The integration host factor (IHF) of Pseudomonas putida connects cell growth to transcriptional activity of distinct promoters. The IHF site of the σ54 promoter Pu of the TOL (m-xylene biodegradation) plasmid pWW0 of P. putida has been examined to define experimentally a relationship between occupation of the promoter by this factor, the biological activity of the protein and the tolerance of the target site to single-base changes through the bound DNA core sequence. The use of an in vivo high-intensity UV imprinting procedure to examine such an occupation of Pu by IHF allowed inspection of the interplay between the factor and cognate site variants under the physiologically relevant conditions of monocopy gene dosage. The resulting data were merged in a structural model for establishing key features of the IHF–DNA interaction. A functional consensus for first-order IHF binding was instrumental for a genome-wide survey of sequences with potential regulatory value. This search revealed that very few, if any, of the maximum 330 sites within intergenic regions were placed in locations controlling expression of central metabolic genes. It thus seems that the IHF regulon of P. putida has a degree of functional specialization that is not evenly distributed through all gene categories.
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In this work, we have addressed this issue by revisiting the binding of the IHF of Pseudomonas putida to target sites in vivo in order to create reference for discerning the regulatory scope of the factor. Our approach entirely stems from the single IHF site located at the intervening region between the upstream activating sequences and the RNAP of the σ54 promoter Pu of the TOL (toluene and m-xylene biodegradation) plasmid pWW0 of P. putida (Ramos et al., 1997). The role of IHF in this promoter is to facilitate the interaction between the m-xylene responsive enhancer-binding activator protein XylR and the promoter-bound σ54-RNAP (de Lorenzo et al., 1991; Calb et al., 1996; Pérez-Martín and Lorenzo, 1996a,b). Specifically, IHF binding to Pu fixes an optimal promoter geometry that facilitates contacts between otherwise distant proteins and aids the recruitment of σ54-RNAP (Bertoni et al., 1998). As a consequence, activity of the normally strong Pu promoter is virtually undetectable in P. putida cells lacking IHF (de Lorenzo et al., 1991; Calb et al., 1996; Valls et al., 2002). We have exploited such a dependence of Pu activity on IHF to set up a reliable in vivo system that measures accurately the functional weight of each of the nucleotides of the bound sequence in IHF binding and activity. A combination of site-directed mutagenesis, in vivo UV imprinting in monocopy gene dosage and lacZ reporter technology has allowed us to generate a reliable standard for surveying potential first-order IHFP. putida binding to target sequences in the genome of this bacterium. Despite some remaining uncertainties regarding the predicted vs. actually bound IHF sites, their location in respect to adjacent genes in the chromosome suggests that IHF has become specialized to regulate a number of housekeeping functions in P. putida that conspicuously rules out central metabolic duties.
Results and discussion
Surveying specificity determinants of the IHFP. putida–DNA interplay
The starting point of the present study was the examination of both the DNA sequences and the matching amino acid residues that bring about strong interactions between the IHF protein of P. putida and its target sites. To this end, a dependable tridimensional model of such interactions was made based on the considerable similarity of the primary structure of the P. putida IHF α and β subunits with the counterpart factor from E. coli (Rice et al., 1996; Muñoz et al., 2010). The alignment of Fig. 1 reveals an identity of 87% between the α chains of either bacterium and 74% for the corresponding β. Threading the sequence of the IHFP. putida on the known crystal structure of the E. coli protein yields an rmsd value of 0.09 for all residues of both structures (model and template) as can be expected from the high degree of primary sequence homology. The overall distribution of amino acid residues involved in DNA binding is kept, although there are some noticeable differences (see below). Otherwise, acidic amino acids placed on highly conserved regions of either subunit are identical. This structure accounts for the effective mutual complementation between IHF variants of either bacterium (Calb et al., 1996). If one places the E. coli extended consensus binding sequence at-aatt--attaaWATCAR-aggTTR------a-a (Goodrich et al., 1990) on the IHF structure, three regions become immediately noticeable to be important, namely an upstream AT-rich segment which seems to be indirectly recognized, as well as WATCAR and TTR sequences that account for much of the specificity of the interaction (Rice et al., 1996; Rice, 1997). Comparison of such DNA sequences with the strong IHF-binding site present in the σ54-dependent promoter Pu (de Lorenzo et al., 1991; Bertoni et al., 1998; Fig. 2) verifies the gross correspondence between the P. putida protein sites and the nucleotides that are involved in the interaction. Yet, it also pinpoints manifest differences at the critical WATCAR and TTR motifs which seem to primarily determine first-order DNA–protein recognition (Muñoz et al., 2010). On this basis, we set out to identify the optimal DNA sequence primarily targeted by IHFP. putida by examining the effects of directed changes in each of the nucleotides of such a core motif.
