Disruption of the alsSD operon of Enterococcus faecalis impairs growth on pyruvate at low pH

Diacetyl and acetoin are pyruvate-derived metabolites excreted by many micro-organisms, and are important in their physiology. Although generation of these four-carbon (C4) compounds in Enterococcus faecalis is a well-documented phenotype, little is known about the gene regulation of their biosynthetic pathway and the physiological role of the pathway in this bacterium. In this work, we identified the genes involved in C4 compound biosynthesis in Ent. faecalis and report their transcriptional analysis. These genes are part of the alsSD bicistronic operon, which encodes α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD). Our studies showed that alsSD operon transcription levels are maximal during the exponential phase of growth, decreasing thereafter. Furthermore, we found that this transcription is enhanced upon addition of pyruvate to the growth medium. In order to study the functional role of the alsSD operon, an isogenic alsSD mutant strain was constructed. This strain lost its capacity to generate C4 compounds, confirming the role of alsSD genes in this metabolic pathway. In contrast to the wild-type strain, the alsSD-deficient strain was unable to grow in LB medium supplemented with pyruvate at an initial pH of 4.5. This dramatic reduction in growth parameters for the mutant strain was simultaneously accompanied by the inability to alkalinize the internal and external medium under these conditions. In sum, these results suggest that the decarboxylation reactions related to the C4 biosynthetic pathway give enterococcal cells a competitive advantage during pyruvate metabolism at low pH.