Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction

Xue-Xian Zhang; Jonathan C Gauntlett; Darby G Oldenburg; Gregory M Cook; Paul B Rainey

doi:10.1128/JB.00361-15

. 2015 Jul 31;197(17):2867–2878. doi: 10.1128/JB.00361-15

Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction

Xue-Xian Zhang ^a,^✉, Jonathan C Gauntlett ^a,^b, Darby G Oldenburg ^a,^b,^*, Gregory M Cook ^c, Paul B Rainey ^b,^d

Editor: G A O'Toole

PMCID: PMC4524029 PMID: 26148710

ABSTRACT

CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA. This, coupled with the findings of [³H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction.

IMPORTANCE Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in Pseudomonas represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.

INTRODUCTION

The ability to recognize and convert external environmental stimuli into appropriate physiological responses is of fundamental importance for all organisms. In bacteria, signal transduction is predominantly mediated by two-component regulatory systems (TCSs) consisting of a sensor kinase (SK) and a cognate response regulator (RR) (1, 2). Both proteins are typically composed of two distinct functional domains (3): a variable N-terminal signal input domain and a conserved C-terminal autokinase domain for the SK and a conserved N-terminal receiver (Rec) domain and a variable C-terminal output domain for the RR. Signal transduction is achieved via phosphoryl transfer between the two conserved protein domains (4). Specifically, when SKs are activated by the presence of a stimulatory ligand, dimeric SKs undergo ATP-dependent autophosphorylation at conserved histidine residues (the H box) (5). The resulting high-energy phosphoryl group is then transferred to an aspartate residue on the cognate RR, causing a conformational shift in the regulatory domain. This in turn activates the specific output domain, leading to alteration of transcriptional, enzymatic, or mechanistic properties, ultimately producing a specific cellular response (6).

SKs are involved in the detection of a diverse array of environmental stimuli; their signal input domains are diverse and lack common structural motifs (7). However, in most instances, SKs contain one or two transmembrane (TM) domains in the N terminus, suggesting that these SKs are located in the cytoplasmic membrane and suitable for perception of extracellular signals. While it is generally accepted that the signal input domain regulates SK activity, just how the sensory domain perceives the signal remains largely unknown (8, 9). Current understanding is limited to a few well-studied examples, and in many cases the signal itself is not known (10 –12). For SKs involved in nutrient acquisition, it seems reasonable to assume that specific binding of the nutrient molecule might enable transmission of the nutrient signal into the cytoplasm, leading to activation of genes for subsequent uptake (and degradation, if applicable). Indeed, such an arrangement has been demonstrated in the control and transport of C₄ dicarboxylates (13 –15). For example, in the plant symbiotic bacteria Sinorhizobium meliloti (16) and Rhizobium leguminosarum (17), uptake of succinate is mediated by the DctA permease, whose expression is regulated by the two-component system DctB and DctD. DctB has a typical SK sensor domain with a short N-terminal cytosolic sequence, a transmembrane segment, a periplasmic domain, and a second transmembrane segment. Significantly, the purified periplasmic region binds succinate in vitro in a specific manner, suggesting that the sensor-substrate interaction acts as the stimulus for the DctBD system (18).

Nutrient perception and uptake are thus often coupled processes mediated by membrane-bound receptors and transporters, respectively. It is not uncommon for efficient acquisition to involve direct or indirect interactions between nutrient receptors and related transporters (19 –22). In the case of succinate uptake, DctA sequesters DctB in the cytoplasmic membrane in order to prevent the SK from autophosphorylation in the absence of succinate (16). In the presence of succinate, succinate initially binds to DctA, causing an increase of the substrate-binding specificity of DctB; DctB is then released and forms a functional two-component system with DctD (14).

A deeper involvement of nutrient transport in signaling is evident in a group of transporters, termed transceptors, which have a dual role in both transport and reception (19, 21, 23). For example, transceptor UhpC in Escherichia coli is a homologue of UhpT, a functional transporter for glucose-6-phosphate (Glc6P). UhpC has residual Glc6P transport activities, and its interaction with the UhpB/UhpA two-component system regulates transcription of the primary Glc6P-specific transporter, UhpT (24).

From a physiological perspective, as the process of nutrient transport could also function as a stimulus, it seems reasonable to expect that selection may have led to the evolution of systems in which nutrient sensing is coupled to activation of transport genes. Such coupled systems could, in principle, ensure a rapid response to external nutrients, conferring a potential benefit for bacteria living in nutrient-poor environments. Interestingly, bacterial genome sequencing has revealed the presence of a unique type of SK, which has a typical autokinase domain fused to a transporter-like polypeptide at the N terminus (8, 25). A well-known example is the CbrA sensor kinase in Pseudomonas (26 –30). The N-terminal portion is predicted to contain 14 transmembrane segments and shows a high degree of similarity with the Na⁺/proline symporter PutP, which belongs to the sodium/solute symporter family (SSSF; Transporter Classification Database classification 2.A.21).

CbrA works together with its cognate RR CbrB, which possesses a σ⁵⁴-interacting output domain. Both CbrA and CbrB are essential for the activation of genes involved in nutrient acquisition, including hut genes for the utilization of histidine and urocanate (urocanate is the first intermediate of the histidine degradation pathway) (26, 27). While the biological roles of CbrAB have been documented in different species of Pseudomonas, the molecular modes of CbrA/CbrB action have not been characterized (28, 29, 31). Current evidence shows that CbrAB is capable of maintaining catabolic activities across a wide range of C/N ratios, but the nature of the signal perceived by the CbrA kinase remains elusive (26, 27). The atypical domain structure of CbrA leads to questions as to the role of the N-terminal transporter domain, whether this is involved in signal perception, and, furthermore, whether activation of the SK involves substrate binding and/or substrate translocation across the cytoplasmic membrane (26).

Here we report the findings of a genetic and biochemical analysis of CbrA in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25. The study was motivated by a mutant hunt that aimed to identify genes for histidine transport located outside the hut operon but that repeatedly implicated CbrA—and only CbrA—in histidine uptake. This led to whole-cell [³H]histidine transport assays and further genetic analyses in order to test the hypothesis that CbrA is involved in histidine uptake and, subsequently, both histidine uptake and signaling. Armed with supportive evidence, we constructed alleles of CbrA containing single nucleotide changes at conserved residues with predicted roles in transport and signal transduction. Our combined data implicate the SSSF domain of CbrA in histidine transport and strongly suggest that transport is coupled with/dependent upon signal transduction.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

All bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5α or One Shot TOP10 (Invitrogen, Carlsbad, CA) was used for general cloning purposes. E. coli DH5αλpir was used for cloning with pUIC3, and E. coli XL1-Blue or XL1-Gold (Agilent Technologies, Santa Clara, CA) was used for gene cloning following site-directed mutagenesis. P. fluorescens and E. coli strains were routinely cultivated in Luria-Bertani (LB) medium at 28°C and 37°C, respectively. P. fluorescens strains were also grown in M9 medium with glucose and ammonium as the carbon and nitrogen sources, respectively, or minimal M9 salts medium (MSM) supplemented with either histidine or urocanate at a final concentration of 15 mM. Where appropriate, the following antibiotics were also added to the growth media at the indicated concentrations: ampicillin (Ap), 100 μg ml⁻¹; gentamicin (Gm), 25 μg ml⁻¹; kanamycin (Km), 50 μg ml⁻¹; nitrofurantoin (Nf), 100 μg ml⁻¹; spectinomycin (Sp), 100 μg ml⁻¹; tetracycline (Tc), 15 μg ml⁻¹. The toxic histidine analogue 3-amino-1,2,4-triazole (3AT) was used to enrich for uptake-defective mutants and was added to M9 medium plates at a concentration of 5 mM (the minimum concentration required to prevent the P. fluorescens wild type from forming colonies).

