σ54-Promoter Discrimination and Regulation by ppGpp and DksA*

The σ54-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of σ54-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of σ54-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of σ54-dependent transcription in combination with a series of σ54-promoters with graded affinities for σ54-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted σ54-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous σ54-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive σ54-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity σ54-promoters that is conferred by the strong DNA binding properties of the P. putida σ54-RNA polymerase holoenzyme.

The 54 -factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of 54 -dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of 54 -dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of 54 -dependent transcription in combination with a series of 54 -promoters with graded affinities for 54 -RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted 54 -transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous 54 -RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive 54 -transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity 54 -promoters that is conferred by the strong DNA binding properties of the P. putida 54 -RNA polymerase holoenzyme.
To ensure promoter specificity, the bacterial multisubunit catalytic core RNA polymerase (RNAP) 4 (␣ 2 ␤␤Ј) is guided to the distinct promoter classes within the genome by a dissociable -factor. In addition to the household 70 -factor, most bacteria encode a variable number of alternative -factors that fall into two families based on primary amino acid sequences and regulatory properties. The largest of these is the 70 -family, which encompasses most alternative -factors within four subgroups that all form RNAP holoenzymes that can spontane-ously initiate transcription (1). Orthologs of the structurally distinct 54 -subunit are the only members of the second family. 54 imposes kinetic constrains that lock the 54 -RNAP holoenzyme in a non-productive closed complex. Thus, 54 -dependent transcription always requires activators, called bacterial enhancer-binding proteins or bEBPs, which use ATP catalysis to remodel 54  Many bacteria from different phylogenetic groups use 54 dependent transcription to control numerous environmentalresponsive processes ranging from expression of chemotaxis transducers and assembly of motility organs, through alginate biosynthesis, to nitrogen assimilation and the utilization of different carbon sources (Refs. 2-4 and references therein). The metabolically and environmentally versatile Pseudomonas putida species use 54 -dependent transcription extensively (5). This group of organisms, in combination with resident catabolic plasmids, is notably associated with its ability to use aromatic compounds as growth substrates. In many cases, the expression of these pathways are ultimately dependent on 54 -RNAP and are subject to global regulatory input. This results in suppression of transcription of the specialized catabolic genes when energetically more favorable carbon sources are present (reviewed in Refs. 6 and 7). One such example is the dimethylphenol dmp-system of the plasmid pVI150, in which transcription of the dmp-operon for production of the catabolic enzymes is driven by the 54 -Po promoter. Transcription from 54 -Po is controlled in response to the presence of phenolic dmp-pathway substrates through the action of the divergently transcribed dmpR gene product, which binds the aromatic compounds to achieve its active transcriptional-promoting form (8 -10).
For the DmpR-regulated 54 -Po promoter, the bacterial alarmone ppGpp acts together with DksA to provide a link between 54 -dependent transcription and the metabolic status of the cell. This dominant global regulation results in silencing of Po activity until nutrient depletion at the exponential to stationary growth phase transition (11)(12)(13)(14). In Escherichia coli, the levels of ppGpp vary enormously and are rapidly elevated in response to both nutritional limitation and physicochemical stress to redirect transcriptional capacity toward genes required to counteract adverse conditions (reviewed in Ref. 15). Both ppGpp and DksA directly target RNAP so that the relatively constant levels of DksA sensitize RNAP to the cellular levels of ppGpp to directly inhibit or stimulate transcription from susceptible 70 -dependent promoters of E. coli (16,17). Lack of ppGpp and/or DksA severely reduces 54 -dependent Po activ-ity in both E. coli and P. putida without altering the constant levels of 54 in the cell (11)(12)(13)(14). Artificial synthesis of ppGpp during exponential growth on rich media, on the other hand, results in transcription from Po under conditions where it is normally silent (11)(12)(13). However, whereas ppGpp and/or DksA-deficiency in E. coli severely limit transcription from Po and a range of other 54 -dependent promoters in vivo, they do not have any apparent effects in an E. coli reconstituted in vitro 54 -dependent transcription system (13). Together with the demonstration that 54 -RNAP levels are limiting in the cell (12), and the properties of RNAP mutations that bypass the need for ppGpp for efficient 54 -dependent transcription, these findings have led to a model in which ppGpp synthesis indirectly results in elevated levels of 54 -RNAP through competition between 54 and other -factors for varying levels of core RNAP that are available for holoenzyme formation (12,13,18).
DksA and ppGpp have also been found to play other roles in the DmpR/ 54 -Po regulatory circuit that would also contribute to the abrupt high level transcription observed from Po at the exponential to stationary phase growth transition (14). First, ppGpp and DksA directly stimulate transcription by 70 -RNAP from the Pr promoter of the dmpR gene to elevate DmpR levels (14). Second, ppGpp and DksA might also elevate the levels of IHF (integration host factor) that facilitates productive interaction between Po-bound 54 -RNAP and DmpR bound to its distally located binding sites (upstream activation sites, UASs) (19). Third, DmpR also stimulates its own production through a mechanism in which 54 -RNAP activity at the 54 -Po promoter stimulates 70 -Pr output. This regulation thus generates a feed forward loop that is dependent on not only the direct effects of ppGpp and DksA on Pr activity, but also their effects on IHF and 54 -RNAP levels required for Po activity (14).
