Molecular cloning and functional expression in Lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (en coding D-xylose isomerase), was cloned In Escherichia coli TG1, The sequenc e revealed two open reading frames which could code for the D-xylulose kina se gene (xylB) and another gene (xylT) encoding a protein of 457 amino adds with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symport er of E, coli, AraE (60%), The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xy lABE operon, Southern hybridization performed with the Lactobacillus Lactob acillus xylR gene (encoding the D-xylose repressor protein) as a probe reve aled the existence of a xylR homologue in L. brevis which is not located wi th the xyABT locus. The existence of a functional XylR was further suggeste d by the presence of xylO sequences upstream of xylA and xylT and by the re quirement of D-xylose for the induction of D-xylose isomerase, D-xylulose k inase, and D-xylose transport activities in L, brevis. When L. brevis was c ultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase an d D-xylulose kinase activities were reduced fourfold and the D-xylose trans port activity was reduced by sixfold, suggesting catabolite repression by D -glucose of D-xylose assimilation, The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-li nked D-xylose transport activity. The role of the XylT protein was confirme d bg:the accumulation of D-xylose in L. plantarum 80 cells, and this accumu lation was dependent on the proton motive force generated by either malolac tic fermentation or by the metabolism of D-glucose. The apparent affinity c onstant of XylT far D-xylose was approximately 215 mu M, and the maximal in itial velocity of transport was 35 nmol/min per mg (drg weight), Furthermor e, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the trans port of D-xylose by XylT competitively, with a K-i of 220 mu M.