## 3 Discussion  
Low pH value (\(\leq\)5.5) can lead to demineralized tooth enamel and favor the occurrence and progression of dental caries [7]. Acid tolerance is one of the main cariogenicity factors of _S. mutans_ because its acid tolerance is regarded as a particularly significant factor in its survival at low pH values [5; 6; 7; 8]. Therefore, clarification of the survival mechanism of _S. mutans_ in an acid stress environment is crucial for understanding its cariogenicity. The development of deep sequencing technology has made the identification of sRNAs feasible, and this technique has been successfully applied to the identification of sRNAs in bacterial species, including _S. mutans_[21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33]. Recent studies showed that sRNAs play important roles in the posttranscriptional regulation of gene expression, which can involve virulence factors [16; 17; 18]. However, to the best of our knowledge, few studies have focused on sRNAs with small size in _S. mutans_ under acid stress conditions, particularly at different acid stress levels. Here, we describe sRNAs (18-50 nt) in _S. mutans_ induced at an initial pH of 5.5. Furthermore, the vitality of bacteria and functions of two of these sRNAs under different acid conditions were evaluated. Given that there is no common secondary structure or naming method for sRNAs, all of the reads mapped to intergenic regions of the reference genome of _S. mutans_ UA159 with high sequence similarity (mismatch \(\leq\)1) from three sequencing cDNA libraries were preserved to prevent us from missing any important sRNAs. All of the sRNA candidates were named according to the starting location in the reference genome [34; 35]. The results of deep sequencing and qRT-PCR suggest the existence of a family of sRNAs in _S. mutans_. Only a small proportion of sRNAs were mapped to previous studies, and the expression levels of the common sRNAs presented in this study and the previous study were not the same [32; 33]. These data revealed that some sRNAs were especially induced in acid stress conditions. Several virulence genes including _ffl_, _brpA_, _relA_, _recA_, and the rest of _S. mutans_ are associated with acid tolerance [36; 37; 38]. It is worth noting that virulence genes (_glnQ_, _glnM_, _ffl_, _brpA_, _relA_) are a candidate target site for sRNAs according to the prediction results. _glnQ_ and _glnM_ are the composition of glutamate transporter operon _glnQHMP_ involved in acid tolerance response (ATR) by transporting of glutamate into _S. mutans_[14]. Fflh, encoded by _ffl_, is a 54-kDa subunit homologue of the signal recognition particle of _Escherichia coli_ involved in acid tolerance by altering membrane composition [6; 36]. BrpA, encoded by _brpA_, is a predicted surface-associated protein with high levels of similarity to LytR of _Bacillus subtilis_ and CpsX of _Streptococcus agalatie_ affects the regulation of acid tolerance [12]. The _relA_ gene of _S. mutans_ codes for a guanosine tetraphosphate and guanosine pentaphosphate ((p)ppGpp) synthetase/hydrolase in biofilm formation and acid tolerance [15]. These results are consistent with our hypothesis that sRNAs induced under acid stress conditions might regulate the expression level of virulence genes responsible for the acid tolerance of _S. mutans_. The analysis of the growth characteristics and vitality indicated that cells not only survive but proliferate at pH 7.5, 6.5, and 5.5. These data are in accordance with a previous study on _S. mutans_ grown at steady state in continuous culture at pH 7.0 or pH 5.0 [38]. The cells stopped growing and gradually died off at pH 4.5. The decrease in the survival capacity at pH 4.5 may be due to autolysis under acidic conditions, which has been observed for _Streptococcus pneumoniae_, another Gram-positive _Streptococcus_[39]. Target genes present relatively higher expression at pH 5.5 and 4.5 compared with their expression at pH 7.5, which induced cells' growth and survival.