Much of the analysis of the mechanisms of ppGpp/DksAmediated control of 54 -transcription have been identified using mutants of E. coli, which has a very different lifestyle than the environmentally and nutritionally adaptable soil Pseudomonads. In addition to bouts of feast and famine, soil bacteria are frequently exposed to extremes in physicochemical parameters (temperature, water content etc). When faced with aro-matic compounds, bacteria are confronted with chemicals that, can potentially be catabolized as a carbon and energy source for growth, but are also toxic stress agents (Ref. 20 and references therein). Transcription from 54 -promoters, as with any other type of promoter, functions within the context of the evolutionary selected regulatory network of the host bacteria. In this work we sought to test if regulatory mechanisms that couple 54 -dependent transcription to cellular physiology in E. coli also operate in P. putida. In particular, we were interested to determine whether the properties of the 54 -RNAPs from E. coli and P. putida underlie differences in ppGpp/DksA control of 54 -promoter activity that we observed in vivo in these two organisms.

EXPERIMENTAL PROCEDURES
General Procedures-E. coli and P. putida strains (Table 1) were grown in Luria-Bertani medium (LB), (21) supplemented with the following antibiotics as appropriate for the strain and resident plasmid selection: carbenicillin (Cb, 100 g ml Ϫ1 for E. coli and 2 mg ml Ϫ1 for P. putida), kanamycin (Km, 50 g ml Ϫ1 for both E. coli and P. putida), tellurite (Tel, 40 g ml Ϫ1 for P. putida), tetracycline (Tc, 5 g ml Ϫ1 for E. coli and 50 g ml Ϫ1 for P. putida), and trimethoprim (Tp, 100 g ml Ϫ1 for E. coli). M9 minimal media plates (21) supplemented with 10 mM glucose and 100 g ml Ϫ1 thiamine were used for prototrophy tests. E. coli DH5 was used for construction and maintenance of all but R6K-based suicide plasmids for which the replication permissive S17pir host was used.
Plasmids and DNA Manipulations-Plasmids were constructed by using standard DNA techniques. The fidelity of all DNA sequences of PCR-generated fragments and linkers was confirmed. Luciferase (luxAB) transcriptional reporters and plasmids used in in vitro transcription assays are listed in Table  2, whereas primers used are listed in supplemental Table S1.
luxAB Transcriptional Reporter Plasmids-Key DNA features of the different luciferase transcriptional reporters used in this study are shown in Fig. 1. Plasmids were constructed by a common procedure of assembly on pBluescript SKϩ (Stratagene) followed by subcloning into an RSF1010-based plasmid to generate the broad host range (16 to 20 copies per cell) transcriptional reporters (13). Construction of pBluescript-based plasmids pVI769 and pVI733 has previously been described (13). Plasmid pVI769 carries the dmpR-Po promoter region with unique NdeI and BamHI sites located Ϫ39 relative to the transcriptional start of Po for insertion of 54 -promoter regions upstream of the promoterless luxAB genes. Plasmid pVI733 carries the same DNA as pVI769 but with a non-native XhoI site engineered at Ϫ122 to Ϫ117 relative to the transcriptional start of Po. The XhoI to NdeI Po promoter upstream region of pVI733 was replaced by the equivalent region of Pu using a custom designed linker, resulting in pVI778. In the next step, linkers with the desired 54 -promoter sequences were introduced into pVI778, as previously described (13), to give pVI779 (xh-Pu/Po-luxAB), pVI780 (xh-Pu/Pu-luxAB), pVI781 (xh-Pu/ PpspA-luxAB), and pVI782 (xh-Pu/PglnA-luxAB). The promoter-luxAB fusions of pVI779 to pVI782 were then excised as BglII to HindIII fragments and cloned between the BamHI and HindIII sites of the RSF1010-based broad host range plasmid pVI432 (13), resulting in pVI789 to pVI792, respectively. To generate a monocopy luxAB transcriptional reporter of the dksA promoter in P. putida, we used the Km R R6K-based suicide plasmid pKm705L that carries the promoterless luxAB genes downstream of a polylinker (22). The promoter region of P. putida dksA was then amplified as a KpnI to SacI fragment using primers dksApro-f and dksApro-r and inserted between these sites of pKm705L to generate pVI906 for subsequent single site recombination into the chromosome of P. putida as previously described (22).
