TcyR regulates L-cystine uptake via the TcyABC transporter in Streptococcus mutans

Abstract

Streptococcus mutans, a primary dental pathogen, has a remarkable capacity to scavenge nutrients from the oral biofilm for its survival. Cystine is an amino acid dimer formed by the oxidation of two cysteine residues that is required for optimal growth of S. mutans, which modulates l-cystine uptake via two recently identified transporters designated TcyABC and TcyDEFGH, which have not been fully characterized. Using a nonpolar tcyABC-deficient mutant (SmTcyABC), here, we report that l-cystine uptake is drastically diminished in the mutant, whereas its ability to grow is severely impaired under l-cystine starvation conditions, relative to wild type. A substrate competition assay showed that l-cystine uptake by the TcyABC transporter was strongly inhibited by dl-cystathionine and l-djenkolic acid and moderately inhibited by S-methyl-l-cysteine and l-cysteine. Using gene expression analysis, we observed that the tcyABC operon was upregulated under cystine starvation. TcyABC has been shown to be positively regulated by the LysR-type transcriptional regulator CysR. We identified another LysR-type transcriptional regulator that negatively regulates TcyABC with homology to the Bacillus subtilis YtlI regulator, which we termed TcyR. Our study enhances the understanding of l-cystine uptake in S. mutans, which allows survival and persistence of this pathogen in the oral biofilm.

FIGURE 1. Organization and promoter map of the tcyABC tricistronic operon in S. mutans.

FIGURE 2. Time course for l-[¹⁴C]cystine uptake in S. mutans UA159 wild-type strain and its SmTcyABC mutant

Cystine uptake was examined in the presence of 4 µM l-[¹⁴C]cystine and 200 µM of unlabeled l-cystine. Briefly, at time zero, l-[¹⁴C]cystine and cold l-cystine were added at a concentration of 4 µM (2.6 mCi/mmol) and 200 µM, respectively, and the reaction mixtures were incubated at 37°C. Samples (100 µl) were removed at regular intervals, immediately filtered and the filters washed and transferred scintillation vials for determination of radioactivity. Tansport experiments were carried out using three independent cultures and each time point was sampled in duplicate. Dry cell weights (mg) were used for normalization. Final rate of uptake was calculated as nmol of substrate per mg of cells per minute.

FIGURE 3. Relative transport % of l-[¹⁴C]cystine uptake in S. mutans UA159 in the presence of a 100-fold excess of non-radioactive compounds

The specificity of tcyABC-mediated amino acid uptake was examined using an amino acid competition assay. Briefly, the uptake of l-[¹⁴C]cystine (4 µM) was measured in the presence of 400 µM of the following cold l-amino acids: arginine, cysteine, glutamine, glutamate, leucine, and methionine. As a positive control and to determine total l-[¹⁴C]cystine uptake, cells were incubated with radio-labeled cystine with no competing substrate. As a negative control, a reaction containing no cells was incubated with l-[¹⁴C]cystine and no radioactivity was detected. Rate of uptake was calculated using three independent experiments, each subjected to duplicate sampling procedures. Uptake was normalized using dry cell weights and final uptake was calculated as nmol of substrate per mg of cells per minute.

FIGURE 4. Relative transcription of genes quantified using real-time PCR

S. mutans UA159 and SmTcyR cultures were grown in Modified Minimal medium in the presence and absence of 1 mM l-cystine and used for RNA isolation and cDNA synthesis. For each strain, fold expression was calculated relative to that in cystine-added culutres whose expression was set at an user-defined value of 1.0. Results represent the mean expression from three independent experiments plus/minus std, error, each conducted in triplicate.

FIGURE 5. Growth of S. mutans UA159, SmTcyABC, and SmTcyR strains in modified minimal medium in the presence or absence of l-cystine

Results are representative of multiple experiments and error bars represent std. deviation.

FIGURE 6. Growth of S. mutans UA159, SmTcyABC, SmTcyA, SmTcyB, and SmTcyC strains in modified minimal medium in the absence of l-cystine

Results are representative of multiple experiments and error bars represent std. deviation.