ABSTRACTWe have previously characterized fromLactobacillus caseiBL23 three α-l-fucosidases, AlfA, AlfB, and AlfC, which hydrolyzein vitronatural fucosyl-oligosaccharides. In this work, we have shown thatL. caseiis able to grow in the presence of fucosyl-α-1,3-N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly,L. caseiexcretes thel-fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only theN-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence ofL. caseiBL23 shows that downstream fromalfB, which encodes the α-l-fucosidase AlfB, a gene,alfR, that encodes a transcriptional regulator is present. Divergently fromalfB, three genes,alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of eitheralfBoralfFabolishes the growth ofL. caseion Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded byalfEFGparticipates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded byalfEFGis not coupled to phosphorylation of the disaccharide. Transcriptional analysis withalfRandccpAmutants shows that the two gene clustersalfBRandalfEFGare regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α-l-fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria.