The Regulation of the AdcR Regulon in Streptococcus pneumoniae Depends Both on Zn2+- and Ni2+-Availability

By using a transcriptomic approach, we have elucidated the effect of Ni2+ on the global gene expression of S. pneumoniae D39 by identifying several differentially expressed genes/operons in the presence of a high extracellular concentration of Ni2+. The genes belonging to the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE) and the PsaR regulon (pcpA, prtA, and psaBCA) were highly upregulated in the presence of Ni2+. We have further studied the role of Ni2+ in the regulation of the AdcR regulon by using ICP-MS analysis, electrophoretic mobility shift assays and transcriptional lacZ-reporter studies, and demonstrate that Ni2+ is directly involved in the derepression of the AdcR regulon via the Zn2+-dependent repressor AdcR, and has an opposite effect on the expression of the AdcR regulon compared to Zn2+.


INTRODUCTION
In bacteria, the transition metal ions play an important role in the proper functioning of many enzymes, transporters, and transcriptional regulators. Transition metal ions are the prerequisite for the proper bacterial growth at low concentrations, but metal ions can be lethal at higher concentrations (Blencowe and Morby, 2003;Finney and O'Halloran, 2003;Ge et al., 2012). Therefore, proper homeostasis of metal ions is very important for the survival of bacteria, which is maintained by the dedicated metal transport-and efflux-systems (Tottey et al., 2008;Waldron and Robinson, 2009;Lisher et al., 2013). These systems are tightly regulated by metal-responsive transcriptional regulators to ensure the proper functioning of the cell by maintaining the minimum levels of metal ions inside the cell.
Streptococcus pneumoniae is one of the most common human pathogens that reside asymptomatically in the human nasopharynx (Mitchell, 2003). However, it may occasionally translocate to the lungs, the eustachian tube, the blood, and the nervous system, causing pneumoniae, otitis media, bacteremia, and meningitis, respectively (Obaro and Adegbola, 2002;Bogaert et al., 2004). During translocation from the nasopharynx to other infection sites, S. pneumoniae may encounter different environmental conditions including varying metal ions concentrations, which might affect the expression of different genes including virulence genes (Gupta et al., 2009;Shafeeq et al., 2011bShafeeq et al., , 2013Plumptre et al., 2014a). However, the exact conditions that S. pneumoniae might face during infections, are poorly understood.
Ni 2+ is an essential micronutrient for certain bacteria, due to its role in various cellular processes like methane formation, hydrolysis of urea, and consumption of molecular hydrogen (Chen and Burne, 2003;Mulrooney and Hausinger, 2003;Rodionov et al., 2006;Anwar et al., 2007). In Escherichia coli, the nik operon (nikABCDE) involved in the transport of Ni 2+ is shown to regulate by transcriptional regulator NikR (De Pina et al., 1999). Moreover, the expression of NmtA, an ATPdependent transporter involved in the efflux of Ni 2+ and Co 2+ , is tightly regulated by Ni 2+ -responsive transcriptional regulator NmtR in Mycobacterium tuberculosis (Cavet et al., 2002). Ni 2+ is also shown to regulate the expression of urease activity in Streptococcus salivarius and Helicobacter pylori (van Vliet et al., 2001;Chen and Burne, 2003). The amount of Ni 2+ in the human blood is estimated to be 0.83 ng ml −1 (Alimonti et al., 2005) and it is likely that S. pneumoniae may encounter Ni 2+ during infection in blood. So far, very little is known about the impact of Ni 2+ on the global gene expression of S. pneumoniae. Previously, the role of Ni 2+ in the regulation of the Zn 2+ -efflux system czcD was reported (Kloosterman et al., 2007). It was shown that the SczA-mediated expression of czcD was highly increased in the presence of Zn 2+ , Co 2+ , or Ni 2+ (Kloosterman et al., 2007). Moreover, a number of proteins and motif with Co 2+ -and Ni 2+ -binding capacity has been identified by Immobilized metal affinity column (IMAC) and LTQ-Orbitrap mass spectrometry (MS) that have diverse functions in the S. pneumoniae (Sun et al., 2013). In a recent study, we demonstrated the role of Ni 2+ in regulation of the PsaR regulon and showed that Ni 2+ not only alleviates the Mn 2+ -dependent binding of PsaR to the promoter regions of the PsaR regulon genes, but also cause Mn 2+ deficiency possibly by blocking Mn 2+ -uptake via PsaA, hence leading to the high expression of the PsaR regulon in the presence of Ni 2+ (Manzoor et al., 2015b).
In this current study, we used a transcriptomic analysis approach for the identification of differentially expressed genes/operons in response to high extracellular Ni 2+ in S. pneumoniae. The expression of genes belonging to the AdcR regulon and the PsaR regulon was highly upregulated in the presence of Ni 2+ . We further studied the role of Ni 2+ in the AdcR-mediated regulation of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE by using transcriptional lacZ-reporter studies, inductively coupled plasma-mass spectrometry (ICP-MS) analysis and electrophoretic mobility shift assays (EMSAs), and showed that Ni 2+ and Zn 2+ play an opposite role in the regulation of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE.

