Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

Laetitia Abdou; Han-Ting Chou; Dieter Haas; Chung-Dar Lu

doi:10.1128/JB.00164-11

. 2011 Jun;193(11):2784–2792. doi: 10.1128/JB.00164-11

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa^{^▿}^,^†

Laetitia Abdou ¹, Han-Ting Chou ², Dieter Haas ^1,^*, Chung-Dar Lu ²

PMCID: PMC3133114 PMID: 21478360

Abstract

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ⁵⁴ RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.

INTRODUCTION

Pseudomonas aeruginosa, like other fluorescent pseudomonads, is a metabolically versatile bacterium; it utilizes more than 100 different organic substrates for growth (37). This versatility requires a complex regulatory network that ensures the cellular carbon-nitrogen balance and determines the order in which growth substrates are degraded. In general, substrates that yield high energy and promote fast growth are degraded preferentially. For instance, intermediates of the tricarboxylic acid (TCA) cycle such as succinate prevent glucose degradation in P. aeruginosa (21). As a result, growth on a mixture of succinate and glucose is biphasic (diauxic) because the bacterium utilizes first succinate and then glucose (39). The underlying mechanism of carbon catabolite repression involves the CbrA/CbrB two-component system (16, 30), the small RNA (sRNA) CrcZ (36), and the RNA-binding protein Crc (6, 22, 25, 26, 33) as key regulatory elements. They are conserved in fluorescent pseudomonads (38; Pseudomonas Genome Database).

Mutational inactivation of the CbrA/CbrB two-component system has vast consequences in fluorescent pseudomonads. In P. aeruginosa, Pseudomonas fluorescens, and Pseudomonas putida, cbrA and cbrB mutants no longer grow on a large number of carbon and nitrogen sources, suffer from a carbon-nitrogen imbalance, and are affected in biofilm development and stress tolerance (1, 19, 29, 30, 40, 41). The signals that interact with the membrane-bound CbrA sensor are unknown, but it appears that TCA cycle intermediates have an inhibitory effect on CbrA/CbrB activity (16). The CbrB protein is a transcriptional activator for σ⁵⁴ RNA polymerase and belongs to the NtrC family of response regulators (30). Genetic evidence indicates that CbrB positively controls the expression of the crcZ sRNA gene in P. aeruginosa (36), the lipA (lipase) gene in Pseudomonas alcaligenes (7, 18), and the hutU (histidine utilization) operon of P. aeruginosa and P. fluorescens (16, 40). The fact that cbrB and crcZ mutants of P. aeruginosa have similar, but not identical, phenotypes suggests that most, but not all, activities of the CbrA/CbrB system are mediated by the sRNA CrcZ (36). CrcZ has high affinity for the Crc protein and, when in excess, prevents Crc from acting as a translational repressor of target mRNAs. Typical Crc targets are mRNAs encoding porins, uptake systems, and enzymes involved in the degradation of less-preferred substrates (20, 27, 28). In P. aeruginosa, CrcZ levels are lower during growth on succinate than during growth on less-preferred substrates such as glucose or mannitol, reflecting the activity of the CbrA/CbrB system as a master regulator of carbon catabolite repression (36). The growth handicap of a crcZ mutant on numerous substrates appears to be a consequence of permanent translational repression of target mRNAs by the Crc protein.

The CbrA/CbrB two-component system has moderate amino acid sequence similarity to the well-characterized NtrB/NtrC regulatory system, which controls nitrogen assimilation in Gram-negative bacteria (14, 30, 32). Importantly, mutations that partially suppress a cbrAB deletion have been mapped to ntrB or ntrC in P. aeruginosa (16, 19), suggesting that both two-component systems may functionally overlap. NtrC is a bacterial enhancer binding protein (bEBP). When activated by phosphorylation, NtrC binds to upstream activating sequences (UASs) in NtrC-dependent σ⁵⁴ promoters (2, 32). Mechanistic aspects of this process have been studied mainly in enteric bacteria (10). In P. putida, NtrC binding sites have been found at −170 to −40 relative to the transcription start sites of various genes whose expression is activated by NtrC (13, 14). The interaction between NtrC bound to a UAS and σ⁵⁴ RNA polymerase is often facilitated by the integration host factor (IHF) protein, which bends the DNA between the UAS and the −24 and −12 promoter sequences recognized by σ⁵⁴ RNA polymerase (10). In the present study, we have obtained evidence for specific CbrB-DNA interaction, allowing us to localize CbrB recognition sites in the crcZ and lipA promoters of P. aeruginosa. We have also found that activation of the crcZ promoter requires IHF.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The strains used in this study are listed in Table 1. P. aeruginosa was grown in Luria broth (LB) (34) at 37°C or in a basal salts medium (BSM) containing 30.8 mM K₂HPO₄, 19.3 mM KH₂PO₄, 15 mM (NH₄)₂SO₄, 1 mM MgCl₂, and 2 μM FeSO₄ amended with either 40 mM succinate or 40 mM mannitol (36). When required, antibiotics were added to the medium at the following concentrations: ampicillin, 100 μg ml⁻¹; tetracycline, 30 μg ml⁻¹ for Escherichia coli or 100 μg ml⁻¹ for P. aeruginosa.

Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristic(s)^a	Source or reference
P. aeruginosa
PAO1	Wild type	ATCC 15692
PAO6358	ΔrpoN	15
PAO6711	ΔcbrB	36
PAO6766	himA::Tn5 Gm^r	C. Reimmann
E. coli
DH5α	recA1 endA1 hsdR17 thi-1 supE44 gyrA96 relA1 deoR Δ(lacZYA-argF)U169 [ϕ80lacZΔM15]	34
XL1-Blue	recA1 endA1 hsdR17 thi-1 supE44 gyrA96 relA1 Δlac [F′::Tn10 proAB⁺lacI^qlacZΔM15] Tc^r	Stratagene
Plasmids
pBAD/HisB	Expression vector for recombinant proteins with His₆ tag at N terminus; Ap^r	Invitrogen
pME6013	pVS1-p15A shuttle vector for translational lacZ fusions; Tc^r	35
pME6016	pVS1-p15A shuttle vector for transcriptional lacZ fusions; Tc^r	12
pME6032	pVS1-p15A shuttle vector with Ptac under lacI^q control; Tc^r	12
pME7260	pME6013 derivative carrying lipA′-′lacZ with 549 bp upstream of lipA transcription start site; Tc^r	This study (Fig. 5)
pME7263	pME7260 derivative with mutated σ⁵⁴ promoter; Tc^r	This study (Fig. 5)
pME9805	Transcriptional crcZ-lacZ fusion on pME6016 derivative with 161 bp upstream of crcZ transcription start site; Tc^r	This study (Fig. 1)
pME9806	Transcriptional crcZ-lacZ fusion on pME6016 derivative with 1,561 bp upstream of crcZ transcription start site; Tc^r	36
pME9812	Vector for delivery of crcZ-lacZ in mini-Tn7; Gm^r	36
pME9814	pME6013 derivative carrying cbrB′-′lacZ	This study (Fig. 4)
pME9825	pUC28 derivative carrying the 161-bp crcZ promoter region with mutated −24 box of the σ⁵⁴ promoter; Ap^r	This study
pME9826	pME6016 derivative carrying crcZ-lacZ with 161-bp promoter region and mutated −24 box of the σ⁵⁴ promoter; Tc^r	This study (Fig. 1)
pME9831	pME6013 derivative carrying lipA′-′lacZ with 134 bp upstream of lipA transcription start site; Tc^r	This study (Fig. 5)
pME9834	pME6016 derivative carrying crcZ-lacZ with 116-bp promoter region; Tc^r	This study (Fig. 1)
pME9835	pUC28 derivative carrying the 161-bp crcZ promoter region; Ap^r	This study
pME9837	pUC28 derivative carrying the 161-bp crcZ promoter region with mutated downstream motif (Mut2) in the UAS; Ap^r	This study
pME9838	pUC28 derivative carrying the 161-bp crcZ promoter region with mutated upstream motif (Mut1) in the UAS; Ap^r	This study
pME9839	pUC28 derivative carrying the 161-bp crcZ promoter region with mutated up- and downstream motifs (Mut1,2) in the UAS; Ap^r	This study
pME9842	pME6016 derivative carrying crcZ-lacZ with 138-bp promoter region; Tc^r	This study (Fig. 1)
pME9843	pME6016 derivative carrying crcZ-lacZ with 161-bp promoter region and mutated downstream motif (Mut2) in the UAS; Tc^r	This study (Fig. 1)
pME9844	pME6013 derivative carrying lipA′-′lacZ with 105 bp upstream of lipA transcription start site; Tc^r	This study (Fig. 5)
pME9845	pME6016 derivative carrying crcZ-lacZ with 161-bp promoter region and mutated upstream motif (Mut1) in the UAS; Tc^r	This study (Fig. 1)
pME9846	pME6016 derivative carrying crcZ-lacZ with 161-bp promoter region and mutated up- and downstream motifs (Mut1,2) in the UAS; Tc^r	This study (Fig. 1)
pME9848	pME6032 carrying truncated, His-tagged ′cbrB; Tc^r	This study (Fig. 2)
pSU300	pBAD/HisB carrying truncated, His-tagged ′cbrB; Ap^r	This study
pUC18	Cloning vector; Ap^r	34
pUC28	Cloning vector; Ap^r	4

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Ap^r, ampicillin resistance; Gm^r, gentamicin resistance; Tc^r, tetracycline resistance.

Construction of plasmids.

The primers used for plasmid construction are listed in Table S1 in the supplemental material. Plasmids carrying a transcriptional crcZ-lacZ fusion were obtained as follows. Primers V3_pcnBlacZfw and crcZ-lacZ rev were used to amplify a 183-bp PCR product from chromosomal DNA of strain PAO1. The resulting crcZ promoter fragment was digested with EcoRI and PstI and cloned into pME6016 cut with the same enzymes to give pME9805. Primers crcZ8fw_EcoRI and crcZ-lacZ rev were used to amplify a 160-bp PCR product from PAO1 chromosomal DNA. Similarly, this PCR product was digested with EcoRI and PstI and inserted into pME6016 to produce pME9842. Primers crcZ7fw_EcoRI and crcZ-lacZ rev were used to amplify a 138-bp PCR product from PAO1 chromosomal DNA. Again, the PCR product was digested with EcoRI and PstI and inserted into pME6016 to produce pME9834.

To construct plasmids carrying mutated motifs in the crcZ UAS, the 171-bp crcZ promoter region was subcloned as an EcoRI-PstI fragment into vector pUC28 to give pME9835. Mutations in the upstream motif (Mut1) were introduced into pME9835 using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with mutagenesis primers Motif_1_fw and Motif_1_rev. The parental DNA template was digested with DpnI, and the mutated plasmid was introduced into E. coli XL1-Blue by transformation, generating pME9838. Mutations in the downstream motif (Mut2) were introduced into pME9835 similarly, using mutagenesis primers Motif_2_fw and Motif_2_rev. The mutated plasmid was termed pME9837. Mutations in both motifs (Mut1,2) were introduced into pME9837 using mutagenesis primers Motif_1_fw and Motif_1_rev, generating pME9839. Plasmids pME9838, pME9837, and pME9839 were digested with EcoRI and PstI, and the resulting 171-bp restriction fragments were inserted into pME6016 at the corresponding sites to give pME9845, pM9843, and pME9846. The −24 box mutations in the σ⁵⁴-dependent crcZ promoter were introduced into pME9835, again by the QuikChange site-directed mutagenesis method, with mutagenesis primers rpoN-mut1_dir and rpoN-mut1_rev; this generated pME9825, which was digested with EcoRI and PstI. The mutated 171-bp restriction fragment was cloned into pME6016 at the same sites, giving pME9826.