DksA Expression Plasmid-The expression plasmid pVI905 encoding P. putida His-DksA was constructed as described for E. coli His-DksA that is functionally indistinguishable from its native counterpart in in vivo and in vitro assays (16). The P. putida dksA coding region (TIGR PP4693) was amplified as an NheI to HindIII fragment using primers dksA-f and dksA-r and cloned between these sites of pET28a (Novagen), to give pVI905.
pfrA Gene Inactivation Plasmid-To generate a gene replacement mutant of the pfrA gene of P. putida (TIGR PP0191), an XhoI to BamHI fragment spanning the 5Ј-and 3Ј-ends of the pfrA gene with a unique BglII site replacing codons 17 to 156 was generated by overlapping PCR using primers pfrAup-f, pfrAup-r, pfrAdo-f, and pfrAdo-r. The resulting XhoI to BamHI fragment was cloned between these sites of pBluescript SKϩ (Stratagene) to generate pVI909. A BamHI fragment carrying the Km gene of p34S-Km3 (23) was then cloned into the BglII site of pVI909, with the antibiotic resistance gene transcribing in the same direction as the pfrA gene, generating pVI910. Finally, the resulting ⌬pfrA::Km gene replacement was cloned as an XhoI to BamHI fragment between the XhoI and BglII sites of the R6K suicide plasmid pDM4-Tc (22) resulting in pVI911.
Strain Generation-Introduction of the lacI Q /P tac -ihfAB mini-Tc transposon carried on the suicide plasmid pUTtetPF (24) into the chromosome of P. putida KT2440::dmpR-Tel was by conjugation from the E. coli donor host S17pir. Tellurite and tetracycline were used as counter selection for the donor and recipient parental strains, respectively. To generate the ⌬pfrA::Km kanamycin gene replacement mutant of P. putida KT2440::dmpR-Tel, pVI911 was introduced by conjugation from S17pir, with tellurite and kanamycin used as counter selection for the donor and recipient parental strains. Double site recombinants were identified by screening for loss of the plasmid-encoded Tc resistance marker, and verified by diagnostic PCR using primers specific for the intact pfrA gene and the ⌬pfrA::Km kanamycin gene replacement.
Luciferase Reporter Gene Assays-Luciferase assays of the luxAB gene product were performed on cultures grown and assayed at 30°C. To ensure balanced growth, overnight cultures were diluted and grown into exponential phase prior to a second dilution to A 600 of 0.05 to 0.08 and initiation of the experiment by addition of the DmpR effector 2-methylphenol or the XylR effector 3-methylbenzylalcohol to a final concentration of 2 mM for P. putida strains and 0.5 mM for E. coli strains. When used, IPTG (isopropyl ␤-D-thiogalactopyranoside) was present throughout growth from overnight cultures. Light emission from 100 l of whole cells using 1:2000 dilution of decanal was measured using a PhL Luminometer (Aureon Biosystems). Data points are the average of duplicate determinations from each of two or more independent experiments.
Immunoblot Analyses-Crude extracts of soluble cytosolic proteins, SDS-PAGE separation, and electrotransfer to membranes for immunodetection of proteins were as described previously (25). Rabbit polyclonal antiserum against E. coli DksA (gift from D. Downs, Wisconsin) was pre-cleared against total cell lysate of an E. coli DksA null mutant (RK201) prior to use. Other polyclonal antisera used were directed against P. putida IHF (26), the amino-terminal 232 residues of DmpR (13) and against P. putida RpoN (14). Antibody-decorated bands were revealed using Amersham Biosciences polyvinylidene difluoride membrane and ECLϩPlus reagents as directed by the manufacturer.
ppGpp and Purified Proteins for in Vitro Assays-The nucleotide ppGpp was synthesized from ribosome-associated RelA as previously described (27). E. coli core RNAP and 54 were as

Reporter and in vitro transcription plasmids carrying hybrid promoter regions
Promoter designations, Px/Px, indicate the origin and status of the upstream IHF binding site containing region (Ϫ122 to Ϫ40)/downstream (Ϫ39 to ϩ2) 54 -RNAP binding region as shown in Fig. 1, with Xh denoting the presence of a non-native XhoI site at Ϫ122 to Ϫ117.

-RNA Polymerase Promoter Discrimination
described in Ref. 13. P. putida IHF was a gift from F. Bartels. Native P. putida KT2440 core RNAP was purified as described in Ref. 28, whereas P. putida 54 was purified after overexpression as previously described (14). P. putida His-DksA was expressed in BL21(DE3) pLysS harboring the P T7 -dksA plasmid pVI905. Cells were grown at 30°C to A 650 of 0.7 and expression induced by culturing in the presence of 0.5 mM IPTG for 3 h prior to harvesting the cells and purification of His-DksA by nickel affinity chromatography essentially as described previously for DmpR-His (9).
