3,5-Dimethylpyrazin-2-ol (DPO) is the active form of Autoinducer-3 (AI-3), a quorum-sensing molecule in Escherichia coli that regulates virulence, motility, and biofilm formation. DPO is synthesized during high cell density, a hallmark of quorum sensing, where bacteria coordinate gene expression based on population size. The biosynthesis of DPO begins with L-threonine, which is converted to aminoacetone (AA) via the enzyme threonine dehydrogenase (Tdh). This reaction involves the oxidation of L-threonine, followed by decarboxylation to produce aminoacetone. Next, aminoacetone condenses with L-alanine in a reaction catalyzed by alanyl-tRNA synthetase (AlaRS), forming N-alanyl-aminoacetone. This intermediate undergoes spontaneous cyclization, dehydration, and oxidation to yield 3,5-dimethylpyrazin-2-ol (DPO), the active AI-3 molecule. DPO functions as a signaling molecule by binding to the QseC receptor, a membrane-bound histidine kinase. Upon binding, QseC undergoes autophosphorylation and transfers the phosphate group to its cognate response regulator, QseB. Phosphorylated QseB then activates the transcription of flhDC, the master regulator of flagellar biosynthesis. The FlhD4C2 complex encoded by flhDC upregulates the fliLMNOPQR operon, which encodes components of the flagellar motor switch and export apparatus. These proteins are transported to the inner membrane via the flagellar export apparatus and assembled into the flagellar structure, enabling bacterial motility. Thus, DPO biosynthesis and signaling play a central role in coordinating bacterial behavior, including virulence and motility, through the QseC-QseB regulatory system and the flagellar gene hierarchy, particularly under conditions of high cell density when quorum-sensing pathways are activated.

Quorum Sensing: Biosynthesis of Autoinducer-3 (AI-3) References