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. 2008 Aug;190(15):5382-93.
doi: 10.1128/JB.00307-08. Epub 2008 May 30.

Opposite effects of Mn2+ and Zn2+ on PsaR-mediated expression of the virulence genes pcpA, prtA, and psaBCA of Streptococcus pneumoniae

Tomas G Kloosterman  1 , Robert M Witwicki, Magdalena M van der Kooi-Pol, Jetta J E Bijlsma, Oscar P Kuipers
Affiliations

Opposite effects of Mn2+ and Zn2+ on PsaR-mediated expression of the virulence genes pcpA, prtA, and psaBCA of Streptococcus pneumoniae

Tomas G Kloosterman et al. J Bacteriol. 2008 Aug.

Abstract

Homeostasis of Zn(2+) and Mn(2+) is important for the physiology and virulence of the human pathogen Streptococcus pneumoniae. Here, transcriptome analysis was used to determine the response of S. pneumoniae D39 to a high concentration of Zn(2+). Interestingly, virulence genes encoding the choline binding protein PcpA, the extracellular serine protease PrtA, and the Mn(2+) uptake system PsaBC(A) were strongly upregulated in the presence of Zn(2+). Using random mutagenesis, a previously described Mn(2+)-responsive transcriptional repressor, PsaR, was found to mediate the observed Zn(2+)-dependent derepression. In addition, PsaR is also responsible for the Mn(2+)-dependent repression of these genes. Subsequently, we investigated how these opposite effects are mediated by the same regulator. In vitro binding of purified PsaR to the prtA, pcpA, and psaB promoters was stimulated by Mn(2+), whereas Zn(2+) destroyed the interaction of PsaR with its target promoters. Mutational analysis of the pcpA promoter demonstrated the presence of a PsaR operator that mediates the transcriptional effects. In conclusion, PsaR is responsible for the counteracting effects of Mn(2+) and Zn(2+) on the expression of several virulence genes in S. pneumoniae, suggesting that the ratio of these metal ions exerts an important influence on pneumococcal pathogenesis.

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Figures

FIG. 1.
FIG. 1.
Identification of a putative PsaR operator. (A) Weight matrix of the identified PsaR operator as present in the promoter regions of pcpA, prtA, and psaB. (B) Positions of the PsaR operator (shaded) in the promoter regions of pcpA, prtA, and psaB. Putative core promoter sequences are in bold. The ribosome binding sites are in bold and underlined. Start codons are in italic.
FIG. 2.
FIG. 2.
Subcloning of PpcpA. A schematic overview of the PpcpA truncations is shown. Numbers indicate the positions of the truncations, which were fused to lacZ, relative to the putative pcpA start site (+1). The flag indicates the position of the core promoter, and the oval indicates the putative PsaR operator. Gray-shaded areas indicate regions of similarity with PprtA (89% identity for the short stretch and 82% identity for the long stretch). The table on the right gives β-galactosidase activities (Miller units) of the promoter truncations in wild-type D39 (wt, strains RW131 to RW134) and the ΔpsaR mutant (psaR, strains RW141 to RW144) grown in GM17 and in GM17 plus 0.5 mM Zn2+. Standard deviations of three measurements are given in parentheses.
FIG. 3.
FIG. 3.
In vitro interaction of PsaR-Strep with the pcpA (A), psaB (B), and prtA (C) promoter regions and with a truncated PpcpA fragment lacking the PsaR operator (D). Purified PsaR-Strep was added at concentrations of 5 nM (PpcpA), 11 nM (PprtA), and 25 nM (PpsaB). Metal ions were added as indicated above the lanes at a concentration of 50 μM. X, free probe. The horizontal bar above lanes 3 to 8 indicates the presence of Mn2+, and the horizontal bar above lanes 2 to 8 indicates the presence of PsaR-Strep. Arrows indicate the positions of the shifted probes, and asterisks indicate the position of the free probe. The presence of weaker bands which run higher than the free probe in the gels is a phenomenon that has also been seen by others in similar experiments. These bands may represent unspecific PCR products or single-stranded DNA (1, 15).
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