In Vitro Transcription Assays-Transcription assays employed supercoiled plasmids pVI736 to pVI741, which carry the Ϫ578 to ϩ2 region of the 54 -promoters on pTE103 (29). Reactions were carried out at 30°C in 20 l of T-buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mM EDTA, and 0.275 mg/ml bovine serum albumin) as described in Ref. 13, except that P. putida transcriptional components were used. In brief, core RNA polymerase was mixed with the indicated molar excess of 54 and incubated for 5 min to allow holoenzyme formation. When present, ppGpp (200 M) and DksA (2 M) were added and incubation continued for a further 5 min. Open complex formation was initiated by the addition of 0.5 nM supercoiled plasmid DNA, IHF (10 nM), DmpR-His (50 nM), the aromatic effector 2-methylphenol (0.5 mM) and ATP (4 mM) that are required for DmpR activity. Transcription was initiated by adding 2.5 l of a mixture of ATP (final concentration, 0.4 mM), GTP and CTP (final concentration, 0.2 mM each), UTP (final concentration, 0.08 mM), and [␣-32 P]UTP (5 Ci at Ͼ3,000 Ci/mmol, Amersham Biosciences). In multiple round assays, heparin (0.125 mg/ml final concentration) was added after 5 min to prevent re-initiation and incubation continued for a further 5 min. In single round assays, heparin was added simultaneously with NTPs and reactions incubated for 10 min. After termination of the reactions, transcripts were analyzed on a 4% acrylamide gel containing 7 M urea, followed by quantification using phosphorimaging.
Plasmon Resonance Assays-Plasmon resonance experiments were performed using a Biacore 3000 system (Uppsala, Sweden). Biotin-labeled double-stranded DNA fragments (121 bp) encompassing the PnifH, Po, and PglnA 54 -promoters were coupled to streptavidin immobilized on a CM4 chip by amine coupling. The DNA fragments were generated from Po/Px-luxAB fusions by PCR amplification using primer 2200 (homologous to the Po upstream region) and the biotin-labeled primer 2201 (complementary to the luxAB genes). The experiments were run using buffer B (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mM EDTA, 0.054% P-Surfactant (Biacore)) at 25°C. 54 -RNAP holoenzymes were formed by preincubation of core RNAP and an 8-fold excess of 54 for 5 min in buffer B, then injected at a flow rate of 20 l/min.

RESULTS AND DISCUSSION
Growth Profiles of ppGpp-or DksA-deficient P. putida-We have previously found that whereas the behavior of the 54 -Po promoter in E. coli generally reflects that observed in P. putida, the ppGpp-dependent profiles in the two strains differ (30).
Likewise, the growth properties of ppGpp-deficient P. putida differ from those of its E. coli counterpart. Consistent with its nutritional adaptability, ppGpp-deficient P. putida remains prototrophic, whereas ppGpp-deficient E. coli is polyauxotrophic (30). That this is a genuine difference between the two organisms, rather than due to a prototrophy-restoring mutation in the ␤ or ␤Ј (RpoBC) subunits of the core RNAP (31), was ensured both by the strategy used to generate the P. putida mutants and by confirming the wild-type sequence of the rpoBC genes in three independent isolates (30). 5 This difference between E. coli and P. putida prompted us to also test DksA-deficient derivatives of P. putida. Like ppGpp-deficient P. putida, 10 independent derivatives of DksA-deficient P. putida were found to be prototrophic, which again contrasts the polyauxotrophic phenotype of DksA-deficient E. coli (32).
Genetic Organization of Reporter Systems to Monitor 54transcription in P. putida-Artificial manipulation of ppGpp levels or natural elevation of ppGpp in response to nutrient depletion at the exponential-to-stationary growth phase transition result in efficient transcription from the 54 -Po promoter in both E. coli and P. putida (11,33). All that is needed to reproduce this metabolic coupling is the Po regulatory region and DmpR, either encoded in its native divergent configuration with respect to the Po promoter, or encoded on a separate replicon (30). To probe ppGpp-and DksA-mediated regulation of 54 -transcription in P. putida KT2440, we used derivatives that express the substrate-responsive bEBPs DmpR or XylR from their native cistrons introduced into the host chromosome via mini-transposons. These strains were used in conjunction with a series of luciferase transcriptional reporter plasmids that were constructed within the framework of the 54 -Po promoter to maintain critical promoter architecture and relative spacing of the DNA binding sites of the proteins that control 54 -dependent transcription (13).
As depicted in Fig. 1, these promoters, designated by the Po/Px series, have the upstream region of the Po promoter (Po/, Ϫ122 to Ϫ40) combined with different 54 -RNAP binding motifs in the Ϫ39 to ϩ2 (/Px) regions. The Ϫ39 to ϩ2 regions are from well studied 54 -promoters and include the P. putidaderived XylR-regulated Pu promoter of toluene/xylene catabolism, the Klebsiella pneumoniae-derived nifH promoter and its mutant derivative nifH049, in which substitution of the Ϫ17 to Ϫ15 CCC by TTT increases affinity and transcriptional output, and the strong E. coli-derived pspA and glnA promoters (13). The relative affinities of E. coli 54 -RNAP for these six Po/Px promoters have previously been defined (13). The six Ϫ39 to ϩ2 promoter sequences were further combined with either a mutated Po upstream region in which the IHF binding site consensus had been disrupted but that maintains the same base composition of Po to result in the xh-Po(-IHF)/Px series (xh indicates the presence of a non-native XhoI cloning site), or with the Pu promoter upstream region to result in the xh-Pu/Px series of transcriptional reporters.
