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. 2015 Dec 8:5:91.
doi: 10.3389/fcimb.2015.00091. eCollection 2015.

The Regulation of the AdcR Regulon in Streptococcus pneumoniae Depends Both on Zn(2+)- and Ni(2+)-Availability

Irfan Manzoor  1 , Sulman Shafeeq  2 , Muhammad Afzal  1 , Oscar P Kuipers  3
Affiliations

The Regulation of the AdcR Regulon in Streptococcus pneumoniae Depends Both on Zn(2+)- and Ni(2+)-Availability

Irfan Manzoor et al. Front Cell Infect Microbiol. .

Abstract

By using a transcriptomic approach, we have elucidated the effect of Ni(2+) 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 Ni(2+). 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 Ni(2+). We have further studied the role of Ni(2+) in the regulation of the AdcR regulon by using ICP-MS analysis, electrophoretic mobility shift assays and transcriptional lacZ-reporter studies, and demonstrate that Ni(2+) is directly involved in the derepression of the AdcR regulon via the Zn(2+)-dependent repressor AdcR, and has an opposite effect on the expression of the AdcR regulon compared to Zn(2+).

Keywords: AdcR; AdcR regulon; Pht family proteins; PsaR regulon; metal homeostasis; nickel; pneumococcus; zinc.

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Figures

Figure 1
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 Ni2+. 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).
Figure 2
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-Zn2+ (Zn2+-depleted medium) (B) with different added concentrations of Ni2+. 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).
Figure 3
Figure 3
Expression level (in Miller units) of the D39 wild-type containing transcriptional lacZ-fusions to PadcR (A), PadcAII (B), PphtA (C), PphtB (D), and PphtE (E), grown in CDM with or without addition of different concentrations of Ni2+ and Zn2+. 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 (*P < 0.05, **P < 0.001, and ***P < 0.0001).
Figure 4
Figure 4
Expression level (in Miller units) of the adcR mutant containing transcriptional lacZ-fusions to PadcR, PadcAII, PphtA, PphtB, and PphtE grown in CDM with or without addition of 0.5 mM Ni2+. 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).
Figure 5
Figure 5
In vitro interaction of Ad-Strep tag with the promoter regions of adcR (A), adcAII (B), phtA (C), phtB (D), and pcpA (E). Ad-Strep was added at a concentration of 30 nM as indicated above panel, while lane 1 is without added protein. Arrows indicate the position of shifted probe and asterisks indicate the position of free probe. 0.2 mM Zn2+ was added in lanes 3, 6, and 7. Whereas, Ni2+ was added at the concentration of 0.2 mM in lane 4 and 6, and 0.4 mM in lanes 5 and 7.
Figure 6
Figure 6
Expression level (miller units) of the D39 wild-type containing transcriptional lacZ-fusion to PczcD grown in CDM with different added concentrations of Ni2+. 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 and ***P < 0.0001).
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