In vivo activity of Pu variants containing mutant IHF sites
Integration host factor is strictly required for transcriptional activation of the m-xylene responsive Pu promoter. Figure 2 shows the location of the target site between −47 and −86 upstream of the transcription initiation site. IHF not only assists formation of the right promoter geometry (Pérez-Martín et al., 1994b), but it also helps recruitment of σ54-RNAP (Bertoni et al., 1998; Carmona et al., 1999; Macchi et al., 2003) and suppresses promiscuous activation by other enhancer-binding proteins (Pérez-Martín and de Lorenzo, 1995). This provides an reliable genetic system to examine and quantify the effects caused by replacing one at a time each of the bases directly involved in IHF action as exposed in the structural model discussed above. For this, we generated nine single-base mutant of the IHF site of Pu by replacing the positions with the same co-ordinates of the WATCAR and TTR motifs by the second most frequent nucleotide of the E. coli consensus (Goodrich et al., 1990), as shown in Fig. 2. For the sake of comprehension, we have numbered 1 to 13 each of the bases AATCAAtaatTTA of the Pu promoter that correspond to the E. coli consensus W1A2T3C4A5R6-aggT11T12R13. Note that the UP region of Pu maps further upstream of the IHF site (Bertoni et al., 1998) and therefore none of these mutants interfere with that component of the activation mechanism. This granted, mutant promoters generated as explained in Experimental procedures were passed to vector pBK16 vector for delivery into the same unique genomic site of a specialized reporter P. putida strain bearing a chromosomal copy of the xylR regulator (P. putida KT2442 hom.fg. xylR/S; Kessler et al., 1992). Conjugative transfer of pBK16 derivatives followed by plating on Sm/X-gal plates led to the generation of mono-copy transcriptional Pu-lacZ fusions that maintain the gene dosage and the stoichiometry of the native system (Fig. 3). With this procedure, we generated 10 entirely isogenic reporter strains, which differed only by the sequence of the IHF site at the Pu promoter. They were then separately cultured in LB medium and exposed to saturating vapours of the XylR effector (and thus Pu inducer) m-xylene (Abril et al., 1989). The accumulation of β-galactosidase by each of the strains after 4 h of induction is plotted in Fig. 3 as a percentage of the levels found in the wild type (4538 ± 630 Miller units). The basal activity of the strains without induction or lacking IHF (i.e. in an ihfA mutant; Valls et al., 2002) was < 200 Miller units. LacZ activities of the strains examined break down the collection of mutants into two groups. In one case, mutants in positions 2, 4, 5, 12 and 13 (numbered according to the co-ordinates of the core sequence: see Fig. 2) appear to lose, to different extents, much of the responsiveness to IHF, the most dramatic being 4, 5 and 12. Mutants 2 and 13 keep a residual activity which is in any case very low compared to the wild-type promoter. The second group of mutants (1, 3, 6, 11 in Fig. 2) do keep Pu activity at high levels but display also some differences. While mutants 1 and 3 came down by a mere 20% in respect to the reference promoter, mutants 6 and 13 displayed an activity indistinguishable from the promoter with an intact IHF site – if not even higher. Alignment of these results with the core consensus site of E. coli (Fig. 2) suggested dissimilarities in the way IHFP. putida and IHFE. coli interact with their respective sites. In fact, the trend is that of a relaxation of the consensus that can be recognized by IHF in P. putida. For instance, mutant Pu-1, which undergoes only a ∼ 30% reduction in promoter output, enters a C residue in a position where the consensus accepts only A or T. Similarly, Pu variant 3 affects a very conserved T residue (exchanged by an A in the mutant), but the promoter still works within the range of the wild type. This is in contrast with mutations in adjacent, equally conserved bases that are changed in Pu variants 2, 4 and 5 which, as expected, lost much of their activity. Expectedly also, replacement of the existing A by a G in mutant Pu-6 originated a 100% active promoter. The most surprising occurrence, though, happened at the downstream TTA motif (TTR in the consensus). While changes T→C and A→C of mutants 12 and 13 respectively did bring down Pu output, replacement of the very conserved T by an A in mutant 11 had no consequences.