Both ppGpp and DksA Are Required for Robust Stationary Phase 54 -Transcription-Comparison of the transcriptional output from the luciferase 54 -Po/Po promoter reporter plasmid in P. putida strains devoid of ppGpp or DksA shows that, as is the case in E. coli (13), both ppGpp and DksA are required for efficient 54 -dependent transcription from this promoter ( Fig.  2A). Consistent with our recent findings (14), the levels of DmpR vary across the growth curve in P. putida, reaching their highest levels in the post-exponential phase of growth ( Fig. 2B and Ref. 14). Again, similar to the case in E. coli, the absence of either ppGpp or DksA did not influence the levels of 54 , whereas ppGpp deficiency (but not DksA deficiency) results in an ϳ2-fold decrease in DmpR levels in both organisms (Fig. 2C and Ref. 13).
P. putida contains a homologue of both the E. coli RelA ppGpp synthetase I and the bifunctional SpoT ppGpp-hydrolase/synthetase II (30). It has previously been experimentally demonstrated that ppGpp levels are dramatically increased at the exponential to stationary phase transition in LB cultured P. putida (11). In E. coli, the levels of the DksA protein are relatively constant across the growth curve and under different growth conditions (16,32,34), whereas in P. aeruginosa, they have been found to vary (35). Therefore, it was of interest to compare transcriptional profiles and DksA protein levels in P. putida and E. coli. To this end we monitored transcription across the growth curve with derivatives of each species that each carried a transcriptional fusion of the luxAB reporter genes to the respective promoter of dksA on their chromosomes. As shown in Fig. 2D, transcription from the P dksA promoters in both species peak during exponential growth then slowly trail off. However, these differences in transcription did not result in more than a 2-fold difference in DksA levels (Fig.  2E). Thus, similar to E. coli, regulation of 54 -dependent transcription by ppGpp in P. putida is primarily mediated by changes in ppGpp levels (11) rather than DksA levels (Fig. 2E).
IHF Levels Do Not Restrict 54 -transcription in P. putida-Physical interaction of the bEBP and 54 -RNAP is aided by IHF-mediated DNA-bending. IHF protein levels increase at the exponential-to-stationary phase growth transition in both E. coli and P. putida and have been shown to be partially under the control of ppGpp in E. coli (26,36,37). Therefore, we considered that IHF levels may contribute to ppGpp/DksA mediated control of 54 -dependent transcription in P. putida.
To examine this possibility, we first used the same genetic systems as described under Fig. 2 to monitor transcriptional output from the xh-Po(-IHF)/Px series of 54 -promoters ( Fig.  1) that differ from the xh-Po/Px series only in minimal changes that abolish IHF binding (13). We found that the temporal transcription profiles of the xh-Po/Px series and cognate xh-Po(-IHF)/Px plasmids were indistinguishable from that shown for the Po/Po-luxAB reporter in Fig. 2A, except that maximal transcriptional output was reduced by lack of IHF-binding  (19) and Pu (52) are shown in bold and aligned with the consensus sequence that includes the core 5Ј-WATCAR-TTR-3Ј motif (where W is A or T and R is A or G) separated from a less conserved A/T-rich tract of 4 to 6 bp (lowercase letters) (53). The residues shuffled to disrupt the core IHF consensus in the xh-Po(-IHF)/Px promoter series are underlined, as are the bases rendered hydroxyl radical hypersensitive by binding of the ␣-subunits of 54 -RNAP in the Pu UP-element (40). The lower part of the figure shows the DNA sequences of the Ϫ39 to ϩ2 regions of the indicated 54 -promoters, with nucleotides that differ from Po in the Ϫ33 to ϩ2 region underlined. Note the sequence of the NdeI site (shown in italics) is destroyed upon introduction of linker DNA specifying the sequences of the promoters to control transcription of the promoterless luxAB genes (13). capacity (Fig. 3A, and data not shown). With the exception of xh-Po/Pu, maximal output from the 54 -promoters was reduced 11-13-fold by lack of the capacity to bind IHF. The higher (ϳ25-fold) dependence on IHF seen with the xh-Po/Pu promoter is consistent with data from Pu in its native context, which is also highly dependent on IHF both in vitro and in P. putida (Ref. 26 and references therein).
Next we tested the temporal expression profiles of the xh-Po(-IHF)/Px reporters in ppGppand DksA-deficient P. putida KT2440 derivatives. Similar to the data for the IHF-binding proficient Po/Po-luxAB ( Fig. 2A), deficiency of either of these two regulatory molecules decreased transcription in a similar manner from reporters that lack the capacity to bind IHF (supplemental Fig. S1), suggesting that ppGpp and DksA primarily mediate their effects at other levels. However, because this series of 54 -promoter exhibited a higher degree of dependence on IHF in P. putida than when assessed in E. coli (13), we were prompted to test if the in vivo levels of P. putida IHF restrict transcription from any of the 54 -promoters. To this end we generated a derivative of the KT2440::dmpR-Tel strain with an additional copy of the P. putida ihfAB genes under the control of the P tac -promoter. IPTG induction in this strain produces IHF levels in the exponential phase at least as high as those found in the stationary phase of the wild-type counterpart (Fig. 3B, compare lanes 1s and 3e). The activity of the 54 -promoters under these conditions, however, only differed in the post-exponential phase, with excess IHF giving rise to a modest 1.1-1.3-fold higher maximal expression level with the different promoters ( Fig. 3B and data not shown). We conclude from this data that the native postexponential phase levels of IHF are near saturating for even extremely IHF-dependent 54 -dependent promoters, and that IHF levels per se are not a limiting factor for 54 -promoter output during any phase of growth in P. putida.