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Genotype and growth phenotype on histidine and urocanate	Reference(s) or source
P. fluorescens strains
SBW25	His⁺ Uro⁺, wild-type strain isolated from sugar beet	42, 57
PBR855	His⁺ Uro⁻ ΔhutT_u	35
PBR856	His⁺ Uro⁺ ΔhutT_h	35
PBR857	His⁺ Uro⁺ ΔhutXWV	35
PBR858	His⁺ Uro⁺ ΔhutT_hXWV	35
PBR859	His⁺ Uro⁻ ΔhutT_hT_uXWV	35
DH128	His⁻ Uro⁻ ΔhutT_h cbrA::Tn Km^r	This work
DH128Cre	His⁻ Uro⁻ ΔhutT_h cbrA::189 nt	This work
DH128Cre-3	His⁻ Uro⁺, a spontaneous Uro⁺ mutant of DH128Cre	This work
MU6-80	His⁻ Uro⁺ ΔhutT_h cbrA::189 nt Tn-cbrB	This work
MU7-83	His⁻ Uro⁺ ΔhutT_h cbrA::189 nt(pME6032-cbrB), derivative of DH128Cre	This work
MU7-86	His⁻ Uro⁻ ΔhutT_h cbrA::189 nt(pME6032), derivative of DH128Cre	This work
MU8-88	His⁻ Uro⁺ ΔhutT_hXWV cbrA::189 nt Tn-cbrB	This work
PBR870	His⁻ Uro⁻ ΔcbrA-TM	This work
PBR871	His⁻ Uro⁻ PBR870(pME6032::cbrA-TM)	This work
PBR876	His⁺ Uro⁺ PBR870(pME6032::cbrA)	This work
PBR872	His⁻ Uro⁻ ΔhutT_hXWV ΔcbrA	This work
PBR873	His⁻ Uro⁺ PBR872 with Tn7-cbrA::189 nt Gm^r	This work
PBR874	His⁺ Uro⁺ ΔhutT_h(pME6032::cbrA-TM), derivative of PBR856, Tc^r	This work
PBR875	His⁺ Uro⁺, strain MU8-88 with Tn7-cbrA(H766G), Gm^r	This work
PBR963	His⁺ Uro⁺ PBR872 with Tn7-cbrA, Gm^r	This work
PBR964	His⁻ Uro⁻ PBR872 with Tn7-cbrA(H766G), Gm^r	This work
PBR1014	His⁺ Uro⁺ PBR872 with Tn7-cbrA(W55G), Gm^r	This work
PBR1006	His⁺ Uro⁺ PBR872 with Tn7-cbrA (G172A) Gm^r	This work
PBR1009	His⁺ Uro⁺ PBR872 with Tn7-cbrA (N876G) Gm^r	This work
Plasmids
pRK2013	Helper plasmid, Tra⁺ Km^r	58
pUIC3	Integration vector with promoterless ′lacZ, Mob⁺ Tc^r	59
pME6032	Shuttle vector for gene expression in Pseudomonas, Tc^r	60
pUC18T-mini-Tn7T-Gm-LAC	A Tn7-based cloning vector for gene expression, Gm^r	39
pUX-BF13	Helper plasmid for transposition of the Tn7 element, Ap^r	61
pME6032::cbrA	pME6032 containing the cbrA wild-type allele	This work
pME6032::cbrA-TM	pME6032 containing the N-terminal CbrA transporter	This work
pME6032-cbrB	pME6032 containing the entire cbrB gene	This work
pUIC3-8	pUIC3 containing the P_hutU-lacZ fusion, Tc^r	41
pSCR001	Plasmid carrying the IS-Ω-Km/hah element, Km^r	33
pCre	Plasmid for Cre-loxP-mediated deletion of the insertion sequence element, Tc^r	34
pCM639	ISphoA/hah delivery plasmid, Tc^r	32

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Growth curves of P. fluorescens strains were generated using a Synergy 2 multimode microplate reader equipped with Gen5 (v1.04.5) software (BioTek, Winooski, VT). To ensure that strains in comparisons were physiologically equivalent, cells stored in glycerol saline at −80°C were used for the preparation of inoculants. They were first inoculated into LB broth and allowed to grow for ∼18 h before subculturing in M9 broth for 24 h. To set up the growth experiments, the M9 medium cultures were adjusted to a similar optical density (A₆₀₀, ∼0.8), and cells were pelleted by centrifugation and washed once using the same amount of MSM salt solution. After starvation at 28°C for 2 h, 2 μl was inoculated into 200 μl of the tested medium (per well) in a 96-well microtiter plate. Turbidity was measured at a wavelength of 450 nm at 5-min intervals for up to either 48 or 72 h, and data from 2-h intervals are plotted for clarity.

Tn mutagenesis analysis.

The transposon (Tn) used in this study, IS-Ω-Km/hah, was a modified version of ISphoA/hah (32) in which phoA and the chloramphenicol resistance (Cm^r) gene were replaced with an Ω-Km cassette (33). P. fluorescens strains were mutagenized by conjugation with E. coli S17-1λpir(pSCR001) (pSCR001 is a plasmid carrying IS-Ω-Km/hah). Conjugation was performed on a filter in an LB agar plate by mixing 500 μl each of overnight cultures of the donor and recipient strains. To increase the conjugation efficiency, the recipient cells were treated at 45°C for 20 min before they were mixed. The conjugation mix was incubated at 28°C for 4 h. Then, the cells on the filter were resuspended in 1 ml sterile water and inoculated onto selective plates, e.g., M9 salts medium supplemented with urocanate and nitrofurantoin (to counterselect E. coli) and Km. The genomic location of the Tn was determined by sequencing the DNA fragments obtained by arbitrarily primed PCR (AP-PCR), as previously described by Manoil (34). IS-Ω-Km/hah possesses the loxP sites, and thus, it can be excised from the mutants by introducing plasmid pCre, which carries the phage P1 gene for Cre recombinase. Excision of the transposon generates a 189-nucleotide (nt) insertion on the site.

Transport assays in whole cells and CbrA-proteoliposomes.

Uptake of [³H]histidine by P. fluorescens cells was performed as previously described (35). Briefly, cells at log phase were harvested by centrifugation and washed twice in 50 mM potassium phosphate buffer containing 2 mM MgCl₂ (pH 7.2, 4°C). Cells in 200-μl aliquots were suspended in the same buffer to a density of an A₆₀₀ of 1.0 to 1.5 and energized for 10 to 15 min with the addition of glucose (10 mM). To initiate transport, [³H]histidine was added to a final concentration of 1 μM (100 nCi). After specific time intervals, transport was terminated by the addition of 2 ml ice-cold LiCl (100 mM) and rapid filtration through cellulose nitrate filters (pore size, 0.45 μm). The filters were washed again with 2 ml of LiCl and dried, and the counts per minute were determined using an LKB Wallac 1214 RackBeta liquid scintillation counter. The protein content of each cell suspension was determined by using a bicinchoninic acid (BCA) protein assay kit from Sigma-Aldrich and bovine serum albumin as the standard.

