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 The aciduricity of Streptococcus mutans is an important virulence factor of the organism, required to both out-compete commensal oral microorganisms and cause dental caries. In this study, we monitored transcriptional changes that occurred as a continuous culture of either an acid-tolerant strain (UA159) or an acid-sensitive strain (fabM::Erm) moved from steady-state growth at neutral pH, experienced glucose-shock and acidification of the culture, and transitioned to steady-state growth at low pH. Thus, the timing of elements of the acid tolerance response (ATR) could be observed and categorized as acute vs. adaptive ATR mechanisms. Modulation of BCAA biosynthesis, DNA/protein repair mechanisms, ROS metabolizers, and PTS occurred in the initial acute phase, immediately following glucose-shock, while up-regulation of F1F0-ATPase did not occur until the adaptive phase, after steady-state growth had been re-established. In addition to the archetypal ATR pathways mentioned above, glucose-shock led to differential expression of genes suggesting a re-routing of resources away from synthesis of fatty acids and proteins, and towards synthesis of purines, pyrimidines and amino acids. These adjustments were largely transient, as upon establishment of steady-state growth at acidic pH, transcripts returned to basal expression levels. During growth at steady-state pH 7, fabM::Erm had a transcriptional profile analogous to that of UA159 during glucose-shock, indicating that even during growth in rich media at neutral pH, the cells were stressed. These results, coupled with a recently established collection of deletion strains (Quivey et al., 2015), provide a starting point for elucidation of the acid tolerance response in S. mutans.

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.