Plasmids carrying a translational lipA′-′lacZ fusion were constructed as follows. Primers lipA4 and lipA1 were used to amplify a PCR product of 665 bp using chromosomal PAO1 DNA as the template. The PCR product obtained was digested with EcoRI and BamHI and cloned into pME6013 at the corresponding sites, resulting in pME7260; in this construct, the lipA′ gene was fused at its 5th codon to ′lacZ. Similarly, a 248-bp PCR product amplified with primers lipA9_EcoRI and lipA1 from PAO1 chromosomal DNA was digested with EcoRI and BamHI and inserted into pME6013 to give pME9831. A 219-bp PCR product obtained with primers lipAFfw_EcoRI and lipA1 was cut with EcoRI and BamHI and cloned into pME6013 to give pME9844. To construct pME7263, an inverse PCR was done using pME7260 as the template and primers 660 M-F1 and 660 M-R1. The PCR product obtained was digested with PstI and recircularized. The resulting plasmid was digested with EcoRI and BamHI, and the 651-bp restriction fragment was subcloned into pME6013 at the corresponding sites. To construct a cbrB′-′lacZ translational fusion, primers cbrB1fw_EcoRI and cbrBrev_PstI were used to amplify a 545-bp PCR product, which was digested with EcoRI and PstI and inserted into pME6013 at the corresponding sites, resulting in pME9814.

For the overexpression of His-tagged, truncated ′cbrB, primers ArgU-3 and ArgU-4 were used to amplify a PCR product of 1,446 bp. This PCR product was digested with BglII and EcoRI and inserted into pBAD/HisB at the corresponding sites to give pSU300. The resulting recombinant ′CbrB protein lacks the 13 N-terminal amino acid residues of the native CbrB protein and carries an N-terminal His₆ tag. To construct pME9848, we used primers cbrBpSUdir_EcoRI and cbrBpSUrev_KpnI to amplify a 1,549-bp PCR product with plasmid pSU300 as the template. The PCR product was digested with EcoRI and KpnI and cloned into pME6032 at the corresponding sites. This placed the cloned ′cbrB gene behind the tac promoter.

Purification of His-CbrB.

The His-tagged, truncated ′CbrB protein (His-CbrB) was expressed from pSU300 in E. coli TOP10 (Invitrogen). After induction with 0.2% (wt/vol) arabinose for 3 h, cells were collected by centrifugation and ruptured by passage through a French pressure cell at 8,500 lb/in². Cell debris was removed by centrifugation at 25,000 × g for 30 min, and the resulting cell-free crude extract was loaded onto a HisTrap HP column (GE Healthcare) in an AKTA fast protein liquid chromatography system, following a purification protocol suggested by the manufacturer. The recombinant His-CbrB protein was eluted by 175 mM imidazole. The purity of His-CbrB in the collected fractions was verified by SDS-PAGE (see Fig. S1 in the supplemental material). The protein concentration was determined by the Bradford method with bovine serum albumin as the standard.

Electrophoretic mobility shift assays.

DNA fragments covering wild-type or mutated promoter regulatory regions were PCR amplified using designed primers (see Table S1 in the supplemental material). The DNA probes were prepared by labeling with [γ-³²P]ATP and T4 polynucleotide kinase (34). Radioactively labeled DNA probes were allowed to interact with different concentrations of purified His-CbrB in a 20-μl reaction mixture containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 5% (vol/vol) glycerol, 150 μg/ml acetylated bovine serum albumin, and 40 ng unlabeled pUC18 vector as nonspecific DNA. The reaction mixtures were incubated at room temperature for 30 min before being applied to a 4% polyacrylamide gel (bisacrylamide-acrylamide ratio, 1:60) in 0.5× Tris-borate-EDTA buffer, pH 7.5 (34). The gel was dried, autoradiographed by exposure to a phosphorimager plate, scanned using FLA-7000 version 1.1, and analyzed by Multi Gauge version 3.0 (FUJIFILM).

β-Galactosidase assays.

β-Galactosidase assays were performed as previously described (24), with 20-ml cultures of P. aeruginosa strains grown in LB or BSM supplemented with different carbon sources. Data are mean values of three independent samples ± standard deviations.

RESULTS

Mutational analysis of the crcZ promoter region.

The crcZ promoter of P. aeruginosa contains conserved −24/−12 boxes (TGGCACGN₄CTGCT) that are typical of σ⁵⁴ promoters (consensus, TGGCACGN₄TTGC^T/_A, where the most highly conserved nucleotides are in boldface and N indicates any nucleotide [3]) (Fig. 1A). A transcriptional lacZ fusion to the predicted +1 transcription start site of the crcZ gene (on pME9805) was highly expressed in wild-type PAO1 (Fig. 1B), whereas the expression of the same construct was <1% in both an rpoN and a cbrB mutant (Fig. 1C). The lacZ construct used contained 161 bp of the crcZ promoter region. When the crcZ promoter region was extended to 1,561 bp, which includes the upstream cbrB gene except for the first 12 codons (on pME9806; Fig. 1A), lacZ expression was unchanged in the wild type (data not shown), indicating that the crcZ promoter activity depends essentially on the 161-bp segment. Mutation of the −24 box in the crcZ-lacZ fusion construct (pME9826; Fig. 1A) resulted in <1% expression (Fig. 1C), confirming that the crcZ promoter is recognized by σ⁵⁴ RNA polymerase.