Bacterial Strains and Media
Bacterial strains used in this study are listed in Table 1. Growth of bacteria and DNA manipulation were performed as described (Shafeeq et al., 2011a;Manzoor et al., 2015a). All experiments in this study were performed in chemically defined medium (CDM).

DNA Microarray and Data Analysis
For microarray analysis in response to Ni 2+ , S. pneumoniae D39 wild-type was grown in two biological replicates in CDM with and without the addition of 0.5 mM NiSO 4. 6H 2 O. To analyze the impact of adcR deletion on the transcirptome of S. pneumoniae in the presence of Ni 2+ , D39 wild-type and adcR (SS200) (Shafeeq et al., 2011a) were grown in two biological replicates in CDM with 0.3 mM of NiSO 4. 6H 2 O. All other procedures regarding microarray experiments and data analysis were done as described before (Shafeeq et al., 2011b;Afzal et al., 2015). For the identification of differentially expressed genes a Bayesian p < 0.001 and a fold change cut-off of 2 was applied. The DNA microarray data have been submitted to gene expression omnibus (GEO) database under the accession number GSE73852.

Construction of Transcriptional lacZ-fusions and β-galactosidase Assays
Chromosomal transcriptional lacZ-fusions to the promoter regions of adcR, adcAII, phtA, phtB, and phtE were constructed in plasmid pPP2 (Halfmann et al., 2007) with the primer pairs listed in Table 2, resulting in pIM501-505. These plasmids were introduced into D39 wild-type and adcR (SS200) (Shafeeq et al., 2011a) resulting in strains IM501-505 and IM551-554, respectively. All plasmids were checked for the presence of correct insert by means of PCR and DNA sequencing. For βgalactosidase activity, the derivatives of S. pneumoniae were grown in triplicate in CDM supplemented with different metal ion concentrations (w/v) mentioned in the Results and harvested at the mid-exponential growth phase. The β-galactosidase activity was measured as described before (Kloosterman et al., 2006). Standard deviations were calculated from three independent replicates of each sample.

Inductively Coupled Plasma-mass Spectrometry (ICP-MS) Analysis
To determine the cell-associated concentration of metal ions, an ICP-MS analysis was performed on the cells grown in triplicates in CDM with and without the addition of 0.5 mM Ni 2+ till the mid-exponential growth phase. Cell cultures were centrifuged at 4 • C and washed twice with overnight Chelex (Sigma) treated phosphate-buffered saline (PBS) with 1 mM nitrilotriacetic acid. Cells were dried overnight in a Speedvac at room temperature. The dried cells were dissolved in 2.5% nitric acid (Ultrapure, Sigma Aldrich) and lysed at 95 o C for 10 min by vigorous vortexing after each 30 s. The lysed cell samples were used for ICP-MS analysis as described (Jacobsen et al., 2011). Metal ion concentrations were expressed as µ g g −1 dry weight of cells.

Overexpression and Purification of Strep-tagged AdcR
The nisin-inducible (NICE) expression system (Kuipers et al., 1998) in Lactococcus lactis strain NZ9000 was used for the overexpression of C-terminally Strep-tagged AdcR (Shafeeq et al., 2011a). Cells were grown until an OD 600 of 0.4 in 1 L culture followed by the induction with 10 ng ml −1 nisin. The purification of AdcR-Strep tag was performed using the Streptactin column from IBA according to the supplier's instructions (www.iba-go.com). The purified protein was eluted in buffers without EDTA and stored at a concentration of 0.5 mg/ml in the elution buffer (100 mM Tris-HCl [pH 8], 150 mM NaCl, 2.5 mM desthiobiotin, and 1 mM β-mercaptoethanol) with 10% glycerol at −80 • C.

