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J Bacteriol. 2010 January; 192(1): 217–224.
Published online 2009 October 23. doi:  10.1128/JB.01124-09
PMCID: PMC2798258

Metabolic Control of Virulence Genes in Brucella abortus: HutC Coordinates virB Expression and the Histidine Utilization Pathway by Direct Binding to Both Promoters [down-pointing small open triangle]

Abstract

Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (PvirB). Using different procedures, we isolated a 27-kDa protein that binds specifically to PvirB. This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses.

Type IV secretion systems (T4SS) are multicomponent machineries central to the pathogenesis of many bacterial genera (e.g., Brucella, Agrobacterium, Helicobacter, Legionella, and Bordetella) (4). T4SS function comprises recognition and translocation of specific substrates across the bacterial cell envelope. The nature of the translocated substrates varies from proteins to DNA-protein complexes. In addition to the well-studied Agrobacterium transferred DNA (T-DNA) and Bordetella pertussis toxin, several translocated effectors have been identified for Helicobacter, Legionella, and Brucella (7). In every case, the translocated molecules alter cellular processes in such a way that allows the pathogen to overcome host defenses.

Brucella is a gram-negative bacterium that causes brucellosis, a worldwide zoonosis that affects domestic mammals. Different Brucella species vary in their host preferences. Brucella abortus, Brucella suis, and Brucella melitensis infect cattle, pigs, and goats, respectively, but also infect humans. In animals, the symptoms of the disease are sterility in males and abortion in pregnant females (6). In humans, brucellosis causes undulant fever during the acute phase and, if it reaches chronicity, can lead to endocarditis, osteoarthritis, and neurological damage.

Brucella is an intracellular parasite that persists and replicates within host macrophages. After internalization, the bacterium actively controls the maturation of the so-called Brucella-containing vacuole (BCV) by modulating the formation of transient interactions with endoplasmic reticulum (ER)-derived membranes and late endosomes/lysosomes (26). Such events are crucial for establishing the intracellular replication niche and determine the intracellular fate of the bacterium. The T4SS of Brucella, which is encoded by the virB operon, plays a central role in this process, since virB mutants fail to reach the intracellular replication compartment and are completely degraded by lysosomes (5, 15, 24).

The virB operon of B. suis is induced after internalization in macrophage-like cell lines (1). In B. abortus, it was observed that transcription of the virB operon reaches the maximum activity at 5 h postinfection (p.i.). Subsequently, virB expression is rapidly repressed before intracellular bacterial replication starts (23). This tightly controlled pattern of expression suggests the existence of regulatory pathways that probably act in response to environmental stimuli sensed by the bacterium during its intracellular trafficking. Therefore, the identification of the transcription factors involved in this process may lead to the understanding of the signals that govern the regulation of the VirB T4SS of Brucella within the eukaryotic host cell.

The virB promoter (PvirB) of B. abortus comprises 430 bp that participate in the intracellular activity of the system (23). The histone-like integration host factor (IHF) binds to PvirB and induces a DNA bending that is necessary for the intracellular regulation of the virB genes (23). The quorum sensing-related protein VjbR was also shown to interact with PvirB, directly controlling virB expression in a positive manner (7, 9). In addition, many other regulators affect virB expression (18). However, no direct interactions with PvirB were reported for these factors, indicating that such influences may be indirect.

Based on the knowledge of the architecture of PvirB, we designed a strategy to identify additional factors that specifically bind to this promoter. Here we report the identification of HutC, a protein known to regulate expression of the histidine utilization (hut) genes, as a transcription factor that directly controls the activity of PvirB. Using electrophoresis mobility shift assays (EMSA) and DNase I footprinting, we identified the HutC-binding sites and analyzed the interaction of this regulator with both hut and virB promoters. Our results show that HutC coactivates virB expression while it represses transcription of the hut genes, both intracellularly and in bacteria incubated under conditions that resemble the intracellular replication niche. These findings demonstrate that transcriptional regulation of the virB operon is linked to the induction of the histidine utilization pathway, probably as a way to synchronize activation of virulence genes with signals that involve internal metabolic variables and external stimuli perceived within the eukaryotic host cell.

MATERIALS AND METHODS

Media and culture conditions.

Brucella strains were grown in tryptic soy broth (TSB) or in minimal medium 1 (MM1), which is derived from medium MM (21). The composition of MM1 is 33 mM KH2PO4, 60.3 mM K2HPO4, and 0.1% yeast extract. Urocanic acid (UCA) (Sigma) or l-histidine (Sigma) was added to a final concentration of 5 mM. After the addition of the different carbon sources, the pH was adjusted to 4.5, 5.5, or 7.0 with HCl, and the media were sterilized by filtration through a 0.2-μm filter (Orange Scientific). Bacteria were cultured at 37°C in a rotary shaker (250 rpm). Media were supplemented with kanamycin (50 μg ml−1) as needed.

Construction of plasmids. pK18mob-sacB-ΔhutC.

Two PCRs were carried out using Pfx (Invitrogen), genomic DNA of B. abortus 2308 as the template, and primers dCSpeA (5′-GGACTAGTATCATCGCGCCCGCAATGT-3′) and comdCA (5′-CTGCAAGAAGGAGAGTATCGACCAGAATACGCTGATGCAG-3′) or dCSpeB (5′-GGACTAGTCAGGCCTTGCCGATTACTG-3′) and comdCB (5′-TCGATACTCTCCTTCTTGCAGTATCCCGGTGACAGCCATG-3′). Both PCR products, corresponding to two 400-bp flanking regions of hutC, were annealed and used as templates for a PCR performed with primers dCSpeA and dCSpeB. The product was digested with SpeI and cloned into the kanamycin-resistant plasmid pK18mob-sacB (22).

pK18mob-PvirB-lacZ.

A 5-kb DNA fragment from plasmid pBluescript-PvirB::lacZ (23), which contains the PvirB-lacZ transcriptional fusion, was excised with BamHI and EcoRI and cloned into pK18mob in the same restriction sites.

pK18mob-Phut-lacZ.

A 4.5-kb fragment that contains a lacZ promoter-probe cassette was released from plasmid pAB2001 after digestion with SphI. The resulting fragment was cloned into plasmid pK18mob at the same restriction site, generating plasmid pK18mob-lacZ. A PCR performed with Taq (Invitrogen), B. abortus 2308 genomic DNA as the template, and primers PhutA (5′-TGAAATGCTGGCTGGATTG-3′) and PhutB (5′-TCGATCAAGCCGAGATTTG-3′) was cloned into pGEM-T Easy (Promega). The resulting plasmid (pGEM-Phut) was digested with EcoRI, releasing a 170-bp fragment that contains the hut promoter sequences. The Phut-containing fragment was cloned into pK18mob-lacZ in the EcoRI site, generating plasmid pK18mob-Phut-lacZ. Clones with the transcriptional fusion Phut-lacZ oriented in the direction of the hutFC operon were identified by PCR using primers PhutA and lacZ6143 (5′-CAGGGTTTTCCCAGTCACG-3′) and checked by DNA sequencing with the same primers.

pK18mob-sacB-hutC-KI.

A PCR was performed using Pfx, genomic DNA of B. abortus 2308 as the template, and primers dCSpeA and dCSpeB. The PCR product, which contains the wild-type hutC gene and two 400-bp flanking regions, was cloned into pGEM-T easy (Promega). A 1.4-kb fragment excised from the resulting plasmid with StuI and HincII was cloned into the SmaI site of pK18mob-sacB.

Expression vector pQE-31-hutC.

PCRs were performed using Pfx and primers uprHut (5′-CGGGATCCGATGGCTGGCGAAGATTCGA-3′) and downrHut (5′-GGTACCTCAGCCGCGAGATGGCGT-3′). The PCR products were digested with KpnI and BamHI and cloned into plasmid pQE-31 (Qiagen).

Construction of strains B. abortus ΔhutC and B. abortus ΔhutC-KI.

Plasmid pK18mob-sacB-ΔhutC was transferred to B. abortus 2308 by biparental conjugation. Kanamycin-resistant colonies were selected as single-homologous recombinants. Selection with sucrose, excision of plasmids, and generation of deletion mutants were performed as described previously (23). PCR analyses of kanamycin-sensitive colonies were carried out with primers dCSpeA and dCSpeB to identify clones that contain the deletion of hutC.

Plasmid pK18mob-sacB-hutC-KI was transferred to B. abortus ΔhutC by biparental conjugation. Kanamycin-resistant colonies were selected as single-homologous recombinants. Selection with sucrose and excision of the plasmid were performed as described previously (23). PCR analyses of kanamycin-sensitive colonies were carried out with primers dCSpeA and dCSpeB. Colonies that generated the wild-type pattern were selected as hutC knock-in (KI) strains.

Construction of strains containing single chromosomal transcriptional fusions.

