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J Bacteriol. Nov 2008; 190(22): 7567–7578.
Published online Aug 22, 2008. doi:  10.1128/JB.01532-07
PMCID: PMC2576675

Functional Definition and Global Regulation of Zur, a Zinc Uptake Regulator in a Streptococcus suis Serotype 2 Strain Causing Streptococcal Toxic Shock Syndrome[down-pointing small open triangle]

Abstract

Zinc is an essential trace element for all living organisms and plays pivotal roles in various cellular processes. However, an excess of zinc is extremely deleterious to cells. Bacteria have evolved complex machineries (such as efflux/influx systems) to control the concentration at levels appropriate for the maintenance of zinc homeostasis in cells and adaptation to the environment. The Zur (zinc uptake regulator) protein is one of these functional members involved in the precise control of zinc homeostasis. Here we identified a zur homologue designated 310 from Streptococcus suis serotype 2, strain 05ZYH33, a highly invasive isolate causing streptococcal toxic shock syndrome. Biochemical analysis revealed that the protein product of gene 310 exists as a dimer form and carries zinc ions. An isogenic gene replacement mutant of gene 310, the Δ310 mutant, was obtained by homologous recombination. Physiological tests demonstrated that the Δ310 mutant is specifically sensitive to Zn2+, while functional complementation of the Δ310 mutant can restore its duration capability, suggesting that 310 is a functional member of the Zur family. Two-dimensional electrophoresis indicated that nine proteins in the Δ310 mutant are overexpressed in comparison with those in the wild type. DNA microarray analyses suggested that 121 genes in the Δ310 mutant are affected, of which 72 genes are upregulated and 49 are downregulated. The transcriptome of S. suis serotype 2 with high Zn2+ concentrations also showed 117 differentially expressed genes, with 71 upregulated and 46 downregulated. Surprisingly, more than 70% of the genes differentially expressed in the Δ310 mutant were the same as those in S. suis serotype 2 that were differentially 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.

Streptococcus suis 2 is a gram-positive pathogenic bacterium capable of infecting both piglets and humans and causing serious diseases such as arthritis, meningitis, and septicemia (58). It has spread to nearly 20 countries, resulting in more than 400 human cases of severe infection worldwide (38). In particular, two recent outbreaks (in 1998 and 2005) of severe human S. suis serotype 2 infections in China were characterized by streptococcal toxic shock syndrome, implying that highly invasive variants of S. suis serotype 2 might be emerging in Asia (63, 72). Virulence factors related to the pathogenesis of S. suis serotype 2 have been partially elucidated and include capsular polysaccharide (56), suilysin (37), muraminidase-released protein (42), and extracellular factor (59). Very recently, our group (8) reported the whole genome sequences of two virulent S. suis serotype 2 isolates (05ZYH33 and 98HAH12).

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

The zinc uptake regulator, Zur, is one of the key components contributing to the molecular systems by which zinc homeostasis is controlled (47). In fact, Zur has been classified as a subgroup of the Fur (ferric uptake regulator) family, which comprises at 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 Escherichia coli (47), the functional members of the Zur family have been extended 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 regulates not only zinc uptake but also the mobilization of zinc through ribosomal proteins (44). Very recently, ribosome proteins were revealed to be regulated by Zur proteins in both Streptomyces coelicolor (46, 55) and M. tuberculosis (40). Tang et al. (62) showed that Zur is involved in extracellular polysaccharide production and is required for full virulence in Xanthomonas campestris. Similarly, Zur was found to be involved in the pathogenesis of Salmonella enterica (7) and Xanthomonas campestris (62). In contrast, zur fails to exhibit obvious roles in the pathogenicity of S. aureus (34). Following the solution of the crystal structure of Fur from Pseudomonas aeruginosa in 2003 (50), Lucrelli et al. (36) characterized the architecture of Zur that is central to zinc homeostasis in M. tuberculosis, providing structural insights into the interplay between Zur and its target DNA sequence (the Zur box). Intriguingly, Zur has also been suggested to act as an indirect activator by repressing two regulatory RNAs (rhyA and rhyB) (36), which is distinct from its general role as a repressor (25, 53).

In attempts to explore the functional genomics of S. suis serotype 2 and the molecular pathogenesis of invasive streptococcal toxic shock syndrome infection, we identified a Fur/Zur-like homologue referred to as gene 310. Subsequently, we carried out systematic investigations to characterize gene 310. Our data demonstrate clearly that gene 310 is a functional member of the Zur family from S. suis 05ZYH33. Moreover, global regulation of Zur in S. suis serotype 2 is presented, for the first time, highlighting its relationship with zinc homeostasis at the genomic level.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Bacterial strains and plasmids are detailed in Table Table1.1. Streptococcus suis strains (e.g., 05ZYH33) were grown in Todd-Hewitt broth (THB) (Difco Laboratories, Detroit, MI) or plated on THB agar containing 5% (vol/vol) sheep blood (63); 100 μg/ml of spectinomycin (Spc) (Sigma) was used to select for S. suis transformants. E. coli strains DH5α and BL21 were cultured in Luria-Bertani (LB) liquid medium or plated on LB agar. If necessary, either 50 μg/ml of ampicillin (Sigma) or 50 μg/ml of kanamycin (Sigma) was utilized to screen E. coli transformants (16).

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/fur gene sequence as search models (62). One putative open reading frame (ORF) with the coding number 310 was recognized as a possible zur/fur-like gene and was subjected to further bioinformatics analysis, including Blast-P and ClustalW.

Overexpression and purification of protein 310.

A pair of primers (310-F and 310-R) (Table (Table2)2) was designed to amplify gene 310, which was then cloned into pGEM-T vector (Takara) for direct DNA sequencing. Subsequently, it was subcloned into the two kinds of prokaryotic expression vectors [pET28(a) (Novagen) and pGEX-6P-1 (Amersham)], generating the recombinant plasmids pET28::310 and pGEX-6P::310, respectively (Table (Table1).1). pET28::310 and pGEX-6P::310 both were transformed into competent cells of E. coli BL21(DE3) for production of recombinant 310 protein.

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

A soluble protein encoded by gene 310 (named protein 310) was obtained as described previously (16) with some minor modifications. After sonication, the clarified bacterial supernatant was loaded onto 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 elution buffer with 50 mM imidazole after removing the contaminant proteins with washing buffer containing 20 mM imidazole. The GST-fused 310 protein was washed with 1× phosphate-buffered saline (PBS) and then eluted with 20 mM reduced glutathione. PreScission proteinase was used to cleave the N-terminal GST tag to obtain the 310 protein with GST removed.

Both versions of the acquired 310 protein (310 with GST removed and His-tagged 310) were concentrated by ultrafiltration (5-kDa cutoff) and exchanged from 1× PBS buffer into the exclusion buffer (20 mM Tris, 150 mM NaCl, pH 8.0). Subsequently, they were subjected to gel filtration analysis using a Superdex 200 column (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 gel electrophoresis (SDS-PAGE), and then stained with Coomassie brilliant blue R250 (Sigma, St. Louis, MO). The apparent molecular weights 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 identify the natural state of protein 310. In brief, the purified recombinant protein (approximately 10 mg/ml) was subjected to chemical cross-linking with ethylene glycol bis-succinimidylsuccinate (EGS) (Pierce). The reaction mixtures were incubated for 1 h on ice at different concentration of EGS (0, 2, 5, and 10 mM) and quenched with 50 mM glycine. Finally, cross-linked samples were analyzed by 15% 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 suicide plasmid integration. We constructed a gene 310 knockout vector, pMD::Spc-310LR, carrying the Spc resistance gene (Spcr) (Table (Table1)1) and electrotransformed it into competent cells of 05ZYH33 as described by Smith et al. (57) and Li et al. (32) with minor modifications. Colony PCR assay was used to examine Spcr transformants with a series of specific primers (Table (Table2).2). To further confirm the mutant, total RNA was extracted from S. suis serotype 2 cultures at an optical density at 600 nm (OD600) of 0.8 with Trizol reagent (Invitrogen) and purified using the RNeasy minikit (Qiagen). Reverse transcription-PCR (RT-PCR) was utilized as described by Chen et al. (8). A mutant of interest, the Δ310 mutant was eventually obtained, which was also verified by direct DNA sequencing.

