Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity

Abstract

Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum.

FIG 1

Growth of L. plantarum ST-III in CDM either alone (no added sugar) or supplemented with 1% commercial FOS or 1% glucose. To account for the glucose, fructose, and sucrose in the commercial FOS, cells were also grown in CDM containing equivalent amounts of these sugars. The two sampling points are indicated by dashed lines. Sampling point 1 was chosen for transcriptomic analysis; both of the sampling points were chosen for lipid extraction, the detection of membrane fluidity, and the analysis of metabolites. RT-qPCR was used for the conformation of the expression levels of the key genes at the two sampling points. Data are mean values based on at least three replicates. Error bars indicate standard deviations.

FIG 2

Functional classification according to COGs of genes differentially expressed by L. plantarum ST-III grown in the presence of FOS versus glucose. The profile of each functional classes is shown as the percentage of all genes in the class whose expression was significantly upregulated (open bars) or downregulated (filled bars).

FIG 3

Genes differentially expressed in the carbohydrate utilization and metabolite production pathways in L. plantarum ST-III during growth on FOS compared with growth on glucose. Genes that were upregulated are shown in red. PTS, phosphotransferase system; SacA, β-fructofuranosidase; SacK, fructokinase; LDH, lactate dehydrogenase; POX, pyruvate oxidase; ACK, acetate kinase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate lyase; PTA, phosphotransacetylase; ACDH, acetaldehyde dehydrogenase; ADHE, alcohol dehydrogenase; CoA, coenzyme A.

FIG 4

Growth of mutant strains of L. plantarum ST-III in CDM either alone (no added sugar) or supplemented with 1% commercial FOS or glucose. (A) BD1101CM, a ΔsacA mutant; (B) BD1102, a Δpts1 mutant; (C) BD1103CM, a Δpts26 mutant; (D) BD1104CM, a Δpts1 Δpts26 mutant. Data are mean values based on at least three replicates. Error bars indicate standard deviations. CHO, carbohydrate.

FIG 5

Differences in the distribution of FAs in L. plantarum ST-III cells grown in the presence of FOS from that in cells grown in the presence of glucose. The proportions of total-membrane FAs were determined in early-logarithmic (OD₆₀₀, 0.65) and mid-logarithmic (OD₆₀₀, 1.5) cultures of L. plantarum ST-III grown in CDM containing either 1% commercial FOS or glucose. The data are mean values based on at least three replicates. Error bars indicate standard deviations.

FIG 6

Differences in membrane fluidity between L. plantarum ST-III cells grown on FOS and those grown on glucose. Fluorescence anisotropy values were determined in early-logarithmic (OD₆₀₀, 0.65) and mid-logarithmic (OD₆₀₀, 1.5) cultures of L. plantarum ST-III grown in CDM containing either 1% commercial FOS (shaded bars) or glucose (open bars). Data are mean values based on at least three replicates. Error bars indicate standard deviations. Values that indicated statistically significant differences (P ≤ 0.05) between cells grown on FOS and those grown on glucose are marked with an asterisk or an octothorpe at an OD₆₀₀ of 0.65 or 1.5, respectively.

FIG 7

Prediction of the cre and operator sites in the sacPTS1 (A) and sacPTS26 (B) clusters of L. plantarum ST-III. Putative functions are indicated by color as follows: green, PTS; black, transcriptional regulators; red, glycoside hydrolases; yellow, enzymes involved in the glycolytic pathway. Predicted transcription terminators are shown as hairpin loops. The potential cre sites are underlined, and the specific operators are shaded. The presumed start codon of each gene is shown in capital letters, and the putative −10 and −35 promoter regions and possible ribosome-binding sites (RBS) are marked.