A deletion removing the region from −161 to −116 (on pME9834) abolished crcZ-lacZ expression almost entirely (Fig. 1B). This region contains a sequence of dyad symmetry (Fig. 1A). Base change mutations in both parts of this palindrome (Mut1,2; on pM9846) also had a drastic negative effect on crcZ-lacZ expression (Fig. 1B). Deletion of the region extending from −161 to −138 (pME9842) or mutations in the upstream element of the palindrome (Mut1; on pME9845) reduced crcZ-lacZ expression by about 70%, compared with the wild-type value. Mutations in the downstream element (Mut2; on pME9843) resulted in the loss of about half of the lacZ activity (Fig. 1B). These data qualify the palindromic sequence as a UAS and suggest that it may be recognized by the CbrB response regulator.

Construction and activity of His-tagged CbrB.

An amino acid sequence alignment of CbrB with related bEBPs reveals an N-terminal regulatory domain with a conserved aspartate residue that is predicted to accept phosphate from CbrA, a central domain, and a C-terminal helix-turn-helix domain for DNA binding (Fig. 2A). The central domain contains all of the motifs that are typical of bEBPs interacting with σ⁵⁴ RNA polymerase: Walker A and Walker B motifs, which are required for ATP binding and hydrolysis, respectively; a GAFTGA element, which defines a sequence interacting with σ⁵⁴; and a switch Asn and an R finger motif, which are involved in oligomerization and nucleotide binding, respectively (10). The former three subdomains are identical in CbrB and NtrC of P. aeruginosa, whereas the latter two subdomains are similar (Fig. 2A). Overall, CbrB and NtrC have 43% amino acid sequence identity in a window of 387 residues. In some members of the NtrC family, the regulatory domain inhibits the activity of the bEBP and truncation of the N terminus can enhance this activity (11, 31). We therefore constructed a full-length, as well as a truncated, version of His-tagged CbrB. The truncated ′CbrB protein lacks the 13 N-terminal amino acid residues of CbrB (Fig. 2A), and its construction is described in Materials and Methods. Whereas the full-length construct in the expression vector pME6032 was active in vivo, we were unable to demonstrate specific DNA binding in vitro (data not shown). In contrast, the truncated His₆-′cbrB construct, when expressed from the induced tac promoter in pME6032, had partial activity as an activator of crcZ expression in vivo (Fig. 2B) and allowed detection of CbrB binding to the UAS (see below).

Fig. 2. — Domains of the CbrB protein and activity of CbrB truncated at the N terminus. (A) Domain map with conserved sequences in the CbrB and NtrC proteins of *P. aeruginosa* PAO1, based on the domain map of *Salmonella enterica* NtrC (10). CbrB is predicted to contain an N-terminal regulatory domain, a central σ⁵⁴ activation domain, and a C-terminal DNA-binding domain with a helix-turn-helix motif (HTH). The conserved phosphorylation site in the regulatory domain is presumed to enable activation of CbrB by the sensor kinase CbrA (5, 30). Gray boxes below show that the 13 N-terminal amino acid (aa) residues of the native CbrB protein are replaced with 35 different amino acids, including a His₆ tag in the truncated CbrB protein. (B) Truncated His-CbrB is partially active in the expression of a *crcZ-lacZ* transcriptional fusion. The *crcZ-lacZ* fusion in mini-Tn7 was introduced into the chromosomes of strains PAO1 and PAO6711 (Δ*cbrB*) via pME9812 as previously described (36). The β-galactosidase activities of the *crcZ-lacZ* fusion were determined in wild-type PAO1 containing the empty vector pME6032 (track 1, black bar), in the *cbrB* mutant PAO6711 containing pME6032 (track 2, basal level), and in PAO6711 containing the plasmid pME9848 (track 3, gray bar), which carries the truncated His₆-′*cbrB* gene. Cells were grown to an optical density at 600 nm of ∼2.2 in LB amended with 0.5 mM isopropyl-β-d-thiogalactopyranoside to induce the *tac* promoter expressing His₆-′*cbrB*.

CbrB protein binds to a palindromic UAS located at −151 to −125 from the crcZ transcription start site.

A wild-type crcZ promoter fragment of 183 bp interacted with the purified truncated His-CbrB protein in an electrophoretic mobility shift assay, and the formation of protein-DNA complexes was seen (Fig. 3). Deletion of both elements of the palindromic UAS (ΔM1,2) abolished CbrB binding, whereas either deletion of the upstream element (ΔM1) or mutation of the downstream element (Mut2) resulted in diminished CbrB binding (Fig. 1A and 3). These data confirm the results obtained with the crcZ transcriptional fusions and indicate that a single element in the UAS is sufficient for CbrB binding. However, both elements are needed for strong binding and full activation of the crcZ promoter.

Fig. 3. — His-CbrB binds to the UAS of the *crcZ* promoter *in vitro*. Interactions of radioactively labeled wild-type and mutated *crcZ* regulatory regions (0.75 nM) with different concentrations of purified His-CbrB were analyzed by electrophoretic mobility shift assays. WT, wild-type probe, corresponding to the *crcZ* promoter region of pME9805; Mut2, mutated motif M2, corresponding to pME9843; ΔM1, deletion of motif M1, corresponding to pME9842; ΔM1,2, deletion of motifs M1 and M2, corresponding to pME9834. The concentrations of His-CbrB in lanes 1 to 5 were 760 nM, 380 nM, 76 nM, 15 nM, and 0 nM, respectively.

IHF is required for crcZ expression.

Contact between bEBPs and σ⁵⁴ RNA polymerase is often facilitated by IHF, a DNA-bending protein that is structurally and functionally conserved between enteric bacteria and P. aeruginosa (8). A sequence resembling the IHF consensus binding site, AATCAAN₄TTG (23), occurs in the crcZ promoter region at −60 to −48 (Fig. 1A). We therefore tested the crcZ-lacZ fusion (on pME9805) in the IHF-negative mutant PAO6766. At the end of exponential growth in LB, the expression was very low (100 ± 15 Miller units), compared to that in the wild type (35,600 ± 2,600 Miller units), indicating that IHF is required for the activation of the crcZ promoter. It is likely that this activation is due to direct binding of IHF to the proposed IHF binding site, but indirect effects of IHF on crcZ expression also remain possible.

