Functional analysis of the fructooligosaccharide utilization operon in \u3ci\u3eLactobacillus paracasei\u3c/i\u3e 1195

The fosABCDXE operon encodes components of a putative fructose/mannose phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a β- 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 a LPQAG cell wall anchor motif at 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 contained 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 β-fructosidase activity was observed when cells were grown on FOS, inulin, sucrose, or fructose, but not 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