Identification of Ni 2+ -dependent Genes in S. pneumoniae
To investigate the impact of Ni 2+ on the transcriptome of S. pneumoniae, a DNA microarray-based comparison of D39 wildtype grown in CDM with 0.5 mM Ni 2+ to the same strain grown in CDM with 0 mM Ni 2+ was performed. Table 3 summarizes the list of differentially expressed genes in the presence of 0.5 mM Ni 2+ . The PsaR regulon consisting of the operon psaBCA (encoding Mn 2+ -dependent ABC transporters, PsaBCA), pcpA (encoding a choline binding protein, PcpA), and prtA (encoding a serine protease PrtA) were highly upregulated in the presence of  (Hoskins et al., 2001;Lanie et al., 2007).
Ni 2+ . The Ni 2+ -dependent upregulation of the PsaR regulon in the presence of Ni 2+ is consistent with our recent study, where we have explored the Ni 2+ -dependent regulation of the PsaR regulon in more details (Manzoor et al., 2015b). Expression of a gene cluster including the cation efflux system gene czcD, the MerR family transcriptional regulator, and the Zn 2+ -containing alcohol dehydrogenase adhB was increased more than 35-fold in FIGURE 1 | Cell-associated metal ion concentrations (expressed ug g −1 ) of S. pneumoniae D39 wild-type when grown in CDM with either 0 mM or 0.5 mM Ni 2+ . The statistical significance of the differences in the mean metal ion concentrations was determined by One-way ANOVA (NS not significant, *P < 0.05, and **P < 0.001).
the presence of Ni 2+ . The cation efflux system CzcD was shown to protect S. pneumoniae against the intracellular Zn 2+ -stress (Kloosterman et al., 2007). A novel TetR family transcriptional regulator SczA has been shown to activate the expression of czcD in the presence of Zn 2+ , Co 2+ , or Ni 2+ (Kloosterman et al., 2007). Therefore, the upregulation of czcD in our transcriptomic analysis is consistent with the finding presented in previous study (Kloosterman et al., 2007). Furthermore, genes encoding a heat shock protein (HtpX) and a Dpr homolog (spd_1402) were also differentially expressed. The Dpr protein has been shown to protect bacterial cells from oxidative stress (Pulliainen et al., 2003). The genes belonging to the AdcR regulon were also upregulated in the presence of Ni 2+ . The expression of the adc operon was 4-fold upregulated. The expression of adcAII-phtD operon was upregulated 2-fold. The expression of other genes encoding for Pht family proteins (PhtA and PhtE), was upregulated more then 7-fold. Previously, it was shown that the expression of the AdcR regulon is repressed by the transcriptional regulator AdcR in the presence of Zn 2+ (Shafeeq et al., 2011a). Transcriptome data was further validated by qRT-PCR analysis (Supplementary data: Table S1). Upregulation of the AdcR regulon in the presence of Ni 2+ might also indicate the putative role of Ni 2+ in the regulation of the AdcR regulon by the transcriptional regulator AdcR. Therefore, we decided to further explore the role of Ni 2+ in the regulation of the AdcR regulon and to determine the intracellular concentrations of metal ions in S. pneumoniae D39 grown in the presence of either 0.5 mM Ni 2+ or 0 mM Ni 2+ in CDM.
S. pneumoniae Accumulates More Ni 2+ When Grown in the Presence of 0.5 mM Ni 2+ To investigate whether the observed transcriptomic responses correlated with high cell-associated concentration of Ni 2+ , we performed an ICP-MS analysis on the same conditions used for performing the transcriptome analysis, i.e., cells grown either in the presence of 0.5 mM Ni 2+ or 0 mM Ni 2+ in CDM. Our ICP-MS data revealed that the cells grown in the presence of 0.5 mM Ni 2+ accumulate 30-fold more cell-associated Ni 2+ compared to the cells grown in 0 mM Ni 2+ (30 µg g −l dry mass of cells vs. <1 µg g −l dry mass of cells) (Figure 1). Moreover, 2.6fold decrease in the cell-associated concentration of Mn 2+ was observed. The cell-associated concentration of other metal ions was not changed in the presence of 0.5 mM Ni 2+ compared to 0 mM Ni 2+ . Therefore, it is likely that the transcriptomic changes observed in the presence of 0.5 M Ni 2+ are due to the high intracellular concentration of Ni 2+ .

