Functional Analysis of the Fructooligosaccharide Utilization Operon in Lactobacillus paracasei 1195

Abstract

The fosABCDXE operon encodes components of a putative fructose/mannose phosphoenolpyruvate-dependent phosphotransferase system and a ␤-fructosidase precursor (FosE) that are involved in the fructooligosaccharide (FOS) utilization pathway of Lactobacillus paracasei 1195. The presence of an N-terminal signal peptide sequence and an LPQAG cell wall anchor motif in the C-terminal region of the deduced FosE precursor amino acid sequence predicted that the enzyme is cell wall associated, indicating that FOS may be hydrolyzed extracellularly. In this study, cell fractionation experiments demonstrated that the FOS hydrolysis activity was present exclusively in the cell wall extract of L. paracasei previously grown on FOS. In contrast, no measurable FOS hydrolysis activity was detected in the cell wall extract from the isogenic fosE mutant. Induction of ␤-fructosidase activity was observed when cells were grown on FOS, inulin, sucrose, or fructose but not when cells were grown on glucose. A diauxic growth pattern was observed when cells were grown on FOS in the presence of limiting glucose (0.1%). Analysis of the culture supernatant revealed that glucose was consumed first, followed by the longer-chain FOS species. Transcription analysis further showed that the fos operon was expressed only after glucose was depleted in the medium. Expression of fosE in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS, inulin, sucrose, and levan.The consumption of fermented food products or dietary supplements containing probiotic species of Lactobacillus and Bifidobacterium has been suggested to promote gastrointestinal (GI) health in humans and other animals by increasing the population of these microorganisms in the GI tract (10, 40). However, the beneficial effects of these bacteria may be transient due to colonization resistance by the commensal microbiota, which restricts the ability of probiotic bacteria to become well established in the intestinal environment (3, 15). An approach to overcome this limitation is to include prebiotics in the host diet. Prebiotics are specific nondigestible dietary sugars that are selectively metabolized by certain probiotic bacteria and that enhance their survival and colonization in the GI tract (12). Such an approach would also enrich the population of indigenous bifidobacteria and lactobacilli, allowing them to occupy a more dominant position in the gut ecosystem.Fructooligosaccharides (FOS) are among the prebiotic substances that have been shown to selectively stimulate the growth and activity of certain strains of Lactobacillus and Bifidobacterium (4,11,13,16,47). Two types of FOS, which differ based on their methods of preparation, are commercially available and are widely used in food. One type, referred to as the GFn type of FOS, is enzymatically produced from sucrose and consists of a glucose monomer (G) linked by ␣-1,2 linkages to two or more ␤-2,1-linked fructose units (F), forming a mixture of GF 2 , GF 3 , and GF 4 (16,17). The ot...

Comparison of Fructooligosaccharide Utilization byLactobacillusandBacteroidesSpecies

“…31) From these facts, it has been proposed that FOS are not metabolized by extracellular enzymes in L. acidophilus NCFM. 30,31) However, we found here that L. acidophilus JCM1023, JCM1028, and JCM5342 metabolized FOS by extracellular enzymes. If we are to clear up this discrepancy, further studies on the extracellular enzymes of these L. acidophilus strains are now essential.…”

“…30) In one route, FOS are transported intact into the cytoplasm where they are hydrolyzed, in the other, they are hydrolyzed by extracellular enzymes, then the hydrolysates are transported into cytoplasm. Among Lactobacillus strains, only the L. acidophilus strains able to degrade GF 3 had extracellular enzymes, suggesting that relatively longer-chain FOS tend to be hydrolyzed by extracellular enzymes rather than being transported intact.…”

Synbiotic functional drink from Jerusalem artichoke juice fermented by probiotic Lactobacillus plantarum PCS26

“…The diauxic growth of L. plantarum when cultivated on complex carbohydrates was also noticed by Hasan and Durr (1974). Goh et al (2007) observed the same growth pattern for L. paracasei in the presence of fructooligosaccharides and limited amount of glucose in the medium (0.1 %). They concluded that oligosaccharides are hydrolyzed extracellularly with the cell wall associated enzyme β-fructosidase, induced by the presence of inulin, sucrose and fructose in the medium.…”

Metabolism and physiology of Lactobacilli: a review

“…However, other authors believe that FOS transport through the bacterial cell occurs through PTS (phosphotransferase system) (Goh et al, 2006(Goh et al, , 2007Chen et al, 2015). In the cytoplasm, FOS is hydrolyzed to fructose and glucose-6-phosphate by the action of the enzyme β-fructofuranosidase.…”