<i>In vitro</i> Manganese-Dependent Cross-Talk between <i>Streptococcus mutans</i> VicK and GcrR: Implications for Overlapping Stress Response Pathways

<div><p><i>Streptococcus mutans</i>, a major acidogenic component of the dental plaque biofilm, has a key role in caries etiology. Previously, we demonstrated that the VicRK two-component signal transduction system modulates biofilm formation, oxidative stress and acid tolerance responses in <i>S. mutans</i>. Using i<i>n vitro</i> phosphorylation assays, here we demonstrate for the first time, that in addition to activating its cognate response regulator protein, the sensor kinase, VicK can transphosphorylate a non-cognate stress regulatory response regulator, GcrR, in the presence of manganese. Manganese is an important micronutrient that has been previously correlated with caries incidence, and which serves as an effector of SloR-mediated metalloregulation in <i>S. mutans</i>. Our findings supporting regulatory effects of manganese on the VicRK, GcrR and SloR, and the cross-regulatory networks formed by these components are more complex than previously appreciated. Using DNaseI footprinting we observed overlapping DNA binding specificities for VicR and GcrR in native promoters, consistent with these proteins being part of the same transcriptional regulon. Our results also support a role for SloR as a positive regulator of the <i>vicRK</i> two component signaling system, since its transcription was drastically reduced in a SloR-deficient mutant. These findings demonstrate the regulatory complexities observed with the <i>S. mutans</i> manganese-dependent response, which involves cross-talk between non-cognate signal transduction systems (VicRK and GcrR) to modulate stress response pathways.</p></div

<i>in vitro</i> phosphorylation of VicK in the presence of various metal cations.

<p>VicK (1 µM) was incubated in 100 mM Tris-HCl, pH 7.5 containing 1 mM of the designated cations and 0.10 µM [γ-³²P] ATP at room temperature for 15 minutes. The relative autophosphorylation of VicK was quantified using Image Quant 5.0 software (Molecular Dynamics) and is represented by the histogram above the scanned gel. The gels shown are representative of at least three independent experiments. Error bars represent ± std. errors of the average phosphorylation values derived from at least 3 independent experiments.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

<i>In vitro</i> model of manganese-independent (A) and –dependent cross-regulation involving <i>S. mutans</i> SloR, VicRK and GcrR.

<p>A) In the absence of Mn²⁺ (approximating conditions of free-floating planktonic cells) <i>S. mutans gcrR</i> expression is de-repressed (VicR expression is not induced in the absence of Mn²⁺). Even though GcrR is the more abundant substrate, VicR is the favored species for VicK phosphorylation under these conditions. B) During this so-called “transition stage” (approximating conditions of an early biofilm) a “spike” in Mn²⁺ renders GcrR (still the predominant species) the favored substrate for phosphorylation by VicK, but only transiently. C) As the biofilm matures and Mn²⁺ concentrations increase, SloR is activated to repress <i>gcrR</i> expression, thereby reducing the availability of GcrR as a substrate. The activated SloR-Mn²⁺ complex encourages <i>vicR</i> expression, and hence VicR becomes the favored substrate for VicK phosphorylation once again.</p

VicK has a significant impact on transcription of known ATR-related genes in <i>S. mutans</i>.

<p>qRT-PCR was performed to reveal fold-change in gene expression at pH 5.5 versus 7.5 with cDNAs derived from <i>S. mutans</i> UA159 (solid black bars) and a <i>vicK</i> insertion-deletion mutant (SmuvicK) (striped bars). Error bars represent ± std. errors of the average expression values derived from at least 3 independent experiments. Student t-tests confirm that all genes are significantly down-regulated in the VicK mutant relative to the UA159 wild-type progenitor strain (p<0.001).</p

<i>in vitro</i> phosphorylation of VicK in the combined presence of Mn²⁺ and other various metal cations.

<p>VicK (1 µM) was incubated in 100 mM Tris-HCl, pH 7.5 containing 1 mM MnCl₂ plus 1 mM of the designated cation and 0.10 µM [γ-³²P] ATP at room temperature for 15 minutes. The relative autophosphorylation of VicK was quantified using Image Quant 5.0 software (Molecular Dynamics). The sample containing only VicK and MnCl₂ was set at 100% for comparison and the results are shown in the histogram above the scanned gel. The gels shown are representative of at least three independent experiments. Error bars represent ± std. errors of the average phosphorylation values derived from at least 3 independent experiments.</p

<i>in vitro</i> transphosphorylation of VicR and GcrR by VicK.

<p>A) Phosphorylation of VicR and GcrR by VicK in the presence of MgCl₂. For each reaction 1 µM of each of the following proteins were included in the reaction: Lane 1: VicK; Lane 2: ComE; Lane 3: VicR; Lane 4: GcrR; Lane 5: VicK and ComE; Lane 6: VicK and VicR; Lane 7: VicK and GcrR; Lane 8: VicK, ComE and VicR; Lane 9: VicK, ComE and GcrR. B) Phosphorylation of VicR and GcrR by VicK in the presence of MnCl₂. For each reaction 1 µM of each of the following proteins were included in the reaction unless otherwise indicated: Lane 1: VicK; Lane 2: VicR; Lane 3: GcrR; Lane 4: VicK and VicR; Lane 5: VicK and GcrR; Lane 6: VicK, GcrR and 0.01 µM VicR; Lane 7: VicK, GcrR and 0.02 µM VicR; Lane 8: VicK, GcrR and 0.04 µM VicR; Lane 9: VicK, GcrR and 1 µM VicR. The gels shown are a representative set of replicate gels run for each experiment.</p

DNaseI footprinting of the <i>gtfC</i> and <i>gcrR</i> promoter regions.

<p>(A) VicR or GcrR at increasing concentrations (0.25 and 0.5 µM) or a combination of VicR and GcrR at an equimolar concentration (0.5 µM) were incubated with labeled <i>gtfC</i> DNA substrate. The S above the fifth lane indicates the DNA substrate incubated in the absence of VicR/GcrR. The arrows designate the areas of enhanced cleavage by DNaseI. (B) Labeled <i>gcrR</i> DNA substrate was incubated with increasing concentrations of VicR or GcrR (0.125, 0.25, and 0.5 µM) or a mixture of VicR and GcrR at equimolar concentrations (0.25 and 0.5 µM). The S above the first and eleventh lanes indicates the DNA substrate incubated in the absence of VicR/GcrR. The solid line represents the region of protected nucleotides by VicR and the dashed line represents the region of protection by GcrR. The VicR consensus sequence is shown to the left of the solid lines.</p

Construction of the <i>S. mutans</i> fusion strains GMS905, GMS906, and GMS907.

<p>The integration of the P<i>gcrR:cat</i> fusion that is resident on plasmid pLM1 occurred via a double cross-over event into the chromosome of <i>S. mutans</i> UA159, GMS584 and SmuvicK at the <i>phnA</i> and <i>mtlA</i> loci. Sequencing across the <i>S. mutans</i> chromosome-pLM1 junction confirmed appropriate insertion of the P<i>gcrR:cat</i> fusion.</p