Expression of fol genes during folate biosynthesis by Streptococcus thermophilus CRL803 in controlled and non-controlled pH conditions

LAIÑO, J. E.; JUAREZ DEL VALLE, M.; SAVOY DE GIORI, G.; LEBLANC, JEAN GUY

Congreso; XI Congreso Argentino de Microbiología General; 2015

Folates play a key role in many metabolic pathways such as DNA and RNA synthesis. Folate deficiency is very frequent, reason for which many countries have adopted mandatory fortification programs; however, high intakes of folic acid (the synthetic form of folate used in fortification) can mask vitamin B12 deficiency, and alter the activity of the hepatic dihydrofolate reductase enzyme. These adverse health effects do not occurs with natural folates. Certain strains of lactic acid bacteria such as Streptococcus (S.) thermophilus is able to synthetize folates de novo. However, there are no reports about expression of fol genes (folE, folQ, folK, folP, folA, and folC) during folate synthesis. In this study, expression of genes involve in folate synthesis by Streptococcus thermophilus CRL803 in low-folate culture medium was studies batch fermentations with uncontrolled pH and constant pH. This medium was based in LAPTg culture medium without yeast extract (the main source of folate). The temperature was maintained at 42ºC and the pH was kept automatically at 6.0 and 5.0 with 3 M NaOH. To perform gene expression analysis by qRT-PCR, samples were aseptically withdrawn at 4, 6, and 10 h of incubation from the fermentation vessel and immediately cooled on ice, using as reference condition the strain grown at free pH at 42ºC. Optimal condition was selected based on highest folate concentration in shortest time of incubation. Cell populations (live, damaged, and death cells) during incubation (4, 6, and 10 h) were evaluated by Flow cytometry (FACS). Folate production (µg/L), cell viability (log cfu/mL) and turbidity (OD 580nm) were evaluated for 12 h of incubation in both conditions. Folate levels were estimated by the microbiological assay using Lactobacillus rhamnosus NCIMB 10463 as the indicator strain. The results showed that folate production and yields were higher under constant pH condition compared to fermentations at free pH. The highest folate production was reached at 6 h of incubation at pH 6.0 (237±14 µg/L) compared to uncontrolled pH conditions (89±8 µg/L). Besides, at pH 6.0, highest values of viability (log cfu/mL 9.1±0.3) were observed, compared to uncontrolled pH (log cfu/mL 7.9±0.4). Based on these results, optimal condition for qRT-PCR was pH 6.0 at 42ºC. A significant increase in de novo fol genes expression (folE=3.7±0.2, folQ=2.7±0.1, folK=4.1±0.2, and folP=2.6±0.2 fold changes pH 6.0 vs free pH) was observed after 6 h of incubation, without significant modifications in folA and folC expression. Flow cytometry analysis revealed an increased sensibility to culture medium pH as is reflected by increase in damaged (15.4±2.4%) cell population at uncontrolled pH respect to pH 6.0 (4.1±0.7%). Conclusion. Streptococcus thermophilus CRL803 grown at pH 6.0 was able to increase almost 3 times the folate concentration by a highest biosynthesis of vitamin as was reflected by increase of de novo fol genes and lower percentage of damaged cells compared to free pH.