CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Bacterial strains and growth conditions.The strains used in this work are described in Table 1. C. crescentus was grown in PYE or M2 minimal medium (5) at 30°C with agitation, and media were supplemented with 20 μg/ml nalidixic acid, 1 μg/ml tetracycline, 5 μg/ml kanamycin, 1 μg/ml chloramphenicol, or 20 μg/ml spectinomycin as necessary. E. coli strains were grown in LB medium, supplemented with 12.5 μg/ml tetracycline, 50 μg/ml kanamycin, 30 μg/ml chloramphenicol, 50 μg/ml spectinomycin, or 100 μg/ml ampicillin as necessary.

Growth of strains NA1000 and SP2012 in liquid PYE and M2 media was carried out in 10 ml of each medium at 30°C with agitation. Preinocula grown in the same medium were inoculated into fresh medium to obtain an initial optical density at 600 nm (OD₆₀₀) of 0.1 to 0.2, and growth was monitored by measuring absorbance at 600 nm at several time points. Experiments were performed at least in triplicate.

Construction of cztR- and cztA-null mutant strains and complementation.Plasmids used in all cloning procedures are listed in Table 1, and primers are listed in Table 2. cztR and cztA were deleted by allelic replacement of each gene by a spectinomycin resistance cassette obtained from plasmid pHPΩ45. Two fragments containing the regions downstream and upstream of the cztR gene (1,160 bp and 620 bp, respectively) were amplified by PCR from genomic DNA with primers Lys1/Lys2 and Lys3/Lys4, and the products were cloned sequentially into the pGEM-T Easy vector. The resultant 1.8-kb SalI/PstI fragment was then cloned into the pNTPS138 suicide vector, and the 2.0-kb Ωspec cassette was inserted between the two fragments. The same was done for the cztA gene: a 2.0-kb HindIII/SalI fragment resulting from the ligation of two 1.0-kb cztA-flanking fragments (amplified with primers Lys7/Lys12 and Lys9/Lys11) was cloned into pNPTS138 and was disrupted by the Ωspec cassette. The pNPTΔcztR::Ωspec and pNPTΔcztA::Ωspec plasmids were then introduced into C. crescentus NA1000 by conjugation with E. coli S17-1, and Kan^s Spec^r clones generated by double recombination were selected, generating strains SP2012 (ΔcztR::Ωspec) and SP2014 (ΔcztA::Ωspec). Gene replacements were confirmed by PCR and Southern blot experiments.

Each mutation was complemented by cloning a 1.26-kb cztR DNA fragment (amplified with primers Lys5/Lys7) or a 1.2-kb cztA DNA fragment (amplified with primers Lys14/Lys15) containing the promoter and coding regions of each gene into the pMR20 plasmid, generating pMRcztR and pMRcztA, respectively. A second vector containing the cztR gene (amplified with primers Lys7/Lys8) under the regulation of a xylose-inducible promoter was constructed by cloning the cztR gene into plasmid pUJ142, generating pUJcztR, and expression of the gene was induced by adding 0.2% xylose.

Determination of the cztR transcription start site.The transcription start site (TSS) of cztR was determined by primer extension assays using a ³²P-labeled primer and total RNA from strain NA1000. Total RNA was isolated from exponential-phase cells with the Trizol reagent by following the manufacturer's instructions. Primer Lys3 was end labeled with 30 μCi [γ-³²P]ATP using the T4 polynucleotide kinase enzyme (GE Healthcare Life Sciences) and was extended with SuperScript III reverse transcriptase (Invitrogen). The DNA sequencing ladder was obtained by cycle sequencing with the M13 universal primer and the M13 single-stranded DNA (ssDNA) as the template by using the Thermosequenase cycle sequencing kit (USB).

Cloning of the promoter regions and gene expression analysis.The katG promoter region was previously cloned into vector pRKlacZ290, generating vector pRKkatG (15). The fragments containing the cztR and cztA regulatory regions were amplified from the C. crescentus chromosome by PCR with primers Lys5 and Lys6 (see Table 2) using C. crescentus NA1000 genomic DNA as the template. The amplified fragments were cloned into the pGEM-T vector (Promega), confirmed by DNA sequencing, and cloned into vector pRKlacZ290, generating vectors pRKcztR and pRKcztA. The plasmids were introduced into C. crescentus NA1000 and SP2012 by conjugation. Promoter activities were determined by measuring the β-galactosidase activity by the method of Miller (23).