Autoregulation of cbrB expression.

It has been noted previously that cbrB expression is not constitutive but varies depending on the growth medium, with succinate exerting catabolite repression (30). We found that a cbrB′-′lacZ fusion carried by pME9814 in wild-type PAO1 was expressed more strongly at the end of growth than during the exponential phase in rich medium (Fig. 4). In the cbrB mutant PAO6711, the same fusion was expressed at elevated levels during exponential growth (Fig. 4), suggesting negative autoregulation of cbrB. The cbrB promoter region contains a potential CbrB binding site, which is located between −21 and +7 (relative to the +1 transcription start site; reference 30) and which is weakly conserved (ATGGAAGN₁₄GATAACCA [conserved nucleotides are in boldface]) in comparison with the CbrB binding site found in the crcZ upstream region (CTGTTACN₁₄GTAACAG). However, we did not investigate the role of the putative autoregulatory site further and autoregulation might involve additional regulatory elements.

Fig. 4. — CbrB negatively autoregulates its own expression. The β-galactosidase activities of a translational *cbrB*′-′*lacZ* fusion carried by pME9814 were measured in wild-type PAO1 (filled diamonds) and in the *cbrB* deletion mutant PAO6711 (filled circles). Cells were grown in LB.

Positive CbrB control of the lipA promoter.

In P. alcaligenes, the expression of the lipA gene coding for secreted lipase is positively controlled by RpoN and by the LipQ/LipR two-component system. LipQ and LipR are homologues of P. aeruginosa CbrA and CbrB, respectively (9, 18). At −130 to −112 of the lipA promoter, a UAS has been proposed as a LipR binding site (7). In P. aeruginosa, lipA expression also requires RpoN and LipR (= CbrB) (17), but molecular details of the lipA promoter and its putative UAS have not been studied. We constructed several translational lipA′-′lacZ fusions, with and without the UAS (Fig. 5A). Two constructs containing the UAS (pME7260 and pME9831) showed ≥7-fold higher expression than did a construct lacking the UAS (pME9844) in wild-type PAO1 (Fig. 5B). In the cbrB mutant PAO6711, lacZ expression directed by pME7260 was low, confirming the requirement for CbrB (Fig. 5B). The requirement for RpoN was corroborated both by mutation of the host rpoN gene and by mutations in the −24 box of the lipA promoter. Both types of mutation strongly diminished lipA expression to <1% of the wild-type level (Fig. 5B).

Fig. 5. — Sequence of the *lipA* promoter region, recognition site for CbrB, and catabolite repression of the *lipA* promoter. (A) In the nucleotide sequence of the *lipA* promoter, the UAS region is highlighted in gray. The *ligT* stop codon, the −24 and −12 elements of the σ⁵⁴ promoter, and the *lipA* start codon are boxed. Inverted arrows below boldface nucleotides show the palindromic sequence in the UAS region. The arrow at +1 shows the start of *lipA* transcription. Different translational *lipA*′-′*lacZ* fusions carried by plasmids are illustrated below. σ⁵⁴mut indicates mutations in the −24 promoter element of pME7263. (B) β-Galactosidase activities were determined in wild-type PAO1 carrying a *lipA*′-′*lacZ* fusion on pME7260 (filled circles), pME7263 (filled circles connected with dotted lines), pME9831 (filled triangles), or pME9844 (filled squares). β-Galactosidase activities of *lipA*′-′*lacZ* carried by pME7260 were also measured in the *cbrB* deletion mutant PAO6711 (filled diamonds) and in the *rpoN* deletion mutant PAO6358 (open diamonds). Cells were grown in LB. (C) Catabolite repression of *lipA* gene expression was demonstrated by measuring β-galactosidase activities of *lipA*′-′*lacZ* carried by pME9831 (triangles) and pME9844 (squares). Cells were grown in minimal medium amended with either 40 mM succinate (filled symbols) or 40 mM mannitol (open symbols).

We then asked whether lipA expression was under catabolite repression control, as would be predicted from the involvement of CbrB as an activator of the lipA promoter. When wild-type cells harboring a lipA′-′lacZ fusion with an intact UAS (on pME9831) were grown in succinate minimal medium, expression was about 10-fold lower than that in mannitol-grown cells (Fig. 5C). We verified that this differential expression was not due to translational regulation via Crc (data not shown). Importantly, a lipA′-′lacZ construct lacking the UAS (on pME9844) did not give rise to derepressed β-galactosidase levels in mannitol-grown cells and was expressed at low levels throughout growth (Fig. 5C). We conclude that CbrB mediates catabolite repression of lipA gene expression, probably by interacting directly with the UAS, although the possibility of an indirect activation mechanism is not entirely excluded.

In an electrophoretic mobility shift assay, the purified His-CbrB protein interacted with the wild-type lipA promoter region (corresponding to pME9831) at the highest protein concentration used but caused no band shift of a promoter fragment lacking the UAS (corresponding to pME9844) (see Fig. S2 in the supplemental material). We suspect that the relatively low affinity of His-CbrB for the UAS in the lipA promoter reflects a suboptimal recognition sequence in comparison with the UAS in the crcZ promoter (Fig. 6).

Fig. 6. — The proposed consensus CbrB recognition site is similar to the consensus NtrC recognition site. The CbrB binding sites in the *crcZ* and *lipA* promoters of *P. aeruginosa* were determined in this work (Fig. 1, 3, and 5; see Fig. S2 in the supplemental material). Mutational analysis of the *lipA* promoter of *P. alcaligenes* (7) and preliminary data on the *hutU* promoter of *P. aeruginosa* (16) reveal similar conserved palindromic sequences. Alignment of their sequences leads us to propose a consensus CbrB binding site, in which the spacer consists of 3 or 12 variable nucleotides (designated N₃ and N₁₂, respectively). The consensus CbrB binding site has similarity to the consensus NtrC binding sites of enteric bacteria (2) and *P. putida* (13, 14).

