The Branched-Chain Amino Acid Aminotransferase Encoded by ilvE Is Involved in Acid Tolerance in Streptococcus mutans

Abstract

Here we constructed and characterized an ilvE deletion mutant of S. mutans UA159. Growth experiments revealed that the ilvE mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of ilvE causes a decrease in acid tolerance. The ilvE strain exhibits a defect in F 1 -F o ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the ilvE promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acidadaptive repertoire of S. mutans.

The Streptococcus mutans Aminotransferase Encoded by ilvE Is Regulated by CodY and CcpA

“…The reduced expression in the presence of leucine also suggests that, like other biosynthetic genes, ilvE could be subject to regulation by the level of leucine. The observable effect on the transcriptional activity occurred at a low pH, which we had previously shown is a condition that triggers an increase in ilvE transcription (28). The results suggest that signaling by bcAAs can modulate transcription of ilvE, demonstrating that both pH and the nutritional state of the cell are signals that regulate ilvE transcription.…”

“…CAT assays were performed following a previously described protocol (3,28,39). Briefly, 50-ml cell pellets were resuspended in 1 ml 10 mM Tris-Cl, pH 7.8.…”

“…A genetic complement strain of S. mutans was created using primers (Table 2) to amplify the codY coding and preceding intergenic regions from UA159 chromosomal DNA. A codY complement plasmid, pSUGBcodY, was constructed as previously described (28,35). The ⌬codY mutant strain was transformed with the integration vector pSUGBcodY, and colonies were selected on brain heart infusion (BHI) agar containing kanamycin (1,000 g/ml).…”

“…For cat transcriptional fusions, full-length ilvE promoter-cat fusions were cloned as previously described (28). In addition, a truncated ilvE promoter-cat construct (NCB [no Cod box]) in which the predicted native CodY-binding domain was absent was created ( Fig.…”

“…Our work has shown that IlvE is involved with branched-chain amino acid metabolism and plays a role in acid adaptation. Loss of ilvE results in an acid-sensitive phenotype and reduced levels of F-ATPase activity when the organism is grown at low pH (28). In several organisms, such as Streptococcus pneumoniae (11), L. lactis (29), and S. aureus (17), ilvE has been identified as a CodY-regulated gene.…”