Expression of cztR was determined in C. crescentus NA1000 and SP2012 during the exponential-growth and stationary phases (24-h and 48-h cultures). Expression of cztA in strains NA1000 and SP2012 was assessed in rich (PYE) and minimal (M2) media at the exponential and stationary phases. Expression was also evaluated during exponential phase in M2 growth medium after 1 h in the presence of different metal chelators at the following concentrations: 300 μM and 500 μM EDTA, 100 μM and 300 μM 2,2-dipyridyl (Sigma), and 300 μM and 500 μM EGTA. In order to identify the divalent cation whose scarcity was inducing the gene, an exponentially growing culture of C. crescentus NA1000 harboring plasmid pRKcztA was divided into several aliquots. To each culture were added 300 μM EDTA and one of the following metals: 10 μM, 25 μM, 50 μM, or 100 μM ZnCl₂; 25 μM CdCl₂; 60 μM or 80 μM CuCl₂; or 100 μM NiCl₂. The cultures were incubated for 24 h at 30°C, and expression was measured by β-galactosidase assays.

Electrophoretic mobility shift assays.Electrophoretic mobility shift assays (EMSA) were carried out using total-protein extracts from strains NA1000 and SP2012. Exponential- or stationary-phase cultures were grown in PYE medium, and total-protein extracts were obtained by sonication in lysis buffer (100 mM sodium phosphate [pH 7.0]. 50 mM NaCl, 1 mM EDTA, 10% glycerol) (34). A 370-bp fragment corresponding to the regulatory region was obtained by PCR using primers Lys5 and Lys6 (Table 2). The fragment was end labeled with [γ-³²P]ATP using T4 polynucleotide kinase (Invitrogen), and unincorporated nucleotides were removed with a Qiaquick PCR purification kit (Qiagen). A DNA binding assay was performed in a 50-μl reaction volume containing binding buffer (10 mM Tris-HCl [pH 7.5], 120 mM KCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin, 10% glycerol), salmon sperm DNA (0.1 mg/ml) or 1 μg poly(dI-dC) as a competitor, 50 ng of the labeled DNA probe, and 30 μg of the protein extract. In competition assays, 0.2 to 2.5 μg of a cold probe was used to challenge each of the labeled probes. After incubation at 25°C for 20 min, the samples were loaded onto a native 5% polyacrylamide gel and were electrophoresed in 1× Tris-borate EDTA (TBE) buffer for 3 h at 30 mA. Radioactive species were detected by autoradiography.

Semiquantitative RT-PCR.Total RNA was isolated from strains NA1000 and SP2012 from exponential- and stationary-phase cells. Semiquantitative reverse transcription-PCR (RT-PCR) was carried out as previously described (16), with the following modifications: RNA was treated with 0.6 U of RQ1 DNase (Promega) for 30 min at 37°C, followed by phenol extraction and ethanol precipitation. Control reactions without reverse transcriptase were carried out to confirm the absence of DNA in the RNA samples. Reactions were carried out with 1 μg RNA and 10 pmol (each) primers AhpC1 and AhpC2 (Table 2) under the conditions described by the manufacturer. The PCR conditions were as follows: 55°C for 30 min, followed by 94°C for 2 min and 30 cycles of 94°C for 45 s, 55°C for 1 min, and 72°C for 1 min. After completion, the tubes were incubated at 72°C for 5 min, and the fragments were analyzed by electrophoresis in 2% agarose or 8% acrylamide TBE gels. As a positive control, genomic DNA from strain NA1000 was used instead of RNA.

