Measurements of Intra- and Extra-Cellular 5-Methyltetrahydrofolate Indicate that Bifidobacterium Adolescentis DSM 20083T and Bifidobacterium Pseudocatenulatum DSM 20438T Do Not Actively Excrete 5-Methyltetrahydrofolate In vitro

Abstract

Certain intestinal bifidobacteria have the ability to synthesize folates. In vitro experiments revealed a high production, cellular accumulation, and release of reduced folate vitamers like 5-methyltetrahydrofolate and tetrahydrofolate in folate-free medium (FFM). However, it is still unclear to which extent synthesized folates are polyglutamylated and probably not available for transport, and if they are actively released by excretion. To address these questions, we characterized intra- and extra-cellular pteroylmonoglutamates and polyglutamylated 5-methyltetrahydrofolate (5-CH3-H4PteGlu2−4) in Bifidobacterium adolescentis DSM 20083T and Bifidobacterium pseudocatenulatum DSM 20438T in vitro. Folates were measured by means of stable isotope dilution assays (SIDA) coupled with LC-MS/MS analysis using [2H4]-5-methyltetrahydrofolic acid, [2H4]-tetrahydrofolic acid, and [2H4]-5-formyltetrahydrofolic acid as internal standards. Cell viability was examined by fluorescence microscopy. Quantitation of folate production by B. adolescentis during the stationary phase revealed a linear increase of dead cells paralleled by increasing concentration of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate (100% 5-CH3-H4PteGlu4) in FFM, whereas the intracellular concentrations of these vitamers remained constant. After 24 h, B. adolescentis (125 mg cells, wet weight) produced a total amount of 0.846 nmol 5-CH3-H4folate: 0.385 ± 0.059 nmol (46 ± 7%) and 0.461 ± 0.095 nmol (54 ± 11%) measured in the intracellular (viable cells; 52 ± 3% measured by fluorescence microscopy) and extracellular (lysed cells; 48 ± 3%) fraction, respectively. For B. pseudocatenulatum (124 mg cells, wet weight), 1.135 nmol 5-CH3-H4folate was produced after 24 h, and a similar proportionality between intra- and extra-cellular folate concentrations and viable/lysed cells was observed. These results indicate that the strains tested produce and accumulate 5-CH3-H4PteGlu4 for cellular metabolism, and that extracellular concentrations of the vitamer arise from cell lysis.

A Structural Basis for Biguanide Activity

“…The likely variation of biguanide–DHFR K i values for different species of gut bacteria provides one possible basis for species selection in metformin-treated patients. , Indeed, the first A–B loop in DHFR derived from different species shows extreme sequence variability, and as discussed above, the binding affinity for pABG differs substantially between the E. coli and L. casei enzymes. Another effect mediated by gut bacteria is based on observations that these microorganisms provide a portion of the vitamins utilized by their host. , Perturbation of folate metabolism in these organisms by biguanides may directly impact the composition of the available folate pool and indirectly influence the availability of other nutrients.…”

“…their host 76,77. Perturbation of folate metabolism in these organisms by biguanides may directly impact the composition of the available folate pool and indirectly influence the availability of other nutrients.The study of E. coli-fed C. elegans by Cabreiro et al,…”

A Novel Synthesis of a Magnetic Porous Imprinted Polymer by Polyol Method Coupled with Electrochemical Biomimetic Sensor for the Detection of Folate in Food Samples

“…First, the solution was sonicated for 15 min and was then diluted to 100 mL with deionized water. The working solutions were prepared using different concentrations (2,4,6,8,10,12, and 14 mg L −1 ) of the standard stock solution. All other solutions used in the experiments-including sulphonamides, curcumin, biotin, and tetracycline solutions-were prepared in the same way.…”

“…Folate or vitamin B9 refers to a group of water-soluble vitamins and a large number of structurally similar compounds. Folates are essential nutrients that can be synthesized by plant species [1], as well as by some bifidobacterial strains [2] and yeast strains [3][4][5]. An inadequate folate amount in pregnant and breastfeeding women can lead to birth defects in newborns [6]; the development of cardiovascular diseases [6], cancer, and Alzheimer's disease [7,8].…”

Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5‐methyltetrahydrofolate

“…The bioproduction of folates by microbial fermentation has been investigated in various production hosts, including Bacillus subtilis, Lactococcus lactis, Bifidobacterium adolescentis, Bifidobacterium pseudocatenulatum , and Ashbya gossypii (Kopp, Dürr, Steigleder, Clavel, & Rychlik, 2017; Serrano‐Amatriain et al, 2016; Sybesma et al, 2003; Zhu et al, 2005). However, the total folate titers in B. subtilis, L. lactis, B. adolescentis , and B. pseudocatenulatum are low, ranging from 78.49 to 163 μg/L, which has increased the cost of FA production and reduced economic competitiveness compared to chemical synthesis.…”