DISCUSSION

The CbrA/CbrB two-component system was discovered as a master regulator that is necessary for growth of P. aeruginosa on a large range of carbon and nitrogen sources such as histidine, proline, arginine, alanine, polyamines, benzoate, or mannitol (30). In independent work, the homologous LipQ/LipR two-component system was described as an activator of lipase expression in an industrial strain of P. alcaligenes (9), which is typically grown in soybean oil medium for lipase production (18). All of the substrates mentioned sustain the growth of pseudomonads to high cell population densities but are energetically less favorable than C₄ dicarboxylates, glutamate, and aspartate, which are preferred substrates of pseudomonads (30, 33). An initial simple model pictured CbrB as a general transcriptional activator of genes that encode catabolic enzymes for the mineralization of less-preferred substrates (16). As it appears now, this regulatory model still applies to the lipAH operons of P. alcaligenes and P. aeruginosa and to the hutUH₁TH₂IG operon of P. aeruginosa. Previous genetic studies have indicated that CbrB recognizes a UAS and activates σ⁵⁴ RNA polymerase in the expression of the lipAH operon of P. alcaligenes and of the hut operon of P. aeruginosa (16, 18). However, it has recently become clear that a majority of catabolic pathways are not under direct transcriptional control by CbrB but are regulated indirectly via the sRNA CrcZ and the RNA-binding protein Crc in P. aeruginosa (36). CbrB strongly activates the promoter of the crcZ gene, and the resulting elevated levels of CrcZ account for sequestration of the Crc protein, which then no longer translationally represses mRNAs coding for catabolic enzymes (36).

The powerful activation of the crcZ promoter by CbrB and the strict dyad symmetry in the UAS have provided us with a lead to define the CbrB recognition sequence in P. aeruginosa. By introducing point mutations and deletions into the UAS of crcZ, we have demonstrated that a palindromic sequence with a 12-bp spacer binds His-CbrB both in vivo and in vitro (Fig. 1 and 3). In the lipA promoter of P. aeruginosa, a similar palindrome occurs but with a 3-bp spacer (Fig. 5 and 6). Our deletion analysis confirms that this sequence is indeed required for CbrB-dependent lipA promoter activation (Fig. 5). The lipA promoter of P. alcaligenes also contains a similar palindrome with a 3-bp spacer, and mutational analysis has previously suggested its function as a UAS (7). In the case of the P. aeruginosa hutU operon, a CbrB binding site has been tentatively localized to the region extending from −240 to −160 (16). In this UAS, only the upstream element is fully conserved and the downstream element, which is separated by a 3-bp spacer, has three mismatches (Fig. 6). Nevertheless, this UAS may be expected to bind CbrB, as it appears that a single upstream or downstream element is sufficient for some CbrB binding in the crcZ promoter (Fig. 1 and 3). The combined analysis of these promoters allows us to propose a consensus CbrB recognition sequence in which the spacer may consist of either 12 bp or 3 bp (Fig. 6). The consensus NtrC binding site, as defined in enteric bacteria and in P. putida (2, 14), is similar to the CbrB recognition site: four nucleotides in each half-site are identical. However, the spacer for NtrC (N₅) differs from the spacer for CbrB (N₃ or N₁₂) (Fig. 6). The overall similarity of CbrB and NtrC recognition sites may explain why a mutant form of ntrC has been found that can suppress a cbrB deletion for growth of P. aeruginosa on histidine (16).

In some members of the NtrC family, such as DctD and XylR, the N-terminal signal input domain inhibits the function of these proteins as bEBPs. Truncation of the N terminus can overcome this inhibition and result in (partially) constitutive activation of the σ⁵⁴-dependent promoters involved (11, 31). We speculate that CbrB might show a similar mechanism and that this might be a reason why the truncated CbrB protein used in our study has facilitated in vitro binding experiments (Fig. 3; see Fig. S2 in the supplemental material). Further studies are necessary to understand the function of the N-terminal regulatory domain and its interaction with the CbrA sensor kinase.

By showing that CbrB activates the lipA promoter of P. aeruginosa and in this way controls succinate-dependent catabolite repression of lipase expression (Fig. 5; see Fig. S2 in the supplemental material), we have obtained further support for the model of Itoh et al. (16), which stipulates that C₄-dicarboxylates and other preferred carbon sources inhibit the activity of the CbrA/CbrB two-component system, whereas growth media containing energetically suboptimal carbon sources are conducive to CbrA/CbrB activity. In turn, high CbrA/CbrB activity favors the expression of catabolic pathways for less-preferred substrates (including lipase-dependent utilization of lipids), whereas low CbrA/CbrB activity results in global catabolite repression of these pathways. As a corollary, the model proposes that the CbrA sensor must be able to integrate a multitude of nutritional signals. The molecular mechanisms by which CbrA achieves this function are not clear yet. Conceivably, the CbrA/CbrB system might receive input from accessory sensors and engage in cross talk with other two-component systems, especially NtrB/NtrC, in order to optimize carbon source utilization and to maintain the carbon-nitrogen ratio.

Supplementary Material

[Supplemental material]

supp_193_11_2784__index.html^{(951B, html)}

ACKNOWLEDGMENTS

We thank Cornelia Reimmann for generously supplying a himA mutant of P. aeruginosa and Sorana Crivii for constructing plasmids pME7260 and pME7263.

We thank the Swiss National Science Foundation (grant 3100A0-120365 to D.H.) and the National Science Foundation (grant 0950217 to C.-D.L.) for support. Han-Ting Chou was supported by the Molecular Basis of Diseases Program at Georgia State University.

Footnotes

^†

Supplemental material for this article may be found at http://jb.asm.org/.

^▿

Published ahead of print on 8 April 2011.