A provisional conclusion of all of the above is that the IHF–DNA interplay paradigm of E. coli may not be automatically translated to P. putida (and perhaps to any other species either) and that a specific consensus sequence has to be raised for this bacterium. Still, the data above were generated through an indirect procedure for measuring the action of IHF on Pu and it says little on the actual occupation of the sites in vivo. To examine whether promoter output was faithfully correlated with IHF binding to the various mutant promoters we resorted to a direct method to assess protein–DNA interactions in vivo, as explained below.
Monitoring IHF binding in vivo to Pu promoter variants with high-intensity UV imprinting
The degree of occupancy of the IHF site of the Pu promoter by the factor under the same induction conditions employed to measure β-galactosidase above was inspected with a non-disruptive in vivo footprinting/imprinting procedure. The method employed (which is explained in detail in Valls and de Lorenzo, 2003) is based on the pattern of [32P] 5′-end-labelled primer extension products caused by the arrests of the proceeding DNA polymerase when it finds intra-strand pyrimidine dimers originated by UV irradiation of whole cells. Since formation of such T^T or T^C dimers is greatly influenced by the neighbouring structure of the DNA, in particular by its local curvature, the binding (or lack of it) of a DNA-bending protein can easily be assessed (Engelhorn et al., 1995; Valls et al., 2002). To have a reliable indication of the attachment of IHF to its target site in Pu with this method we first run the procedure in vitro with plasmid DNA encompassing the region of interest and the purified IHF protein of P. putida. The [32P]-labelled oligonucletide was designed to prime upstream of the transcription initiation site for having a maximum (i.e. single nucleotide level) resolution of the IHF binding sequence. Figure 4 shows the result of such an in vitro experiment, where the gel loaded with the extension products displayed clear-cut descriptor of IHF binding in the relative ratio of bands −58/57 vs. −66/65 as well as in the protection of five out of the nine bands corresponding to the long T-rich track at −75/86. These patterns are identical to those found with an alternative high-energy UV laser imprinting procedure (Valls et al., 2002). With this reference in hand, we set out to run the same protocol with live cells. To this end, the strains under examination (carrying either the wild-type Pu sequence or its mutant variants integrated in the chromosome) were grown in LB plus m-xylene up to early stationary phase, the condition where IHF concentration in the cells is maximal (Valls et al., 2002). The cultures were then irradiated with a non-coherent high-intensity UV light beam (Experimental procedures) and the genomic DNA extracted for primer extension. Figure 4 shows a gel loaded with each of the corresponding labelled products. The control sample with the wild-type Pu promoter exhibited intense bands at −66/65, very faint signals at −58/57 and a considerable protection of the upstream T-track, all indicating a complete occupation of the promoter by IHF. The same was true for Pu mutant 1, whose change in respect to the E. coli consensus was as irrelevant for IHF binding as it was for promoter activity (Fig. 3). In contrast, mutant 2 gave a pattern of bands virtually identical to that of the wild-type promoter in vitro in the absence of IHF, indicating that this sequence cannot bind (or binds poorly) IHF in vivo either. In the case of mutants 3 and 4, the bands corresponding to positions −66/65 disappeared because the native TC duplet is changed to AC and TA respectively, which cannot yield pyrimidine dimers with UV light. Yet, the other descriptors indicated that Pu mutant 3 was bound by IHF while variant 4 was not at all. This is also in good agreement with the Pu activity data of Fig. 3. Mutant Pu-5 diverged considerably from the previous ones, because both doublets −66/65 and −58/57 were produced with similar intensities. While highlighting of TC doublet at −66/65 does indicate IHF binding, the bands of the upstream T track match those of the unbound form of the sequence. This pattern is not trivial to explain structurally, but it suggests that IHF can interact with such a Pu variant yet it fails to bend DNA. This could explain the virtual lack of activity of the cognate Pu-lacZ fusion (Fig. 3). Pu mutants 6 and 11 yield interaction patters virtually indistinguishable from the wild type, even more pronounced in the bands that demonstrate binding. It comes as no surprise that promoter activity is also indistinguishable from the non-mutated Pu. Finally, the extension products of variants 12 and 13 are very similar to those of Pu mutant 5 discussed above (i.e. possible IHF interaction with DNA, but little or bending) and the transcriptional activities that they endow are low as well.