To further perform the functional complementation of the Δ310 mutant, a DNA fragment of gene 310 together with its upstream promoter was amplified by PCR using a pair of primers with specific restriction enzyme sites (C310-F and C310-R) (Table (Table1)1) and then was cloned directionally into the E. coli-S. suis shuttle vector pSET1, generating the recombinant plasmid pSET::C310 (Table (Table1).1). After the verification of direct DNA sequencing, pSET1::C310 was electrotransformed into the Δ310 mutant. The complemented strains of the Δ310 mutant (CΔ310 mutant) were screened on THB agar 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 three strains of S. suis 2, i.e., the wild type (WT) (05ZYH33), the Δ310 mutant, and the Δ310 mutant complemented strain (CΔ310 mutant). They were then separated by 15% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane for the Western blot-based detection of protein 310 as we described previously (16). Here, the first antibody is polyclonal anti-310 protein rabbit serum, which was obtained according to the customary immunization protocol for New Zealand White rabbits (33).

Tests of the sensitivity of the Δ310 mutant to divalent cations.

Since 310 is a member of the zur/fur family, we aimed to test its function. Salts as sources of six different divalent cations, i.e., MnCl2, FeSO4, MgCl2, CaCl2, CuSO4, and ZnSO4 were mixed with THB liquid medium in appropriate concentrations. The growth of the Δ310 mutant cultivated in this above mixed medium was compared 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 defined medium (CDM).

Measurement of zinc ions.

To examine whether protein 310 contains zinc, the purified protein 310 after the desalting treatment was tested by inductively coupled plasma atomic emission spectrometry (ICP-AES) (model Vista-Mpx; Varin, Japan). For the measurement of bacterial zinc content, both strains (the WT and the Δ310 mutant) were grown in 100 ml of THB (and CDM) liquid medium with 150 mM ZnSO4. Bacterial cells harvested by centrifugation were washed thrice with PBS containing 0.5 mM EDTA to remove externally bound Zn2+ as described by Tang et al. (62). The cell density was adjusted with sterilized double-distilled water to an OD600 of 1.0. The bacterial zinc content was determined by ICP-AES. Finally, the zinc contents in double-distilled water, THB, and CDM were tested using the same instrument.

Proteomics analysis.

Two bacterial strains (the WT and the Δ310 mutant) were cultivated in 200 ml of THB liquid medium and colleted by centrifugation at 4°C to an OD600 of 0.8. The total proteins were extracted from pellets as described by Wang et al. (68). To optimize two-dimensional electrophoresis (2-DE), a preliminary experiment was first performed on 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 triplicate via 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 (Amersham Biosciences) in transmission mode. The image analysis was carried out by a combination of manual visualization and software analysis with Image Master Elite version 4.1 (Amersham Biosciences). To gain comparable data for quantitative analysis, several key parameters in the image analysis were fixed as constant (67). The average spot intensity was normalized to the total spot volume with a multiplication factor of 100 (68). Spots on 2-DE with different intensity were carefully excised, dehydrated with acetonitrile, and digested in gel with trypsin solution. The digested products were subjected to further analysis by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Based on the acquired peptide sequences, database searches were conducted with MASCOT software 1.9 (Matrix Science) against the protein database of S. suis serotype 2 in the Beijing Genomics Institute.

Expression microarray-based analysis.

On the basis of the 05ZYH33 genome sequence, oligonucleotide probes with an average length of 35 nucleotides were designed to match all the putative ORFs (2,194 in total) using Array Designer 2.0 and then synthesized onto complementary metal oxide semiconductor matrix utilizing an in situ electrochemical synthesis technique (Combimatrix). To gain insights into the response of S. suis serotype 2 to Zn2+, we selected another isolate that behaved like the WT in the presence of 200 μM Zn2+ (referred to as WT/Zn2+). Total RNAs from these three samples (WT, Δ310 mutant, and WT/Zn2+) were utilized to perform RT-PCR, and the generated cDNAs were labeled with Cy3 dCTP during reverse transcription. After prehybridization, microarray slides were hybridized with the relevant samples at 65°C for 4 to 16 h (13). This was repeated four times for three samples.

