Its biochemical properties have been studied in detail because of the considerable biotechnological interest, since it occurs after the alcohol fermentation during wine making affecting the flavour of the wine. MleR and L-malate were required to achieve maximal expression of all genes, includingmleRitself. Deletion ofmleRresulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation. == Conclusion == This study shows that the MLF gene cluster is usually part of the early acid tolerance response inS. mutansand is usually induced by both low pH and L-malate. == Background == S. mutansis considered the major etiological agent of dental caries due to its strong aciduric and acidogenic capacities. During the metabolism of dietary carbohydrates and subsequent formation of acid ABH2 end-products, acidogenic bacteria can shift the plaque pH to 4 or lower within minutes and can retain it at this value for up to one hour, depending on the age of the plaque biofilm [1-4]. Demineralisation of the tooth enamel caused by Secretin (rat) low pH is the beginning of caries development. To withstand these pH fluctuations and to compete with other oral bacteriaS. mutanshas evolved an effective acid tolerance response (ATR). The ATR is usually induced under acidic conditions and has an optimal pH between 5.5-5. Several proteomic studies showed that more than sixty proteins were involved in this response and that many of them appeared within the first 30 minutes after acid shock, whereas full induction occurred after 90-120 minutes [5-8]. General determinants are the induction of general stress proteins, the reduction of membrane proton permeability, increased glycolytic activity and a shift to homo-fermentative metabolism, resulting in elevated lactate production. Anabolic reactions are in return down-regulated, which results in slower growth and lower cell yield [6,8-10]. The concomitant surplus of ATP is used to drive the H+/ATPase, which leads to an increased translocation of protons across the membrane. More specific reactions that contribute to the aciduricity are e.g. the agmatine deiminase system (AgDS). Agmatine is usually secreted by other bacteria in response to low pH but is usually internalised and deaminated byS. mutansto ammonia and carbamoylputrescine. The latter is usually further decarboxylated to putrescine, yielding carbon dioxide and ATP, which again can be used for proton extrusion [11]. Another mechanism for gaining ATP is usually malolactic fermentation (MLF), which is a secondary fermentation that lactic acid bacteria can carry out when L-malate is present in the medium. Its biochemical properties have been studied in detail because of the considerable biotechnological interest, since it occurs after the Secretin (rat) alcohol fermentation during wine making affecting the flavour of the wine. In MLF the dicarboxylic acid L-malate is converted to L-lactate and carbon dioxide by the malolactic enzyme (MLE) in a two Secretin (rat) step reaction without releasing intermediates. Since malic acid (pKa = 3.4, 5.13) is a stronger acid than lactic acid (pKa = 3.85) decarboxylation of L-malate leads to an alkalinization of the cytoplasm. This effect is usually further enlarged by diffusion of H2CO2/CO2out of the cell into the gas phase. The concomitant pH gradient drives the electrogenic malate/lactate antiporter and is coupled to ATP synthesis, which is used to maintain the intracellular pH more alkaline than the environment by extrusion of protons [12,13].S. mutansUA159 possesses a malolactic fermentation gene cluster, that is oriented in opposite direction to the putative regulatormleR[14]. A homologue of this regulator was the first lysR-type transcriptional regulator (LTTR) described in Gram positive bacteria and was shown to positively regulate MLF inLactococcus lactis. A seven-fold induction of L-malate decarboxylation activity and a three-fold increase of gene expression determined by amleR-lacZfusion was observed in the presence of L-malate [15]. However, inOenococcus oenimalolactic fermentation activity was not enhanced by the presence of MleR or L-malate [16]. Recently Sheng and Marquis showed thatS. mutans possesses MLF activity with a pH optimum of pH 4 in planktonic cells [17]. Significant intracellular ATP maintenance and enhanced protection against lethal pH values were observed in the presence of L-malate [17]. Since this study showed that MLF has a great impact on the aciduric capacities ofS. mutans, we were interested if this mechanism is part of the general ATR of the cell or if it is specifically induced by MleR and the presence of L-malate. Deletion ofmleRand luciferase reporter strains formleRandmleSand RT-PCR revealed insights into the expression and regulation of themlegene cluster and especially the effect of pH. Electrophoretic mobility shift assays (EMSA) indicated several binding sites for.