IHF-mediated Recruitment of 54 -RNAP Alone Cannot Relieve Tight Exponential Phase Control of P. putida 54 Transcription-
The high dependence of the Pu promoter on IHF relates to its role in aiding promoter occupancy by 54 -RNAP. At Pu, IHF-mediated changes in DNA architecture provide a thus far unique 54 -RNAP recruitment mechanism by providing a DNA architecture that allows simultaneous binding of the two ␣-subunits of 54 -RNAP to a distally located DNA UP element. This additional DNA binding element thus increases the affinity of the Pu promoter for 54 -RNAP (38 -40) and would be anticipated to impart higher affinity for 54 -RNAP to any 54 -promoter. To test if the 54 -RNAP recruitment mechanism of Pu was sufficient to allow detectable transcription from other 54 -pro-   lanes 1e and 1s), and KT2440::dmpR-Tel-Ptac-ihfAB grown in the absence (lanes 2e and 2s), and in the presence of IPTG (lanes 3e and 3s). Cells were harvested at A 600 ϭ 0.35 (e) and 3.5 (s) as indicated by the arrows in panel B intervening lanes contain 15, 7.5, 3.8, and 1.9 ng of purified P. putida IHF. moters in exponentially growing P. putida, we generated the series of xh-Pu/Px promoters, which maintain the UAS binding sites for DmpR in exactly the same location relative to the Ϫ24,Ϫ12 sequences but have the appropriately positioned Pu UP-element and IHF binding site that is located one helical turn from that in the Po upstream region (see Fig. 1, upper). However, as shown in Fig. 4A, whereas the xh-Pu/Px derivatives exhibit a modest increase in maximal output in the ppGppproficient strain, and increase the expression in the ppGppdeficient strain as compared with their cognate xh-Po/Px counterparts in the stationary phase, no transcription in the exponential phase could be detected (data not shown). Furthermore, no further increase in maximal transcription levels or exponential phase expression was seen with any of the xh-Pu/Px promoters upon overexpression of IHF using the P tac -IHF P. putida strain described above (data not shown). Because transcriptional initiation at 54 -promoters requires physical interaction between the bEBP and 54 -RNAP, these results suggest that even with additional IHF and the IHF-dependent 54 -RNAP recruitment device, the levels of DmpR and/or 54 -RNAP are too low for sufficient co-occupancy of the promoters to mediate detectable transcription during the exponential phase of growth.
Tight Exponential Phase Control in P. putida Reflects 54 -RNAP and bEBP Promoter Co-occupancy-Consistent with the proposed limitation imposed by the co-occupancy outlined above, we have recently shown that artificial elevation of DmpR protein levels in LB-cultured P. putida does allow some 54 -Po output during the exponential phase of growth (14), whereas similar manipulations in E. coli do not (13). However, even in P. putida expressing excess DmpR, ppGpp synthesis triggered by nutrient depletion still greatly enhanced transcription at the exponential to stationary phase growth transition (14). Hence, whereas DmpR levels are a major limiting factor for 54 -dependent transcription in exponentially growing P. putida, 54 -RNAP levels also appear to limit transcription in this phase of growth (14).
To determine whether naturally occurring levels of a bEBP in P. putida could allow exponential phase 54 -dependent transcription, we used strains expressing the bEBP XylR, which is highly homologous to DmpR and can efficiently bind and activate transcription from the UASs of Po in response to its natural effectors (41,42). The levels of XylR in P. putida, like those of DmpR, increase as cells enter stationary phase (43). However, XylR is naturally produced from its native P. putida pWW0 plasmid in higher levels than those of DmpR from pVI150 (30) and the P. putida strains used here maintain XylR levels at ϳ65% of those from the pWW0 plasmid in this host (30).
As shown in Fig. 4, B-F, XylR regulation results in higher transcription levels than DmpR in the P. putida ppGpp-deficient strain with all the test 54 -promoters. Notably, the additional 54 -RNAP recruitment device of the xh-Pu/Px promoter series both increases the transcription levels in the absence of ppGpp as compared with cognate xh-Po/Px promoters (compare Fig. 4, E and F, with C and D), and results in transcription during the exponential phase of growth (Fig. 4, E and F, and data not shown). These results clearly demonstrate that the IHFmediated 54 -RNAP recruitment devices is operational in these promoters, and provide evidence that the level of the 54 -RNAP holoenzyme is a critical parameter that can restrict exponential phase 54 transcription in P. putida. However, the results also clearly suggest that given sufficient levels of a bEBP, some 54dependent systems could still be expressed during rapid high energy growth conditions in P. putida.