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

Real-time quantitative RT-PCR.

To validate the results of the microarray data, 10 randomly selected genes (Table (Table2)2) were subjected in triplicate to quantitative RT-PCR utilizing Sybr green detection in a Rotor Gene 6000 (Corbett) (75). For each sample (WT, Δ310 mutant, and WT/Zn2+), 2 μg of total RNA was used for first-strand cDNA synthesis with a commercial RT kit (Promega) as recommended by the manufacturer, and the primers (Table (Table2)2) were designed according to the published genomic sequence of 05ZYH33 (8). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. Bacterial DNAs in a dilution series, as standard samples, were applied to generate the standard quantitative curve, and double standard curves were utilized to perform the quantitative analysis of the genes of interest. The average ratio of target genes’ transcription levels was calculated with the software of Rotor Gene 6.0.

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 facilitated the further development of functional genomics. Bioinformatics analysis identified an ORF termed 310 located at bp 302393 to 301941 on the negative strand of the genome (not shown). The deduced product of 310 (named protein 310 here) consisted of 151 amino acids (aa) exhibiting 28.4% and 35.1% amino acid similarity to the Zur proteins of E. coli and M. tuberculosis, respectively (Fig. (Fig.1).1). Further analysis suggested that protein 310 contains a potential DNA-binding motif (helix-turn-helix) at its N terminus (not shown) and possesses nearly 38% α-helices in its secondary structure (Fig. (Fig.1),1), implying that it is possibly a transcription factor.

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 ...

Biochemical characterization of protein 310.

To better understand protein 310, the complete coding sequence of 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 showed that they eluted at approximately same position at ~16.2 ml (~35 kDa) (Fig. (Fig.2A).2A). The samples collected from these two peaks (with and without chemical cross-linking) were separated by 15% SDS-PAGE, which showed that (i) protein 310 has a molecular mass of ~17 kDa, which is consistent with the theoretical estimation of the amino acid content of a monomer (Fig. 2A and B), and (ii) protein 310 exists as a dimer after the chemical cross-linking assay (Fig. (Fig.2A).2A). Protein 310 before and after gel filtration was also subjected to further analysis of chemical cross-linking. The position of protein 310 shifted from monomer to a mixture of monomer and dimer (an intermediate state) to dimer after addition of EGS (Fig. (Fig.2C),2C), suggesting that the 310 protein is indeed a dimer, a typical biochemical characteristic of the Zur family (26). Subsequently, ICP-AES measurement confirmed that protein 310 contains zinc (Table (Table3),3), which is in consistent with structural findings for M. tuberculosis Zur protein (36). The above data provide indirect biochemical evidence that protein 310 is a member of the Zur family.

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 ...
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 Δ310 mutant was obtained from more than 200 S. suis transformants. The correct genotype of the Δ310 mutant was systemically confirmed by multiple approaches, such as PCR (Fig. 3A and B), RT-PCR (Fig. (Fig.3C),3C), Western blotting (Fig. (Fig.3D),3D), and direct DNA sequencing.

FIG. 3.
Identification of the Δ310 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 ...

Several biological properties were addressed, including morphology, hemolytic activity, and sensitivity to H2O2. No obvious morphological difference was observed between the Δ310 mutant and the WT under the culture conditions used (not shown). To further investigate the physiological function of gene 310, six divalent metal ions (Mg2+, Fe2+, Zn2+, Ca2+, Mn2+, and Cu2+) were assessed for their toxicity to S. suis serotype 2. For each of the five ions Mg2+, Ca2+, Mn2+, Cu2+, and Fe2+, both strains (WT and Δ310 mutant) were found to grow well even at an abnormally high concentration (800 μM), indicating that gene 310 was not involved in the tolerance to them. The Δ310 mutant was highly resistant to Fe2+ at the same level the WT, suggesting that 310 is not involved in iron metabolism.