REFERENCES

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20. Linares J. F., et al. 2010. The global regulator Crc modulates metabolism, susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Environ. Microbiol. 12:3196–3212 [DOI] [PubMed] [Google Scholar]
21. Liu P. 1952. Utilization of carbohydrates by Pseudomonas aeruginosa. J. Bacteriol. 64:773–781 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. MacGregor C. H., Wolff J. A., Arora S. K., Phibbs P. V., Jr 1991. Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa. J. Bacteriol. 173:7204–7212 [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Mangan M. W., et al. 2006. The integration host factor (IHF) integrates stationary-phase and virulence gene expression in Salmonella enterica serovar Typhimurium. Mol. Microbiol. 59:1831–1847 [DOI] [PubMed] [Google Scholar]
24. Miller J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [Google Scholar]
25. Moreno R., Ruiz-Manzano A., Yuste L., Rojo F. 2007. The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. Mol. Microbiol. 64:665–675 [DOI] [PubMed] [Google Scholar]
26. Moreno R., Marzi S., Romby P., Rojo F. 2009. The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation. Nucleic Acids Res. 37:7678–7690 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Moreno R., Martínez-Gomariz M., Yuste L., Gil C., Rojo F. 2009. The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: evidence from proteomic and genomic analyses. Proteomics 9:2910–2928 [DOI] [PubMed] [Google Scholar]
28. Moreno R., Fonseca P., Rojo F. 2010. The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing translation of regulatory and structural genes. J. Biol. Chem. 285:24412–24419 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Müsken M., Di Fiore S., Dötsch A., Fischer R., Häussler S. 2010. Genetic determinants of Pseudomonas aeruginosa biofilm establishment. Microbiology 156:431–441 [DOI] [PubMed] [Google Scholar]
30. Nishijyo T., Haas D., Itoh Y. 2001. The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa. Mol. Microbiol. 40:917–931 [DOI] [PubMed] [Google Scholar]
31. Pérez-Martín J., de Lorenzo V. 1995. The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor. Proc. Natl. Acad. Sci. U. S. A. 92:9392–9396 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Porter S. C., North A. K., Kustu S. 1995. Mechanisms of transcriptional activation by NtrC, p. 147–158 In Hoch J. A., Silhavy T. J. (ed.), Two-component signal transduction. American Society for Microbiology, Washington, DC [Google Scholar]
33. Rojo F. 2010. Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and the interaction with the environment. FEMS Microbiol. Rev. 34:658–684 [DOI] [PubMed] [Google Scholar]
34. Sambrook J., Russell D. W. 2001. Molecular cloning: a laboratory manual, 3rd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Google Scholar]
35. Schnider-Keel U., et al. 2000. Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J. Bacteriol. 182:1215–1225 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Sonnleitner E., Abdou L., Haas D. 2009. Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 106:21866–21871 [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Stanier R. Y., Palleroni N. J., Doudoroff M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159–271 [DOI] [PubMed] [Google Scholar]
38. Stover C. K., et al. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964 [DOI] [PubMed] [Google Scholar]
39. Wolff J. A., MacGregor C. H., Eisenberg R. C., Phibbs P. V., Jr 1991. Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J. Bacteriol. 173:4700–4706 [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Yeung A. T. Y., Bains M., Hancock R. E. W. 2011. The sensor kinase CbrA is a global regulator that modulates metabolism, virulence and antibiotic resistance in Pseudomonas aeruginosa. J. Bacteriol. 193:918–931 [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Zhang X.-X., Rainey P. B. 2008. Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25. Genetics 178:185–195 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental material]

supp_193_11_2784__index.html^{(951B, html)}

supp_193_11_2784__TABLE_S1__Figs__S1_and_S2_22_03_11.doc^{(703KB, doc)}

[B1] 1. Amador C., Canosa I., Govantes F., Santero E. 2010. Lack of CbrB in Pseudomonas putida affects not only amino acid metabolism but also different stress responses and biofilm development. Environ. Microbiol. 12:1748–1761 [DOI] [PubMed] [Google Scholar]

[B2] 2. Ames G. F.-L., Nikaido K. 1985. Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus sequence. EMBO J. 4:539–547 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Barrios H., Valderrama B., Morett E. 1999. Compilation and analysis of σ⁵⁴-dependent promoter sequences. Nucleic Acids Res. 27:4305–4313 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Benes V., Hostomský Z., Arnold L., Pačes V. 1993. M13 and pUC vectors with new unique restriction sites for cloning. Gene 130:151–152 [DOI] [PubMed] [Google Scholar]

[B5] 5. Bourret R. B. 2010. Receiver domain structure and function in response regulator proteins. Curr. Opin. Microbiol. 13:142–149 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Collier D. N., Hager P. W., Phibbs P. V., Jr 1996. Catabolite repression control in the pseudomonads. Res. Microbiol. 147:551–561 [DOI] [PubMed] [Google Scholar]

[B7] 7. Cox M., Gerritse G., Dankmeyer L., Quax W. J. 2001. Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene. J. Biotechnol. 86:9–17 [DOI] [PubMed] [Google Scholar]

[B8] 8. Delic-Attree I., Toussaint B., Froger A., Willison J. C., Vignais P. M. 1996. Isolation of an IHF-deficient mutant of a Pseudomonas aeruginosa mucoid isolate and evaluation of the role of IHF in algD gene expression. Microbiology 142:2785–2793 [DOI] [PubMed] [Google Scholar]

[B9] 9. Gerritse G., Quax W. J. 6 November 2001. Expression system for altered expression levels. U.S. patent 6,313,283 B1

[B10] 10. Ghosh T., Bose D., Zhang X. 2010. Mechanisms for activating bacterial RNA polymerase. FEMS Microbiol. Rev. 34:611–627 [DOI] [PubMed] [Google Scholar]