Lack of Rsd or PfrA Does Not Influence the Growth Phase Transition Checkpoint of 54 -Transcription-The data in the preceding sections are consistent with the previous proposal that ppGpp/DksA in part mediate their effects on 54 -transcription by enhancing the levels of the 54 -RNAP holoenzyme and thus promoter occupancy at the exponential to stationary phase transition (12,13,18). In E. coli, the levels of the 70binding protein Rsd are partially under ppGpp control (44,45), and Rsd can sequester both free 70 and actively remove 70 from the 70 -holoenzyme in vitro (46,47). The finding that overexpression of Rsd in E. coli enhances transcription from stress-response promoters dependent on alternative -factors such as S and 54 (12,48,49) led to the speculation that Rsd may be involved in ppGpp-mediated control of 54 -transcription (13). To test this idea we compared 54 -transcription in wild-type and the Rsd null mutant of E. coli using the transcriptional reporter plasmid pVI466 (dmpR-Po-luxAB), and generated a null mutant of the Rsd P. putida homologue PfrA in the KT2440::dmpR-Tel strain. Transcription in the P. putida isogenic pair was monitored using the Po/Po-luxAB reporter pVI708. However, lack of these proteins in either organism did not significantly alter temporal or maximal 54 -transcription (supplemental Fig. S2). Hence, we conclude that the physiologically relevant levels of these proteins do not significantly impact 54 -transcription under our test conditions where in vivo ppGpp-mediated effects are prominent (Fig. 2).
Robust Stationary Phase Transcription in P. putida Correlates with Reduced Promoter Discrimination by Its 54 -RNAP-When analyzed with E. coli 54 -RNAP in gel-shift assays, the affinities of the six Po/Px 54 -promoters shown in Fig. 1 were found to lie in the order Po/PnifH Ͻ Po/Pu Ͻ Po/Po Ͻ Po/PnifH049 Ͻ Po/PglnA and Po/PpspA (13). This order is maintained in similar assays using purified P. putida 54 -RNAP (data not shown). Transcriptional reporter gene assays in E. coli revealed an 18-fold difference between the lowest affinity promoter (Po/PnifH) and the highest affinity promoters (Po/ PpspA and Po/PglnA), which was reproduced in vitro using E. coli . This E. coli data contrast our findings in P. putida where we observe only an ϳ3-fold difference between the maximal outputs from the highest and lowest affinity promoters (Fig. 5A). However, similar to E. coli (13), all the promoters were dependent on both ppGpp and DksA for efficient output in P. putida. Lack of either ppGpp or DksA in P. putida leads to 3-7-fold reduced transcription, with the low affinity promoters being notably more dependent on ppGpp and DksA than high affinity promoters (Fig. 5A). This data supports the previous proposal, based on extensive E. coli data, that ppGpp and DksA deficiency results in reduced levels of 54 -RNAP, which will most severely affect occupancy of low affinity 54promoters (13).
The apparent relatively high stationary phase transcription from even the lowest affinity promoters of the Po/Px series of 54 -promoters in wild-type P. putida suggested to us that either (i) the stationary phase levels of 54 -RNAP are sufficiently high to allow frequent activation from even very low affinity promoters, or (ii) that the transcriptional apparatus of P. putida is less discriminative of sequence differences in the Ϫ33 to ϩ2 region than its E. coli counterpart. To test the latter possibility, we employed multiple round transcription assays with P. putidaderived proteins under the same conditions previously used with E. coli-derived proteins and that recapitulate the known negative and positive effects of these molecules at 70 -promoters (13). As was previously found with E. coli-derived proteins (13), addition of ppGpp and DksA had little, if any, direct effect on in vitro transcription from any of the promoters (Fig. 5B). Importantly, the P. putida in vitro reconstituted system reproduced the in vivo differences in transcriptional output from these promoters in wild-type P. putida, namely only an ϳ3-fold difference between the lowest (Po/PnifH) and highest affinity promoter (Po/PglnA), compare Fig. 5, A and B.
To directly compare the activities of the 54 -holoenzymes of E. coli and P. putida, we determined transcription from the Po/Po promoter in the presence of a constant level (5 nM) of the E. coli or P. putida core enzymes and increasing concentrations of the 54 -subunit from each organism in single round in vitro transcription assays (Fig. 5, C and D). The data show that the subunits from both organisms saturate the core at similar concentrations, i.e. the core association properties of the 54 preparations from the two organisms are essentially the same. However, the absolute transcript levels with saturating concentrations of the holoenzymes are markedly different (Fig. 5D). It follows that these differences cannot be attributed to differences in holoenzyme concentrations, but reflect the binding and complex formation of the holoenzyme with the Po promoter DNA. Taken together, the data suggests that ppGpp and DksA regulation of 54 -transcription in E. coli and P. putida follow the same general rules in the two organisms. However, the ϳ18-fold difference in transcription from these promoters in in vivo and in vitro assays with E. coli 54 -RNAP (13) as compared with ϳ3-fold with P. putida (Fig. 5) suggests that the P. putida 54 -RNAP has evolved DNA-binding properties that likely explain the robust stationary phase transcription seen with even comparatively low affinity 54 -promoters in this organism.