However, the Δ310 mutant grew slowly in THB medium supplemented with 150 and 200 μM Zn2+, whereas the WT and complemented (CΔ310) strains grew well under the same conditions (Fig. (Fig.4A).4A). When the concentration of Zn2+ was increased to 250 μM, the growth rates of both the WT and CΔ310 strains were also hampered, and they all were killed when the concentration of Zn2+ was higher than 300 μM (Fig. (Fig.4A).4A). On the other hand, the biomass of the Δ310 mutant was decreased greatly (Fig. (Fig.4B).4B). Similar sensitivity of the Δ310 mutant to Zn2+ was found in CDM (not shown).

FIG. 4.
Role of gene 310 in the sensitivity of the Δ310 mutant to Zn2+. (A) Sensitivity of the Δ310 mutant to Zn2+. An overculture of each S. suis serotype 2 strain (60 μl, cell density adjusted to an OD600 of 0.7) was ...

Quantitative analysis of bacterial zinc revealed that the zinc content in the Δ310 mutant is obviously higher than that in the WT (growing in either THB or CDM with suppression of Zn2+). This implies that deletion of 310 is responsible in part for the dysfunction of zinc metabolism in Streptococcus suis cells (Table (Table3).3). Indeed, a similar effect has been noted in the zur isogenic mutant of Xanthomonas campestris pv. campestris (62). These data strongly demonstrate that gene 310 is a functional member 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 transcription factor, it was of interest to investigate Zur-mediated, genome-wide regulation in S. suis serotype 2. 2-DE was first applied to compare the differential expression profiles of the Δ310 mutant and the WT. Protein spot 352 was found only in the Δ310 mutant and was identified as an acetyl coenzyme A carboxylase (ACC) beta subunit (Table (Table4).4). Eight other protein spots whose densities in the Δ310 mutant were three times greater than those in the WT were also observed (Table (Table44).

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

The limited information acquired by 2-DE prompted us to employ DNA microarray analysis to further investigate this issue. An expression microarray of S. suis serotype 2 that covers 2,194 genes of 05ZYH33 was designed. In this study, a twofold difference in relative transcription level was selected as a threshold for analysis of microarray data, as described by Ichikawa et al. (27). The effectiveness of the microarray data was confirmed by real-time quantitative RT-PCR (Table (Table5).5). Globally, 5.6% of the genes represented on the microarray (n = 121) were differentially transcribed in the Δ310 mutant compared with the WT. Among them, 72 genes were upregulated, and 49 genes were downregulated (Fig. (Fig.5A).5A). These genes were involved in metabolism, information storage/process, and cellular signaling/defense. For positively regulated genes in the Δ310 mutant, there were 23 genes categorized as proteinase genes, 8 genes related to DNA metabolism/repair, and 5 surface-associated protein genes, as well as many genes for unknown/hypothetical proteins (Table (Table6).6). Likewise, the negatively regulated genes in the Δ310 mutant included the above members. Of note, 10 ABC transporters, which are usually associated with influx and efflux of metabolic molecules (71) and metal ions (6), were positively regulated in the Δ310 mutant, and 2 ABC-type transporters were negatively regulated in the Δ310 mutant (Table (Table6).6). A Zn-dependent NADPH:quinone reductase was revealed to be upregulated in the Δ310 mutant, posing the possibility that zinc ions may be directly competed by protein 310 in bacterial cells (Table (Table5;5; see Table S1 in the supplemental material). Additionally, metal-responsive transcription factors were involved in Zur regulation (Table (Table6;6; see Tables S1 and S2 in the supplemental material). Combined with genome-wide search for zinc-related genes (Table (Table7),7), semiquantitative RT-PCR analysis of the Δ310 mutant prompted us to link gene 310 directly to a zinc ABC transporter (05SSU0112), implying that protein 310 probably functions as a zinc-responsive negative regulator in this case (Fig. (Fig.6),6), which would be in close agreement with E. coli Zur regulator (47).