Oxidative-stress survival tests.Cultures were grown at 30°C with agitation in PYE medium to early-stationary phase (24 h), and 0.5 ml of culture was spread over PYE plates. Resistance to tert-butyl hydroperoxide was assessed by measuring the halos of growth inhibition formed by paper discs containing 0.5% (vol/vol) tert-butyl hydroperoxide placed on top of the cultures. Tests for survival of hydrogen peroxide and superoxide were performed by adding to 10-ml stationary-phase cultures in PYE medium either paraquat to a final concentration of 10 mM (vol/vol) or H₂O₂ to a final concentration of 5 mM (vol/vol). The cultures were incubated at 30°C with agitation, and at several time points, aliquots were removed, serial dilutions were plated in PYE medium, and plates were incubated at 30°C. Survival was determined as the percentage of CFU relative to the CFU count before the addition of the agent (time zero). The results are averages for two independent experiments performed in duplicate.

CztR is negatively autoregulated and is required for the expression of cztA. Three independent mutant strains with insertions in the cztR gene were previously isolated from a mini-Tn5/lacZ transposon library as sensitive to CdCl₂ (4). In order to perform expression assays using lacZ as a reporter gene, it was necessary to obtain a new cztR-null mutant strain devoid of the lacZ gene. This was achieved by exchanging the cztR gene in wild-type strain NA1000 for a spectinomycin resistance cassette, generating strain SP2012. This strain also presented a Cd- and Zn-sensitive phenotype.

In order to investigate the pattern of expression of cztR, a lacZ fusion was introduced into strains NA1000 and SP2012, and expression was determined by β-galactosidase activity assays. Expression of cztR was also evaluated during growth, and it was observed that the gene is expressed to a higher level in stationary phase, showing approximately 2-fold induction (Fig. 1 A) both in NA1000 and in SP2012. It can be observed that the levels of expression in the cztR mutant are 2-fold higher than those in the wild type at exponential phase (Fig. 1B). This indicates that CztR negatively regulates its own expression, in agreement with the findings reported for regulators of this family (3, 19). The introduction of the cztR gene in a multicopy plasmid under the control of a xylose-inducible promoter in SP2012(pUJcztR) restores the β-galactosidase activity to levels comparable to those of the wild-type strain, even in the absence of xylose in the medium (Fig. 1B). The leakage of cztR transcription observed in the absence of xylose was apparently enough to ensure negative autoregulation.

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FIG. 1.

Autoregulation and expression of cztR at different growth phases. (A) The promoter of cztR was cloned into plasmid pRKlacZ290, generating plasmid pRKcztR. Cultures of strain NA1000 (open bars) and strain SP2012 (shaded bars) carrying pRKcztR were grown for as long as 48 h, and the promoter activity was determined by measuring β-galactosidase activity at the exponential (exp) and stationary phases (24 h and 48 h). (B) pRKcztR was introduced into strains NA1000, SP2012. and SP2012(pUJcztR). β-Galactosidase activity was assayed at late-exponential phase with (+) or without (−) the addition of 0.2% xylose. The results shown are the averages for at least three experiments. Error bars indicate standard deviations.

Transcriptional regulators of the LysR family usually regulate the divergent gene (3, 19). The gene divergent to cztR, cztA, encodes a putative permease of the SbtA family (31). To test whether cztA is regulated by CztR, a cztA-lacZ fusion was introduced into strains NA1000 and SP2012. As can be observed in Fig. 2, the gene is induced in stationary-phase NA1000 cells, both in PYE and M2 media, though the levels of expression are 2- to 3-fold higher in M2 medium. The expression of cztA was also determined in strain SP2012 in M2 medium, and it was observed that expression was diminished at both growth phases. More importantly, the stationary-phase induction was lost in the cztR mutant strain. These results showed that CztR is required for the increase in cztA expression that occurs with the shift from rich to minimal medium or upon entry into stationary phase.

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FIG. 2.

Growth phase- and medium-dependent expression of cztA. C. crescentus strains NA1000 and SP2012 carrying plasmid pRKcztA were grown in PYE or M2 medium, as indicated, and β-galactosidase activity was determined during the exponential-growth (open bars) and stationary (shaded bars) phases. The results shown are the averages for at least three experiments. Error bars indicate standard deviations.

CztR binds to the divergent regulatory region.The apparent transcription start site (TSS) of cztA was previously determined within a 5-bp interval by tiled microarray analysis (20). In order to characterize the cis-regulatory elements controlling the expression of each gene, we determined the TSS of the cztR gene by primer extension analysis (Fig. 3 A). Two labeled fragments can be observed; the stronger one corresponds to an adenine 68 bp upstream of the ATG, and the weaker to a cytosine 60 bp upstream of the ATG. In this work we considered only the stronger TSS (adenine) in order to determine the cis-regulatory elements of the cztR promoter.

