9 and pH 7.5). The asterisk indicates statistically CB-839 order significant difference (p ≤ 0.005; Student’s t-test) in comparison to the other conditions. C: Effect of different tyrosine concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 2, 5 and 10 mM) on tyrS expression AR-13324 purchase at pH 4.9 The strength of these environmental conditions on tyrS expression was quantified by RT-qPCR. Data in Figure 1B confirmed that tyrS is maximally transcribed in absence of tyrosine and at pH 4.9, showing a greater than 10-fold induction in mRNA levels over levels occurring in presence of tyrosine. Even when tyrosine was not added to the media,

no induction was detected at pH 7.5. These results confirmed that both conditions (acidic pH and absence of tyrosine) are needed

for expression of tyrS gene. Next, we examined whether intermediate tyrosine concentrations have an effect on tyrS expression. Therefore, we investigated at optimal pH 4.9, the effect that different tyrosine concentrations in the media (0, 0.01, 0.05, 0.1, 0.5, 1, 2, 5 and 10 mM) exert on gene expression by comparing RT-qPCR results obtained in each condition. As indicated in Figure 1C, tyrS expression showed an inverse correlation with the increased tyrosine concentration and exhibited a great sensitivity to very low tyrosine levels, since the maximal expression level was reached in absence of tyrosine and an increase of 0.01 mM tyrosine in the media was enough to reduce this level to the half. Not significant changes in transcription were observed above 2 mM Selleckchem JIB04 tyrosine, probably because of saturating concentrations of tyrosine. Such concentrations were assayed because tyrosine can reach very high concentrations in some cheeses and even precipitate forming crystals [20]. Mapping of the tyrS transcription initiation site To map the precise start point of the transcription of tyrS, primer extension was performed using

RNA samples extracted under optimal conditions of expression (pH 4.9 and absence of tyrosine). A single band of 322 PIK3C2G bp was observed, indicating that the position +1 of the mRNA corresponds to a T residue located 322 nucleotides upstream of the ATG codon (Figure 2). Seven nucleotides upstream this point, it was localized the -10 sequence TATGAT spaced 17 nucleotides downstream of the -35 sequence TTGACA, that nearly matched the consensus sequence for LAB promoters [21]. In a position 9-14 nucleotides upstream the ATG codon of this gene, it was identified the Shine-Dalgarno region (CGGAGG) (bases fitting with the consensus sequences are underlined). Figure 2 Primer extension identification of transcription start site (*) of tyrS and transcriptional regions -10 and -35 (boxes).