[B11] 11. Gu B., Lee J. H., Hoover T. R., Scholl D., Nixon B. T. 1994. Rhizobium meliloti DctD, a σ⁵⁴-dependent transcriptional activator, may be negatively controlled by a subdomain in the C-terminal end of its two-component receiver module. Mol. Microbiol. 13:51–66 [DOI] [PubMed] [Google Scholar]

[B12] 12. Heeb S., et al. 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant Microbe Interact. 13:232–237 [DOI] [PubMed] [Google Scholar]

[B13] 13. Hervás A. B., Canosa I., Santero E. 2008. Transcriptome analysis of Pseudomonas putida in response to nitrogen availability. J. Bacteriol. 190:416–420 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Hervás A. B., Canosa I., Little R., Dixon R., Santero E. 2009. NtrC-dependent network for nitrogen assimilation in Pseudomonas putida. J. Bacteriol. 191:6123–6135 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Heurlier K., Dénervaud V., Pessi G., Reimmann C., Haas D. 2003. Negative control of quorum sensing by RpoN (σ⁵⁴) in Pseudomonas aeruginosa PAO1. J. Bacteriol. 185:2227–2235 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Itoh Y., Nishijyo T., Nakada Y. 2007. Histidine catabolism and catabolic regulation, p. 371–395 In Ramos J.-L., Filloux A. (ed.), Pseudomonas—a model system in biology, volume 5 Springer, Dordrecht, Netherlands [Google Scholar]

[B17] 17. Jaeger K. E., et al. 1996. Lipase of Pseudomonas aeruginosa: molecular biology and biotechnical application, p. 319–330 In Nakazawa T., Furukawa K., Haas D., Silver S. (ed.), Molecular biology of pseudomonads. ASM Press, Washington, DC [Google Scholar]

[B18] 18. Krzeslak J., Gerritse G., van Merkerk R., Cool R. H., Quax W. J. 2008. Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system. Appl. Environ. Microbiol. 74:1402–1411 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Li W., Lu C.-D. 2007. Regulation of carbon and nitrogen utilization by CbrAB and NtrBC two-component systems in Pseudomonas aeruginosa. J. Bacteriol. 189:5413–5420 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Linares J. F., et al. 2010. The global regulator Crc modulates metabolism, susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Environ. Microbiol. 12:3196–3212 [DOI] [PubMed] [Google Scholar]

[B21] 21. Liu P. 1952. Utilization of carbohydrates by Pseudomonas aeruginosa. J. Bacteriol. 64:773–781 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. MacGregor C. H., Wolff J. A., Arora S. K., Phibbs P. V., Jr 1991. Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa. J. Bacteriol. 173:7204–7212 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Mangan M. W., et al. 2006. The integration host factor (IHF) integrates stationary-phase and virulence gene expression in Salmonella enterica serovar Typhimurium. Mol. Microbiol. 59:1831–1847 [DOI] [PubMed] [Google Scholar]

[B24] 24. Miller J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [Google Scholar]

[B25] 25. Moreno R., Ruiz-Manzano A., Yuste L., Rojo F. 2007. The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. Mol. Microbiol. 64:665–675 [DOI] [PubMed] [Google Scholar]

[B26] 26. Moreno R., Marzi S., Romby P., Rojo F. 2009. The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation. Nucleic Acids Res. 37:7678–7690 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Moreno R., Martínez-Gomariz M., Yuste L., Gil C., Rojo F. 2009. The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: evidence from proteomic and genomic analyses. Proteomics 9:2910–2928 [DOI] [PubMed] [Google Scholar]

[B28] 28. Moreno R., Fonseca P., Rojo F. 2010. The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing translation of regulatory and structural genes. J. Biol. Chem. 285:24412–24419 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Müsken M., Di Fiore S., Dötsch A., Fischer R., Häussler S. 2010. Genetic determinants of Pseudomonas aeruginosa biofilm establishment. Microbiology 156:431–441 [DOI] [PubMed] [Google Scholar]

[B30] 30. Nishijyo T., Haas D., Itoh Y. 2001. The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa. Mol. Microbiol. 40:917–931 [DOI] [PubMed] [Google Scholar]

[B31] 31. Pérez-Martín J., de Lorenzo V. 1995. The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor. Proc. Natl. Acad. Sci. U. S. A. 92:9392–9396 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Porter S. C., North A. K., Kustu S. 1995. Mechanisms of transcriptional activation by NtrC, p. 147–158 In Hoch J. A., Silhavy T. J. (ed.), Two-component signal transduction. American Society for Microbiology, Washington, DC [Google Scholar]

[B33] 33. Rojo F. 2010. Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and the interaction with the environment. FEMS Microbiol. Rev. 34:658–684 [DOI] [PubMed] [Google Scholar]

[B34] 34. Sambrook J., Russell D. W. 2001. Molecular cloning: a laboratory manual, 3rd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Google Scholar]

[B35] 35. Schnider-Keel U., et al. 2000. Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J. Bacteriol. 182:1215–1225 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Sonnleitner E., Abdou L., Haas D. 2009. Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 106:21866–21871 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. Stanier R. Y., Palleroni N. J., Doudoroff M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159–271 [DOI] [PubMed] [Google Scholar]

[B38] 38. Stover C. K., et al. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964 [DOI] [PubMed] [Google Scholar]

[B39] 39. Wolff J. A., MacGregor C. H., Eisenberg R. C., Phibbs P. V., Jr 1991. Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J. Bacteriol. 173:4700–4706 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40. Yeung A. T. Y., Bains M., Hancock R. E. W. 2011. The sensor kinase CbrA is a global regulator that modulates metabolism, virulence and antibiotic resistance in Pseudomonas aeruginosa. J. Bacteriol. 193:918–931 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41. Zhang X.-X., Rainey P. B. 2008. Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25. Genetics 178:185–195 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa^{^▿}^,^†

Laetitia Abdou

Han-Ting Chou

Dieter Haas

Chung-Dar Lu

Abstract

INTRODUCTION

MATERIALS AND METHODS