To express CbrA with six histidine residues at the C terminus, the coding region was amplified by PCR and cloned into the expression vector pTrc99A at the NcoI and SalI restriction sites, generating plasmid pTrc-CbrAHis. Expression and purification of CbrA were performed using protocols previously described (36). Briefly, after induction with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), E. coli C43(DE3) cells were lysed by sonication and fractionated by high-speed centrifugation, and the cell membranes were solubilized in 1% n-dodecyl-β-d-maltoside (DDM). The soluble membrane fraction (1% DDM) was loaded on a Ni²⁺-nitrilotriacetic acid (NTA) affinity column. The column was washed in 20 mM KPO₄, 5% (vol/vol) glycerol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 500 mM NaCl, 75 mM imidazole (HCl), and 0.05% (wt/vol) DDM. CbrA-His₆ was eluted in 300 mM imidazole (HCl), and the identity of CbrA was confirmed by SDS-PAGE and mass spectrometry analysis. After the CbrA identity was confirmed, the Ni²⁺-NTA affinity-purified CbrA was reconstituted into phosphatidylcholine liposomes with a lipid-to-protein ratio of 50:1 (wt/wt) as previously described (36). The CbrA-proteoliposomes were then incubated in transport buffer (50 mM Na phosphate, pH 7.4, 2 mM MgCl₂) in the presence or absence of 5 mM ATP as an energy source. A valinomycin-induced potassium diffusion potential of 100 mV was generated by the addition of 200 mM KCl plus 0.5 μM valinomycin to the dilution buffer. Transport was initiated by the addition of [³H]histidine or 0.5 μM valinomycin (potassium diffusion potential) and stopped by addition of a 10-fold volume of 0.1 M LiCl and rapid filtration through a 0.45-μm-pore-size filter (Millipore). Uptake of [³H]histidine into the lumen of the proteoliposomes was determined by scintillation counting (LKB Wallac). For isothermal titration calorimetry (ITC) analysis, CbrA proteins were further purified by gel filtration. Ni²⁺-NTA affinity-purified CbrA was concentrated using a centrifugal filter (Amicon) and injected onto a Superdex 200 gel filtration column (GE Healthcare) equilibrated in 20 mM KPO₄, 5% glycerol, 1 mM DTT, 150 mM NaCl, 0.1 mM PMSF, 0.05% DDM. ITC was performed with 5 μM purified CbrA protein, and 10 μl of 50 μM histidine was injected at 480-s intervals.

Strain construction.

All molecular techniques were conducted according to standard procedures (37). Restriction or DNA-modifying enzymes were obtained from Invitrogen (Auckland, New Zealand). Routine PCR and PCR for TA cloning were conducted using Taq DNA polymerase from Invitrogen, and PCR for direct cloning was conducted using Phusion high-fidelity DNA polymerase from Finnzymes, Thermo Scientific Ltd. Details about the oligonucleotide primers are available on request. Gene mutation in vivo was achieved by a previously described procedure of splicing by overlapping extension PCR in conjunction with a two-step allelic exchange strategy using the integration vector pUIC3 (38).

All the cbrA mutant alleles were constructed in vitro using a QuikChange site-directed mutagenesis kit from Agilent Technologies Ltd. (Auckland, New Zealand) according to the manufacturer's instructions. The cbrA coding region with its own promoter was PCR amplified from wild-type SBW25 using primers cbrA-GWF and cbrA-GWR (data not shown) and cloned into the pCR8 vector from Invitrogen. The resultant plasmid, pCR8-cbrA⁺, was then used as the template for PCR using mutagenic primers. The cbrA mutant allele obtained was transferred to vector pUC18T-mini-Tn7T-Gm-GW (39), using the Gateway LR Clonase enzyme mix from Invitrogen following the manufacturer's recommendations. The resulting construct was then mobilized into the appropriate P. fluorescens strain by conjugation in the presence of two E. coli strains containing pRK2013 and pUX-BF13, respectively (40). Integration of the Tn7 element harboring the cbrA mutant allele at the single attTn7 site downstream of the glmS gene of P. fluorescens SBW25 was confirmed by PCR with primers SBW25-glmS and Tn7R109 (40).

RESULTS

Identification of transporters involved in histidine uptake.

P. fluorescens SBW25 is capable of growth on histidine (and urocanate) as the sole source of carbon and nitrogen (41, 42). As outlined in Fig. 1, the five hut enzymes required for the breakdown of histidine are encoded by genes in the hutU to hutG (hutU-hutG) and hutF operons. Expression of the hutU-hutG operon is induced by the presence of histidine and requires activation mediated by CbrAB, as well as derepression of the HutC repressor by urocanate (41). The hut operon harbors three transport systems, two permeases (HutT_u and HutT_h), and an ABC-type transporter, HutXWV. In a previous study (35), we showed that hutT_u encodes a urocanate-specific transporter, deletion of which abolishes growth on urocanate (Uro⁻). HutT_h is a high-affinity histidine-specific permease, but a mutant devoid of hutT_h shows significant, albeit reduced, growth on histidine as the sole source of carbon and nitrogen (it is phenotypically His⁺). Deletion of hutXWV results in a detectable but minor reduction in growth on histidine. Importantly, a mutant lacking all three transport systems grows in minimal medium with histidine as the sole carbon and nitrogen source (35). This indicates the presence of an additional histidine transporter(s) located elsewhere in the P. fluorescens SBW25 genome.

To identify the predicted transporter(s), we adopted a transposon (Tn) mutagenesis strategy to screen for mutants unable to grow on histidine as the sole source of carbon and nitrogen (His⁻). Mutagenesis was performed using IS-Ω-Km/hah (33), which encodes resistance to kanamycin. Strains of two His⁺ genotypes (ΔhutT_uXWVT_h and ΔhutT_h) were subject to analysis. His⁻ mutants were detected by replica plating transconjugants (Km^r) onto agar plates containing M9 salts medium supplemented with histidine (15 mM). Approximately 10,000 mutants were screened, and a total of 22 His⁻ mutants were obtained.

Additional His⁻ mutants were selected using the toxic histidine analogue 3-amino-1,2,4-triazole (3AT) to enrich for uptake-defective mutants (such mutants were expected to take up less 3AT and thus show a higher 3AT tolerance than the wild-type strain). Transconjugants were selected on M9 medium plates supplemented with nitrofurantoin (to counterselect E. coli), kanamycin, and 3AT. The 3AT-resistant Tn mutants were subsequently examined for their ability to grow on histidine as the sole carbon and nitrogen source. About 6 × 10⁵ Tn mutants were screened, and 7 His⁻ mutants were obtained.

The genomic location of the Tn insertion was determined for all mutants using an arbitrarily primed PCR (AP-PCR) strategy followed by DNA sequencing. A total of 29 mutants (7 mutants obtained from 3AT enrichment plus 22 mutants from general screening) were successfully mapped. No obviously identifiable transporters were identified (Table 2). Of particular note were eight Tn insertions located within cbrA (Fig. 2) and two located within the adjacent response regulator, cbrB. Significantly, among the seven His⁻ Tn mutants obtained from the 3AT enrichment, six mapped to the cbrAB locus (the single other mutant mapped to thiI, a gene with a predicted role in thiamine biosynthesis). All cbrAB mutants were more resistant to the toxic histidine analogue 3AT than wild-type strain SBW25 and mutant PBR859 (ΔhutT_uXWVT_h), as revealed in a growth assay (data not shown), suggesting that these cbrAB mutants are unable to import histidine.

TABLE 2.

