Chaillou S, Bor Y C, Batt C A, Postma P W, Pouwels P H
EC Slater Institute, BioCentrum, University of Amsterdam, 1018 TV Amsterdam, The Netherlands.
Appl Environ Microbiol. 1998 Dec;64(12):4720-8. doi: 10.1128/AEM.64.12.4720-4728.1998.
A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids 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+ symporter 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 xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport 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-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, 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 of D-xylose by XylT competitively, with a Ki of 220 microM.
将短乳杆菌木糖A基因（编码D - 木糖异构酶）下游的一个3 kb区域克隆到大肠杆菌TG1中。该序列显示有两个开放阅读框，它们可能编码D - 木酮糖激酶基因（xylB）和另一个基因（xylT），后者编码一种由457个氨基酸组成的蛋白质，与大肠杆菌的D - 木糖 - H⁺同向转运蛋白XylE（相似度57%）、巨大芽孢杆菌的XylT（相似度58%）、嗜盐四联球菌的D - 木糖 - Na⁺同向转运蛋白XylE（相似度57%）以及大肠杆菌的L - 阿拉伯糖 - H⁺同向转运蛋白AraE（相似度60%）具有显著相似性。短乳杆菌的xylABT基因呈现出与巨大芽孢杆菌的xylABT操纵子和嗜盐四联球菌的xylABE操纵子相似的排列方式。以戊糖乳杆菌木糖R基因（编码D - 木糖阻遏蛋白）为探针进行的Southern杂交表明，短乳杆菌中存在木糖R同源物，但其并不位于xyABT基因座处。木糖A和木糖T上游存在木糖O序列，以及短乳杆菌中D - 木糖异构酶、D - 木酮糖激酶和D - 木糖转运活性的诱导需要D - 木糖，这些进一步表明存在功能性的XylR。当短乳杆菌在D - 葡萄糖和D - 木糖的混合物中培养时，D - 木糖异构酶和D - 木酮糖激酶活性降低了四倍，D - 木糖转运活性降低了六倍，这表明D - 葡萄糖对D - 木糖同化存在分解代谢物阻遏作用。木糖T基因在植物乳杆菌80中实现了功能性表达，该菌株缺乏与质子动力相关的D - 木糖转运活性。植物乳杆菌80细胞中D - 木糖的积累证实了XylT蛋白的作用，并且这种积累依赖于苹果酸 - 乳酸发酵或D - 葡萄糖代谢产生的质子动力。XylT对D - 木糖的表观亲和常数约为215 μM，最大初始转运速度为35 nmol/（min·mg干重）。此外，在测试的多种糖类中，只有6 - 脱氧 - D - 葡萄糖竞争性抑制XylT对D - 木糖的转运，其抑制常数Ki为220 μM。