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 Δ310 mutant compared with the WT. (B) Genome-wide response of S. suis serotype ...
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 ...
TABLE 5.
Real-time quantitative RT-PCR assays of microarray data
TABLE 6.
Genome-wide display of the genes differentially expressed in the Δ310 mutant and S. suis serotype 2 WT/Zn2+
TABLE 7.
Genome-wide search for genes with involvement of zinc ions

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 analyzed by DNA microarray analysis. In total, there were 117 genes whose transcription profiles were affected during the process in response to high levels of Zn2+. Dual regulation was confirmed to relate to stimulus of zinc ion, which included 71 positively regulated genes and 46 negatively regulated genes (Table (Table66 and Fig. Fig.5B).5B). To our surprise, more than 70% of genes affected by zinc in the specific transcriptome of WT/Zn2+ were also affected by deletion of 310, which in turn provides evidence that gene 310 is a novel zinc-dependent regulator. In detail, 52 of the upregulated genes in WT/Zn2+ overlapped with those in the Δ310 mutant (Fig. (Fig.5C),5C), and the downregulated genes in WT/Zn2+ covered 73.7% of those in the Δ310 mutant (Fig. (Fig.5D5D).

DISCUSSION

Streptococcus suis infections are one of the primary and secondary bacterial diseases causing serious economic losses in pig industries worldwide each year (58, 64). These pathogens are generally categorized into 35 serotypes on the basis of the difference in their capsular antigens, of which serotype 2 is a prevalent serotype most frequently isolated from diseased piglets and human patients (58). There is an increasing amount of data suggesting that 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 and antimicrobials has been hampered partly due to the increased tolerance of S. suis serotype 2 to antimicrobials together with current limited knowledge on its pathogenesis (38). Therefore, a better understanding of the different aspects of S. suis serotype 2, especially pathogenesis, will facilitate the development of effective therapeutics which can prevent and/or treat S. suis serotype 2 infections.

We identified gene 310 from Streptococcus suis, encoding a functional member of the Zur family. Bioinformatics analysis revealed that this protein comprises two conserved domains in the family of Fur/Zur proteins: a hypothetical DNA-binding motif at its N terminus and a putative C-terminal dimerizing module (2). Sensitivity assays with divalent transition metal ions demonstrated that gene 310 is a zinc-responsive regulator gene, referred to as zur (Fig. (Fig.4).4). Our biochemical data (Fig. (Fig.2)2) showed that the full-length product of gene 310 exists as a dimer, which is consistent with the crystal structures (48) and previous biochemical analysis (2, 12) of other members of this superfamily. Although the deletion of fur/zur was found to impair the growth of Actinobacillus pleuropneumoniae (28), the Δ310 mutant exhibited growth similar to that of its parental strain (not shown). Subsequent microarray results provided one possible explanation for this, in that the overexpression of Era, which is required for bacterial growth (45), may remedy the defect caused by the deletion of gene 310 in the Δ310 mutant to some extent. The inactivation of gene 310 seemed to not affect the bacterial phenotype, such as hemolytic activity. In contrast to those regulators sensing oxidative stress in this family, e.g., PerR (31, 69), our data did not support the involvement of gene 310 in H2O2 sensitivity (not shown). Retrospectively, Dpr (Dps-like peroxide resistance protein) was suggested to confer on S. suis serotype 2 resistance to H2O2 (52). However, the role of zur in pathogenesis is controversial (7, 34, 62) In our piglet infections, zur had no apparent effect on S. suis serotype 2 virulence, which is consistent with results seen for S. aureus (34).

