Global Regulation by (p)ppGpp and CodY inStreptococcus mutans

The RelA, RelP, and RelQ enzymes are responsible for the production of the alarmone (p)ppGpp inStreptococcus mutans . A strain lacking all three synthetases (ΔrelAPQ ) does not grow in minimal medium lacking the branched-chain amino acids (BCAA) leucine or valine but grows well if isoleucine is also omitted. Here, we investigated whether there was a correlation between growth in the absence of leucine and valine with (p)ppGpp pools and the activation of CodY. By using a combination of single, double, and triple mutants lacking the (p)ppGpp synthetase enzymes, we demonstrated that the ability to grow in the absence of leucine or valine required basal levels of (p)ppGpp production by RelP and RelQ. The introduction of acodY mutation into the ΔrelAPQ strain fully restored growth in medium lacking leucine or valine, revealing that the growth-defective phenotype of ΔrelAPQ was directly linked to CodY. Lowering GTP levels through the addition of decoyinine did not alleviate CodY repression or affect the expression of genes involved in BCAA biosynthesis, suggesting thatS. mutans CodY is not activated by GTP. The results of phenotypic studies revealed that thecodY mutant had a reduced capacity to form biofilms and that its growth was more sensitive to low pH, showing a role for CodY in two key virulence properties ofS. mutans . Microarray results revealed the extent of the CodY regulon. Notably, the identification of putative CodY-binding boxes upstream of genes that were downregulated in thecodY mutant indicates that CodY may also function as a transcriptional activator inS. mutans .