The 29 His⁻ transposon mutants identified in this work

Mutant	Genotype of parent strain	3AT enriched	Tn insertion site
Mutant	Genotype of parent strain	3AT enriched	Genomic location	Target gene	Gene product
3-68a	ΔhutT_uXWVT_h	No	6254632	pgk (pflu5705)	Phosphoglycerate kinase
5-1DH2	ΔhutT_uXWVT_h	No	∼6374420	ptsP (pflu5819)	Phosphoenolpyruvate phosphotransferase
5-4DH15	ΔhutT_uXWVT_h	No	6091968	rpe (pflu5563)	Ribulose 5-phosphate 3-epimerase
5-14DH17	ΔhutT_uXWVT_h	No	6111860	apaH (pflu5580)	Diadenosine tetraphosphatase
5-18DH23	ΔhutT_uXWVT_h	No	3457101	pflu3160	Putative quinone oxidoreductase
5-21DH30	ΔhutT_uXWVT_h	No	1990887	sucC (pflu1823)	Succinyl coenzyme A synthetase, beta subunit
5-72DH124	ΔhutT_uXWVT_h	No	4382956	ilvE (pflu3968)	Branched-chain amino acid aminotransferase
4-57-10	ΔhutT_uXWVT_h	No	6313495	pyrC (pflu5759)	Dihydroorotase and related cyclic amidohydrolases
5-20DH26	ΔhutT_uXWVT_h	No	6091969	rpe (pflu5563)	Ribulose 5-phosphate 3-epimerase
4-55-8	ΔhutT_uXWVT_h	No	5779648	carB (pflu5265)	Carbamoylphosphate synthase large subunit
3-69b	ΔhutT_uXWVT_h	No	406113	hutH₂ (pflu0367)	Histidine ammonia-lyase
5-2DH6	ΔhutT_uXWVT_h	No	409020	hutI (plfu0369)	Imidazolonepropionase
5-16DH21	ΔhutT_uXWVT_h	No	405510	hutH₂ (pflu0367)	Histidine ammonia-lyase
5-53DH94	ΔhutT_uXWVT_h	No	404166	hutH₁ (pflu0362)	Histidine ammonia-lyase (nonfunctional)
5-15DH19	ΔhutT_uXWVT_h	No	1692425	pdxB (pflu1546)	Erythronate-4-phosphate dehydrogenase
5-64DH110	ΔhutT_uXWVT_h	No	∼1691840	pdxB (pflu1546)	Erythronate-4-phosphate dehydrogenase
4-25-3AT-3	ΔhutT_uXWVT_h	Yes	6000485	ribH (pflu5470)	Riboflavin synthase beta chain
5-55DH97	ΔhutT_uXWVT_h	No	381703	thiI (plfu0349)	Thiamine biosynthesis ATP pyrophosphatase
5-63DH109	ΔhutT_uXWVT_h	No	4336061	clpX (pflu3928)	ATP-dependent protease Clp
5-19DH25	ΔhutT_uXWVT_h	No	5744602	cbrA (pflu5236)	Sensor kinase
5-35DH44	ΔhutT_uXWVT_h	No	5742901	cbrA (pflu5236)	Sensor kinase
5-57DH100	ΔhutT_uXWVT_h	No	5742472	cbrA (pflu5236)	Sensor kinase
5-70DH122	ΔhutT_uXWVT_h	No	5743360	cbrA (pflu5236)	Sensor kinase
DH128	ΔhutT_h	Yes	5743495	cbrA (pflu5236)	Sensor kinase
5-77DH129	ΔhutT_h	Yes	5743496	cbrA (pflu5236)	Sensor kinase
5-78DH130	ΔhutT_h	Yes	5743647	cbrA (pflu5236)	Sensor kinase
3AT-4	ΔhutT_uXWVT_h	Yes	5743740	cbrA (pflu5236)	Sensor kinase
3AT-1	ΔhutT_uXWVT_h	Yes	5746559	cbrB (pflu5237)	Response regulator
3AT-2	ΔhutT_uXWVT_h	Yes	5746001	cbrB (pflu5237)	Response regulator

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FIG 2 — Outline of the Tn mutagenesis analysis with the domain structure of CbrA and a schematic map of the *cbrAB* locus in mutant DH128Cre. (A) The first round of Tn mutagenesis involved selection of His⁻ mutants, and one resultant mutant (DH128 His⁻ Uro⁻) was subjected to the second round of Tn analysis for either His⁺ or Uro⁺ mutants. (B) The total length of CbrA is 980 amino acids, and its N-terminal half (504 amino acids) possesses 14 putative transmembrane domains (rectangles). Vertical lines depict the locations of IS-Ω-Km/hah in eight His⁻ mutants generated from the first round of Tn mutagenesis and, accordingly, the 189-nt in-frame insertion sites after Cre-mediated excision of the Tn. Like DH128Cre, all eight mutants from which the Tn was excised remained His⁻ and Uro⁻. Arrows indicate the locations of the five CbrA mutant alleles constructed *in vitro*. (C) Circles denote transposons mapped to the *cbrAB* promoter regions in the 28 Uro⁺ mutants of DH128Cre.

While the Tn insertions clearly implicate cbrAB in histidine uptake, cbrAB is required for transcriptional activation of the hutU-hutG operon (41). The His⁻ phenotype of the cbrA insertion mutants could therefore be either a direct consequence of the inability of CbrA to take up histidine (which would implicate CbrA in histidine uptake) or an indirect effect attributable to the fact that histidine, brought into the cell via some secondary transport system, cannot be metabolized by the hut operon (which would mean that CbrAB-mediated regulation of hut is inoperative). A third possibility is that CbrA plays a role in both histidine uptake and regulation of the hut locus.

Histidine uptake independent of known histidine-specific transporters.

If CbrAB functions in both uptake (of histidine) and signaling (activation of transcription of the hut locus), then the physiological capacity to take up histidine is likely to be constitutive. In other words, when grown in the absence of histidine, the CbrAB system should nonetheless be capable of transporting the substrate should it suddenly appear. In contrast, when grown in a histidine-free environment, mutants lacking cbrAB should be unable to transport histidine, because in histidine-free environments the histidine transporters in the hut locus are not induced (41).

To test this prediction, histidine transport assays were performed for wild-type strain SBW25 grown in M9 medium (with glucose and ammonium as the sole carbon and nitrogen sources, respectively). As shown in Fig. 3, histidine uptake was detected. Histidine transport exhibited increasing rates of uptake as the external histidine concentration increased. At [³H]histidine concentrations of >5 μM, the rate of histidine uptake continued to increase (Fig. 3), whereas uptake was saturated at these concentrations for cells grown on histidine, as revealed in a previous study (35). The apparent K_m was 1.66 μM, and on average, this was 2-fold higher than the K_m for cells grown on histidine (0.78 μM). The V_max of histidine uptake was also higher than that of histidine-grown cells: 0.90 nmol min⁻¹ mg protein⁻¹ for cells grown on glucose and ammonium and 0.66 nmol min⁻¹ mg protein⁻¹ for cells grown on histidine. The rate of histidine uptake was not dependent on sodium ions; in addition, monensin, a sodium ionophore, had no effect on histidine uptake (data not shown).

FIG 3 — Kinetics of [³H]histidine uptake by *P. fluorescens* SBW25. Transport assays were performed using cells grown in M9 minimal medium containing glucose and ammonium. The initial rates of histidine uptake, expressed as nanomoles of [³H]histidine per minute per milligram of SBW25 protein, were measured over 40 s at various concentrations of histidine ranging from 1 to 20 μM. (Inset) Lineweaver-Burk plot of the data. v, reaction velocity; [S], substrate concentration. Experiments were performed two or more times, and the values reported are the means from biological duplicates.

Next, we measured the rates of histidine uptake in mutants defective in the permeases of the hut operon (ΔhutT_h, ΔhutT_u, ΔhutXWV, ΔhutXWVT_h, and ΔhutT_uXWVT_h mutants). The assay was performed using cells grown in M9 medium, and the results are shown in Table 3. All hut transporter mutants exhibited rates of [³H]histidine uptake that were comparable to the rate for the wild-type strain; however, when either cbrA or cbrB was additionally mutated, no significant [³H]histidine uptake was detected (Table 3).

TABLE 3.

Rates of [³H]histidine uptake in P. fluorescens SBW25 and derived mutants for cells grown in MSM with glucose and ammonium as the sole carbon and nitrogen sources

Genotype	Strain	[³H]histidine uptake (nmol · min⁻¹ mg protein⁻¹)^a
Wild type	SBW25	0.12 ± 0.04
ΔhutT_u	PBR855	0.17 ± 0.06
ΔhutT_h	PBR856	0.15 ± 0.03
ΔhutXWV	PBR857	0.29 ± 0.03
ΔhutXWVT_h	PBR858	0.30 ± 0.05
ΔhutT_hT_uXWV	PBR859	0.14 ± 0.02
ΔhutT_uXWVT_h cbrA::Tn	3AT-4	0.04 ± 0.02
ΔhutT_uXWVT_h cbrB::Tn	3AT-2	0.03 ± 0.01

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The rate of histidine uptake was measured with external [³H]histidine at a concentration of 1 μM. Results are means and standard errors from between two and four independent experiments.