Given that Zur is a global regulator in bacteria, we tried to dissect its regulatory networks at the levels of the proteome and transcriptome. At the proteomics level, we identified only nine protein spots (Table (Table4).4). One protein dot (no. 352) that is present only in the Δ310 mutant was confirmed to be the beta subunit of ACC. ACC is a central metabolic enzyme that catalyzes the committed step in fatty acid biosynthesis. Recently, the ACC structures of both S. aureus and E. coli revealed a zinc-binding motif (5). Therefore, it is reasonable that Zur-mediated downregulation of ACC can be attributed to its interaction with zinc. On the other hand, there are five other proteins whose expression level in the Δ310 mutant was three times higher than that in the WT. First, it is reasonable that the two proteins (ribosome-binding factor A [spot 24] and GTP-binding translation factor [spot 427]) were derepressed in the Δ310 mutant. Several studies have indicated that zinc is required for the structural stability of translation initiation factors that generally contain ribosome-binding domains (22, 35). It is possible that ribosome-binding factors compete with a translation initiation factor for the resources of ribosomes (19, 21). Second, Era (a GTP-binding protein) was revealed by 2-DE to be negatively regulated by Zur. Era has been demonstrated to interact with 16S rRNA (23) and mediate the assembly of the 30S ribosome unit (54), and ribosomal proteins have been suggested to be related to Zur protein and zinc ions (46, 55). Therefore, it is logical that Era can be linked to Zur in S. suis serotype 2. Certainly, the total number of affected proteins elucidated by 2-DE seemed to be much less than that one might expect.

Further microarray analysis showed that the gene 310-mediated dual-regulation network is globally involved in 121 genes, of which 72 were upregulated and 49 were downregulated in the Δ310 mutant (Fig. (Fig.5A).5A). Most genes affected by zur encode putative proteinases, of which 23 were upregulated and 12 were downregulated (Table (Table6).6). They included some Zn2+-dependent members (e.g., NADPH:quinone reductase and lipase) involved in the basic metabolism of carbohydrates, nucleic acids, and fatty acids (10). There were more than 20 unknown genes which are influenced by gene 310. Membrane/surface proteins have been suggested to contribute greatly to the invasiveness and immunogenicity of pathogens (16, 41). We observed that they are also subject to dual regulation by Zur (some are stimulated, and others are repressed). In particular, a hyaluronidase with an LPXTG motif, which plays roles in pathogenicity of group A streptococci (60), was also found to be negatively regulated by Zur. However, cps2C, which is involved in the production of capsular polysaccharide, a virulence determinant of S. suis serotype 2 (56), is positively regulated by Zur. It seemed that virulence manifestation is linked to the regulation of Zur in S. suis. Transcription factors and activators (e.g., alsR and plcR) were also affected by Zur, implying the existence of indirect regulation mediated by Zur in S. suis serotype 2 (40, 74).

The transcriptome of S. suis serotype 2 under the control of Zn2+ exhibited 117 differentially expressed genes compared with that under normal culture condition (Fig. (Fig.5B).5B). Intriguingly, we noticed that more than 70% of the genes regulated in response to Zn2+ overlapped with those regulated by Zur protein in S. suis serotype 2 (Fig. (Fig.5C).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 greatly in response to the emerging stress of zinc ions at a high level (73), which is similar to what is observed in the Δ310 mutant. Second, not only were most ABC-type transporters regulated by Zn2+, but membrane proteins, proteinases, and unknown proteins were also affected, which in turn validates the Zur-mediated regulation network. It is well known that MscS (the mechanosensitive channel of small conductance) plays a critical role in osmoregulation in prokaryotic microorganisms (3, 65). Similarly, microarray results revealed that treatment with Zn2+ can repress the transcription of mscS in S. suis serotype 2. Unexpectedly, MreC, a cell shape determinant (29, 30), was found to be positively regulated but not negatively regulated by Zur, suggesting that S. suis serotype 2 has evolved morphological machineries to adapt to the dramatic change of environmental zinc ions. In addition, transcription regulators were affected by zinc, suggesting that their target genes may be indirectly controlled by Zur (40).

In summary, we defined a functional zur gene from S. suis serotype 2. We attempted to define the genome-wide regulation network of Zur by 2-DE and DNA microarray analysis. Based on microarray analysis of the Δ310 mutant compared with S. suis serotype 2 treated with zinc ions, we gained, for the first time, a glimpse of the cross talk between gene 310 and Zn2+, which in turn provides genome-wide evidence that gene 310 is a functional member of the zur family.

Supplementary Material

[Supplemental material]

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 Daisuke Takamatsu, National Institute of Animal Health, Japan, for kindly providing 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 Technologies R&D Program (2006BAD06A04). George F. Gao is a distinguished young investigator of the NSFC (grant no. 30525010).

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 August 2008.

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

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