Ni 2+ -dependent Expression of the AdcR Regulon
To explore the transcriptional regulation of the genes/operons belonging to the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE) found in our microarray analysis, transcriptional lacZ-fusions were constructed to the promoter regions of adcR, adcAII, phtA, phtB, and phtE in plasmid pPP2 (Halfmann et al., 2007) and transferred to S. pneumoniae D39 wild-type. The expression of PadcR-lacZ, PadcAII-lacZ, PphtA-lacZ, PphtB-lacZ, and PphtE-lacZ was measured in CDM and CDM-Zn 2+ (Zn 2+ depleted medium) with the addition of 0, 0.1, 0.3, or 0.5 mM Ni 2+ . As AdcR represses the expression of the AdcR regulon in the presence of Zn 2+ , we also used Zn 2+ -depleted medium (CDM-Zn 2+ ). β-galactosidase activity (Miller Units) showed that the elevated concentration of Ni 2+ led to the high expression of all these promoters in CDM and CDM-Zn 2+ (Figures 2A,B). However, the expression of these promoters was much higher in CDM-Zn 2+ compared to CDM. The full CDM contains minor amounts of Zn 2+ (around 883 µg l −1 ) (Manzoor et al., 2015a), which could explain the lower expression of these promoters in CDM compared to CDM-Zn 2+ . This data not only suggests the role of Ni 2+ in the regulation of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE, but also indicate the ability of Ni 2+ to derepress the Zn 2+ -dependent repression of these genes.
Opposite Effect of Zn 2+ and Ni 2+ on the Expression of the AdcR Regulon β-galactosidase activities shown above indicate that Ni 2+ might compete with Zn 2+ and that both metal ions have opposite effects FIGURE 2 | Expression level (in Miller units) of the D39 wild-type containing transcriptional lacZ-fusions to PadcR, PadcAII, PphtA, PphtB, and PphtE, grown in CDM (A) and CDM-Zn 2+ (Zn 2+ -depleted medium) (B) with different added concentrations of Ni 2+ . Standard deviation of three independent replications is indicated with error bars. Statistical significance of the differences in the expression levels was determined by One-way ANOVA (NS, not significant, *P < 0.05, **P < 0.001, and ***P < 0.0001).
To elucidate the Ni 2+ -dependent role of AdcR in more details and find more targets of AdcR in the presence of Ni 2+ , microarray comparison of the adcR mutant with D39 wildtype was performed in CDM with 0.3 mM Ni 2+ . As expected, the expression of genes belonging to the AdcR regulon was highly upregulated (Table 4), except for the adc operon, which was downregulated in our transcriptome analysis ( Table 4). For creating an adcR mutant in previous study, an erythromycinresistance gene cassette was used to replace the adcR gene (Shafeeq et al., 2011a). Therefore, downregulation of the adc operon might be due to the polar effect of adcR deletion on the downstream genes of adcR (Shafeeq et al., 2011a). We further validated our DNA microarray data by qRT-PCR. qRT-PCR data is also in agreement with our transcriptome data (Supplementary data: Table S2).