These data show that SBW25 possesses a functional histidine transporter that is not subject to histidine induction. This unknown transporter is encoded by either cbrA itself or an unknown gene under the control of CbrAB. If CbrA were to be directly involved in histidine transport, then the fact that cbrB mutants are defective in the uptake of histidine would suggest that uptake might be tied to signal transduction (in which CbrB plays a seminal role).

Suppressor analysis implicates CbrA in histidine transport.

Support for the conjecture that CbrA is the cryptic histidine transporter would mount if it were possible to show the absence of histidine transporters, beyond those already known within the hut operon, under the control of CbrA (and CbrB). In principle, this could be achieved by mutagenizing a cbrA::Tn (His⁻) mutant of a ΔhutT_h strain containing a constitutively active hut locus, followed by a screen for His⁺ mutants.

To obtain a mutant of a cbrA::Tn ΔhutT_h strain carrying a constitutively expressed hut locus, we identified spontaneous Uro⁺ mutants arising from the cbrA mutant strain DH128 (ΔhutT_h cbrA::Tn; Fig. 2); however, before doing so—and to aid with subsequent rounds of Tn mutagenesis—IS-Ω-Km/hah was first excised from the cbrA locus. Excision of the transposon left a 189-nucleotide in-frame insertion in transmembrane domain number 10 of CbrA (Fig. 2B). The resultant mutant (named DH128Cre) retained the original phenotype of Uro⁻ and His⁻ (when the Tn in the other seven cbrA mutants was also excised, all genotypes remained Uro⁻ and His⁻).

To select for spontaneous Uro⁺ mutants, an overnight culture of DH128Cre (ΔhutT_h cbrA::189 nt) was plated onto minimal M9 salts medium with urocanate as the sole carbon and nitrogen source. Following 3 days of incubation, 10 spontaneous Uro⁺ mutants were obtained, indicating that the genes for urocanate utilization, which is encoded by the hut locus, were expressed. All 10 Uro⁺ mutants were phenotypically His⁻.

Next, we took one spontaneous Uro⁺ mutant (DH128Cre-3) and mutagenized this with IS-Ω-Km/hah (IS-Ω-Km/hah can activate the transcription of genes from an internal npt promoter [33]). Transconjugants were plated on M9 salts medium with histidine as the sole carbon and nitrogen source. His⁺ mutants could, in principle, arise as a result of the activation of any additional existing histidine transporter by promoters carried on the transposon. No His⁺ mutants were found within 3 days of incubation at 28°C, despite a saturation screen of the genome. This result provides little support for the possibility that there is an additional transporter(s) controlled by cbrAB.

Success in obtaining spontaneous Uro⁺ mutants of DH128Cre (ΔhutT_h cbrA::189 nt) suggested that such mutants might be obtained by Tn mutagenesis; moreover, the capacity to map the genomic location of relevant insertions could provide valuable information. To this end, DH128Cre (His⁻ Uro⁻) was mutagenized with IS-Ω-Km/hah to select for Uro⁺ mutants on minimal medium with urocanate as the sole carbon and nitrogen source. Approximately 6.1 × 10⁵ transconjugants were screened in three independent rounds of mutagenesis (∼20 times coverage of the genome). A total of 28 Uro⁺ mutants were obtained, and the position of the transposon was determined using AP-PCR. All insertions mapped to the cbrAB locus: 13 were located immediately upstream of the ATG start codon of cbrA::189 nt, while the other 15 were located upstream of the ATG start codon of cbrB (Fig. 2C). Significantly, all 28 Uro⁺ mutants were incapable of growth on histidine (His⁻): no growth was detected during the first 24 h, and only minor growth was observed by 48 h (Fig. 4). The minor growth was attributed to HutXWV: when hutXWV was deleted from one Uro⁺ His⁻ mutant, MU6-80 (ΔhutT_h cbrA::189 nt Tn-cbrB), the resulting mutant strain, MU8-88 (ΔhutXWVT_h cbrA::189 nt Tn-cbrB), showed no growth over a period of 48 h (Fig. 4), providing further confirmation of the involvement of HutXWV in histidine uptake (35).

FIG 4 — Growth dynamics of the transposon mutants of *P. fluorescens* SBW25. Bacteria were grown in minimal salts medium with histidine (A) or urocanate (B) as the sole source of carbon and nitrogen. Results are means and standard errors for 6 independent cultures. Mutant DH128Cre is a His⁻ mutant of an Δ*hutT_h* strain with a 189-nt insertion in *cbrA*. MU6-80 is a Uro⁺ mutant of DH128Cre with a Tn insertion in the promoter region of *cbrB*. MU8-88 was derived from MU6-80 with the deletion of *hutXWV* genes. WT, wild type.

The fact that the His⁻ and Uro⁻ phenotype of DH128Cre could revert to His⁻ and Uro⁺ upon insertion of IS-Ω-Km/hah into the promoter region of either cbrA or cbrB led us to consider that activation of CbrB is sufficient to activate enzymes of the hut operon and, by logical extension, any other genes under the control of CbrAB, including cryptic transporters of histidine. To test this prediction, cbrB was cloned into a shuttle vector, pME6032, and expressed under the control of an IPTG-inducible P_tac promoter. The recombinant plasmid pME6032-cbrB was then introduced into DH128 (ΔhutT_h cbrA::189 nt His⁻ Uro⁻), and the resulting transconjugant, MU7-83, was examined for the ability to grow in minimal medium with urocanate or histidine as the sole carbon and nitrogen source. As expected, overexpression of cbrB restored the Uro⁺ phenotype of DH128, but this genotype remained His⁻.

Should a histidine transporter(s) exist under the control of cbrAB, it ought to have been activated, either by virtue of Tn-generated promoter activity or as a consequence of CbrB overexpression. The fact that no Uro⁺ mutant grew on histidine means that it is highly unlikely that the genome contains genes for additional histidine transporters under the control of CbrAB. Nonetheless, further attempts to obtain His⁺ mutants via Tn mutagenesis of DH128Cre (ΔhutT_h cbrA::189 nt His⁻ Uro⁻) and MU8-88 (ΔhutXWVT_h cbrA::189 nt Tn-cbrB Uro⁺ His⁻), using ISphoA/hah (32), were made. Despite saturation screening of the genome, no His⁺ mutants were obtained. This further confirms that all the spontaneous Uro⁺ mutants of DH128Cre are His⁻ and further implicates CbrA as the cryptic transporter of histidine. Moreover, given that in-frame insertions of 189-nt tags (left after excision of IS-Ω-Km/hah) into seven different regions of the putative transporter domain of CbrA (Fig. 2B) abolished expression of the hut operon (the mutants were unable to grow on either histidine or urocanate), the transporter domain is further implicated in not just transport but also signal transduction. This suggests that signaling and transport may be intimately coupled.

Physical coupling between the N- and C-terminal domains of CbrA is required for function.

If transport and signaling are coupled processes, then destroying the physical linkage between the N-terminal transporter region of CbrA and the C-terminal autokinase domain should abolish the ability to grow on histidine and urocanate. To test this prediction, the DNA region encoding the CbrA transporter domain (cbrA-TM) was deleted from wild-type P. fluorescens SBW25, leaving the autokinase domain under the control of the native cbrA promoter. The resultant strain (PBR870 [ΔcbrA-TM]) was unable to grow on histidine or urocanate. Further, a promoterless lacZ fusion to the hutU operon was not expressed under these growth conditions (data not shown), indicating that the autokinase domain alone is incapable of activating the transcription of hut. Such a finding is not surprising, given that the N-terminal region is likely to be involved in sensory perception and necessary for activation of the autokinase domain.

Introduction into PBR870 (ΔcbrA-TM) of a cloned and overexpressed copy of the deleted cbrA N-terminal region (to generate PBR871 [ΔcbrA-TM, carrying pME6032::cbrA-TM]) did not restore the ability of the mutant to grow on histidine or urocanate (nor did it activate transcription of the hut operon). A control experiment in which wild-type cbrA was introduced into PBR870 (to generate strain PBR876) restored the ability to grow on both histidine and urocanate. Physical coupling between the two domains is thus required for CbrA function.

Although the above-described experiment provided no evidence of histidine transport independent of the autokinase domain, we reasoned that a strain carrying wild-type cbrA might, if provided with additional copies of the N-terminal domain, have an increased capacity for growth on histidine. We therefore took PBR856 (ΔhutT_h), which, carrying a deletion of the histidine-specific permease hutT_h, is significantly impaired in its ability to grow on histidine, and introduced the N-terminal region of cbrA (carried on plasmid pME6032 [pME6032::cbrA-TM]) into the cell (to generate strain PBR874). Despite the presence of additional copies of the N-terminal region, the strain with this genotype (which carries a wild-type copy of cbrA in its native context) showed no improvement in growth on histidine compared to that of the ΔhutT_h mutant (PBR856). While it is unknown whether truncated CbrA is folded properly and is stable, whether it is correctly integrated into the cytoplasmic membrane, or whether there is some obligate interaction between domains, including possible coupling between transport and signaling, the genetic data indicate a lack of functionality of the N-terminal domain in the absence of a direct connection to the autokinase domain.

Dissecting the relationship between signal transduction and transport.

If histidine transport is dependent upon signaling, then a mutant with a mutated allele of cbrA carrying a single amino acid change at the predicted phosphorylation site (H766) should be incapable of growth on histidine. To test this hypothesis and facilitate additional analyses of the phenotypic effects of a range of site-directed cbrA mutants, we developed a complementation strategy that involved first construction of a mutant lacking hutT_h, hutXWV, and cbrA (strain PBR872). Strain PBR872 was unable to grow on either histidine or urocanate (it was fully defective for both histidine transport and signaling). Into this background we introduced cbrA alleles at the unique chromosomal attTn7 site via the mini-Tn7 cloning system (pUC18-mini-Tn7T-Gm-LAC [see Materials and Methods]). To check that complementation in trans was possible, we introduced a wild-type allele of cbrA and showed that growth on histidine and urocanate was restored (strain PBR963; Fig. 5).

FIG 5 — Growth dynamics of mutants carrying *cbrA* mutant alleles in the autokinase domain (A and B) and the transport-like signal input domain (C and D). CbrA function was assessed in the genetic background of PBR872 (Δ*cbrA* Δ*hutT_h* Δ*hutXWV*), which was not able to grow on histidine and urocanate (C and D). Bacterial cells were grown in minimal medium with histidine (A and C) or urocanate (B and D) as the sole carbon and nitrogen source. Data are means and standard errors for six independent cultures.

Next, a cbrA allele carrying a single amino acid change at the predicted phosphorylation site (H766G) was generated and incorporated into the chromosome via the mini-Tn7 cloning system. This single amino acid change completely abolished growth on histidine and urocanate (strain PBR964; Fig. 5A and B). Moreover, a lacZ fusion to the hutU operon confirmed that the locus remained inactive. This finding shows that growth on histidine (which requires the transport of histidine into the cell) is dependent on the capacity of CbrA to undergo phosphorylation. However, while this result is consistent with the prediction that transport is dependent upon signaling, it does not exclude the possibility that the H766G allele is transport proficient (histidine may enter the cell but is not metabolized due to an inability of the allele to phosphorylate CbrB and, thus, activate the hut locus).

To test whether the H766G allele of CbrA is transport defective and signaling defective or transport proficient but signaling defective, we obtained a spontaneous Uro⁺ mutant of strain PBR964 (PBR964 harbors the CbrA H766G allele in a background devoid of hutT_h, hutXWV, and cbrA) and asked whether growth on histidine had been restored. A total of 24 spontaneous Uro⁺ colonies of PBR964 were picked, and all were His⁻ (data not shown). These data strongly suggest that the transport function of CbrA is dependent upon the capacity to engage in signal transduction.

Next, we sought a signaling-impaired mutant of cbrA via introduction of a mutation in the C-terminal signaling domain that would retain some capacity for signal transduction. We reasoned that if histidine enters the cell via a transporter other than CbrA, as is the case for urocanate, then a reduction in signaling would have an equally proportionate effect on growth on both histidine and urocanate. However, if CbrA is also involved in transport, then impairment in signal transduction could have a disproportionate effect on growth on histidine. To this end, a single amino acid change was made at position 876 (N876G), one of several amino acid positions predicted to be involved in binding to inorganic phosphate and to the adenosine moiety of ATP in the active site of the kinase (43). PBR872, carrying the N876G allele of CbrA, grew on urocanate to a final density commensurate with that of the control strain carrying wild-type cbrA, albeit with a reduction in the growth rate (Fig. 5B). However, on histidine, this allele resulted in a greatly reduced capacity for growth (Fig. 5A). This finding once again implicates CbrA in transport, but it strongly suggests that histidine transport is dependent upon signal transduction.

Next, we considered the possibility that intramolecular interactions are important. We therefore took strain MU8-88 (ΔhutT_h XWV cbrA::189 nt Tn-cbrB), which is His⁻ (due to a lack of known histidine transport systems; it also carries an insertion in the 10th membrane-spanning domain of CbrA) and is Uro⁺ (as a consequence of IS-Ω-Km/hah driving expression of cbrB [see above]). Into this background we introduced (via the Tn7-based chromosomal integration strategy) the signaling-dead allele of cbrA [cbrA(H766G)]. Growth on histidine was restored (Fig. 6). Thus, the presence of two full-length but mutant alleles of cbrA, one carrying a defect in the N-terminal domain and the other carrying a defect in signaling, allows restoration of functionality. This effect of complementation is most likely due to intermolecular interactions via formation of a heterodimer, whereby the transport deficiency of cbrA::189 nt is complemented by the functional N terminus of the H766G allele and the signaling defect of H766G is complemented by the functional autokinase domain of cbrA::189 nt. That such complementation occurs makes a compelling case for the dependency of histidine transport on signaling.

FIG 6 — Genetic complementation between *cbrA*::189 nt and *cbrA*(H766G). (A) The Uro⁺ His⁻ mutant MU8-88 (*cbrA*::189 nt) was able to grow on histidine as the sole source of carbon and nitrogen upon the introduction of *cbrA*(H766G). (B) Both strains grew when urocanate was the sole source of carbon and nitrogen. Data are means and standard errors for six independent cultures.

To further explore the functionality of the CbrA N-terminal domain, we introduced site-specific changes at two invariant residues adjacent to the predicted Na⁺ and substrate-binding sites, which have been experimentally characterized in PutP from E. coli (44). Residues W55 and G172 are located in transmembrane (TM) domains TM2 and TM5, respectively (Fig. 2). When the tryptophan residue (W55) was converted to glycine (W55G), the mutant displayed a delay in growth on histidine (Fig. 5C) and a slight reduction in its growth rate, whereas growth on urocanate was not affected (Fig. 5D). We constructed a total of three G172 variants in which the glycine residue was replaced by alanine, serine, or histidine (i.e., G172A, G172S, and G172H, respectively). As shown in Fig. 5C and D, no discernible effect of the G172A allele was observed when the strain was grown on either histidine or urocanate; the G172S and G172H mutants showed a delay in growth on histidine, but their ability to grow on urocanate was largely unchanged. Together, the negligible growth effects on urocanate but larger effects on histidine are consistent with the suggested dual role of the CbrA N terminus in histidine transport and signal transduction.

Biochemical analysis of histidine transport by CbrA-proteoliposomes.

Finally, we sought to demonstrate in vitro the predicted function of CbrA in histidine transport. Recombinant CbrA with a C-terminal hexahistidine tag was expressed in E. coli C43(DE3). SDS-PAGE analysis of purified CbrA (in 0.5% DDM after concentration) indicated overproduction of a protein with a predicted size of His₆-tagged CbrA (∼109 kDa). Its identity was subsequently confirmed by mass spectrometry. Purified CbrA was reconstituted into phosphatidylcholine liposomes to study [³H]histidine transport. CbrA was successfully reconstituted, as determined by SDS-PAGE analysis of CbrA-proteoliposomes (data not shown). However, CbrA-proteoliposomes energized with either ATP or a valinomycin-induced potassium diffusion potential showed no histidine uptake, and the rate of binding was comparable to that of nonenergized proteoliposomes in both the presence and absence of sodium ions. Isothermal titration calorimetry (ITC) of CbrA was performed to test if CbrA binds to histidine. The assay was performed with 5 μM purified CbrA protein, and 10 μl of 50 μM histidine was injected at 480-s intervals. No heat of binding of histidine to CbrA was detected.

DISCUSSION

We aimed to identify and characterize the one or more unknown, but experimentally evident, transporters of histidine (26, 35). A hefty weight of data from mutagenesis screens, from mutant enrichment, from site-directed mutagenesis, and from whole-cell histidine transport assays implicates CbrA in histidine transport. The close similarity between CbrA and the sodium/solute symporter family of transporters adds further substance (20, 45, 46). Moreover, exhaustive attempts to test the hypothesis that histidine uptake is determined by additional histidine transporters under the control of CbrA were negative. Nonetheless, direct biochemical proof that CbrA transports histidine (e.g., via transport of [³H]histidine by His₆-tagged CbrA) is lacking. However, failure to detect histidine transport in CbrA-proteoliposomes does not exclude this possibility. Such transport assays depend on the ability to energize CbrA with an appropriate driving force (e.g., ATP and/or the proton or sodium motive force), and this may not have been fulfilled. Further, if, as is suspected (see below), CbrA is involved in both transport and signaling and if signaling is required for transport, then transport assays in proteoliposomes may require signal transduction from CbrA to CbrB. It is also possible that CbrA did not fold into an active form under our assay conditions.

Given the weight of support for the hypothesis that CbrA is directly involved in the transport of histidine, combined with the fact that CbrA is a histidine kinase with a known role in signal transduction (26, 27, 30, 31), the second part of our study explored the connection between signal transduction and transport. Several lines of evidence suggest that the two are coupled, as found in the Saccharomyces cerevisiae Gap1 amino acid transceptor (47). The most compelling evidence comes from showing that two alleles of cbrA, one with a mutation in the N terminus (cbrA::189 nt) and the other with a mutation in the C terminus [cbrA(H766G)], complement one another. Furthermore, the fact that it was possible to generate, via site-specific changes, single amino acid mutant alleles of CbrA with unequal impairment in urocanate versus histidine growth phenotypes strongly suggests that CbrA not only transports histidine but also does so in a manner that depends on signal transduction. This was most clearly apparent with the N876G variant of CbrA, which had a minimal impact on growth on urocanate but brought about a significant impairment of growth on histidine (Fig. 5B).

Numerous questions concerning the nature of the signal perceived by CbrA arise: whether this signal is histidine, the transport of histidine, or the movement (either into or out of the cell) of some secondary or coupled factor, such as ammonium. Also unknown is the range of compounds transported by CbrA. Given the high-level regulatory control of CbrA over the uptake and catabolism of many carbon and nitrogen sources, including proline, arginine, xylose, and mannose (26), it is inconceivable that histidine is the sole transported molecule. One possibility is that CbrA is a general transporter with a broad substrate specificity whose primary role is to signal to operons dedicated to the uptake and breakdown of various amino and organic acids information sufficient to prime these pathways (via CbrB), thus ensuring that each pathway is ready for activation should the compound specific to that pathway be transported. Such a strategy would ensure a rapid and coordinated response to the presence of and changes in the presence of a diverse array of nutritional sources.

It has recently been shown that the CbrAB system plays a key regulatory role in carbon catabolite repression (CCR) in various species of Pseudomonas (48), including P. fluorescens SBW25 (49). CCR is a mechanism that allows bacteria to acclimate rapidly to preferred carbon and energy sources, thereby maximizing the growth rate (50). For example, expression of xut genes for xylose utilization is repressed in the presence of succinate (49). The role of CbrAB in the CCR of P. fluorescens SBW25 was first noted in previous work (26), which showed that CbrAB is functionally required for xylose utilization (a cbrB deletion mutant cannot grow on xylose). Our current model of CCR in P. fluorescens SBW25 shows that, in the presence of succinate, CbrAB activates the expression of two noncoding small RNAs (CrcY and CrcZ) which sequestrate the Crc/Hfq protein complex, relieving Crc- and Hfq-mediated repression of mRNA transcripts of catabolic enzymes and transporters, including those for the uptake and degradation of xylose (49). Similar mechanisms have been reported in model strains of P. aeruginosa and P. putida (51 –53). While CbrAB is positioned at the top of the CCR regulatory hierarchy, how CbrAB perceives and processes complex nutrient signals remains elusive.

Data presented here indicate high sensitivity with regard to the transcriptional levels of cbrA relative to those of cbrB. For example, when the cbrA::189 nt mutant allele was placed in the original cbrA locus rather than the Tn7 insertion site (located downstream of glmS), a significant difference in growth on urocanate was observed (Uro⁻ for mutant DH128Cre versus Uro⁺ for mutant PBR873; Table 1). Additionally, during the second round of Tn mutagenesis of DH128Cre (Uro⁻), Tn insertions in the promoter regions of either cbrA::189 nt or cbrB led to significant growth on urocanate (Fig. 2B). Notably, in previous work, we also showed that cbrB is subject to regulation by its own promoter, with its expression fluctuating with changes to external nutrients (cbrA is expressed at similar lower levels by cells grown on different carbon sources) (26). Together, the data suggest that CCR in Pseudomonas is likely achieved via modulation of cbrB promoter activity through the effects of a yet unknown transcriptional regulator(s). CbrA plays a seemingly minor role in the perception of CCR nutrient signals, but it is capable of maintaining the promoter activities of catabolic genes over a wide range of C/N ratios (26).

Membrane-bound sensor kinases and transporters have separate but analogous functions for nutrient utilization: the former transport nutrient information from surface receptor to the cytoplasm, whereas the latter transport nutrient molecules from outside the cell to the inside. The two dedicated proteins work in a coordinated manner to ensure that expression of transporters is specifically induced by the presence of nutrients in the extracellular environment. Transporters can participate in nutrient sensing via intramembrane protein-protein interactions with the sensor kinase (54). This has been documented in bacteria, such as for the transporter and kinase genes dctA and dctB (or dcuS) for the utilization of C₄ dicarboxylate (15, 16), as well as the ABC-type transport system PtsSABC and sensor kinase PhoR for the specific uptake of inorganic phosphate (55). However, from a physiological perspective, proteins with a dual role of nutrient sensing and uptake could have evolved, and moreover, nutrient uptake can potentially act as the stimulus that activates a given kinase (19 –21). The E. coli UhpC transceptor is the only such example of a transporter-like kinase so far found in bacteria (20, 54). UhpC is capable of transporting and sensing glucose-6-phosphate (56). However, transport and signaling are two separable functions encoded by a single polypeptide, and there is no evidence to indicate that the two functions are coupled (24). CbrA represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain (at the N terminus) via a cytoplasmic linker domain (19). Data presented here suggest that CbrA plays a dual role in histidine transport and sensing and, significantly, that the transport function is dependent on signal transduction.

ACKNOWLEDGMENTS

We thank Sieu Tran and David Hsiao for technical assistance.

X.-X.Z. acknowledges support from the Guangdong Innovative and Entrepreneurial Research Team Program (no. 2013S033) and the Auckland Medical Research Foundation (AMRF project no. 4114009). P.B.R. is the recipient of an International Blaise Pascal Research Chair of the French State and the IIe-de-France. This work was funded by the Marsden Fund Council from government funding administered by the Royal Society of New Zealand (UOA0505/3606398).

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[B10] 10.Cheung J, Hendrickson WA. 2009. Structural analysis of ligand stimulation of the histidine kinase NarX. Structure 17:190–201. doi: 10.1016/j.str.2008.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Roggiani M, Goulian M. 2015. Oxygen-dependent cell-to-cell variability in the output of the Escherichia coli Tor phosphorelay. J Bacteriol 197:1976–1987. doi: 10.1128/JB.00074-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Krell T, Lacal J, Busch A, Silva-Jimenez H, Guazzaroni ME, Ramos JL. 2010. Bacterial sensor kinases: diversity in the recognition of environmental signals. Annu Rev Microbiol 64:539–559. doi: 10.1146/annurev.micro.112408.134054. [DOI] [PubMed] [Google Scholar]

[B13] 13.Witan J, Bauer J, Wittig I, Steinmetz PA, Erker W, Unden G. 2012. Interaction of the Escherichia coli transporter DctA with the sensor kinase DcuS: presence of functional DctA/DcuS sensor units. Mol Microbiol 85:846–861. doi: 10.1111/j.1365-2958.2012.08143.x. [DOI] [PubMed] [Google Scholar]

[B14] 14.Yurgel SN, Kahn ML. 2004. Dicarboxylate transport by rhizobia. FEMS Microbiol Rev 28:489–501. doi: 10.1016/j.femsre.2004.04.002. [DOI] [PubMed] [Google Scholar]

[B15] 15.Steinmetz PA, Worner S, Unden G. 2014. Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of Escherichia coli: function of DctA as an activity switch and of DcuS as the C4-dicarboxylate sensor. Mol Microbiol 94:218–229. doi: 10.1111/mmi.12759. [DOI] [PubMed] [Google Scholar]

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[B18] 18.Nan B, Liu X, Zhou Y, Liu J, Zhang L, Wen J, Zhang X, Su XD, Wang YP. 2010. From signal perception to signal transduction: ligand-induced dimeric switch of DctB sensory domain in solution. Mol Microbiol 75:1484–1494. doi: 10.1111/j.1365-2958.2010.07069.x. [DOI] [PubMed] [Google Scholar]

[B19] 19.Tetsch L, Jung K. 2009. The regulatory interplay between membrane-integrated sensors and transport proteins in bacteria. Mol Microbiol 73:982–991. doi: 10.1111/j.1365-2958.2009.06847.x. [DOI] [PubMed] [Google Scholar]

[B20] 20.Tetsch L, Jung K. 2009. How are signals transduced across the cytoplasmic membrane? Transport proteins as transmitter of information. Amino Acids 37:467–477. doi: 10.1007/s00726-009-0235-x. [DOI] [PubMed] [Google Scholar]

[B21] 21.Thevelein JM, Voordeckers K. 2009. Functioning and evolutionary significance of nutrient transceptors. Mol Biol Evol 26:2407–2414. doi: 10.1093/molbev/msp168. [DOI] [PubMed] [Google Scholar]

[B22] 22.Falord M, Karimova G, Hiron A, Msadek T. 2012. GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. Antimicrob Agents Chemother 56:1047–1058. doi: 10.1128/AAC.05054-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Hundal HS, Taylor PM. 2009. Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling. Am J Physiol Endocrinol Metab 296:E603–E613. doi: 10.1152/ajpendo.91002.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Schwoppe C, Winkler HH, Neuhaus HE. 2003. Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli. Eur J Biochem 270:1450–1457. doi: 10.1046/j.1432-1033.2003.03507.x. [DOI] [PubMed] [Google Scholar]

[B25] 25.Galperin MY. 2004. Bacterial signal transduction network in a genomic perspective. Environ Microbiol 6:552–567. doi: 10.1111/j.1462-2920.2004.00633.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Zhang XX, Rainey PB. 2008. Dual involvement of CbrAB and NtrBC in the regulation of histidine utilization in Pseudomonas fluorescens SBW25. Genetics 178:185–195. doi: 10.1534/genetics.107.081984. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.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: 10.1046/j.1365-2958.2001.02435.x. [DOI] [PubMed] [Google Scholar]

[B28] 28.Amador CI, Canosa I, Govantes F, Santero E. 2010. Lack of CbrB in Pseudomonas putida affects not only amino acids metabolism but also different stress responses and biofilm development. Environ Microbiol 12:1748–1761. doi: 10.1111/j.1462-2920.2010.02254.x. [DOI] [PubMed] [Google Scholar]

[B29] 29.Yeung AT, Janot L, Pena OM, Neidig A, Kukavica-Ibrulj I, Hilchie A, Levesque RC, Overhage J, Hancock RE. 2014. Requirement of the Pseudomonas aeruginosa CbrA sensor kinase for full virulence in a murine acute lung infection model. Infect Immun 82:1256–1267. doi: 10.1128/IAI.01527-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Valentini M, Garcia-Maurino SM, Perez-Martinez I, Santero E, Canosa I, Lapouge K. 2014. Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida. Microbiology 160:2243–2252. doi: 10.1099/mic.0.078873-0. [DOI] [PubMed] [Google Scholar]

[B31] 31.Yeung AT, Bains M, Hancock RE. 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: 10.1128/JB.00911-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.Bailey J, Manoil C. 2002. Genome-wide internal tagging of bacterial exported proteins. Nat Biotechnol 20:839–842. doi: 10.1038/nbt715. [DOI] [PubMed] [Google Scholar]

[B33] 33.Giddens SR, Jackson RW, Moon CD, Jacobs MA, Zhang XX, Gehrig SM, Rainey PB. 2007. Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment. Proc Natl Acad Sci U S A 104:18247–18252. doi: 10.1073/pnas.0706739104. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34.Manoil C. 2000. Tagging exported proteins using Escherichia coli alkaline phosphatase gene fusions. Methods Enzymol 326:35–47. [DOI] [PubMed] [Google Scholar]

[B35] 35.Zhang XX, Chang H, Tran SL, Gauntlett JC, Cook GM, Rainey PB. 2012. Variation in transport explains polymorphism of histidine and urocanate utilization in a natural Pseudomonas population. Environ Microbiol 14:1941–1951. doi: 10.1111/j.1462-2920.2011.02692.x. [DOI] [PubMed] [Google Scholar]

[B36] 36.Gauntlett JC, Gebhard S, Keis S, Manson JM, Pos KM, Cook GM. 2008. Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis. J Biol Chem 283:8591–8600. doi: 10.1074/jbc.M709503200. [DOI] [PubMed] [Google Scholar]

[B37] 37.Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [Google Scholar]

[B38] 38.Zhang XX, Rainey PB. 2007. Construction and validation of a neutrally-marked strain of Pseudomonas fluorescens SBW25. J Microbiol Methods 71:78–81. doi: 10.1016/j.mimet.2007.07.001. [DOI] [PubMed] [Google Scholar]

[B39] 39.Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer HP. 2005. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2:443–448. doi: 10.1038/nmeth765. [DOI] [PubMed] [Google Scholar]

[B40] 40.Liu Y, Rainey PB, Zhang XX. 2014. Mini-Tn7 vectors for studying post-transcriptional gene expression in Pseudomonas. J Microbiol Methods 107:182–185. doi: 10.1016/j.mimet.2014.10.015. [DOI] [PubMed] [Google Scholar]

[B41] 41.Zhang XX, Rainey PB. 2007. Genetic analysis of the histidine utilization (hut) genes in Pseudomonas fluorescens SBW25. Genetics 176:2165–2176. doi: 10.1534/genetics.107.075713. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction

Xue-Xian Zhang

Jonathan C Gauntlett

Darby G Oldenburg

Gregory M Cook

Paul B Rainey

Roles

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS