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.

Summary A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. Since S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared to the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and 5 or in vivo in an animal model for oral infection. Taken together, this data represents a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

SUMMARY SMU.1745c, encoding a putative transcriptional regulator of the MarR family, maps to a location proximal to the fab gene cluster in Streptococcus mutans. Deletion of the SMU.1745c (fabTSm) coding region resulted in a membrane fatty acid composition comprised of longer-chained, unsaturated fatty acids (UFA), compared with the parent strain. Previous reports have indicated a role for FabT in regulation of genes in the fab gene cluster in other organisms, through binding to a palindromic DNA sequence. Consensus FabT motif sequences were identified in S. mutans in the intergenic regions preceding fabM, fabTSm and fabK in the fab gene cluster. Chloramphenicol acetyltransferase (cat) reporter fusions, using the fabM promoter, revealed elevated transcription in a ΔfabTSm background. Transcription of fabTSm was dramatically elevated in cells grown at pH values of 5 and 7 in the Δ fabTSm background. Transcription of fabTSm was also elevated in a strain carrying a deletion for the carbon catabolite repressor CcpA. Purified FabTSm and CcpA bound to the promoter regions of fabTSm and fabM. Hence, the data indicate that FabTSm acts as a repressor of fabM and fabTSm itself and the global regulator CcpA acts as a repressor for fabTSm.

). The phenotype suggested that amino acid metabolism is important for acid adaptation, as turnover of branched-chain amino acids (bcAAs) could provide important signals to modulate expression of genes involved in the adaptive process. Previous studies have demonstrated that ilvE is regulated in response to the external pH, though the mechanism is not yet established. CodY and CcpA have been shown to regulate expression of branched-chain amino acid biosynthetic genes, suggesting that the ability to sense carbon flow and the nutritional state of the cell also plays a role in the regulation of ilvE. Electrophoretic mobility shift assays using the ilvE promoter and a purified recombinant CodY protein provided evidence of the physical interaction between CodY and ilvE. In order to elucidate the signals that contribute to ilvE regulation, cat reporter fusions were utilized. Transcriptional assays demonstrated that bcAAs are signaling molecules involved in the repression of ilvE through regulation of CodY. In a codY deletion background, ilvE transcription was elevated, indicating that CodY acts a repressor of ilvE transcription. Conversely, in a ccpA deletion background, ilvE transcription was reduced, showing that CcpA activated ilvE transcription. The effects of both regulators were directly relevant for transcription of ilvE under conditions of acid stress, demonstrating that both regulators play a role in acid adaptation.

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