Oligosaccharide Metabolism and Lipoteichoic Acid Production in Lactobacillus gasseri and Lactobacillus paragasseri

Abstract

Lactobacillus gasseri and Lactobacillus paragasseri are human commensal lactobacilli that are candidates for probiotic application. Knowledge of their oligosaccharide metabolic properties is valuable for synbiotic application. The present study characterized oligosaccharide metabolic systems and their impact on lipoteichoic acid (LTA) production in the two organisms, i.e., L. gasseri JCM 1131^T and L. paragasseri JCM 11657. The two strains grew well in medium with glucose but poorly in medium with raffinose, and growth rates in medium with kestose differed between the strains. Oligosaccharide metabolism markedly influenced their LTA production, and apparent molecular size of LTA in electrophoresis recovered from cells cultured with glucose and kestose differed from that from cells cultured with raffinose in the strains. On the other hand, more than 15-fold more LTA was observed in the L. gasseri cells cultured with raffinose when compared with glucose or kestose after incubation for 15 h. Transcriptome analysis identified glycoside hydrolase family 32 enzyme as a potential kestose hydrolysis enzyme in the two strains. Transcriptomic levels of multiple genes in the dlt operon, involved in D-alanine substitution of LTA, were lower in cells cultured with raffinose than in those cultured with kestose or glucose. This suggested that the different sizes of LTA observed among the carbohydrates tested were partly due to different levels of alanylation of LTA. The present study indicates that available oligosaccharide has the impact on the LTA production of the industrially important lactobacilli, which might influence their probiotic properties.

Keywords: Lactobacillus gasseri; Lactobacillus paragasseri; RNAseq; glycoside hydrolase; kestose; lipoteichoic acid.

Figure 1

Growth characteristics of L. gasseri JCM 1131^T and L. paragasseri JCM 11657 cultured in the presence of glucose, kestose, or raffinose. Bars show averaged cell growth expressed as optical density at 660 nm (OD_{660 nm}), and error bars indicate standard deviations (n = 3). ^* Sugar-free medium.

Figure 2

Immunoblot detection of lipoteichoic acid (LTA) in L. gasseri JCM 1131^T and L. paragasseri JCM 11657 cells (a), and amount of LTA in 1 µL of suspended (OD_{660 nm} = 1) L. gasseri cells (b), and L. paragasseri cells (c) cultured with glucose (denoted by G), kestose (denoted by K), or raffinose (denoted by R). Lane M in (a) is the size marker. Each lane in (a) is the μL of cell suspensions (OD_{660 nm} = 5) was used for each test, except OD_{660 nm} = 0.5 was used for L. gasseri cells cultured with raffinose because of the high LTA quantity. One-way ANOVA and Tukey’s post hoc test were applied to compare the amount of LTA among the cells cultured with different carbohydrates.

Figure 3

Sequences around the NDPNG motif (red boxed) of glycoside hydrolase (GH) 32 sucrose-6-phosphate hydrolase (S6PHs) found in L. gasseri JCM 1131^T and L. paragasseri JCM 11657. GH32 S6PH of L. gasseri strain 224-1, whose crystal structure has been determined (PDB ID, 6NU8), was included as a reference.

Figure 4

Hierarchical clustering (a) and (b) and Principal component analysis (PCA, c and d) based on transcriptome analysis of L. gasseri JCM 1131^T (a) and (c) and L. paragasseri JCM 11657 (b and d) in cultures with glucose (denoted by g), kestose (denoted by k), or raffinose (denoted by r). The transcriptome analysis was performed in triplicate using RNA isolated from three different cultures for each carbohydrate.