Structural understanding of the IHFP. putida–DNA interplay
Figure 5 summarizes the results of both the Pu-lacZ experiments and the UV in vivo imprinting assays. The gross effects of the mutations examined in this work match the role of the corresponding nucleotide in the structural model, although some differences with the E. coli case become apparent. First, exchange A1→C1 results in a still active promoter (Fig. 3), perfectly able to bind IHF (Fig. 4). That position thus tolerates a base other than a W (A,T) perhaps because – according to the structural model – there may not be direct interactions of the protein with that DNA position. In contrast, location 2 of the target sequence is not permissive, for the reason that a A2→G2 change dramatically decreased both Pu promoter occupation and transcriptional activity. The basis of this requirement could be that the phosphate group of A2 is predicted to directly interact with amino acid residues H54 and R56 of the IHF chain β (Fig. 1). In position 3 (Fig. 2), most known E. coli sequences have T, although an A is occasionally found (Muñoz et al., 2010). This fits the behaviour of the T3→A3 change, which causes only a small reduction of both IHF binding and Pu promoter activity. Threading the IHFP. putida–DNA sequences in the crystal structure of Rice et al. (1996) and Rice (1997) prognoses a DNA bending of 32° at this location as a result of protein-induced buckling, and either T or A in position 3 tolerates a narrower minor groove as predicted. In addition, the base at position 3 interacts with IHF indirectly through a water molecule, what may increase its tolerance to mutation. According to Rice et al. (1996) and Rice (1997) the DNA molecule buckles up upon insertion of the P64 of IHF subunit α in the positions 4 and 5 of the consensus. The C and A nucleotides in these positions are conserved in every known IHF site (Muñoz et al., 2010), probably because these two base pairs are the only ones of the E. coli consensus involved in direct interactions with the protein. This seems to be the case of the P. putida protein, because Pu variants C4→A4 and A5→C5 mutations lack both IHF binding and transcriptional activity. Position 6 is believed to have a strong preference for an A (and less for G, thereby the W in the consensus code), because mutation of its paired T reduces binding by fivefold (Muñoz et al., 2010). Our data indicate this not to be essential in P. putida, because Pu mutant 6 behaves in a fashion impossible to tell apart from the wild type. It thus seems that either A or G at position 6 further favour intercalation of IHFα P64, allowing extensive hydrophobic contacts among the proline and base rings. Next, according to the crystal structure of the E. coli's protein (Rice et al., 1996), the second portion of the consensus (T11T12R13) is recognized by the core body of IHF rather than its distinct upwards extensions (Fig. 1). In this case, amino acid R46 of IHF chain β reaches out the minor groove of DNA to make both direct and water-mediated hydrogen bonds to the three conserved bases and van der Waals interactions with the ribose moieties. Such contacts through a single protein residue require a narrower minor groove and explain the bias against bases with bulky amino groups (G, C) at positions 11–12 but tolerance to T→A transversions. Our results with variants T11→A11 mutation (full activity, full binding) and T12→C12 (unproductive binding, no activity) are consistent with this view. Finally, stable IHF–DNA interactions require a somewhat odd kink between bases 12 and 13 of the consensus that adds to the overall sharp bending. Therefore, an easily deformable duplet such as T12 R (A,G)13 often occurs in existing native IHF sites. Consistently with this, our Pu mutant 13 (A13→C13) had only a marginal transcriptional activity, which surely resulted from a suboptimal binding to the factor.
Figure 5 shows the apparent core consensus WWCARnnnnWTR for IHFP. putida binding that recapitulates all of the above results and analyses. Note that the sequence is somewhat more permissive than the one proposed for E. coli (WATCARnnnnTTR). The P. putida's sequence was then employed to survey its chromosome for such sites and thus examine the potential regulatory landscape of the factor in this bacterium. To this end, it should be noted that recognition of IHF target sites is driven by indirect readout (Rice et al., 1996; Muñoz et al., 2010), which involves also interactions with less defined adjacent sequences (see above). Although such extensions (typically AT-rich) are important, their role in mediating binding vs. bending vs. functionality is not straightforward (Goodman et al., 1999). Also, since only single-base mutations were employed to produce the consensus, then we cannot rule out that given combinations of other changes could also accommodate IHF binding. This sets some limitations on the use of the consensus IHFP. putida first-order binding motif just proposed for identifying sites at a genome scale. Yet, the above core sequence is expected to generally rule out false negatives, although not false positives. With these considerations in mind we set out to employ the core sequence WWCARnnnnWTR as a reference to estimate the maximum extent of the IHF regulon in P. putida.
Distribution of possible IHF binding sites in the P. putida genome
Estimations of the number of IHF binding sites in the genome of E. coli have yielded disparate results ranging from 70 to over 600 locations (Boffini and Prentki, 1991; Freundlich et al., 1992; Ussery et al., 2001; Grainger et al., 2006; Grainger and Busby, 2008). In our case, we resorted to use the functional information regarding the IHF site of the Pu promoter presented above to estimate the upper limit of this figure in the chromosome of P. putida KT2440. Using the consensus sequence determined before, we found 2653 sites, which matched the motif WWCARnnnnWTR and thus represent the maximum edge of the functional extent of the factor. Evidently, these reflect core target DNA sequences that are necessary for specific recognition, but functional binding may not occur unless there are other less defined characteristics in the adjacent sequences, e.g. an upstream AT-rich track and overall bendability of the region (see above). However, given the increasing number of IHF molecules of P. putida along growth (approx. from 100 to > 4000; Valls et al., 2002) it could well happen that many of the sites are in fact occupied in cells grown to stationary phase. Out of them, 330 WWCARnnnnWTR sequences were found in intergenic regions (Table S1) and are thus likely to have a role in regulating expression of contiguous genes. The set of sites encompasses ORFs for functions belonging to different categories (Table S2), including amino acid transport/metabolism, cell envelope and outer membrane biogenesis, coenzyme metabolism, energy production and conversion, signal transduction mechanisms, inorganic ion transport and metabolism, transcription and translation, ribosomal structure and biogenesis (summary in Fig. 6). Also, a large share of IHF sites includes hypothetical proteins within or close to mobile elements, suggesting an important role of the factor in the occurrence of horizontal gene transfer (Fass and Groisman, 2009). Surprisingly, we did not find IHF sites in any regulatory position near genes encoding central carbon metabolism (i.e. core pathways for consumption/production of carbohydrates and amino acids), an issue that is discussed below. Finally, we examined the positioning of IHF binding sites in respect to repetitive extragenic palindromic (REP) sequences from P. putida. Enterobacterial REP sequences are associated to IHF (Boccard and Prentki, 1993; Oppenheim et al., 1993), but the counterparts found in P. putida may not follow the E. coli paradigm (Aranda-Olmedo et al., 2002; Ramos-Gonzalez et al., 2006). Inspection of the ∼ 800 REP sites of P. putida (Aranda-Olmedo et al., 2002) in respect to predicted IHF sites revealed that there was not one case where the two occurred on top of each other. Moreover, only in 49 instances the distance between IHF and REP sites was < 100 bp (not shown). This indicates that the arraying of REP sequences with IHF sites that seem to be important for nucleoid structure and evolution in some bacteria (van Belkum et al., 1998) has little importance in P. putida. Perhaps this is related to the high GC content found in REP sequences (Aranda-Olmedo et al., 2002), which stands in contrast with the elevated AT share of IHF sites.
Each bacterial group seems to possess a limited number of global regulators. Some of them are associated to the nucleoid and regulate or co-regulate expression of large sets of genes regardless of their specific function (Martinez-Antonio and Collado-Vides, 2003). In E. coli, as little as 7–10 factors that respond to a suite of environmental and metabolic conditions suffice to submit expression of individual genes to the general physiology of the cells at any given time. IHF appears among the top factors that rule this over-imposed control (Ma et al., 2004) and it has bona fide orthologous in many bacteria, including P. putida. The question at stake is whether sharing the same molecular mechanism of action (i.e. binding and sharply bending DNA) goes necessarily together with sharing the same functional scope in different bacteria as well. While the key properties of IHF in its interplay with DNA may be preserved along the phylogenetic tree, small differences in interaction parameters might entirely change the functional window of activity in vivo. This has been observed, e.g. in Crp proteins borne by non-E. coli species, which are structurally very close to the archetypal enterobacterial regulator, but they run an entirely different biological duty depending on the specific host (Milanesio et al., 2011). We show above that the IHF of P. putida shares much of the overall protein structure and target DNA sites with the E. coli counterpart. Yet, it seems that minor disparities in their preferred bound sequences can shift their window of functionality towards dissimilar regulatory duties. Note that the consensus that we propose on the basis of mutagenizing the IHF site of Pu sets an upper limit to the number of genes likely to be influenced by the factor in P. putida, but does not predict accurately what sites may or may not be occupied at given times. Intuitively, this should depend on the specific affinity of IHF for each site, a parameter surely influenced by the sequences flanking the core recognition site (Goodman et al., 1999) and the concurrent binding of other proteins to overlapping or adjacent DNA segments. In this context, it is remarkable that even an overestimation IHF-controlled genes (Table S1) hardly contains any that participates in regulation of central metabolic functions. This conspicuous absence is intriguing and will be the subject of future studies.
Bacterial growth conditions and general methods
Escherichia coli strains were always grown at 37°C in LB or LB-agar supplemented, when required with 100 µg ml−1 of ampicillin, 50 µg ml−1 of streptomycin or 30 µg ml−1 of chloramphenicol, as needed. Unless indicated otherwise, P. putida was grown in LB at 30°C in the presence of 200 µg ml−1 of streptomycin, when required. Plasmids were transferred from donor E. coli strains into the P. putida receptor strain by mobilization using a filter mating technique (de Lorenzo and Timmis, 1994). Tripartite matings were set with equal amounts of donor, receptor and helper E. coli HB101 (pRK600) strains and incubated for 8 h at 30°C on the surface of LB plates. Cells were resuspended in 10 mM MgSO4 and appropriate dilutions plated on M9 minimal medium with 0.2% citrate supplemented with streptomycin, X-gal and 3 mM 3-methyl-benzyl-alcohol (3-MBA). This medium permitted the counter-selection of E. coli, and the identification of double recombinants as pale blue or blue colonies, depending on the activity of the Pu promoter mutant (see below). Double recombinants were re-streaked and verified by sensitivity to kanamycin. For quantification of promoter output, β-galactosidase activity assay was measured from 100 µl of bacterial cultures following the procedure of Miller (1972). The linearity of the assay within the range of cell densities and the time of reaction with o-nitrophenyl-β-D-galactoside was verified in all cases. β-galactosidase activity values given throughout this study represent the average of two independent experiments using three different clones in duplicate samples.
Construction of site-directed mutants in Pu and introduction into the P. putida chromosome
Plasmid pEZ9 was used as a template for mutagenesis of the IHF-binding site in Pu promoter. This is a pUC18 derivative that carries the entire region between co-ordinates −208 and +93 of the Pu sequence in respect to transcription initiation, inserted as an EcoRI-BamHI fragment (de Lorenzo et al., 1991). Single-base mutations were introduced in pEZ9 by using the QuikChange™ XL site-directed mutagenesis kit (Stratagene) with pairs of complementary 33-mer oligonucleotides bearing the desired mutation in the central position. This mutagenic procedure originated pEZ9 derivatives each bearing a different single-base change in Pu that was verified through DNA sequencing of the insert region. For recombination of the different Pu variants at the same location in the P. putida chromosome, the wild-type Pu sequence and its nine single-base variants were excised from the pEZ9 plasmids with EcoRI/BamHI and re-cloned in the corresponding sites of the delivery plasmid pBK16. This plasmid contains a small polylinker flanked by the aadA streptomycin/spectinomycin resistance cassette and the lacZ gene, both containing an amber codon in their sequences (Kessler et al., 1992). Cloning of the Pu promoter and its mutant derivatives in the EcoRI/BamHI polylinker sites originated a corresponding collection of Pu-lacZ transcriptional fusions. pBK16-Pu and its mutant variants were routinely maintained in an E. coli supF strain that enabled to select plasmid maintenance by resistance to streptomycin (Kessler et al., 1992). pBK16-derived plasmids were mobilized by conjugative transfer in P. putida KT2442 hom. fg. xylR/S– a specialized Pseudomonas strain that bears a Km resistance gene between truncated aadA and lacZ genes in the chromosome (Kessler et al., 1992). P. putida KT2442 hom. fg. xylR/S also bears a chrosomosomal insertion with the xylR gene under the control of its native promoter. Selection of transconjugants (see above) enabled the isolation of double recombinants in which the Pu variants had replaced the KmR cassette in the chromosome (Fig. 3).
In vivo UV footprinting/imprinting
The P. putida strains under examination were grown in liquid LB until an OD600 = 1.0 in the presence of inducer m-xylene. The cultures were then divided in 50 µl of aliquots into 96-well plates and each well was irradiated with UV during 25–30 s as described (Valls and de Lorenzo, 2003). Aliquots were immediately collected, mixed and chilled on ice. Genomic DNA was extracted out of 500 µl of light-exposed bacterial cultures and processed (see Valls et al., 2002, for details), in order to reveal the photo-reactivity of the DNA region under study. To this end, 5–10 µg of the treated genomic DNA were subject to 45 cycles of primer extension (1 min 94°C, 1 min 59°C, 1 min 72°C) using Taq polymerase (Perkin Elmer, MA) and the radioactively [32P]-5′-labelled oligonucleotide 5′CCCGCTTTGAGGATATACATGGCGAAAGC3′. This sequence is complementary to positions +63/+91 on the coding strand of the Pu promoter DNA. Amplifications were performed in a standard PCR buffer with 2.5 mM MgCl2, 5 pmol of labelled primer and five units of polymerase. After a final extension for 10 min at 72°C, the amplified products were ethanol-precipitated, washed with 70% ethanol and resuspended in 5 µl of TE and 10 µl of formamide loading buffer (Ausubel et al., 1994). Half volume of the samples was analysed by electrophoresis in 6% denaturing polyacrylamide gels (Ausubel et al., 1994), which were subsequently dried, exposed for 1 week to a Molecular Imager System ®-sensitive screen and visualized with the Quantity One software (Bio-Rad). For in vitro control experiments, 50 ng of supercoiled pEZ9 plasmid – which contains the Pu promoter – were incubated in the presence or absence of the purified P. putida IHF protein as described (Valls et al., 2002). Ten-microlitre samples were then irradiated for 15–20 s with the UV lamp in 0.5 ml Eppendorf tubes. Each sample was subjected to 10 amplification cycles in a total volume of 20 µl in the same conditions described for genomic DNA (see above). After addition of 6 µl of formamide dye, 2 µl of each sample were loaded for electrophoresis and analysis.
To generate a structural model of the IHF protein of P. putida and its target DNA sequences (Muñoz et al., 2010), we generated multiple alignments Clustal-W on the Psi-Blast list for IHF-like proteins, using the sequence corresponding to both chains A and B from P. putida (according to SMART database). The two chains of IHF were treated independently (one at a time), since experimental information suggests that the implication of both chains is not the same in the binding of DNA: while IHF-α participates in the recognition of the first segment of the DNA-consensus sequence (WATCAA for E. coli), IHF-α interplays with the second portion of the consensus, TTG. Models for each of the chains were generated with the alignments obtained previously from clustal-W and using Swiss-Model, a protein structure homology modelling server (Arnold et al., 2006). The quality of model structures was analysed for packing quality, rotamer normality, bond angles and lengths, side chain planarity, dihedral angles, etc., using the tools available at the What-If package (Vriend, 1990). Four residues not involved in protein–DNA interactions in the crystal structure are observed to be facing the protein groove and therefore were not considered in this study. A complete account of the methods employed for this model is to be found in Muñoz et al. (2010).
Prediction of possible IHF sites in P. putida KT2440 genome
The consensus sequence driven from the structural and functional analysis in vivo (WWCARnnnnWTR, where n = A, T, G or C; W = A or T; R = A or G) was employed to probe the genome of P. putida (accession number NC_002947) for matching sites using the Virtual Footprint server http://prodoric.tu-bs.de/vfp (Munch et al., 2005). Genomic search was restricted, where indicated, to non-coding, putative intergenic regions and manually curated, e.g. sites located at the intergenic region between the 3′ ends of two convergently transcribed (and thus unlike to play a role in gene expression) were removed from the analysis. To examine relationships with REP sequences, the genomic co-ordinates of the IHF sites were compared pair-to-pair with DNA segments previously reported for P. putida (Aranda-Olmedo et al., 2002) using an ad hoc perl script.
This work was defrayed by generous grants of the CONSOLIDER program of the Spanish Ministry of Science and Innovation, by the BACSIN and MICROME Contracts of the EU and by funds of the Autonomous Community of Madrid.