Functional Definition and Global Regulation of Zur, a Zinc Uptake Regulator in a Streptococcus suis Serotype 2 Strain Causing Streptococcal Toxic Shock Syndrome

Zinc is an essential trace element for all living organismsand plays pivotal roles in various cellular processes. However,an excess of zinc is extremely deleterious to cells. Bacteriahave evolved complex machineries (such as efflux/influx systems)to control the concentration at levels appropriate for the maintenanceof zinc homeostasis in cells and adaptation to the environment.The Zur (zinc uptake regulator) protein is one of these functionalmembers involved in the precise control of zinc homeostasis.Here we identified a zur homologue designated 310 from Streptococcussuis serotype 2, strain 05ZYH33, a highly invasive isolate causingstreptococcal toxic shock syndrome. Biochemical analysis revealedthat the protein product of gene 310 exists as a dimer formand carries zinc ions. An isogenic gene replacement mutant ofgene 310, the ${Delta}$ 310 mutant, was obtained by homologous recombination.Physiological tests demonstrated that the ${Delta}$ 310 mutant is specificallysensitive to Zn²⁺, while functional complementation of the ${Delta}$ 310mutant can restore its duration capability, suggesting that310 is a functional member of the Zur family. Two-dimensionalelectrophoresis indicated that nine proteins in the ${Delta}$ 310 mutantare overexpressed in comparison with those in the wild type.DNA microarray analyses suggested that 121 genes in the ${Delta}$ 310mutant are affected, of which 72 genes are upregulated and 49are downregulated. The transcriptome of S. suis serotype 2 withhigh Zn²⁺ concentrations also showed 117 differentially expressedgenes, with 71 upregulated and 46 downregulated. Surprisingly,more than 70% of the genes differentially expressed in the ${Delta}$ 310mutant were the same as those in S. suis serotype 2 that weredifferentially expressed in response to high Zn²⁺ concentration,consistent with the notion that 310 is involved in zinc homeostasis.We thus report for the first time a novel zinc-responsive regulator,Zur, from Streptococcus suis serotype 2.

INTRODUCTION

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INTRODUCTION

Streptococcus suis 2 is a gram-positive pathogenic bacteriumcapable of infecting both piglets and humans and causing seriousdiseases such as arthritis, meningitis, and septicemia (58).It has spread to nearly 20 countries, resulting in more than400 human cases of severe infection worldwide (38). In particular,two recent outbreaks (in 1998 and 2005) of severe human S. suisserotype 2 infections in China were characterized by streptococcaltoxic shock syndrome, implying that highly invasive variantsof S. suis serotype 2 might be emerging in Asia (63, 72). Virulencefactors related to the pathogenesis of S. suis serotype 2 havebeen partially elucidated and include capsular polysaccharide(56), suilysin (37), muraminidase-released protein (42), andextracellular factor (59). Very recently, our group (8) reportedthe whole genome sequences of two virulent S. suis serotype2 isolates (05ZYH33 and 98HAH12).

Similar to other transition metals such as iron, manganese,and nickel, zinc is recognized as an essential trace elementfor all living organisms, including a variety of bacterial pathogens(9). It plays critical roles in various cellular processes andphysiological functions by either serving as a catalytic cofactorfor numerous enzymes or maintaining the structure scaffold ofmetal-proteins (4, 10). However, an excess of zinc ion is toxicto normal physiological processes because it can trigger theformation of hydroxyl radicals, resulting in severe damage toDNA, proteins, and lipids (53). Consequently, the intracellularlevel of zinc must be precisely regulated to reach a dynamicbalance (24, 53). Bacteria have evolved complex machineries(such as efflux/influx systems) to maintain zinc homeostasis(24). To survive and complete their infection cycles, pathogenicbacteria must compete with their hosts for the limited resourceof Zn²⁺ in common niches (20, 24).

The zinc uptake regulator, Zur, is one of the key componentscontributing to the molecular systems by which zinc homeostasisis controlled (47). In fact, Zur has been classified as a subgroupof the Fur (ferric uptake regulator) family, which comprisesat least five members, namely, Fur (for Fe²⁺) (15, 25), Zur(for Zn²⁺) (34, 47), Nur (for Ni²⁺) (1), Mur (also called TroR)(51) (for Mn²⁺) (14, 49), and PerR (for peroxide) (31, 66).Since the first discovery of the Zur protein in Escherichiacoli (47), the functional members of the Zur family have beenextended to a wide range of bacteria, such as Bacillus subtilis(17, 18), Listeria monocytogenes (11), Staphylococcus aureus(34), Salmonella enterica (7), Mycobacterium tuberculosis (40,43), and Xanthomonas campestris (62). In B. subtilis, Zur regulatesnot only zinc uptake but also the mobilization of zinc throughribosomal proteins (44). Very recently, ribosome proteins wererevealed to be regulated by Zur proteins in both Streptomycescoelicolor (46, 55) and M. tuberculosis (40). Tang et al. (62)showed that Zur is involved in extracellular polysaccharideproduction and is required for full virulence in Xanthomonascampestris. Similarly, Zur was found to be involved in the pathogenesisof Salmonella enterica (7) and Xanthomonas campestris (62).In contrast, zur fails to exhibit obvious roles in the pathogenicityof S. aureus (34). Following the solution of the crystal structureof Fur from Pseudomonas aeruginosa in 2003 (50), Lucrelli etal. (36) characterized the architecture of Zur that is centralto zinc homeostasis in M. tuberculosis, providing structuralinsights into the interplay between Zur and its target DNA sequence(the Zur box). Intriguingly, Zur has also been suggested toact as an indirect activator by repressing two regulatory RNAs(rhyA and rhyB) (36), which is distinct from its general roleas a repressor (25, 53).

