Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl ؊ . Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl ؉ strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose. Xylose metabolism has been described for a wide array of microorganisms. Extensively characterized xylose-metabolizing (Xyl ϩ ) bacteria include Escherichia coli (24, 46), Lactobacillus spp. (3,4,(26)(27)(28), Bacillus spp. (16,33,38), and Staphylococcus xylosus (44). Free xylose can be transported via low-affinity symporters (XylE or XylT) or high-affinity binding-protein dependent systems (XylFGH) (1, 6, 9, 41). Xylose isomerase (xylA gene) then converts the aldose sugar to xylulose, which is phosphorylated by xylulokinase (xylB gene). Further metabolism of xylulose-5-phosphate occurs via the pentose-phosphate or phosphoketolase pathways. Xylose metabolism is induced by xylose, mediated via XylR. In Salmonella and E. coli strains, XylR is an activator when xylose is present (42,46). In grampositive organisms, XylR is a repressor which is inactivated when xylose binds (13,26,27,37,44,45). The catabolite repression of xylose metabolism by glucose is mediated primarily through the CcpA protein (40).Lactococcus lactis subsp. cremoris and almost all L. lactis subsp. lactis strains cannot metabolize xylose. These organisms have undergone intense selection for use in dairy fermentations, but plants are thought to be their original ecological niche because L. lactis subsp. lactis isolates have been recovered from many different plants (22,34,35). Also, the plant isolate Lactobacillus xylosus has been reclassified as L. lactis subsp. lactis (39). However, L. lactis subsp. cremoris strains are almost exclusively dairy associated (22).We discovered xylose metabolic genes from both plant (Xyl ϩ ) and dairy (Xyl Ϫ ) isolates of L. lactis. The sequencing of xylRAB from L. lactis strains IO-1, 210, a...