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FIG. 3.

Determination of athe cztR transcriptional start site. (A) Primer extension analysis of the cztR gene was carried out using as templates total RNAs from strain NA1000 at the exponential-growth (lane 1) or stationary (lane 2) phase. A ³²P-labeled primer hybridizing to cztR mRNA was extended with reverse transcriptase. A DNA-sequencing ladder of phage M13mp18 was used as a molecular size marker. The arrow indicates the band corresponding to the main transcription start site observed. (B) Diagram showing the cztA-cztR divergent regulatory regions. The −35 regions of each promoter are indicated by boxes and the respective transcription start sites as bent arrows. The CztR protein, binding downstream of its transcription start site, probably represses its own transcription and activates that of cztA. (C) DNA sequence of the divergent regulatory region. The −35 and −10 regions of each promoter are boxed, and the respective transcription start sites are marked by “+1” in each strand. Arrows on the same strand pointing in the same direction indicate a direct repeat (GATTTC), and arrows on different strands pointing in opposite directions indicate an inverted repeat. The putative LTTR-binding consensus sequence is indicated above the inverted repeat. The ribosomal binding sites (RBS) of CztR and CztA are underlined.

Based on this and on previous results, the localization of putative cis-regulatory elements involved in controlling the expression of each gene could be proposed (Fig. 3B). A putative promoter region can be found upstream of the cztR TSS with similarity to the proposed sigma 70 consensus sequence for C. crescentus (TTGAC-N₁₆-CCTANA) (20) (Fig. 3C). Located about 68 bp upstream of the cztA TSS is a perfect inverted-repeat sequence (CATGATGAG) spaced by an AT-rich 5-bp sequence that could be a putative protein binding site. A consensus binding site that corresponds to a conserved T-N₁₁-A motif within an inverted-repeat sequence has been described for LTTRs (8, 30). The motif found in the C. crescentus cztR/cztA divergent region fulfills these requirements, and it is correctly spaced from the cztA promoter. This sequence is therefore a good candidate for the CztR binding site, and its position relative to the promoters suggests that CztR could activate the cztA promoter and at the same time repress its own transcription, in agreement with the experimental data.

In order to determine whether CztR binds to the regulatory region, a DNA binding assay was carried out using as a probe a labeled 370-bp DNA fragment containing the divergent promoters. Several attempts at purification of a recombinant His-tagged CztR in E. coli were unsuccessful, because we could not obtain the protein in a soluble form (data not shown). In order to circumvent this, we used total-protein extracts from strains NA1000 and SP2012 for the EMSA.

A very distinct retarded band was observed after electrophoresis when the DNA probe was incubated with protein extracts from strain NA1000, but not when it was incubated with protein extracts from SP2012 (Fig. 4 A). A retarded band was observed with both exponential- and stationary-phase cell extracts, with an increase in the intensity of the shifted band when the stationary-phase extract was used. However, no retardation was obtained with the SP2012 extracts in either growth phase, indicating that either the CztR protein or a protein whose activity or expression is dependent on the presence of CztR binds to this region. To confirm that the binding activity was specific, a competition assay was carried out using an excess of unlabeled competitor DNA. It can be observed in Fig. 4B that when the same DNA fragment was used, the shifted probe was competed out, while a nonspecific DNA fragment was not able to compete for CztR binding, even at the highest concentrations.

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FIG. 4.

Assessment of protein binding to the cztR regulatory region by gel electrophoresis mobility shift assays. (A) DNA fragments corresponding to the cztR promoter were ³²P labeled and were then either incubated or not (−) with total-protein extracts from the NA1000 (WT) and SP2012 (Δ) strains at the exponential-growth (exp) and stationary (stat) phases. (B) Competition assays with increasing concentrations of unlabeled fragments of the same region (S) or the cztR coding region (NS) as competitors. The concentrations of DNA competitors are given above each lane.