Plasmid pK18mob-PvirB-lacZ or pK18mob-Phut-lacZ was transferred by biparental conjugation into strain B. abortus 2308, B. abortus ΔhutC, or B. abortus ΔhutC-KI. Kanamycin-resistant colonies were selected as single-homologous recombinants.

EMSA and DNase I footprinting.

Construction of probes PvirB and PvirB-ihf for EMSA and determination of apparent dissociation constants (Kd) were performed as described previously (23). Probe Phut was constructed using primers PhutA and PhutB. The control probe, which contains sequences corresponding to 226 bp of virB10 of B. abortus 2308, was constructed with primers B10Qu (5′-CTATGCAACCCAGAAGGTCGG-3′) and B10d2 (5′-GGGAATTCGTCAGGCACAATAAAGTCAC-3′). The unlabeled competitors b1 and c2 were constructed by PCR using primers pvirdown I and pvu229 and primers pvirdown I and pvu300, respectively (23). Binding reactions were performed with binding buffer (15 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 10 μg ml−1 bovine serum albumin [BSA], 1 mM dithiothreitol [DTT], 30 mM KCl, 6% glycerol) containing 50 μg ml−1 salmon sperm DNA for isolation of HutC or 25 μg ml−1 poly(dI·dC) (Amersham) for Kd determinations and IHF-HutC competition experiments.

Probes PvirB and Phut for DNase I footprinting were constructed using primer pvu144 and 32P-labeled primer pvirdownI and primer PhutA and 32P-labeled primer PhutB, respectively. Labeling reactions, purification of probes, binding reactions, DNase I digestion, purification of fragments, and electrophoresis were performed as described previously (23).

Isolation and identification of HutC.

The isolation of the PvirB-binding protein was performed as described previously (23), with the following modifications. To diminish the risk of working with the virulent wild-type strain, the protein detected by EMSA in B. abortus 2308 was isolated from cultures of the avirulent strain B. abortus Δpgm (27) harvested in exponential phase (optical density at 600 nm [OD600] of 0.5). After disruption of bacteria, ultracentrifugation, filtration, and ammonium sulfate precipitation, the protein fractions were analyzed by EMSA using probe PvirB-ihf or the control probe. The positive fraction (25 to 45% ammonium sulfate saturation) was suspended in 35 mM phosphate buffer (pH 6.8)-3 mM β-mercaptoethanol and dialyzed against the same buffer overnight at 4°C. The solution was loaded onto a Mono-S column and eluted with a linear gradient of 35 mM phosphate buffer (pH 6.8), 3 mM β-mercaptoethanol, and 1 M NaCl. The DNA-binding activity of the fractions was analyzed by EMSA using the probes described above. Positive fractions were subjected to affinity chromatography: the biotinylated probe biot-PvirB-ihf, which corresponds to the region from −201 to +24 of PvirB lacking the IHF-binding site, was constructed by PCR using the 5′-biotinylated primer pvirdown I, the primer pvu229, and plasmid pBluescript-PvirB-IHF-sacB/R (23) as the template. The biotinylated control probe was constructed by PCR using the 5′-biotinylated primer ADP-Biot (5′-GCGATGGGCAGAGCGCCGG-3′), primer PE2P3 (5′-GGATCCGGTGCTCGACGCCAA-3′), and genomic DNA of Mesorhizobium loti as the template. The biotinylated probes were bound to streptavidin paramagnetic spheres (Promega), and a binding reaction was performed using binding buffer and the positive fractions of the Mono-S column. After two washes with binding buffer with 0.2 M NaCl, the DNA-bound proteins were eluted with 0.85 M NaCl and analyzed by 12.5% SDS-PAGE. After Coomassie blue staining, six bands were observed in the sample obtained with the biotinylated probe biot-PvirB-ihf, whereas one of them was absent in the sample obtained with the control probe. The differential band was excised from the gel and analyzed by mass spectrometry at Vital Probes, Inc. (PA).

Expression and purification of recombinant proteins.

Recombinant IHF was prepared as described previously (23). Recombinant HutC (rHutC) was prepared as follows. Plasmid pQE-31-hutC was transferred into Escherichia coli M15(pREP4) (Qiagen) and induced with IPTG (isopropyl-β-d-thiogalactopyranoside). Bacteria were harvested, suspended in lysis buffer (20 mM Tris-HCl [pH 7.6], 1 mM phenylmethylsulfonyl fluoride [PMSF]), and disrupted by sonication. After centrifugation, NaCl was added to a final concentration of 0.5 M and the sample was loaded into a Hi-Trap nickel-chelating column (Amersham Biosciences). After a wash with buffer B (20 mM Tris-HCl [pH 7.6], 0.5 M NaCl, 100 mM imidazole), the column was equilibrated with buffer A (20 mM Tris-HCl [pH 7.6], 0.5 M NaCl) and eluted with a linear gradient of buffer C (20 mM Tris-HCl [pH 7.6], 0.5 M NaCl, 100 mM EDTA). Eluates were analyzed by 12.5% SDS-PAGE, and the fractions containing rHutC (purity near 95%) were pooled and dialyzed against buffer D (20 mM Tris-HCl [pH 7.6], 0.5 M NaCl, 3 mM β-mercaptoethanol). The sample was stored at −20°C with 0.5% sucrose.

β-Gal activity determinations.

For cultured bacteria, measurement of β-galactosidase (β-Gal) activity was carried out with whole cells as described previously (23). β-Gal activity was expressed in Miller units (M.U.) (A420/volume × OD600) × 100. Infection of macrophage-like J774 cells and measurement of intracellular β-Gal activity with 4-methylumbelliferyl-β-d-galactoside were performed as described previously (23). Intracellular β-Gal activity was expressed in relative units (R.U.): {(fluorescence/fluorescence of 200 nM 4-methylumbelliferone)/[(CFU/ml) × dilution]} × 106.

Western blot experiments.

Bacteria grown for 24 h in rich medium (TSB) were centrifuged, washed with phosphate-buffered saline (PBS), and suspended in different media. After culture in each medium for 4 h, bacteria were suspended in sodium dodecyl sulfate (SDS) loading gel buffer at a concentration of 5 × 1010 CFU/ml and incubated at 100°C for 5 min. A portion (10 μl) of each bacterial cell lysate was subjected to electrophoresis in a 12.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences). Proteins were detected using a mouse polyclonal antiserum against recombinant B. abortus HutC (dilution 1:1,000) and a secondary peroxidase-conjugated anti-mouse immunoglobulin antibody (dilution 1:10,000) (DakoCytomation) and developed with SuperSignal West Pico chemiluminescent substrate (Pierce).

RESULTS

Identification and characterization of HutC as a virB promoter-binding protein.

To identify proteins that bind to PvirB, we performed EMSA using crude protein extracts of B. abortus and probe PvirB-ihf (Fig. (Fig.1A),1A), which spans positions −201 to +24 relative to the transcription start site and lacks the ability to bind IHF, due to a replacement of the IHF-binding site by a nonrelated sequence. Using this procedure, we detected a protein that binds to PvirB in a specific manner (data not shown). The DNA-binding protein was subsequently isolated from B. abortus extracts by using EMSA, ionic-exchange columns, and affinity chromatography (see Materials and Methods). The 27.5-kDa isolated protein was identified by mass spectrometry as HutC (NCBI database accession no. YP_418519), a transcriptional regulator of the hut (for histidine utilization) genes, which confer the ability to use histidine as a carbon and nitrogen source on many bacterial species (25). In Brucella, the hut genes are organized as two divergent transcriptional units that code for the regulator HutC and for enzymes that catalyze degradation of histidine to glutamate and formate in five steps.

FIG. 1.
Analysis of binding of rHutC to PvirB. (A) Schematic representation of genomic sequences corresponding to PvirB (gray bar) and the first gene of the virB operon (virB1) (striped bar), radioactive labeled probes used for EMSA (black bars), and unlabeled ...

To analyze the interaction of HutC with PvirB, we performed EMSA experiments using specific or nonspecific probes (schematized in Fig. Fig.1A)1A) and a His-tagged recombinant protein (rHutC). Binding of rHutC to PvirB produced a complex observed by EMSA, whereas incubation with a control probe did not generate any signal, thus indicating that binding of HutC to PvirB is specific (Fig. (Fig.1B).1B). To further characterize the region recognized by HutC within the promoter, EMSA experiments were performed using two unlabeled DNA fragments as competitors (Fig. (Fig.1A).1A). As shown in Fig. Fig.1C,1C, the formation of the rHutC-PvirB complex was impaired by competitor b1 (which spans the region −201 to +24), whereas competitor c2 had no effect. These results indicate that HutC binds to PvirB in a region that is localized between positions −201 and −130, which contains the IHF-binding site.

Taking into account that both IHF and HutC bind to the same 71-bp region, we examined whether these proteins affect each other's binding to the promoter. As shown in Fig. Fig.1D,1D, EMSA performed by coincubating probe PvirB with both proteins did not generate any ternary complex, indicating that IHF and HutC do not bind to PvirB simultaneously. Moreover, binding of IHF was titrated by increasing concentrations of HutC, which indicates that both factors compete for the binding to the promoter.

Analysis of interaction of HutC with the virB and hut promoters.

The binding affinity of HutC to the promoters was studied by determining the apparent dissociation constants (Kd). EMSA were carried out by incubating HutC with probe PvirB or with a 220-bp radiolabeled probe corresponding to the hut promoter (Phut). Kd were determined graphically from the intensity of the bands corresponding to rHutC-PvirB or rHutC-Phut complexes. As shown in Fig. Fig.2,2, the Kd of rHutC-PvirB and rHutC-Phut were 24 nM and 0.75 nM, respectively. Thus, HutC had 30-fold more affinity for its own promoter than for PvirB, which could be mechanistically relevant for the regulatory link exerted by HutC upon the Hut and VirB systems.

FIG. 2.
Analysis of binding affinity of HutC to PvirB or Phut. (A) EMSA performed with probe PvirB and increasing concentrations of rHutC. (B) Determination of the apparent dissociation constant of rHutC for the binding to PvirB. The intensity of the bands obtained ...

The regulation of hut genes has been extensively studied in different organisms. cis-Urocanic acid (UCA), the first intermediate of the hut pathway, is an inducer that interacts with HutC, causing a conformational change that dissociates the repressor from the promoter sequences (12). After dissociation, transcription of the hut genes is derepressed, thus allowing synthesis of the Hut enzymes. In Klebsiella and Pseudomonas, dissociation of the regulator also induces transcription of HutC itself (11, 30). To analyze the effect of UCA on the DNA-binding activity of HutC, EMSA experiments were carried out with rHutC and probe PvirB or Phut in the presence of the inducer. Figure Figure33 shows that higher concentrations of UCA were required for dissociating HutC from Phut (50 μM) than from PvirB (5 μM), which was consistent with the observed differences in Kd. On the other hand, the addition of UCA did not have any effect on the binding of IHF to the promoter, indicating that the susceptibility to the inducer is specific for HutC (Fig. (Fig.3A3A).

FIG. 3.
Effect of UCA on DNA-binding activity of HutC. (A) EMSA performed with rIHF or rHutC incubated with probe PvirB and increasing concentrations of UCA as indicated. Concentration of rIHF, 70 nM; concentration of rHutC, 25 nM. (B) EMSA performed with rHutC ...

To identify the DNA-binding sites of HutC, DNase I footprinting experiments were carried out with probes corresponding to those used for EMSA. Figure Figure4A4A shows that in Phut, HutC protected a 20-bp region that contains the 12-bp dyad symmetric sequence ATGTATATACAT (Fig. (Fig.4C),4C), which is entirely conserved in all hut promoters of the closely related genera Agrobacterium, Ochrobactrum, and Rhizobium (Fig. (Fig.4E).4E). In PvirB, however, HutC protected a 20-bp region that contains 8 out of 12 nucleotides of the conserved symmetric dyad sequence found in Phut (Fig. (Fig.4B).4B). These differences may explain why HutC had 30-fold more affinity for the binding to Phut than for the binding to PvirB. It is worth pointing out that the sequence recognized by HutC in PvirB is more similar to the known hut operators of Klebsiella or Pseudomonas (16, 30) than to the HutC-binding site of Phut in Brucella (Fig. (Fig.4E).4E). It can also be observed that the HutC-protected region of PvirB is centered at position −188 (Fig. (Fig.4D),4D), which is 25 bp upstream of the IHF-binding site and overlaps the IHF-protected region in DNase I footprinting experiments (23), supporting the idea that the observed competition between IHF and HutC was due to steric hindrance.

FIG. 4.
Identification of the HutC-binding sites. (A) DNase I footprinting experiment carried out with probe Phut and increasing concentrations of HutC as indicated. Lanes A and G show results from DNA sequence reactions performed by the Sanger method. The HutC-protected ...

HutC modulates the expression of the virB operon both intracellularly and during bacterial incubation in vitro.

The intracellular expression of the virB operon was analyzed by measuring β-Gal activity of transcriptional fusions between PvirB and the lacZ gene. To this end, single PvirB-lacZ transcriptional fusion constructs (Fig. (Fig.5A)5A) were integrated in the chromosome of the wild-type strain, a hutC deletion mutant, or a complemented knock-in control strain, generating strains B. abortus 2308 PvirB-lacZ, B. abortus ΔhutC PvirB-lacZ, and B. abortus ΔhutC-KI PvirB-lacZ, respectively. In addition, we analyzed β-Gal activity of transcriptional fusions between the hut promoter and lacZ (Fig. (Fig.5B)5B) in the same genetic backgrounds.

FIG. 5.
Role of HutC in control of activity of the virB and hut promoters. (A and B) Schematic representation of the PvirB-lacZ (A) and Phut-lacZ (B) transcriptional fusion constructs. Orientation relative to the hutFC and hutIHUG operons is indicated. (C) Intracellular ...

To analyze the role of HutC in virB expression within the host cell, we infected J774 macrophages and determined β-Gal activity of intracellular bacteria. Figure Figure5C5C shows that, whereas both wild-type and control strains displayed the maximal activation of virB expression at 5 h p.i., the deletion mutant strain B. abortus ΔhutC PvirB-lacZ showed a 60% reduction in β-Gal activity. These results indicated that during the intracellular infection of J774 macrophages HutC functions as a coactivator of virB expression, which is a role that was not previously reported for this transcriptional regulator. The analysis of intracellular activity of the hut promoter showed that, as expected, HutC is a repressor of hut expression (Fig. (Fig.5D).5D). These results demonstrate that HutC participates directly in the intracellular transcriptional regulation of the virB operon and revealed that this transcription factor exerts different roles (as repressor or as coactivator) depending on the target promoter.

Using gentamicin protection assays, we observed that the deletion of hutC did not affect the intracellular multiplication of B. abortus in J774 cells (Fig. (Fig.6A).6A). Thus, although the intracellular activation of virB expression was reduced in the ΔhutC mutant, the level of virB expression reached (40%) was enough for Brucella to overcome the cellular defenses in this experimental model of infection. However, the CFU recovered from spleens of mice infected with B. abortus ΔhutC were reduced by 0.8 log units (Fig. (Fig.6B).6B). As the deletion of hutC turns the hut systems into a constitutively active state, the expression of the Hut enzymes is not abrogated in this mutant, suggesting that the observed reduction of persistence in mice was likely due to the defect in the intracellular expression of the virB genes.

FIG. 6.
(A) Intracellular replication of B. abortus 2308 and the deletion mutant B. abortus ΔhutC in J774 macrophages. Macrophages (1 × 105 per well) were inoculated with 5 × 106 CFU of bacteria. After 1 h of incubation at 37°C, ...

As HutC was involved in the control of the intracellular expression of the virB operon, we attempted to reproduce in vitro the conditions encountered by Brucella within the host cell to examine the role of this regulator in cultured bacteria. Previous studies carried out with B. suis suggested that the intracellular environment within the BCV is acidic and nutrient poor (1, 13). Accordingly, a minimal medium without magnesium, carbon, and nitrogen sources (MM1) was prepared, the pH was adjusted to 4.5, and PvirB activity was examined in the different strains. Figure Figure5E5E shows that, compared to the levels in both wild-type and control strains, virB expression dropped 90% in the ΔhutC mutant after 4 h of incubation in MM1 at pH 4.5 in the presence of 5 mM UCA in the culture medium, indicating that HutC acts as a coactivator of PvirB under these in vitro conditions. It is remarkable that no differences in PvirB activity were observed between strains when the pH was adjusted to 5.5 or when UCA was absent (data not shown), indicating that both acidification and Hut induction are required for the observed HutC-dependent virB expression.

Analysis of the activity of Phut in cultured bacteria showed that hut expression was repressed in the wild-type strain and derepressed in the ΔhutC mutant (Fig. (Fig.5F),5F), which is in agreement with the results obtained with intracellular bacteria (Fig. 5C and D). The fact that HutC represses Phut in the wild-type strain indicates that under such conditions the regulator is bound to the operator site. Thus, the cytoplasmic concentration of UCA within Brucella might be low enough to allow the interaction of HutC with its DNA-binding sites, even in the presence of 5 mM extracellular UCA. This can be explained if, after hut induction, the Hut enzymes metabolize UCA as it is incorporated, which is feasible, given that UCA is a metabolizable inducer. Therefore, to determine whether the Hut system was induced in such conditions, cultured bacteria were analyzed using an anti-HutC antiserum. Western blot analyses of bacteria incubated in MM1 at pH 4.5 revealed that the levels of HutC are higher in the presence of the inducer (Fig. (Fig.7),7), which indicates that the Hut system is induced in response to UCA. The analysis of OD600 during 48 h of incubation in MM1 at pH 4.5 indicated that growth of Brucella was arrested under these conditions, regardless of the addition of UCA or histidine (data not shown). However, by increasing the pH value to 5.5, we observed that Brucella was able to grow using UCA but not histidine (Fig. (Fig.8),8), which is in agreement with previous studies that revealed that many strains of Brucella incorporate and metabolize UCA as efficiently as mesoerythritol, xylose, or ribose (2, 10).

FIG. 7.
Western blot analysis. (A) B. abortus 2308 (wt), B. abortus ΔhutChutC), or B. abortus ΔhutC-KI (KI) was grown in TSB until the stationary phase of growth. Samples corresponding to equal numbers of bacteria were subjected to ...
FIG. 8.
Growth curve of B. abortus 2308 in MM1 at pH 5.5. Bacteria were grown in TSB until the exponential phase (OD600 of 0.5 to 1). Subsequently, bacteria were harvested, suspended, and cultured in MM1 at pH 5.5 supplemented with 5 mM UCA (filled circles) or ...

Taken together, these results demonstrate that the Hut and VirB systems are interrelated by means of a regulatory mechanism that involves induction of a catabolic pathway and the action of HutC upon two different promoters.

DISCUSSION

The aim of this study was to identify signals and transcription factors directly involved in the control of expression of the virB genes. Using EMSA, affinity purification assays, and mass spectrometry, we isolated and identified a protein that binds to PvirB in a specific manner. This protein is homologous to HutC, a well-studied transcriptional regulator of the hut operons. This finding prompted us to hypothesize that the interaction of HutC with the virB promoter could represent a link that coordinates regulation of the virB genes of Brucella with induction of the histidine utilization pathway.

Our results showed that in Brucella, HutC modulates expression of the virB operon both within the eukaryotic host cell and in bacteria incubated under conditions that resemble the cellular intraphagosomal environment (Fig. (Fig.5).5). The analyses revealed that instead of repressing transcription, as occurs in all known hut promoters, HutC coactivates transcription of the virB genes under both conditions tested. Control experiments carried out using the same experimental methods corroborated that HutC indeed represses the hut promoter while it coactivates PvirB (Fig. (Fig.5),5), which demonstrated that HutC performs opposite functions depending on the target promoter.

Until now, the regulatory mechanism reported for HutC was based on the steric hindrance of RNA polymerase (RNApol) holoenzyme access to the hut promoters (12). In the case of PvirB, the fact that the HutC-binding site is localized at position −188 accounts for the different role that HutC exerts on this system. Interestingly, among the list of repressors that were also found to activate transcription (e.g., CytR, LuxR, and Lrp) (17, 19, 28), Fur represses promoters by occluding RNApol access while it activates other promoters through binding to regions far upstream of the transcription start site (8). The latter example highlights similarities with Brucella HutC that deserve to be further analyzed. Although the mechanism of HutC-mediated coactivation remains to be determined, it can be speculated that it operates through the modulation of a downstream-acting primary activator (e.g., VjbR), probably by means of a mechanism such as repositioning (3). Alternatively, HutC may act on PvirB by interfering with the activity of negative regulators.

β-Gal activity assays of bacteria incubated in vitro revealed that PvirB was coactivated by HutC only under specific conditions that required pH 4.5 and a nutrient-deprived medium (Fig. (Fig.5E),5E), which are conditions similar to those encountered by Brucella within the host cell (1, 13). In addition, the HutC-mediated regulation of PvirB required the presence of UCA in the culture medium. Western blot experiments and analyses of OD600 of bacterial cultures revealed that Brucella induces the Hut system in response to UCA and is able to grow using this compound, but not histidine, as a sole carbon source (Fig. (Fig.8).8). Taken together, these findings indicate that Brucella is specially adapted to utilize this metabolite, which is in agreement with previous reports showing that Brucella efficiently incorporates and metabolizes UCA (2, 10). This raises an intriguing question: does Brucella incorporate and metabolize UCA during its life cycle within the host? It is known that UCA is present at high concentrations in mammalian skin and skin secretions, where it acts as a UV photoprotector or as an immunosuppressor (14). Nevertheless, to our knowledge, the published literature does not provide information about the concentration of this compound in tissues other than skin. Therefore, we cannot rule out the possibility that Brucella incorporates and metabolizes UCA at a particular stage of the infection, which could provide a signal for the bacterium to enhance virB expression when it is needed to overcome host defenses.

