Regulatory circuits controlling Spx levels in Streptococcus mutans

Abstract

Spx is a major regulator of stress responses in Firmicutes. In Streptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation of clpP or simultaneous inactivation of clpC and clpE stabilized the GFP::SpxA2_tail fusion protein. Addition of acidic amino acids to the GFP::SpxA2_tail chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1_tail . Promoter reporter fusions revealed that spxA1 transcription is co-repressed by the metalloregulators PerR and SloR while spxA2 transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement with spxA2 being part of the LiaR regulon, SpxA2 was found to be critical for the growth of S. mutans under envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.

Keywords: Streptococcus mutans; ClpP; Spx; oxidative stress.

Figure 1:

ClustalW alignment of SpxA1 (A) and SpxA2 (B) proteins from selected gram-positive bacteria. SpxA1 protein length varies from 131 to 137 amino acids whereas SpxA2 from all streptococcal species are 132 amino acids long. Identical residues are shown in dark shades and similar residues in light shades. A consensus is shown below the sequences. Black boxes indicate the conserved CXXC motif and glycine 52 residue. Red boxes indicate acidic residues present within the last 10 amino acids of each protein.

Figure 2.

Stability of GFP::SpxA1_tail and GFP::SpxA2_tail fusion proteins in S. mutans. (A) Graphical representation of GFP fused to the last 10 C-terminal amino acids of either SpxA1_Smu or SpxA2_Smu. (B) Fluorescence decay of GFP::SpxA1_tail and GFP::SpxA2_tail expressed in UA159 (parent) or ΔclpP strains after addition of chloramphenicol. Asterisks indicate time points showing statistically significant differences (p ≤ 0.01, one-way ANOVA) in decay of GFP expression in the SpxA2_tail construct when hosted in UA159 compared to the ΔclpP strain. (C) Western blot analysis of UA159 or ΔclpP expressing GFP::SpxA1_tail and GFP::SpxA2_tail probed with anti-GFP polyclonal antibody. The images shown are representative of 3 or more independent experiments.

Figure 3.

Stability of GFP::Spx_tail fusion proteins with acidic residues added to the C-terminus of SpxA2 (SpxA2_DDtail) or with acidic residues deleted from SpxA1 (SpxA1_-5tail and SpxA1_-7tail). (A) Fluorescence decay of GFP::SpxA2_tail or GFP::SpxA2_DDtail when expressed in UA159 or ΔclpP host strains. Asterisks indicate time points showing statistically significant differences (p ≤ 0.01, one-way ANOVA) in GFP::SpxA2_tail decay compared to GFP::SpxA2_DDtail when expressed in UA159. (B) Fluorescence decay of GFP::SpxA1_tail, GFP::SpxA1_-5tail or GFP::SpxA1_-7tail expressed in UA159. Asterisks indicate time points showing statistically significant differences (p ≤ 0.01, one-way ANOVA) in decay of the GFP::SpxA1_tail construct as compared to GFP::SpxA1_-5tail or GFP::SpxA1_-7tail. (C) Western blot analysis of S. mutans UA159 expressing GFP::SpxA2_DDtail, GFP::SpxA1_-5tail or GFP::SpxA1_-7tail probed with anti-GFP polyclonal antibody. The images shown are representative of 3 or more independent experiments.

Figure 4.

Stability of GFP::SPNSpx fusion proteins. Fluorescence decay of GFP::SPNSpxA1_tail and GFP::SPNSpxA2_tail expressed in UA159 or ΔclpP strains. Asterisks indicate time points showing statistically significant differences (p ≤ 0.01, one-way ANOVA) when the corresponding fusion protein is expressed in S. mutans UA159 or ΔclpP strains; a single asterisk indicates comparisons with the GFP::SPNSpxA1_tail construct, while two asterisks indicate comparisons with the GFP::SPNSpxA2_tail construct.

Figure 5.

Fluorescence decay of GFP::SpxA2_tail fusion proteins expressed in S. mutans UA159 and a panel of clp single and double mutant strains. Time points showing statistically significant differences (p ≤ 0.01, one-way ANOVA) when GFP::SpxA2_tail expressed in S. mutans UA159 is compared to GFP::SpxA2_tail expressed in ΔclpP (@), ΔclpE (*), ΔclpEΔclpC (#), or ΔclpXΔclpE (&) are indicated in the figure.

Figure 6.

qRT PCR analysis of (A) SpxA1-regulated sodA or (B) SpxA2-regulated smu.11412c genes in different the ΔclpP and ΔclpE mutant background. (*) p< 0.05

Figure 7.

Transcriptional characterization of S. mutans spxA1 and spxA2. (A) Sequence of the P_spxA1 and P_spxA2 promoter regions. The σ^A-type −35 and −10 regions, mapped by 5’ RACE-PCR, are underlined. The transcriptional start site is shown in boldface and labeled with “+1”. The ATG start codon is also shown in boldface. Putative PerR- and SloR-binding sites in P_spxA1 and LiaR-binding site in P_spxA2 are shown inside boxes. (B-E) CAT activity driven from P_spxA1 or P_spxA2. (B-C) Cells were grown to mid-log phase and incubated in the presence of H₂O₂ (0.5 mM), diamide (0.5mM) or pH 6.0 for 10 or 30 minutes. (D-E) Mutant strains harboring the indicated CAT fusions were grown to mid-log phase. Asterisks indicates statistical significance (p value ≤ 0.01) when compared to control (B-C) or UA159 (D-E) by one-way ANOVA.

Figure 8.

CAT activity driven from P_spxA2 under selected cell envelope stress conditions. Cells were grown to mid-log phase and incubated in the presence of bacitracin (64 µg ml⁻¹), daptomycin (2 µg ml⁻¹), ampicillin (0.4 µg ml⁻¹), chlorhexidine (0.5 µg ml⁻¹) or SDS (0.00125% w/v) for either 10 or 30 minutes. Asterisks indicate statistical significance (p value ≤ 0.01) when compared to untreated control by one-way ANOVA.

Figure 9.

Growth curves of S. mutans UA159, Δspx and Spx^SXXC strains in (A) BHI or (B) in the presence of sub-inhibitory concentration of H₂O₂ (0.4 mM).

Figure 10.

Growth curves of S. mutans UA159, ΔspxA2 and SpxA2^SXXC strains in BHI or in the presence of sub-inhibitory concentrations of ampicillin (0.4 µg ml⁻¹), bacitracin (32 µg ml⁻¹), norfloxacin (1 µg ml⁻¹), chloramphenicol (1 µg ml⁻¹), chlorhexidine (125 ng ml⁻¹), daptomycin (2 µg ml⁻¹), or SDS (0.0005%).