Binding of AdcR to Its Target Is Zn 2+ -and Ni 2+ -dependent
To study the direct interaction of AdcR with the promoter regions of the genes belonging to the AdcR regulon in the   (Hoskins et al., 2001;Lanie et al., 2007). c Ratios >2.0 or <2.0 (SS200 + 0.3 mM Ni 2+ /wild-type + 0.3 mM Ni 2+ ).
presence of Ni 2+ , we performed EMSAs with purified Streptagged AdcR (Ad-Strep tag) and 33 P-labeled promoters of adcR, adcAII, phtA, phtB, and pcpA. To prevent the interference of metal ions with Ad-Strep tag, all the experiments were performed in EDTA free gels and buffers. The pcpA promoter region was taken as a negative control. Ad-Strep tag was unable to shift the promoter regions of adcR, adcAII, phtA, and phtB in the absence of metal ions (Lane 2 in Figure 5). However, the addition of 0.2 mM Zn 2+ led to the binding of Ad-Strep tag to the promoter regions of adcR, adcAII, phtA, and phtB (Lane 3 in Figures 5A-D), which is consistent with our previous study (Shafeeq et al., 2011a). Interestingly, 0.2 and 0.4 mM Ni 2+ were unable to stimulate the binding of Ad-Strep tag with the promoter regions of adcR, adcAII, phtA, and phtB (Lane 4 and 5 in Figures 5A-D). In our transcriptome data mentioned above, Ni 2+ showed a derepressive effect on the expression of the AdcR regulon. Therefore, we also decided to check the interaction of Ad-Strep tag with the promoter regions of adcR, adcAII, phtA, and phtB in the presence of both Zn 2+ and Ni 2+ together. The Zn 2+ -dependent interaction of AdcR with these promoters in the presence of 0.2 mM Zn 2+ was alleviated with the addition of 0.2 mM or 0.4 mM Ni 2+ (Lane 6 and 7 in Figures 5A-D).
Under the same conditions, we did not see any band shift with the promoter region of pcpA as a negative control ( Figure 5E). Thus, this data indicates that Zn 2+ and Ni 2+ have an opposite effects on the interaction of AdcR with the promoter regions of adcR, adcAII, phtA, and phtB.
Effect of Ni 2+ on SczA-mediated Expression of the Zn 2+ -efflux system czcD To investigate the regulation of czcD in the presence of Ni 2+ , we studied the transcriptional response of PczcD-lacZ grown in complete CDM with the addition of different concentrations of Ni 2+ . β-galactosidase assays showed that PczcD-lacZ responded to Ni 2+ and its expression was highly increased with an increasing concentration of Ni 2+ (Figure 6). This data is in agreement with our transcriptomic data mentioned above and suggests the putative role of CzcD in Ni 2+ homeostasis.