The analyses of the interaction of HutC with both virB and hut promoters showed that there is a hierarchical order of Kd values, which may have a mechanistic significance for the regulation of both systems (Fig. (Fig.2).2). HutC binds to PvirB with lower affinity (apparent Kd = 24 nM). As the affinity for Phut is 30-fold higher (apparent Kd = 0.75 nM), basal levels of HutC might be sufficient for repressing the hut genes without affecting virB expression. Only after induction by UCA do the levels of HutC increase, reaching the required concentration for the binding to PvirB, as was confirmed by Western blotting (Fig. (Fig.7).7). The different susceptibilities to UCA observed for the hut and virB promoters may also be part of a mechanism by which, as the inducer is catabolized, HutC is enabled to interact sequentially with the operator sequences. If the intracellular concentration of UCA drops below 50 μM, then HutC is able to interact with Phut and repress hut transcription (Fig. (Fig.3B).3B). In this way, as consumption of UCA proceeds, HutC is subsequently enabled to bind to PvirB and activate virB transcription when the inducer concentration decreases below 5 μM (Fig. (Fig.3A3A).

The observed differences in affinity and susceptibility to UCA are likely due to the different architectures observed for the Phut and PvirB HutC-binding sites (Fig. (Fig.4).4). It is interesting to note that the sequence of the HutC-binding site of B. abortus Phut is present in all hut promoters of other closely related alphaproteobacteria (Fig. (Fig.4E),4E), which indicates that the ancestral sequence recognized by HutC is fully conserved among this taxonomic group. Instead, the sequence recognized in PvirB more closely resembles the HutC-binding site of Klebsiella or Pseudomonas than that of alphaproteobacterial hut promoters (Fig. (Fig.4E).4E). This observation could evidence an evolutionary trace of the acquisition of the virB operon and its regulatory sequences, which may have occurred during a horizontal-transfer event in the common ancestor of Brucella (29). It is worth mentioning that both HutC-binding motifs found in PvirB and Phut are unique in the genome of B. abortus. However, we identified additional sequences that bind HutC with high affinity, which are being investigated (R. Sieira, unpublished results). Such sequences are located within intergenic regions, upstream of open reading frames unrelated to the recently discovered T4SS substrates of Brucella VceA and VceC.

EMSA experiments showed that HutC competes with IHF for the binding to PvirB (Fig. (Fig.1D).1D). In our previous work, we found that during vegetative growth the virB expression is IHF dependent at pH 7.0 but not at pH 4.5 (23). Here, the opposite situation was observed for HutC, since virB expression is HutC dependent at pH 4.5 but not at pH 5.5 or 7.6 (data not shown). Based on these observations, we hypothesize that a sequential participation of IHF and HutC may be required for the proper induction of virB expression. IHF probably acts by supporting a promoter structure necessary for recruiting transcription factors. As BCV maturation proceeds, induction of the hut system under acidic conditions may displace IHF from its binding site, coactivating expression of the virB operon in a HutC-dependent manner. In this way, induction of the Hut catabolic pathway probably functions in Brucella as a way to sense homeostatic variations and develop adaptive responses to ensure survival within the host.

Our results showed that in Brucella, a catabolic pathway is directly linked to the regulation of expression of a T4SS. Another example of interrelation between histidine utilization and transcription of virulence genes was evidenced by mutations that alter expression of Hut enzymes in Pseudomonas aeruginosa, which result in a decreased transcription of type III secretion system (T3SS) effectors and a consequent reduction of cytotoxicity of the bacterium (20). Many pathogenic bacteria have acquired genes coding for virulence factors through horizontal transfer, which implies that such foreign genetic information had been integrated into preexisting regulatory networks of the new context. Acquisition of a functional HutC-binding site within PvirB during the evolution of Brucella may eventually have resulted in a cooptation of a negative regulatory protein to perform a new role: coactivating transcription of virulence genes by means of a mechanism that involves the integration of acidic and metabolic stress signals.

Acknowledgments

We thank Jeanette E. Polcz, María Georgina Davies, and Juan E. Ugalde for critical reading of the manuscript. We also thank Joseph Connolly for help with mass spectrometry.

This work was supported by grants PICT05-38207 to R.S., PICT05-38272 to D.J.C., and PICT06-651 to R.A.U. from Agencia Nacional de Promoción Científica y Tecnológica, Buenos Aires, Argentina.

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 October 2009.

This work is dedicated to the memory of Dr. Rodolfo A. Ugalde.

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