Investigation of the role of CztA.CztA has 10 predicted transmembrane segments, and sequence similarity analysis indicates that it has significant similarity to proteins of the Na⁺-dependent bicarbonate transporter (SBT) family. The CztA protein is the only one belonging to this family encoded in the C. crescentus NA1000 genome, and our results showed the induction of cztA in minimal medium and at stationary phase (Fig. 2), indicating that it is important for growth under low-nutrient conditions, so it could be involved in nutrient transport.

To test this hypothesis, a cztA-null mutant strain was generated, and growth in PYE and M2 media was evaluated. Deletion of cztA had no effect on growth in PYE medium, but the mutant strain showed a pronounced delay in growth in minimal medium (generation times, 5.1 h for SP2012 and 3.6 h for the wild-type and complemented strains) (Fig. 5 A and B), indicating that this gene is important for growth under conditions of low nutrient availability. Complementation with a copy of the cztA gene restored growth to levels similar to those of the wild-type strain (Fig. 5B).

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FIG. 5.

Growth of NA1000 and SP2014 in rich and minimal media. Growth curves of strains NA1000 (open squares), SP2014 (open diamonds), and SP2014 carrying plasmid pMRcztA (filled diamonds) were determined for cultures grown in either PYE (A) or M2 (B) medium. Experiments are described in the text, and the curves shown are representative of the results obtained from at least three experiments.

All our results suggested that cztA may respond to nutrient limitation. To determine which nutrient could be the target of CztA transport, we used a strategy to investigate which nutrient could induce cztA expression. Given that a strain with a mutation of the regulator of CztA, CztR, presents a cadmium-sensitive phenotype, we decided to investigate the role of divalent metals in cztA expression. Deprivation of metals was achieved by treating the cells with ion chelators with different metal binding specificities: EDTA, 2,2-dipyridyl, and EGTA. The expression of cztA was measured in exponentially growing NA1000 cultures in M2 medium that were divided into several aliquots; a different chelator was then added to each aliquot, and after incubation for 1 h, the cztA promoter activity was determined. Figure 6 A shows that the expression of cztA is not altered by the addition of 2,2-dipyridyl or EGTA but is increased about 1.5-fold when EDTA is added to the cells.

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FIG. 6.

Effect of metal depletion on cztA expression. (A) C. crescentus NA1000 carrying plasmid pRKcztA was grown up to exponential phase in M2 medium, and cells were incubated in the presence of one of several ion chelators at different micromolar concentrations as indicated. β-Galactosidase activity was determined after 1 h. (B) cztA expression was assessed in strain NA1000 cultures incubated simultaneously with 300 μM EDTA and different divalent cations added to aliquots of the culture at the indicated micromolar concentrations. β-Galactosidase activity was determined after 24 h in the presence of the metals and EDTA. (C) cztR expression was determined as for panel B. The results shown are the averages for at least three experiments. Error bars indicate standard deviations.

EDTA is able to chelate several divalent metal ions with different affinities, so in order to determine which metal's depletion could be inducing cztA expression, each individual metal was added to the cultures simultaneously with EDTA. As shown in Fig. 6B, the expression of cztA in the presence of 10 to 100 μM ZnCl₂ is progressively decreased from that in the presence of EDTA alone or with the addition of other cations, such as Cu²⁺, Ni²⁺, or Cd²⁺. These results suggest that cztA is induced in response to the depletion of zinc ions from the medium; consequently, the CztA transporter could have a relevant role under conditions of low zinc availability. To test whether this difference in expression was mediated by the presence of the CztR regulator, the expression of the divergent cztR gene was measured under the same conditions. As shown in Fig. 6C, the expression of cztR increases progressively with the addition of increasing concentrations of ZnCl₂ in the presence of EDTA. The results indicate that CztR is able to activate the transcription of cztA and to repress its own transcription only when zinc is depleted, which could suggest that zinc ions may affect its binding to the regulatory region.

The results suggest that gene regulation is achieved in response to very low concentrations of zinc ions in the medium. In fact, maximal repression of cztA expression is already observed with 50 μM ZnCl₂, and the estimated amount of ZnCl₂ necessary to inhibit 50% of expression is 30 μM. Further addition of ZnCl₂ does not increase inhibition to levels lower than those observed in the exponential-phase culture (compare Fig. 6A and B). We have measured the expression of both cztR and cztA in stationary phase in the presence of ZnCl₂ and the absence of EDTA, but no difference was observed (data not shown), indicating that there is a basal concentration of zinc in the medium that is sufficient to limit cztA expression.