DISCUSSION
Transition metal ions such as Mn 2+ , Zn 2+ , Cu 2+ , Fe 2+ , Co 2+ , and Cd 2+ have been shown to play a pivotal role in the metabolism and virulence of S. pneumoniae (Brown et al., 2001;Kloosterman et al., 2008;Shafeeq et al., 2011b;Begg et al., 2015). However, the role of Ni 2+ on the global gene expression of S. pneumoniae has not been studied before. In this study, we analyze the transcriptome changes in S. pneumoniae D39 wildtype in response to high Ni 2+ concentration. The expression of a number of important genes and operons with diverse functions, including the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE), the PsaR regulon (pcpA, prtA, and psaBCA) regulon, and the Zn 2+ -efflux system czcD were significantly altered in the presence of Ni 2+ . We further studied the role of Ni 2+ in the regulation of the AdcR regulon and demonstrated that Ni 2+ plays an opposite role compared to Zn 2+ in the regulation of the AdcR regulon.
The AdcR regulon consists of adcRCBA, adcAII-phtD, phtA, phtB, phtE, and adhC in S. pneumoniae. The adc operon (adcRCBA) is involved in Zn 2+ acquisition, and encodes for a Zn 2+ -responsive MarR family transcriptional regulator, AdcR, two ABC transporter proteins AdcC and AdcB, and an extracellular Zn 2+ -binding protein AdcA Bayle et al., 2011). The adcAII gene encodes an adhesion lipoprotein which has an overlapping specificity with AdcA for Zn 2+ (Bayle et al., 2011). AdcAII belongs to the LraI-lipoprotein family and is organized in an operon with a phtD gene encoding pneumococcal histidine triade protein precursor D (PhtD). phtA, phtB, and phtE encodes for pneumococcal histidine triade protein A, B, and E, respectively. Recent studies have demonstrated the role of the PhT family proteins (PhtA, PhtB, PhtE, and PhtD) in intracellular Zn 2+ acquisition and pathogenesis in S. pneumoniae (Hava and Camilli, 2002;Ogunniyi et al., 2009;Plumptre et al., 2014b). The adhC gene encodes for a Zn 2+ -containing alcohol dehydrogenase. Previously, it was demonstrated that the expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE is repressed, while the expression of adhC is activated by the transcriptional regulator AdcR in the presence of Zn 2+ (Shafeeq et al., 2011a). Here, we show that Ni 2+ also plays a role in the regulation of adcRCBA, adcAII-phtD, phtA, phtB, and phtE. Our β-galactosidase assays showed that the expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE was increased with increasing concentrations of Ni 2+ . However, we did not find any significant change in the expression of adhC in our both transcriptome analysis performed in this study. This might exclude the role of Ni 2+ in the AdcR mediated regulation of adhC.
High concentrations of Ni 2+ can be very toxic for bacteria (Macomber and Hausinger, 2011). Therefore, bacteria must limit the toxic amount of Ni 2+ to perform normal cellular functions. In many bacteria, CDF-family efflux pumps help to maintain proper concentrations of heavy metals in the cell. For example, in Bacillus subtilis, the CzcD heavy metal efflux pump is involved in the homeostasis of Zn 2+ , Co 2+ , Cu 2+ , and Ni 2+ , and is regulated by CzrA . It is also important to note that the expression of czcD is highly upregulated in our transcriptome analysis in response to Ni 2+ . Expression of czcD is regulated by the TetR family transcriptional regulator SczA in the presence of Zn 2+ , Co 2+ , or Ni 2+ (Kloosterman et al., 2007). Moreover, Zn 2+ , Co 2+ , or Ni 2+ has been shown to stimulate the binding of SczA to the promoter region of czcD (Kloosterman et al., 2007). In this study, we further confirmed the expression of czcD in the presence of Ni 2+ by transcriptional lacZ-reporter study with PczcD-lacZ and our results are consistent with a previous study (Kloosterman et al., 2007).
The PsaR regulon consists of psaBCA, pcpA, and prtA that encodes for the Mn 2+ uptake system (PsaBCA), a choline binding protein (PcpA), and a serine protease (PrtA), respectively. The expression of the PsaR regulon is shown to be repressed by the DtxR family transcriptional regulator PsaR in the presence of Mn 2+ (Johnston et al., 2006). Notably, Zn 2+ and Co 2+ can bind with PsaR to relieve the Mn 2+ -dependent repression of the PsaR regulon (Kloosterman et al., 2008;Manzoor et al., 2015a). Recently, we have studied the regulation of the PsaR regulon in the presence of Ni 2+ and demonstrated that like Zn 2+ and Co 2+ , Ni 2+ also has the ability to derepress the Mn 2+ -dependent repression of the PsaR regulon, and that high concentrations of Ni 2+ leads to cell-associated Mn 2+ deficiency (Manzoor et al., 2015b). In this study, we have also observed the significant upregulation of the PsaR regulon in our transcriptome analysis performed in the presence of Ni 2+ ( Table 3). Upregulation of the PsaR regulon in our transcriptome further verifies our previous results (Manzoor et al., 2015b). Moreover, we have also observed the cell-associated deficiency of Mn 2+ in our ICP-MS analysis performed in this study (Figure 1), which is also in consistent with our previous results (Manzoor et al., 2015b).
The interplay, or competition, of metal ions plays an important role in the regulation of metal responsive genes. In S. pneumoniae, competition of Mn 2+ with Zn 2+ , Co 2+ , or Ni 2+ in the regulation of the PsaR regulon by transcriptional regulator PsaR has already extensively been studied (Kloosterman et al., 2008;Manzoor et al., 2015a,b). Similarly, the interplay of Cu 2+ and Zn 2+ in the regulation of cop operon by transcriptional regulator CopY was studied before, where Cu 2+ induces and Zn 2+ represses the CopY-mediated expression of cop operon (Shafeeq et al., 2011b). Here, we elaborated for the first time the interplay of Ni 2+ and Zn 2+ in the regulation of genes belonging to the AdcR regulon. Our lacZ-reporter studies determined the ability of Ni 2+ , in derepressing the Zn 2+ -dependent repression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE. Our in vitro data showed that the Zn 2+ -dependent binding of AdcR to the promoter regions of the genes belonging to the AdcR regulon was alleviated by the addition of Ni 2+ . Recently, it has been shown that Cd 2+ -uptake reduces the accumulation of cell-associated Mn 2+ and Zn 2+ (Begg et al., 2015). Our ICP-MS comparison of cells grown in CDM with 0.5 mM to 0 mM Ni 2+ has not shown any difference in the concentration of Zn 2+ or other metal ions, which also indicates the direct role of Ni 2+ in the regulation of adcRCBA, adcAII-phtD, phtA, phtB, and phtE. Moreover, the role of genes belonging to the AdcR regulon in the pathogenesis of S. pneumoniae has already been demonstrated, which also suggests the important role of Ni 2+ in pneumococcal virulence.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fcimb. 2015.00091