In attempts to explore the functional genomics of S. suis serotype2 and the molecular pathogenesis of invasive streptococcal toxicshock syndrome infection, we identified a Fur/Zur-like homologuereferred to as gene 310. Subsequently, we carried out systematicinvestigations to characterize gene 310. Our data demonstrateclearly that gene 310 is a functional member of the Zur familyfrom S. suis 05ZYH33. Moreover, global regulation of Zur inS. suis serotype 2 is presented, for the first time, highlightingits relationship with zinc homeostasis at the genomic level.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids are detailed in Table 1. Streptococcussuis strains (e.g., 05ZYH33) were grown in Todd-Hewitt broth(THB) (Difco Laboratories, Detroit, MI) or plated on THB agarcontaining 5% (vol/vol) sheep blood (63); 100 µg/ml ofspectinomycin (Spc) (Sigma) was used to select for S. suis transformants.E. coli strains DH5 ${alpha}$ and BL21 were cultured in Luria-Bertani(LB) liquid medium or plated on LB agar. If necessary, either50 µg/ml of ampicillin (Sigma) or 50 µg/ml of kanamycin(Sigma) was utilized to screen E. coli transformants (16).

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TABLE 1. Strains and plasmids used in this study

Bioinformatics search of zur/fur-like gene candidate. To identify zur/fur-like genes from the genome of S. suis 2,an in silico search was carried out using the E. coli zur/furgene sequence as search models (62). One putative open readingframe (ORF) with the coding number 310 was recognized as a possiblezur/fur-like gene and was subjected to further bioinformaticsanalysis, including Blast-P and ClustalW.

Overexpression and purification of protein 310. A pair of primers (310-F and 310-R) (Table 2) was designed toamplify gene 310, which was then cloned into pGEM-T vector (Takara)for direct DNA sequencing. Subsequently, it was subcloned intothe two kinds of prokaryotic expression vectors [pET28(a) (Novagen)and pGEX-6P-1 (Amersham)], generating the recombinant plasmidspET28::310 and pGEX-6P::310, respectively (Table 1). pET28::310and pGEX-6P::310 both were transformed into competent cellsof E. coli BL21(DE3) for production of recombinant 310 protein.

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TABLE 2. Primers used for PCR amplification and real time RT-PCR detection

A soluble protein encoded by gene 310 (named protein 310) wasobtained as described previously (16) with some minor modifications.After sonication, the clarified bacterial supernatant was loadedonto affinity columns (for the His tag version, a nickel-ion[Ni⁺] affinity column [Qiagen], for the glutathione S-transferase[GST]-fused version, a glutathione Sepharose 4B column [Amersham]).The recombinant His₆-tagged protein 310 was eluted in elutionbuffer with 50 mM imidazole after removing the contaminant proteinswith washing buffer containing 20 mM imidazole. The GST-fused310 protein was washed with 1x phosphate-buffered saline (PBS)and then eluted with 20 mM reduced glutathione. PreScissionproteinase was used to cleave the N-terminal GST tag to obtainthe 310 protein with GST removed.

Both versions of the acquired 310 protein (310 with GST removedand His-tagged 310) were concentrated by ultrafiltration (5-kDacutoff) and exchanged from 1x PBS buffer into the exclusionbuffer (20 mM Tris, 150 mM NaCl, pH 8.0). Subsequently, theywere subjected to gel filtration analysis using a Superdex 200column (Pharmacia) fixed on an Äkta purifier system (Pharmacia)and monitored at a flow rate of 0.5 ml/min in running buffer(which is same as the exclusion buffer). The peak was collected,visualized by 15% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), and then stained with Coomassiebrilliant blue R250 (Sigma, St. Louis, MO). The apparent molecularweights were estimated by comparison with standard protein markers(Sangon, Shanghai, China) run on the same gel (16).

Chemical cross-linking assays. Chemical cross-linking experiments were performed to identifythe natural state of protein 310. In brief, the purified recombinantprotein (approximately 10 mg/ml) was subjected to chemical cross-linkingwith ethylene glycol bis-succinimidylsuccinate (EGS) (Pierce).The reaction mixtures were incubated for 1 h on ice at differentconcentration of EGS (0, 2, 5, and 10 mM) and quenched with50 mM glycine. Finally, cross-linked samples were analyzed by15% SDS-PAGE (39).

