Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract

Abstract

Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo. In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two β-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway. IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

Phenotypic and genetic characterization of differential galacto-oligosaccharide utilization in Lactobacillus plantarum

“…Not much is known about the mechanism of import and subsequent hydrolysis of GOS in L. plantarum, although a recent comparative transcription analysis of L. plantarum STIII revealed the GOS-growth induced upregulation of two gene clusters harboring various galactose- and lactose utilization genes, including lacS, lacA, lacL and lacM 23 . Similar genes were also identified to be involved in the utilization of GOS in L. reuteri ATCC PTA-6475 22 .…”

“…It remains to be established what the precise roles of LacA and LacLM in GOS hydrolysis are in L. plantarum (i.e., DP and branched or linear structure). Intriguingly, a recent investigation of GOS metabolism in L. reuteri ATCC PTA-6475 established a role of its LacA homologue (~ 37% identity with LacA in L. plantarum ) in hydrolysis of galactose-galactose linkages in GOS, but also showed that this enzyme could not hydrolyze lactose, which was dependent on the LacLM homologue in this species (~ 64% identity with L. plantarum ) 22 . Moreover, a LacS deficient L. reuteri derivative (~ 58% identity with L. plantarum LacS) displayed reduced growth efficiency on GOS as well as lactose, which is similar to the LacS mutant of L. plantarum in this study.…”

“…In lactobacilli in general, it is thought that GOS is imported by lactose permeases (LacS), which was verified in L. acidophilus NCFM to be the sole importer for GOS and lactose 19 . Once imported, lactose is hydrolyzed by β-galactosidases of Glycoside Hydrolase family 2 20 (LacZ/LacLM), whereas GOS is proposed to be digested by GH family 42 (LacA-type) β-galactosidases 21 , 22 . Not much is known about the mechanism of import and subsequent hydrolysis of GOS in L. plantarum, although a recent comparative transcription analysis of L. plantarum STIII revealed the GOS-growth induced upregulation of two gene clusters harboring various galactose- and lactose utilization genes, including lacS, lacA, lacL and lacM 23 .…”

A Phylogenetic View on the Role of Glycerol for Growth Enhancement and Reuterin Formation in Limosilactobacillus reuteri

“…Moreover, although our findings provide important basic information for the generation of hypotheses on the ecological role of pdu cluster, these hypotheses should be evaluated through in vivo experiments in relevant host systems using experimental designs similar to those utilized in our previous studies ( Duar et al, 2017 ; Lin et al, 2018 ; Cheng et al, 2020 ). Finally, pdu cluster functionality should be tested with additional carbohydrates that are therapeutically relevant, such as prebiotics ( Rattanaprasert et al, 2019 ).…”

Comparative Genomic and Functional Evaluations of Bacillus subtilis Newly Isolated from Korean Traditional Fermented Foods

“…Among the five strains, SRCM103571 and SRCM103689 possessed a number of unique genes including cscB, lacF_2, lacG, maa_1, nisB, nisC_2, sacA, and spaS, which are involved in disaccharide permeases, sucrose-5-phosphate hydrolase, nisin biosynthesis, and lantibiotic subtilin synthesis. Disaccharide metabolism-associated proteins including fructooligosaccharide and galactooligosaccharide permeases have been reported to be essential for colonization, have effects on cell membrane fluidity, and be required for prebiotic utilization, providing the bacterial strains to attach and colonize the host intestine [64][65][66]. Additionally, peptides such as nisin and subtilin, encoded by nisB, nisC, and spaS, are bacteriocin effective against many Gram-positive organisms, functioning as probable probiotics with antimicrobial activities [67][68][69].…”