Role of Aminotransferases in Branched-Chain Amino Acid Metabolism: Acid Tolerance and Fatty Acid Synthesis in Streptococcus mutans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.Streptococcus mutans relies on a variety of adaptive mechanisms to successfully colonize tooth surfaces in the human oral cavity and to become a dominant species in dental plaque. Organic acid production, arising from sugar metabolism, results in the accumulation of end‐products that damage tooth surfaces. Previous studies have shown that survival of S. mutans in under acidic conditions is predicated on an acid‐adaptive response. Branched‐chain amino acid (bcAA) metabolism is one of the pathways thought to be important for mitigating acidification. Synthesis of bcAAs allows S. mutans to reroute pyruvate, generate substrates for fatty acid synthesis, modulate carbon flow, and alter gene expression in response to its physiological needs. To elucidate the role of bcAA metabolism in the acid adaptive response of S. mutans, a strain carrying a mutation in the branched‐chain amino acid aminotransferase, ilvE, was characterized. Physiological and transcriptional studies demonstrated that ilvE is regulated by pH and by the global transcriptional regulator CodY. CodY acts a repressor of ilvE transcription, mediated through a physical interaction between the ilvE promoter and CodY protein, via a consensus‐binding domain. Regulation of ilvE is dependent on both CodY and physiological levels of branched‐chain amino acids, which act as signaling molecules to enhance binding affinity of CodY. The carbon catabolite regulator, CcpA, was also demonstrated to regulate ilvE in a positive manner, by activating ilvE transcription. The role of amino acid metabolism in branched‐chain fatty acid (bcFA) synthesis was also determined. Degradation of bcAAs provides the substrates for bcFA synthesis, since increased levels of ATase activity correlated with strains whose membrane fatty acid composition contained bcFAs. Although bcFAs were hypothesized to be a compensatory mechanism, detailed membrane fatty acid analysis demonstrated otherwise, since S. mutans was shown to be unable to incorporate bcFAs within its membrane. The results from this study demonstrated that branched‐chain amino acid metabolism plays an important role in providing the substrates necessary for the changes in gene expression required during acid adaptation and for the synthesis of bcFAs in S. mutans