Deletion and functional complementation of gene 310. To identify the function of gene 310 in S. suis serotype 2,we inactivated gene 310 using the strategy of homologous suicideplasmid integration. We constructed a gene 310 knockout vector,pMD::Spc-310LR, carrying the Spc resistance gene (Spc^r) (Table1) and electrotransformed it into competent cells of 05ZYH33as described by Smith et al. (57) and Li et al. (32) with minormodifications. Colony PCR assay was used to examine Spc^r transformantswith a series of specific primers (Table 2). To further confirmthe mutant, total RNA was extracted from S. suis serotype 2cultures at an optical density at 600 nm (OD₆₀₀) of 0.8 withTrizol reagent (Invitrogen) and purified using the RNeasy minikit(Qiagen). Reverse transcription-PCR (RT-PCR) was utilized asdescribed by Chen et al. (8). A mutant of interest, the ${Delta}$ 310mutant was eventually obtained, which was also verified by directDNA sequencing.

To further perform the functional complementation of the ${Delta}$ 310mutant, a DNA fragment of gene 310 together with its upstreampromoter was amplified by PCR using a pair of primers with specificrestriction enzyme sites (C310-F and C310-R) (Table 1) and thenwas cloned directionally into the E. coli-S. suis shuttle vectorpSET1, generating the recombinant plasmid pSET::C310 (Table1). After the verification of direct DNA sequencing, pSET1::C310was electrotransformed into the ${Delta}$ 310 mutant. The complementedstrains of the ${Delta}$ 310 mutant (C ${Delta}$ 310 mutant) were screened on THBagar plates with double selection pressure of Spc and chloramphenicol,following the method we reported recently (32).

Western blot analysis of protein 310 expression in S. suis 2. Total bacterial protein was prepared by sonication from threestrains of S. suis 2, i.e., the wild type (WT) (05ZYH33), the ${Delta}$ 310 mutant, and the ${Delta}$ 310 mutant complemented strain (C ${Delta}$ 310 mutant).They were then separated by 15% SDS-PAGE and transferred ontoa polyvinylidene difluoride (PVDF) membrane for the Westernblot-based detection of protein 310 as we described previously(16). Here, the first antibody is polyclonal anti-310 proteinrabbit serum, which was obtained according to the customaryimmunization protocol for New Zealand White rabbits (33).

Tests of the sensitivity of the ${Delta}$ 310 mutant to divalent cations. Since 310 is a member of the zur/fur family, we aimed to testits function. Salts as sources of six different divalent cations,i.e., MnCl₂, FeSO₄, MgCl₂, CaCl₂, CuSO₄, and ZnSO₄ were mixedwith THB liquid medium in appropriate concentrations. The growthof the ${Delta}$ 310 mutant cultivated in this above mixed medium wascompared to that of the WT by measuring the OD₆₀₀ (62, 70).Similarly, the assays of toxicity of divalent cations to S.suis serotype 2 strains were performed using chemically definedmedium (CDM).

Measurement of zinc ions. To examine whether protein 310 contains zinc, the purified protein310 after the desalting treatment was tested by inductivelycoupled plasma atomic emission spectrometry (ICP-AES) (modelVista-Mpx; Varin, Japan). For the measurement of bacterial zinccontent, both strains (the WT and the ${Delta}$ 310 mutant) were grownin 100 ml of THB (and CDM) liquid medium with 150 mM ZnSO₄.Bacterial cells harvested by centrifugation were washed thricewith PBS containing 0.5 mM EDTA to remove externally bound Zn²⁺as described by Tang et al. (62). The cell density was adjustedwith sterilized double-distilled water to an OD₆₀₀ of 1.0. Thebacterial zinc content was determined by ICP-AES. Finally, thezinc contents in double-distilled water, THB, and CDM were testedusing the same instrument.

Proteomics analysis. Two bacterial strains (the WT and the ${Delta}$ 310 mutant) were cultivatedin 200 ml of THB liquid medium and colleted by centrifugationat 4°C to an OD₆₀₀ of 0.8. The total proteins were extractedfrom pellets as described by Wang et al. (68). To optimize two-dimensionalelectrophoresis (2-DE), a preliminary experiment was first performedon a 7-cm gel with a linear range of pH (3.0 to 10.0). Then,total proteins of S. suis serotype 2 were separated in triplicatevia 2-DE on 17-cm gels in which the pH ranged from 3.0 to 7.0.

The 2-DE images were acquired with an Image Scanner (AmershamBiosciences) in transmission mode. The image analysis was carriedout by a combination of manual visualization and software analysiswith Image Master Elite version 4.1 (Amersham Biosciences).To gain comparable data for quantitative analysis, several keyparameters in the image analysis were fixed as constant (67).The average spot intensity was normalized to the total spotvolume with a multiplication factor of 100 (68). Spots on 2-DEwith different intensity were carefully excised, dehydratedwith acetonitrile, and digested in gel with trypsin solution.The digested products were subjected to further analysis bymatrix-assisted laser desorption ionization-time-of-flight massspectrometry. Based on the acquired peptide sequences, databasesearches were conducted with MASCOT software 1.9 (Matrix Science)against the protein database of S. suis serotype 2 in the BeijingGenomics Institute.

