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A 3-kb region, located downstream of theLactobacillus brevis xylA gene (encodingd -xylose isomerase), was cloned inEscherichia coli TG1. The sequence revealed two open reading frames which could code for thed -xylulose kinase gene (xylB ) and another gene (xylT ) encoding a protein of 457 amino acids with significant similarity to thed -xylose–H+ symporters ofE. coli , XylE (57%), andBacillus megaterium , XylT (58%), to thed -xylose–Na+ symporter ofTetragenococcus halophila , XylE (57%), and to thel -arabinose–H+ symporter ofE. coli , AraE (60%). TheL. brevis xylABT genes showed an arrangement similar to that of theB. megaterium xylABT operon and theT. halophila xylABE operon. Southern hybridization performed with theLactobacillus pentosus xylR gene (encoding thed -xylose repressor protein) as a probe revealed the existence of axylR homologue inL. brevis which is not located with thexyABT locus. The existence of a functional XylR was further suggested by the presence ofxylO sequences upstream ofxylA andxylT and by the requirement ofd -xylose for the induction ofd -xylose isomerase,d -xylulose kinase, andd -xylose transport activities inL. brevis . WhenL. brevis was cultivated in a mixture ofd -glucose andd -xylose, thed -xylose isomerase andd -xylulose kinase activities were reduced fourfold and thed -xylose transport activity was reduced by sixfold, suggesting catabolite repression byd -glucose ofd -xylose assimilation. ThexylT gene was functionally expressed inLactobacillus plantarum 80, a strain which lacks proton motive force-linkedd -xylose transport activity. The role of the XylT protein was confirmed by the accumulation ofd -xylose inL. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism ofd -glucose. The apparent affinity constant of XylT ford -xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-d -glucose inhibited the transport ofd -xylose by XylT competitively, with aKi of 220 μM.

applied and environmental microbiology

Molecular Cloning and Functional Expression in Lactobacillus plantarum 80 of xylT , Encoding the <scp>d</scp> -Xylose–H + Symporter of Lactobacillus brevis

Molecular Cloning and Functional Expression in Lactobacillus plantarum 80 of xylT , Encoding the d -Xylose–H + Symporter of Lactobacillus brevis

Molecular Cloning and Functional Expression in Lactobacillus plantarum 80 of xylT , Encoding the d -Xylose–H ⁺ Symporter of Lactobacillus brevis