Gene Regulation by the LiaSR Two-Component System in Streptococcus mutans

Abstract

The LiaSR two-component signal transduction system regulates cellular responses to several environmental stresses, including those that induce cell envelope damages. Downstream regulons of the LiaSR system have been implicated in tolerance to acid, antibiotics and detergents. In the dental pathogen Streptococcus mutans, the LiaSR system is necessary for tolerance against acid, antibiotics, and cell wall damaging stresses during growth in the oral cavity. To understand the molecular mechanisms by which LiaSR regulates gene expression, we created a mutant LiaR in which the conserved aspartic acid residue (the phosphorylation site), was changed to alanine residue (D58A). As expected, the LiaR-D58A variant was unable to acquire the phosphate group and bind to target promoters. We also noted that the predicted LiaR-binding motif upstream of the lia operon does not appear to be well conserved. Consistent with this observation, we found that LiaR was unable to bind to the promoter region of lia; however, we showed that LiaR was able to bind to the promoters of SMU.753, SMU.2084 and SMU.1727. Based on sequence analysis and DNA binding studies we proposed a new 25-bp conserved motif essential for LiaR binding. Introducing alterations at fully conserved positions in the 25-bp motif affected LiaR binding, and the binding was dependent on the combination of positions that were altered. By scanning the S. mutans genome for the occurrence of the newly defined LiaR binding motif, we identified the promoter of hrcA (encoding a key regulator of the heat shock response) that contains a LiaR binding motif, and we showed that hrcA is negatively regulated by the LiaSR system. Taken together our results suggest a putative role of the LiaSR system in heat shock responses of S. mutans.

Fig 1. LiaS autophosphorylates and transfers phosphate to LiaR.

(A) MBP-tagged LiaS was incubated in phosphorylation buffer 1 (20 mM Tris-Cl, 50 mM KCl, 10 mM MnCl₂, 1 mM DTT and 10 μCi γ³²P-dATP at pH 7.4) for indicated times. (B) MBP-tagged LiaS, pretreated with γ³²P for 10 min was incubated with His-tagged LiaR (molar ratio 1:2) in phosphorylation buffer 1 for indicated times. Both reaction products were resolved on 10% SDS-PAGE gels.

Fig 2. Mutation D58A in LiaR affects phosphorylation and DNA binding ability.

(A) Phosphorylated LiaS was incubated with LiaR (1:2 molar ratio) in phosphorylation buffer 1. Samples were drawn at indicated time points and resolved on 10% SDS-PAGE gels. (B) ~5 μg of purified His-LiaR / D58A were incubated in phosphorylation buffer 2 [50 mM Tris-Cl, 50 mM KCl, 20 mM MnCl2, 1 mM DTT at pH 7.5 containing either 50 mM acetyl phosphate (Ac-Po4) or 20 mM phosphoamidate (PA) as phosphodonors] for 1 h at 37°C and then resolved by PhosTag-SDS PAGE. (C) ~0.5 pmol P_SMU.2084 end labelled with γ³²P-dATP were incubated with ~5, 10 and 15 pmol of purified His-LiaR / His-D58A in binding buffer (20 mM Tris-Cl, 100 mM NaCl, 0.01 mM DTT, 5% glycerol (vol/vol), 1 mM EDTA, 0.01 mg/ml BSA, 5 mM MgCl₂, and 10 μg/ml Poly(dI-dC) at pH 7.5) for 30 min and then resolved on EMSA gels (5.5% (w/vol) polyacrylamide gels containing 5% glycerol (vol/vol) using 0.5x TBE buffer with 5% glycerol (vol/vol)). Marker F indicates free DNA, while marker B indicates the DNA-protein complex.

Fig 3. LiaR specifically binds the promoters of SMU.753, SMU.1727 and SMU.2084 but is unable to bind P_SMU.485.

(A) ~0.5 pmol of P_SMU.485, P_SMU.753 and P_SMU.1727 end labelled with γ³²P-dATP were incubated with ~5, 10 and 15 pmol of purified His-LiaR in binding buffer for 30 min. (B) Addition of non-radiolabelled P_SMU.2084 as cold competitor, two-fold in excess of radiolabelled P_SMU.2084 abolished the gel shift. Both reaction products were resolved on EMSA gels. Marker F indicates free DNA, while marker B indicates the DNA-protein complex.

Fig 4. A conserved 25 bp motif is essential for LiaR binding and is present upstream of regulons directly under LiaR control.

(A) Predicted LiaR binding motif found using MEME (Multiple Em for Motif Elicitation) in P_SMU.2084, P_SMU.753, and P_SMU.1727. Arrows indicate the position of the inverted repeat while * indicates highly conserved positions. High-scoring motifs found upstream of potential new LiaR regulons SMU.235 and SMU.hrcA are also shown. Bases in the P_SMU.hrcA and P_SMU.235 motifs that vary from conserved positions are underlined. (B) ~0.5 pmol of P_SMU.hrcA, end labelled with γ³²P-dATP was incubated with ~5, 10 and 15 pmol of purified His-LiaR in binding buffer for 30 min and then resolved on EMSA gels. Marker F indicates free DNA, while marker B indicates the DNA-protein complex. (C) Quantification of hrcA expression in the ΔliaR strain IBSA13 relative to the wildtype strain UA159. Data shown is the mean ± SD of triplicate measurements and rpoB was used as an endogenous control. *, significant difference in relation to the wildtype (P<0.05) using a Student’s t-test.

Fig 5. Alterations in conserved residues of the predicted LiaR binding motif affect binding by LiaR variably

(A) The LiaR binding consensus identified in this study was altered by introducing substitutions at fully conserved postions 13,17, 23 and 25. Additionally the binding ability of the inverted repeat alone to LiaR was assessed. (B) ~1 pmol of indicated, annealed oligos (Consensus, IR only, C13A/A17G, and A23G/T25C), end labelled with γ³²P-dATP were incubated with ~15, 20 and 30 pmol of purified His-LiaR in binding buffer for 30 min and then resolved on 7.5% EMSA gels. Marker F indicates free DNA, while marker B indicates the DNA-protein complex. (C) Biotinylated consensus, C13A/A17G and A23G/T25C oligos were immobilized on streptavidin biosensors and then exposed to 1 μM LiaR, prepared in binding buffer for a period of 5 minutes to allow association followed by a 5 min exposure to binding buffer to allow complex dissociation. The maximum binding ability (Rmax and the equilibrium binding ability (Req) were calculated automatically and reported.