Since this study showed that MLF has a great impact on the aciduric capacities of S. 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 of mleR and luciferase reporter strains for mleR and mleS and RT-PCR revealed insights into the expression and regulation of the mle gene cluster and especially the effect of pH. Electrophoretic mobility shift assays (EMSA) indicated several binding sites for the MleR protein which were influenced by the presence of L-malate. Moreover we investigated the role of

MleR for the ability of S. mutans to withstand acid stress. Results Analysis of the mle locus by RT-PCR and EMSA In the genome of S. mutans UA159 [14], the lysR type transcriptional regulator MleR is orientated opposite to a gene cluster #Sotrastaurin purchase randurls[1|1|,|CHEM1|]# encoding the malolactic enzyme (mleS), a malate permease (mleP), and a oxalate decarboxylase (oxdC),

respectively. Additionally a putative prophage repressor is inserted between mleR and mleS (Figure 1). This insertion is unique for the oral streptococci (S. mutans UA159, S. gordonii str. Challis CH1 and S. sanguinis SK36) among all sequenced Lactobacillales. Adjacent to the genes involved in malolactic fermentation is the gene oxdC encoding the oxalate decarboxylase which catalyses the conversion of oxalate to formate and CO2. This gene is unique for S. mutans UA159 PF 01367338 among all sequenced Lactobacillales. RT-PCR disclosed that it is co-transcribed with mleS and mleP since it was possible to amplify overlapping fragments of all three genes (Figure 1A). The putative gluthatione reductase (Smu.140) located downstream of oxdC, which is involved in the removal of reactive oxygen species, could not be assigned to the same operon by the use of RT-PCR. Figure 1 Genetic organization of the mle locus. A:

RT-PCR analysis of mRNA transcripts. The solid arrows indicate the primers used for RT-PCR. The minus RT control is assigned with “”-”"; the positive control, using genomic DNA, is assigned with “”+”". B: Gelshift analysis of the region between mleS and mleR. Arrows indicate primers that were used to amplify PCR products, that were subsequently used for EMSA. Primers are designated at their 5′ end. The box shows CYTH4 a representative selection of gel shift assays with the respective fragment in the presence or absence of L-malate. Thin arrows indicate DNA fragments in the absence of protein. Bold arrows indicate DNA in complex with MleR. Competitor DNA is marked with an asterix. For all EMSAs, 1× binding buffer was loaded on the left and MleR protein on the right lane. In all EMSAs without malate, an internal fragment of mleS was used as competitor DNA. In EMSAs with malate the fragment within the IGS of mleR and Smu.136c, generated by hybridising primers EP10/11 was used (except for EMSA 5, where the internal fragment of mleS was added).