Expression microarray-based analysis. On the basis of the 05ZYH33 genome sequence, oligonucleotideprobes with an average length of 35 nucleotides were designedto match all the putative ORFs (2,194 in total) using ArrayDesigner 2.0 and then synthesized onto complementary metal oxidesemiconductor matrix utilizing an in situ electrochemical synthesistechnique (Combimatrix). To gain insights into the responseof S. suis serotype 2 to Zn²⁺, we selected another isolate thatbehaved like the WT in the presence of 200 µM Zn²⁺ (referredto as WT/Zn²⁺). Total RNAs from these three samples (WT, ${Delta}$ 310mutant, and WT/Zn²⁺) were utilized to perform RT-PCR, and thegenerated cDNAs were labeled with Cy3 dCTP during reverse transcription.After prehybridization, microarray slides were hybridized withthe relevant samples at 65°C for 4 to 16 h (13). This wasrepeated four times for three samples.

All hybridization slides were scanned with a GenePix 4100A scannerafter appropriate washing, and the average pixel intensity valueswere quantified using GenePix Pro 4.1. Statistical analysis(t test and P values) was carried out, and genes with more thantwofold change ratios were regarded as candidate targets (13).

Real-time quantitative RT-PCR. To validate the results of the microarray data, 10 randomlyselected genes (Table 2) were subjected in triplicate to quantitativeRT-PCR utilizing Sybr green detection in a Rotor Gene 6000 (Corbett)(75). For each sample (WT, ${Delta}$ 310 mutant, and WT/Zn²⁺), 2 µgof total RNA was used for first-strand cDNA synthesis with acommercial RT kit (Promega) as recommended by the manufacturer,and the primers (Table 2) were designed according to the publishedgenomic sequence of 05ZYH33 (8). Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) served as an internal control. BacterialDNAs in a dilution series, as standard samples, were appliedto generate the standard quantitative curve, and double standardcurves were utilized to perform the quantitative analysis ofthe genes of interest. The average ratio of target genes’transcription levels was calculated with the software of RotorGene 6.0.

RESULTS

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RESULTS

Bioinformatics-based identification of a zur/fur-like gene, 310, from S. suis serotype 2 strain 05ZYH33. The recently determined genome sequence of S. suis 05ZYH33,a virulent Chinese isolate of S. suis serotype 2 (8), has facilitatedthe further development of functional genomics. Bioinformaticsanalysis identified an ORF termed 310 located at bp 302393 to301941 on the negative strand of the genome (not shown). Thededuced product of 310 (named protein 310 here) consisted of151 amino acids (aa) exhibiting 28.4% and 35.1% amino acid similarityto the Zur proteins of E. coli and M. tuberculosis, respectively(Fig. 1). Further analysis suggested that protein 310 containsa potential DNA-binding motif (helix-turn-helix) at its N terminus(not shown) and possesses nearly 38% ${alpha}$ -helices in its secondarystructure (Fig. 1), implying that it is possibly a transcriptionfactor.

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FIG. 1. Multiple alignment of protein 310 from S. suis 05ZYH33 with the related Zur regulators at the amino acid level. The multiple alignment was conducted using the ESPript software together with ClustalW online. The Zur proteins correspond to S. suis 05ZYH33 (YP_001197678), M. tuberculosis F11 (2O03A), and E. coli W3110 (BAE 78048). Three types of protein secondary structure are as follows: ${alpha}$ , alpha-helix (with cartoon helixes); β, beta-sheet; T, turn. S. suis serotype 2 strain 05ZYH33 is highlighted in red. According to the structural architecture of the M. tuberculosis Zur protein, five conserved residues (H80, C85, C88, C125, and C128) that are critical for the specific binding to zinc ions are indicated with blue arrows.

Biochemical characterization of protein 310. To better understand protein 310, the complete coding sequenceof gene 310 was cloned into pET28(a). The purified, recombinant,soluble 310 protein (before and after chemical cross-linking)was analyzed by gel filtration. The fast protein liquid chromatography(FPLC) profile of both 310 protein samples on Superdex 200 showedthat they eluted at approximately same position at $~$ 16.2 ml ( $~$ 35kDa) (Fig. 2A). The samples collected from these two peaks (withand without chemical cross-linking) were separated by 15% SDS-PAGE,which showed that (i) protein 310 has a molecular mass of $~$ 17kDa, which is consistent with the theoretical estimation ofthe amino acid content of a monomer (Fig. 2A and B), and (ii)protein 310 exists as a dimer after the chemical cross-linkingassay (Fig. 2A). Protein 310 before and after gel filtrationwas also subjected to further analysis of chemical cross-linking.The position of protein 310 shifted from monomer to a mixtureof monomer and dimer (an intermediate state) to dimer afteraddition of EGS (Fig. 2C), suggesting that the 310 protein isindeed a dimer, a typical biochemical characteristic of theZur family (26). Subsequently, ICP-AES measurement confirmedthat protein 310 contains zinc (Table 3), which is in consistentwith structural findings for M. tuberculosis Zur protein (36).The above data provide indirect biochemical evidence that protein310 is a member of the Zur family.

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FIG. 2. Existence of protein 310 as a dimer. (A) FPLC profile of protein 310. The purified protein 310 (before and after chemical cross-linking) was subjected to gel filtration on a Superdex 200HR 10/30 column. The samples from the two different peaks were visualized on a 15% SDS-PAGE gel. Lane 1, protein 310 from before chemical cross-linking; lane 2, protein 310 from after chemical cross-linking. The protein of interest is indicated by the arrow. (B) Elution behavior of standard proteins. Standardized proteins (Pharmacia) were run on a Superdex 200HR 10/30 to relatively determine the molecular mass of protein 310. Protein 310 (same as in panel A) is highlighted circled. (C) Chemical cross-linking assay of protein 310. The samples were separated by 15% SDS-PAGE following chemical cross-linking. GST, a typical dimer protein, was used as a positive control in the chemical cross-linking experiment. The GST monomer is indicated by an asterisk, while the GST dimer is highlighted with double asterisks. 0, 2, 5, and 10, concentrations of EGS (mM). Protein 310 occurs only as a monomer in vitro on SDS-PAGE without addition of EGS. With the increase of EGS, the intensity of the monomer is decreased, while the dimer is enhanced. Protein 310 before and after the gel filtration behaves similarly in the chemical cross-linking experiments. Also, protein 310 was demonstrated to carry zinc ions by ICP-AES (Table 3).

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TABLE 3. Determination of zinc contents in protein 310 and S. suis serotype 2 strains

Physiological function of gene 310. In order to elucidate the role of 310, the ${Delta}$ 310 mutant was obtainedfrom more than 200 S. suis transformants. The correct genotypeof the ${Delta}$ 310 mutant was systemically confirmed by multiple approaches,such as PCR (Fig. 3A and B), RT-PCR (Fig. 3C), Western blotting(Fig. 3D), and direct DNA sequencing.

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FIG. 3. Identification of the ${Delta}$ 310 mutant, the isogenic mutant of gene 310 in S. suis serotype 2. (A) Gene 310 knockout from the S. suis serotype 2 chromosome. pMD::Spc-310LR is the recombinant vector constructed to specifically inactivate gene 310. 310L and 310R indicate the left and right borders of gene 310. A pair of specific primers (P-F and P-R) located in both sides adjacent to gene 310 are indicated by arrows and were used for PCR detection of existence of gene 310 in the genome of S. suis serotype 2. (B) Multiple-PCR analysis of the ${Delta}$ 310 mutant. The PCR products were separated by electrophoresis on a 1.0% agarose gel stained with ethidium bromide. P, PCR product amplified with primers P-F and P-R. Gene 310 in the ${Delta}$ 310 mutant has been replaced by an Spc^r gene, without affecting either boundary sequence (not shown). (C) RT-PCR analysis of the ${Delta}$ 310 mutant. RT-PCR products of gene 310 were separated by electrophoresis on a 1.0% agarose gel (top). Total RNAs of S. suis serotype 2 strains (WT, the ${Delta}$ 310 mutant, and C ${Delta}$ 310) were determined by electrophoresis on a 1.0% agarose gel (bottom). 23 S, 23S ribosome subunit; 16 S, 16S ribosome subunit. (D) Western blot analysis. Protein 310 is shown to be expressed in the WT and the complemented strain, C ${Delta}$ 310, but not in the gene 310 isogenic mutant.

Several biological properties were addressed, including morphology,hemolytic activity, and sensitivity to H₂O₂. No obvious morphologicaldifference was observed between the ${Delta}$ 310 mutant and the WT underthe culture conditions used (not shown). To further investigatethe physiological function of gene 310, six divalent metal ions(Mg²⁺, Fe²⁺, Zn²⁺, Ca²⁺, Mn²⁺, and Cu²⁺) were assessed for theirtoxicity to S. suis serotype 2. For each of the five ions Mg²⁺,Ca²⁺, Mn²⁺, Cu²⁺, and Fe²⁺, both strains (WT and ${Delta}$ 310 mutant)were found to grow well even at an abnormally high concentration(800 µM), indicating that gene 310 was not involved inthe tolerance to them. The ${Delta}$ 310 mutant was highly resistant toFe²⁺ at the same level the WT, suggesting that 310 is not involvedin iron metabolism.

However, the ${Delta}$ 310 mutant grew slowly in THB medium supplementedwith 150 and 200 µM Zn²⁺, whereas the WT and complemented(C ${Delta}$ 310) strains grew well under the same conditions (Fig. 4A).When the concentration of Zn²⁺ was increased to 250 µM,the growth rates of both the WT and C ${Delta}$ 310 strains were also hampered,and they all were killed when the concentration of Zn²⁺ washigher than 300 µM (Fig. 4A). On the other hand, the biomassof the ${Delta}$ 310 mutant was decreased greatly (Fig. 4B). Similar sensitivityof the ${Delta}$ 310 mutant to Zn²⁺ was found in CDM (not shown).

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FIG. 4. Role of gene 310 in the sensitivity of the ${Delta}$ 310 mutant to Zn²⁺. (A) Sensitivity of the ${Delta}$ 310 mutant to Zn²⁺. An overculture of each S. suis serotype 2 strain (60 µl, cell density adjusted to an OD₆₀₀ of 0.7) was inoculated into 3 ml of THB liquid medium supplemented with ZnSO₄ to a final concentration of 0, 50, 100, 150, 200, 250, 300, 400, 500, or 600 µM. After 12 h of stationary culture at 37°C bacterial cells were subjected to 1 min of shaking at 160 rpm to reach density uniformity before measurement of bacterial cell density. The cell density was measured spectrometrically at 600 nm. (B) Defect of ${Delta}$ 310 mutant growth with high concentrations of zinc ions. The dry weights of 200-ml bacterial cultures were measured. Prior to calculating the weight, the cells were harvested by centrifugation, washed with 1x PBS three times, and then air dried for 20 min. The values are expressed as the means ± standard deviation from five repeats. **, P $≤$ 0.001; *, P $≤$ 0.01.

Quantitative analysis of bacterial zinc revealed that the zinccontent in the ${Delta}$ 310 mutant is obviously higher than that in theWT (growing in either THB or CDM with suppression of Zn²⁺).This implies that deletion of 310 is responsible in part forthe dysfunction of zinc metabolism in Streptococcus suis cells(Table 3). Indeed, a similar effect has been noted in the zurisogenic mutant of Xanthomonas campestris pv. campestris (62).These data strongly demonstrate that gene 310 is a functionalmember of the zur family that controls zinc homeostasis.

Global regulation of gene 310 in S. suis serotype 2. Because 310 was characterized as Zur, a zinc-responsive transcriptionfactor, it was of interest to investigate Zur-mediated, genome-wideregulation in S. suis serotype 2. 2-DE was first applied tocompare the differential expression profiles of the ${Delta}$ 310 mutantand the WT. Protein spot 352 was found only in the ${Delta}$ 310 mutantand was identified as an acetyl coenzyme A carboxylase (ACC)beta subunit (Table 4). Eight other protein spots whose densitiesin the ${Delta}$ 310 mutant were three times greater than those in theWT were also observed (Table 4).

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TABLE 4. Mass spectrometry identification of the protein spots identified by 2-DE analysis

The limited information acquired by 2-DE prompted us to employDNA microarray analysis to further investigate this issue. Anexpression microarray of S. suis serotype 2 that covers 2,194genes of 05ZYH33 was designed. In this study, a twofold differencein relative transcription level was selected as a thresholdfor analysis of microarray data, as described by Ichikawa etal. (27). The effectiveness of the microarray data was confirmedby real-time quantitative RT-PCR (Table 5). Globally, 5.6% ofthe genes represented on the microarray (n = 121) were differentiallytranscribed in the ${Delta}$ 310 mutant compared with the WT. Among them,72 genes were upregulated, and 49 genes were downregulated (Fig.5A). These genes were involved in metabolism, information storage/process,and cellular signaling/defense. For positively regulated genesin the ${Delta}$ 310 mutant, there were 23 genes categorized as proteinasegenes, 8 genes related to DNA metabolism/repair, and 5 surface-associatedprotein genes, as well as many genes for unknown/hypotheticalproteins (Table 6). Likewise, the negatively regulated genesin the ${Delta}$ 310 mutant included the above members. Of note, 10 ABCtransporters, which are usually associated with influx and effluxof metabolic molecules (71) and metal ions (6), were positivelyregulated in the ${Delta}$ 310 mutant, and 2 ABC-type transporters werenegatively regulated in the ${Delta}$ 310 mutant (Table 6). A Zn-dependentNADPH:quinone reductase was revealed to be upregulated in the ${Delta}$ 310 mutant, posing the possibility that zinc ions may be directlycompeted by protein 310 in bacterial cells (Table 5; see TableS1 in the supplemental material). Additionally, metal-responsivetranscription factors were involved in Zur regulation (Table6; see Tables S1 and S2 in the supplemental material). Combinedwith genome-wide search for zinc-related genes (Table 7), semiquantitativeRT-PCR analysis of the ${Delta}$ 310 mutant prompted us to link gene 310directly to a zinc ABC transporter (05SSU0112), implying thatprotein 310 probably functions as a zinc-responsive negativeregulator in this case (Fig. 6), which would be in close agreementwith E. coli Zur regulator (47).

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TABLE 5. Real-time quantitative RT-PCR assays of microarray data

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FIG. 5. Global regulation of gene 310 and evaluation of genome-wide response of S. suis serotype 2 to Zn²⁺ suppression. (A) Differentially expressed profile of the ${Delta}$ 310 mutant compared with the WT. (B) Genome-wide response of S. suis serotype 2 to the presence of Zn²⁺. The upregulated genes are in yellow, and the downregulated genes are in gray. The criteria for selecting differentially expressed genes were a fold change of $≥$ 2 and a P value of $≤$ 0.05. For every bacterial sample, the hybridization was repeated in four unique DNA microarrays, and the signal intensity was quantified with GenePix Pro 4.1. Values are expressed as the means from four repeats. (C) Comparative analysis of the upregulated genes from the ${Delta}$ 310 mutant and WT/Zn²⁺. The red circle indicates the total number of genes upregulated in the ${Delta}$ 310 mutant compared with the WT. The blue circle represents the total number of genes upregulated in WT/Zn²⁺ compared with the WT. A total of 52 upregulated genes are shared by the ${Delta}$ 310 mutant and S. suis serotype 2 WT/Zn²⁺. A total of 20 upregulated genes are specific to the ${Delta}$ 310 mutant, and 19 upregulated genes are found only in WT/Zn²⁺ (for details, see Tables S1 and S3 in the supplemental material). (D) Comparative analysis of the downregulated genes from the ${Delta}$ 310 mutant and WT/Zn²⁺. A total of 35 downregulated genes were shared by the ${Delta}$ 310 mutant and S. suis serotype 2 WT/Zn²⁺. A total of 14 downregulated genes are specific to the ${Delta}$ 310 mutant, and 11 negatively regulated genes are present only in WT/Zn²⁺ (for details, see Tables S2 and S4 in the supplemental material).

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TABLE 6. Genome-wide display of the genes differentially expressed in the ${Delta}$ 310 mutant and S. suis serotype 2 WT/Zn²⁺

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TABLE 7. Genome-wide search for genes with involvement of zinc ions

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FIG. 6. Transcriptional analysis of gene 0112, a zinc-binding lipoprotein in ABC-type transporters. On the basis of the combined information, gene 0112, a component of the ABC zinc transporter (also called ZnuA), is proposed to be a possible element which can be directly regulated by gene 310 in Streptococcus suis. Semiquantitative RT-PCR was used for transcriptional analysis of gene 0112. The DNA fragment of interest is 235 bp in length.

Correlation of gene 310 with Zn²⁺ suppression of S. suis serotype 2. The bacterial culture of S. suis serotype 2 treated with Zn²⁺(200 µM) (abbreviated WT/Zn²⁺ here) was also analyzedby DNA microarray analysis. In total, there were 117 genes whosetranscription profiles were affected during the process in responseto high levels of Zn²⁺. Dual regulation was confirmed to relateto stimulus of zinc ion, which included 71 positively regulatedgenes and 46 negatively regulated genes (Table 6 and Fig. 5B).To our surprise, more than 70% of genes affected by zinc inthe specific transcriptome of WT/Zn²⁺ were also affected bydeletion of 310, which in turn provides evidence that gene 310is a novel zinc-dependent regulator. In detail, 52 of the upregulatedgenes in WT/Zn²⁺ overlapped with those in the ${Delta}$ 310 mutant (Fig.5C), and the downregulated genes in WT/Zn²⁺ covered 73.7% ofthose in the ${Delta}$ 310 mutant (Fig. 5D).

DISCUSSION

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DISCUSSION

Streptococcus suis infections are one of the primary and secondarybacterial diseases causing serious economic losses in pig industriesworldwide each year (58, 64). These pathogens are generallycategorized into 35 serotypes on the basis of the differencein their capsular antigens, of which serotype 2 is a prevalentserotype most frequently isolated from diseased piglets andhuman patients (58). There is an increasing amount of data suggestingthat S. suis serotype 2 has evolved into a highly invasive entity,raising great concerns over global public health (38). Unfortunately,the control of S. suis serotype 2 infections by vaccines andantimicrobials has been hampered partly due to the increasedtolerance of S. suis serotype 2 to antimicrobials together withcurrent limited knowledge on its pathogenesis (38). Therefore,a better understanding of the different aspects of S. suis serotype2, especially pathogenesis, will facilitate the developmentof effective therapeutics which can prevent and/or treat S.suis serotype 2 infections.

We identified gene 310 from Streptococcus suis, encoding a functionalmember of the Zur family. Bioinformatics analysis revealed thatthis protein comprises two conserved domains in the family ofFur/Zur proteins: a hypothetical DNA-binding motif at its Nterminus and a putative C-terminal dimerizing module (2). Sensitivityassays with divalent transition metal ions demonstrated thatgene 310 is a zinc-responsive regulator gene, referred to aszur (Fig. 4). Our biochemical data (Fig. 2) showed that thefull-length product of gene 310 exists as a dimer, which isconsistent with the crystal structures (48) and previous biochemicalanalysis (2, 12) of other members of this superfamily. Althoughthe deletion of fur/zur was found to impair the growth of Actinobacilluspleuropneumoniae (28), the ${Delta}$ 310 mutant exhibited growth similarto that of its parental strain (not shown). Subsequent microarrayresults provided one possible explanation for this, in thatthe overexpression of Era, which is required for bacterial growth(45), may remedy the defect caused by the deletion of gene 310in the ${Delta}$ 310 mutant to some extent. The inactivation of gene 310seemed to not affect the bacterial phenotype, such as hemolyticactivity. In contrast to those regulators sensing oxidativestress in this family, e.g., PerR (31, 69), our data did notsupport the involvement of gene 310 in H₂O₂ sensitivity (notshown). Retrospectively, Dpr (Dps-like peroxide resistance protein)was suggested to confer on S. suis serotype 2 resistance toH₂O₂ (52). However, the role of zur in pathogenesis is controversial(7, 34, 62) In our piglet infections, zur had no apparent effecton S. suis serotype 2 virulence, which is consistent with resultsseen for S. aureus (34).

Given that Zur is a global regulator in bacteria, we tried todissect its regulatory networks at the levels of the proteomeand transcriptome. At the proteomics level, we identified onlynine protein spots (Table 4). One protein dot (no. 352) thatis present only in the ${Delta}$ 310 mutant was confirmed to be the betasubunit of ACC. ACC is a central metabolic enzyme that catalyzesthe committed step in fatty acid biosynthesis. Recently, theACC structures of both S. aureus and E. coli revealed a zinc-bindingmotif (5). Therefore, it is reasonable that Zur-mediated downregulationof ACC can be attributed to its interaction with zinc. On theother hand, there are five other proteins whose expression levelin the ${Delta}$ 310 mutant was three times higher than that in the WT.First, it is reasonable that the two proteins (ribosome-bindingfactor A [spot 24] and GTP-binding translation factor [spot427]) were derepressed in the ${Delta}$ 310 mutant. Several studies haveindicated that zinc is required for the structural stabilityof translation initiation factors that generally contain ribosome-bindingdomains (22, 35). It is possible that ribosome-binding factorscompete with a translation initiation factor for the resourcesof ribosomes (19, 21). Second, Era (a GTP-binding protein) wasrevealed by 2-DE to be negatively regulated by Zur. Era hasbeen demonstrated to interact with 16S rRNA (23) and mediatethe assembly of the 30S ribosome unit (54), and ribosomal proteinshave been suggested to be related to Zur protein and zinc ions(46, 55). Therefore, it is logical that Era can be linked toZur in S. suis serotype 2. Certainly, the total number of affectedproteins elucidated by 2-DE seemed to be much less than thatone might expect.

Further microarray analysis showed that the gene 310-mediateddual-regulation network is globally involved in 121 genes, ofwhich 72 were upregulated and 49 were downregulated in the ${Delta}$ 310mutant (Fig. 5A). Most genes affected by zur encode putativeproteinases, of which 23 were upregulated and 12 were downregulated(Table 6). They included some Zn²⁺-dependent members (e.g.,NADPH:quinone reductase and lipase) involved in the basic metabolismof carbohydrates, nucleic acids, and fatty acids (10). Therewere more than 20 unknown genes which are influenced by gene310. Membrane/surface proteins have been suggested to contributegreatly to the invasiveness and immunogenicity of pathogens(16, 41). We observed that they are also subject to dual regulationby Zur (some are stimulated, and others are repressed). In particular,a hyaluronidase with an LPXTG motif, which plays roles in pathogenicityof group A streptococci (60), was also found to be negativelyregulated by Zur. However, cps2C, which is involved in the productionof capsular polysaccharide, a virulence determinant of S. suisserotype 2 (56), is positively regulated by Zur. It seemed thatvirulence manifestation is linked to the regulation of Zur inS. suis. Transcription factors and activators (e.g., alsR andplcR) were also affected by Zur, implying the existence of indirectregulation mediated by Zur in S. suis serotype 2 (40, 74).

The transcriptome of S. suis serotype 2 under the control ofZn²⁺ exhibited 117 differentially expressed genes compared withthat under normal culture condition (Fig. 5B). Intriguingly,we noticed that more than 70% of the genes regulated in responseto Zn²⁺ overlapped with those regulated by Zur protein in S.suis serotype 2 (Fig. 5C). Two points of interest are as follows.First, it is reasonable that the expression of a 60-kDa chaperonin(see Table S3 in the supplemental material) is elevated greatlyin response to the emerging stress of zinc ions at a high level(73), which is similar to what is observed in the ${Delta}$ 310 mutant.Second, not only were most ABC-type transporters regulated byZn²⁺, but membrane proteins, proteinases, and unknown proteinswere also affected, which in turn validates the Zur-mediatedregulation network. It is well known that MscS (the mechanosensitivechannel of small conductance) plays a critical role in osmoregulationin prokaryotic microorganisms (3, 65). Similarly, microarrayresults revealed that treatment with Zn²⁺ can repress the transcriptionof mscS in S. suis serotype 2. Unexpectedly, MreC, a cell shapedeterminant (29, 30), was found to be positively regulated butnot negatively regulated by Zur, suggesting that S. suis serotype2 has evolved morphological machineries to adapt to the dramaticchange of environmental zinc ions. In addition, transcriptionregulators were affected by zinc, suggesting that their targetgenes may be indirectly controlled by Zur (40).

In summary, we defined a functional zur gene from S. suis serotype2. We attempted to define the genome-wide regulation networkof Zur by 2-DE and DNA microarray analysis. Based on microarrayanalysis of the ${Delta}$ 310 mutant compared with S. suis serotype 2treated with zinc ions, we gained, for the first time, a glimpseof the cross talk between gene 310 and Zn²⁺, which in turn providesgenome-wide evidence that gene 310 is a functional member ofthe zur family.

ACKNOWLEDGMENTS

We thank Ben Adler, Department of Microbiology, Monash University,Australia; Min Wu, University of North Dakota; Shibo Jiang,New York Blood Center; and John E. Cronan and Quin Christensin,Department of Microbiology, University of Illinois at Urbana-Champaign,for critical reading of the manuscript. We are grateful to DaisukeTakamatsu, National Institute of Animal Health, Japan, for kindlyproviding pSET1, an E. coli-S. suis shuttle vector.

This work was supported by the National Basic Research Program(973) of the Ministry of Science and Technology (MOST) of China(2005CB523001), the National Natural Science Foundation of China(NSFC) (30670105 and 30600533), and the National Key TechnologiesR&D Program (2006BAD06A04). George F. Gao is a distinguishedyoung investigator of the NSFC (grant no. 30525010).

FOOTNOTES

* Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Phone: 86-10-64807688. Fax: 86-10-64807882. E-mail: gaof{at}im.ac.cn

Published ahead of print on 22 August 2008.

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

Present address: Department of Microbiology, University of Illinoisat Urbana-Champaign, Urbana, IL 61801.

REFERENCES

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