-5508. 6. Perez-Martin, J., Rojo, F. & de Lorenzo, V. (1994) Microbiol. Rev. 58, 268-290. 7. Friedman, D. I. (1988) Cell 55, 545-554. 8. Hoover, T. R., Santero, E., Porter, S. & Kustu, S. (1990) Cell 63, 11-22. 9. Perez-Martin, J., Timmis, K. N. & de Lorenzo, V. (1994) J Bio. Chem. 269, 22657-22662. 10. Marques, S. & Ramos, J. L. (1993) MoL Microbiol. 9, 923-929. 11. Goodman, S. D., Nicholson, S. C. & Nash, H. A. (1992) Proc. Natl. Acad. Sci. USA 89, 11910-11914. 12. de Lorenzo, V., Herrero, M., Metzke, M. & Timmis, K. N. (1991) EMBO J. 10, 1159-1167. 13. Abril, M. A., Buck, M. & Ramos, J. L. (1991) J Biol. Chem. 266, 1583215838. 14. Chandler, M. & Galas, D. (1983) J. Mol. Biol. 170, 61-91. 15. Abril, M. A. & Ramos, J. L. (1993) Mo. Gen. Genet. 239, 281-288. 16. Simons, R. W., Houman, F. & Kleckner, N. (1987) Gene 53, 85-96. 17. Inouye, S., Nakazawa, A. & Nakazawa, T. (1983) J Bacteriol. 155, 11921199. 18. Buchanan-Wollaston, V., Cannon, M. C., Beynon, J. L. & Cannon, F. C. (1981) Nature (London) 294, 776-778. 19. Berger, K. D., Narberhaus, F. & Kustu, S. (1994) Proc. Natl. Acad. Sci. USA 91, 103-107. 20. Amann, E., Ochs, B. & Abel, K J. (1988) Gene 69, 301-315. 21. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Lab. Press, Plainview, NY). 22. Santero, E., Hoover, T. R., North, A. K., Berger, D. K., Porter, S. C. & Kustu, S. (1992) J. Mol. Biol. 227, 602-620. 23. Drummond, M. H., Contreras, A. & Mitchenall, L. A. (1990) Mol. MicrobioL 4, 29-37. 24. Huala, E. & Ausubel, F. M. (1989) J. Bacteriol. 171, 3354-3365. 25. Huala, E. & Ausubel, F. M. (1992) J. Bacteriol. 174, 1428-1431. 26. Fernindez, S., de Lorenzo, V. & Perez-Martin, J. (1995) Mol. Microbiol. 16, 205-213. 27. Wootton, J. C. & Drummond, M. (1989) Protein Eng. 2, 535-543. 28. Dixon, R. (1986) Mol. Gen. Genet. 203, 129-136. 29. Claverie-Martin, F. & Magasanik, B. (1992) J. Mol. Biol. 227, 996-1008. 30. Richet, E., Vidal-Ingigliardi, D. & Raibaud, 0. (1991) Cell 66, 1185-1195. 31. Austin, S., Buck, M., Cannon, W., Eydmann, T. & Dixon, R. (1994) J. Bacteriol. 176, 3460-3465. 32. Gartenberg, M. R. & Crothers, D. M. (1991) J. Mol. BioL 219, 217-230. 33. Giladi, H., Igarashi, K., Ishihama, A. & Oppenheim, A. M. (1992) J. Mol. Biol. 227, 985-990. 34. Higgins, C. F., Hinton, J. C. D., Hulton, C. S. J., Owen-Hughes, T., Pavitt, G. D. & Seirafi, A. (1990) Mol. Microbiol. 4, 2007-2012. 35. Zuber, F., Kotlarz, D., Rimsky, S. & Buc, H. (1994) Mol. Microbiol. 12, 231-240.