Branched-Chain Amino Acids Utilization by Streptococcus mutans

Objectives: Although branched-chain amino acids (BCAA): Leucine, Isoleucine, and Valine (L, I, V), are essential to synthesize microbial protein, their utilization by Streptococcus mutans has not been studied, so far. Computational analysis revealed that S. mutans possesses a putative BCAA transport-related gene cluster (liv cluster: livFGMHK) in which LivK is supposed to be a solute-binding lipoprotein. In addition, genes related to BCAA synthesis pathway, in which IlvE likely plays a significant role, have also been found on the S. mutans genome, implying that not only exogenous BCAA but also synthesized BCAA are utilized in S. mutans. In this study, an IlvE-deficient mutant (ΔilvE) and a series of double deletion mutants (ΔilvE/ΔlivF, G, M, H, or K) were constructed and were examined their contribution to BCAA utilization by S. mutans. Methods: The mutants listed above were constructed by non-polar replacement of each functional gene with antibiotic-resistance gene, respectively. Uptake of exogenous BCAA was assessed using growth assays with the media in the presence (LIV+) or absence (LIV-) of BCAA. Substrate-specificity of LivK was evaluated using competition assay with the media in the presence of either excessive L, I, V, respectively. Results: The ΔilvE grew in the LIV+ media but not in the LIV- media, in addition, none of the double mutants (ΔilvE/ΔlivF, G, M, H, or K) grew in the LIV- nor LIV+ media. These results suggest that IlvE plays a key role in the synthesis of intracellular L, I, V and that all liv cluster components are essential for the uptake of extracellular L, I, V. Competition assays indicate that LivK is responsible for the uptake of all three BCAA. Conclusion: Collectively, these results suggest that BCAA utilization by S. mutans depends not only on IlvE-related intracellular synthesis but on indispensable liv cluster-mediated exogenous uptake.