The cztR mutant is sensitive to oxidative stress.The putative role of CztA in zinc homeostasis and the Cd²⁺-sensitive phenotype of the cztR mutant prompted us to investigate whether CztR and CztA could be important for the oxidative-stress response. The oxidative-stress response of SP2012 was evaluated by testing its resistance to hydrogen peroxide, tert-butyl-hydroperoxide, and superoxide (generated intracellularly by the addition of paraquat). Resistance to tert-butyl hydroperoxide was determined by measuring the halo of growth inhibition, but no difference in the inhibition halo diameters was observed between SP2012 and NA1000 (data not shown). Resistance to H₂O₂ and superoxide was determined by adding each agent to stationary-phase cultures and removing aliquots for CFU counting. As shown in Fig. 7, strain SP2012 is much more sensitive to paraquat and moderately sensitive to H₂O₂, and this phenotype is complemented by a copy of the gene in trans. Taken together, these results indicate that cztR is important for resistance to hydrogen peroxide and superoxide, but not for resistance to organic hydroperoxide. We also investigated whether strain SP2014 was sensitive to paraquat, but it showed the same resistance profile as that observed in the wild-type strain (data not shown). The possible involvement of cztR in the oxidative-stress response prompted us to investigate whether its expression is altered under oxidative conditions. Exponential-phase cells were incubated with oxidative agents for different times, and expression was determined by β-galactosidase activity assays. However, no difference was observed in the presence of CdCl₂, paraquat, or H₂O₂ (data not shown).

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FIG. 7.

Survival of strain SP2012 in response to oxidative stress. Stationary-phase cultures of NA1000 (open squares), SP2012 (open diamonds), or SP2012(pMRcztR) (filled diamonds) were subjected to oxidative stress, and aliquots were taken at each time point to determine cell viability by CFU counts. An aliquot was taken before the addition of the agents (time zero), and the results were determined relative to the CFU at this time (taken as 100%). (A) Superoxide stress was achieved by the addition of paraquat to stationary-phase cultures at time zero to a final concentration of 10 mM. (B) Hydrogen peroxide was added to stationary-phase cultures at time zero to a final concentration of 5 mM. The results are the averages for two independent experiments. Error bars indicate standard deviations.

Considering the oxidative-stress phenotype of SP2012, we decided to investigate whether the expression of some antioxidative enzymes were affected in the mutant. Expression of the catalase-peroxidase, encoded by the katG gene, was determined by β-galactosidase activity assays. We could observe that there was an increase in the transcription of katG in SP2012 over that in NA1000 only in stationary phase (Fig. 8 A). This result was confirmed by RT-PCR and immunoblotting with an anti-KatG antiserum (data not shown). The transcript levels of the ahpC gene, encoding alkyl hydroperoxide reductase subunit C, were measured by semiquantitative RT-PCR. The levels of ahpC transcripts at exponential phase were also higher in SP2012 than in NA1000, and no difference was observed at stationary phase, when the gene was much less expressed (Fig. 8B). The expression of sodA (Mn superoxide dismutase), sodB (Fe superoxide dismutase), and sodC (CuZn superoxide dismutase) was also determined by RT-PCR, and the transcript levels of these genes were not evidently different between the two strains (data not shown).

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FIG. 8.

Expression of oxidative stress response genes. (A) β-Galactosidase activity of the katG promoter in strain NA1000 (open bars) and strain SP2012 (shaded bars) was determined during exponential and stationary growth. The results are the averages for two independent experiments. Error bars indicate standard deviations. (B) mRNA levels of the gene encoding the enzyme alkyl hydroperoxide reductase subunit C (ahpC) were determined by semiquantitative RT-PCR. The cDNAs were amplified by RT-PCR using as templates the total RNAs of cells from strains NA1000 and SP2012 at the exponential-growth (E) and stationary (S) phases. As a positive control (+), PCR was carried out using NA1000 genomic DNA.