The P. putida 54 -Subunit Confers Reduced Promoter Discrimination via Its DNA Binding Properties-To dissect promoter binding and discrimination by E. coli and P. putida-derived 54 -RNAPs, we employed Biacore plasmon resonance analysis of binding to chip-coupled double-stranded DNA encompassing the lowest (Po/PnifH), an intermediate (Po/Po), and the highest affinity (Po/PglnA) promoter, under the same buffer conditions as used in in vitro transcription assays. As illustrated in Fig. 6, dose-dependent responses caused by the 54 -RNAP holoenzymes from the two organisms differed greatly, with that from E. coli exhibiting Ͼ5-fold difference in binding to the lowest versus highest affinity promoter (Fig. 6D), whereas that from P. putida exhibited only a Ͻ2-fold difference (Fig. 6B). This higher binding and reduced discrimination between the different promoters by the P. putida holoenzyme mirror that observed in both the in vivo and in vitro transcription assays shown in the preceding section.
It seemed likely that this difference between the holoenzymes from the two organisms would be mediated by the DNA binding properties conferred by the cognate 54 -subunits. To test this idea we compared the binding of 20 nM native and heterologous 54 -RNAP holoenzymes to each of the three test 54 -promoters. As shown in Fig. 7, the characteristic DNAbinding profiles of holoenzymes to the different promoters were essentially reproduced in an organism-specific manner depending primarily on the origin of the 54 -subunit rather than the core RNAP. For example, note the marked poor binding and slow binding kinetics to both of the two lower affinity promoters (PnifH, dashed line, and Po, dash-dot line) with the E. coli holoenzyme (Fig. 7B) or heterologous 54 -RNAP with E. coli 54 (Fig. 7C), which are comparatively more rapidly bound by the P. putida holoenzyme or heterologous 54 -RNAP with P. putida 54 (Fig. 7, A and D). However, the magnitude of the responses seen with the different promoters, which reflects the absolute levels of binding, also suggests that the core RNAP to some extent contributes to the rates and final level of binding that is achieved by each combination such that the native P. putida combination binds all the promoters most efficiently. FIGURE 6. P. putida and E. coli derived 54 -RNAPs exhibit differential promoter binding. A and C, plasmon resonance assay monitoring binding of increasing amounts of P. putida (PP, panel A) and E. coli (EC, panel C) 54 -RNAP to chip-immobilized double stranded DNA encompassing the 54 -promoters Po/PnifH, Po/Po, or Po/PglnA. Background resonance units from a reference well with no coupled DNA were always less than 10%, and were subtracted from the values shown. The results are representative of two independent overlapping titrations. Concentrations refer to nanomolar core RNAP used to reconstitute the holoenzymes with 8-fold excess of 54 . The rapid binding of both 54 -RNAPs was abolished with control DNA in which the Ϫ24,Ϫ12 motif of either /Po or /PglnA was destroyed, whereas 600 nM 54 alone from either organism exhibit no specific binding under these conditions (data not shown). B and D, P. putida (B) and E. coli (D) 54 -RNAP binding response relative to that of /PnifH set as 1. Bars represent the average relative response at all concentrations assayed in A and C with standard errors. Concluding Remarks-Orthologs of 54 are widely distributed among different bacteria, but the operons that they control are quite diverse and differ from one organism to another. P. putida strains are nutritional and environmentally very adaptable (50), and have to respond to different external conditions and stimuli than the gut-commensal E. coli. In P. putida and related bacteria, 54 -promoters control a variety of processes that are responsive to rapidly changing environmental cues (3), and are prevalent in controlling expression of auxiliary metabolic pathways, such as those for catabolism of aromatic compounds, that feed into the central metabolism (51). The production of specialized enzymes for such pathways is metabolically expensive, and regulatory devices are needed to ensure that these kinds of pathways are not expressed if more favorable sources of carbon and energy are available. The work presented here demonstrates that P. putida 54 -transcription is very tightly controlled under high energy conditions provided by complex media, and a combination of DNA elements was needed to obtain sufficiently high affinity for 54 -RNAP to carry out any appreciable transcription under these conditions. Conversely, in comparison with E. coli, upon nutrient depletion as cells enter the stationary phase, transcription from even comparatively low affinity promoters is robust and this robust transcription is attributable to the reduced promoter discrimination of low affinity promoters conferred by the DNA binding properties of the P. putida 54 -RNAP holoenzyme. Because aromatic compounds are stress agents even for bacteria capable of using them as carbon sources, expression of the enzymes for their catabolism is interfaced with both central metabolism and stress responses (6). With respect to 54 -transcription, our findings suggest that P. putida has evolved and integrated ppGpp/DksA global regulation of 54 -transcription to provide both extremely tight control during rapid growth with vigorous transcription under nutritional stress. It is perhaps for these reasons that 54 -promoters are frequently found in association with control of auxiliary metabolic pathways in this nutritionally adaptable organism.