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. 2014 Feb;196(3):614-23.
doi: 10.1128/JB.01041-13. Epub 2013 Nov 22.

Cyclic di-AMP impairs potassium uptake mediated by a cyclic di-AMP binding protein in Streptococcus pneumoniae

Yinlan Bai  1 , Jun Yang, Tiffany M Zarrella, Yang Zhang, Dennis W Metzger, Guangchun Bai
Affiliations

Cyclic di-AMP impairs potassium uptake mediated by a cyclic di-AMP binding protein in Streptococcus pneumoniae

Yinlan Bai et al. J Bacteriol. 2014 Feb.

Abstract

Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections. However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae, a Gram-positive pathogen. In this study, we identified a c-di-AMP binding protein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal potassium uptake.

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Figures

FIG 1
FIG 1
Identification of CabP as a c-di-AMP binding protein in S. pneumoniae. (A) Test of the column. A mixture containing the same concentrations of purified M. tuberculosis DisA and BSA was loaded onto a c-di-AMP affinity column (Biolog) and separated as described in Materials and Methods. The eluted samples were analyzed using SDS-PAGE and stained with Coomassie bright blue. Lane M, molecular mass standards with sizes (in kDa) noted on the left side of the gel. (B) Purification of c-di-AMP binding proteins in S. pneumoniae. The samples eluted from the affinity chromatography were analyzed using SDS-PAGE and staining with Coomassie bright blue. Lys, bacterial lysates; lane M, molecular mass standards with sizes (in kDa) noted on the left side of the gel; lanes 1 to 6, eluted samples.
FIG 2
FIG 2
Purification of recombinant CabP. (A) SDS-PAGE of purified CabP. Lane M, molecular mass markers; lanes 1 to 3, eluted protein. (B) Gel filtration of CabP monitored at 280 nm. Retention volumes of molecular mass standards (in kDa) are indicated. mAu, milliabsorbence units.
FIG 3
FIG 3
Interaction between CabP and c-di-AMP. (A) CabP protein and S. pneumoniae PdxR control were loaded for SDS-PAGE, followed by staining with Coomassie bright blue (Staining), or transferred onto a PVDF membrane and then incubated with labeled c-di-AMP (Blotting). The membrane was finally exposed on a phosphor screen and scanned with a Storm 860 phosphorimager. (B) Mobility of 32P-labeled c-di-AMP in the presence of 4 μM each purified protein as indicated. M. tuberculosis DisA served as a positive control, and S. pneumoniae PdxR served as a negative control. (C) Mobility of 32P-labeled c-di-AMP in the absence or presence of various concentrations of purified CabP. (D) Mobility of 32P-labeled c-di-AMP in the presence of 1 μM purified CabP and an excessive amount of each unlabeled nucleotide as indicated.
FIG 4
FIG 4
Growth of mutants in CDM with various concentrations of potassium. Bacterial growth was determined at 8 h postinoculation for all panels. (A) Domain and genetic organizations of the annotated Trk proteins in the S. pneumoniae genome. The deleted regions in the mutant strains as listed are indicated with black lines. (B) Growth of WT and the quadruple mutant (ST2811) in a formulated CDM contains 5-fold serial dilutions of KCl. The commercial CDM (CDM), which contains ∼10 mM potassium salts, was used as a control. (C) Growth of the ΔSPD_0076 ΔcabP (ST2798) and ΔSPD_0430 ΔSPD_0429 (ST2809) double mutants in the formulated CDM with indicated concentrations of KCl. ST2811 was used as a control. (D) Growth of ΔcabP (ST2796) and ΔSPD_0076 (ST2797) mutants in the formulated CDM containing the indicated concentrations of KCl. ST2798 was used as a control. All the growth data shown in panels B through D are the means of three independent experiments. The error bars denote the SEMs. (E) Growth of the complemented ΔSPD_0076 and ΔcabP mutants in the formulated CDM. Complemented strains were generated by transformation of the plasmid expressing either SPD_0076 or cabP into the respective “host.” Strains transformed with pVA838 plasmid (Control) served as controls.
FIG 5
FIG 5
Role of c-di-AMP in potassium uptake. (A) Interaction between CabP and SPD_0076 determined in a two-hybrid system. The indicated recombinant E. coli strains were spotted onto LB plates containing 80 μg/ml of X-Gal. ST2838 is a positive control, whereas ST2845 is a negative control. The data shown are representative of three repeat experiments. (B) Detection of bacterial c-di-AMP in the indicated E. coli strains. Note that only ST2860 and ST2861 exhibited detectable c-di-AMP levels. The data shown are the means of three independent experiments. The error bars denote the SEMs. (C and D) Growth of the WT (C) and ST2734 (D) in CDM with indicated concentrations of KCl. The data shown are the means of three independent experiments. The error bars denote the SEMs.
FIG 6
FIG 6
Growth of the LB2003 recombinant strains in minimal medium containing 2 mM KCl. (A) Expression of SPD_0076, cabP, and M. tuberculosis disA (Mtb_disA) in LB2003. These genes were cloned in pMBC664, in which the expression is controlled by an E. coli crp promoter as illustrated, and were transformed into LB2003 to generate the indicated strains. (B) Growth curve of the LB2003 recombinant strains as indicated in panel A in minimal medium containing 2 mM KCl. The data shown are the means of three independent experiments. The error bars denote the SEMs.
FIG 7
FIG 7
Model of c-di-AMP-controlled potassium uptake by S. pneumoniae. In the absence of c-di-AMP, CabP interacts with SPD_0076 and results in efficient potassium uptake. In the presence of c-di-AMP, the interaction between CabP and SPD_0076 is impaired, and the potassium uptake is significantly reduced. Therefore, the requirement of the environmental potassium threshold for bacterial growth is elevated.
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