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

Abstract

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.

FIG. 1

(A) Physical map and organization of the L. brevis xylABT locus. The upper part shows the xylB and xylT cloning strategy. The stem-loop structures indicate the putative transcriptional terminators. The nucleotide (nt) sequences of upstream regions of xylA and xylT are depicted in panels B and C, respectively. The open boxes denote putative −10 and −35 consensus sequences of the promoters. The putative regulatory elements (cre and xylO) are in boldface italic letters. The potential RBSs are indicated by asterisks. The beginning of the deduced amino acid sequence of the xylA and xylT genes is depicted below the nucleotide sequence. The putative transcriptional terminator located downstream of the xylB gene is underlined by thin arrows (panel C). For clarity, only the NlaIII and Sau3AI sites used for cloning are depicted.

FIG. 2

Comparison of the primary sequences of XylT of L. brevis (XylT-Lb); GlfZ of Z. mobilis (GlfZ-Zm, Swiss-Prot accession number P21906); XylT of B. megaterium (XylT-Bm, EMBL gene bank accession number Z71474); GalP, AraE, and XylE of E. coli (GalP-Ec, Swiss-Prot accession number P37021; AraE-Ec, Swiss-Prot accession number P09830; XylE-Ec, Swiss-Prot accession number P09098); and XylE of T. halophila (XylE-Th, EMBL gene bank accession number AB009593). The alignment was done by using the Pileup program (9), and some gaps were introduced to maximize the alignment. The identical amino acids are shown in white letters on a solid background. The 12 putative transmembrane segments are indicated by arrows above the alignment. The numbers on the left of the alignment correspond to the amino acid positions for each protein.

FIG. 3

Structure of the xylT lactobacillus-E. coli shuttle expression vector pLPA9(t). The expression cassette of this vector comprises the strong ldh promoter (P_ldh from L. casei ATCC 393), the xylT gene, the E. coli β-glucuronidase gene (gusA) used as a marker of gene expression, and the terminator sequence of the L. plantarum 80 cbh gene (T_cbh [6]). The two terminator sequences (T_ldh from L. casei ATCC 393 [25]) downstream from the strong P_ldh promoter are used to circumvent instability of the expression vector in E. coli. These two T_ldh sequences can be eliminated by digestion of the plasmid with NotI and religation, yielding pLPA9. After transformation of L. plantarum 80 with plasmid pLPA9, the transformants expressing the gusA reporter gene can be selected as described in Materials and Methods.

FIG. 4

d-Xylose uptake by cells of L. plantarum 80(pLPA9) (○; three independent experiments are plotted) or L. plantarum 80 wild type (◊). Cells were preenergized at 30°C by incubation with 50 mM l-malate for 2 min at an extracellular pH of 4.5 (panel A) or by incubation with 5 mM d-glucose for 5 min at an extracellular pH of 6.5 (panel B). Uptake of 0.1 mM d-[U-¹⁴C]xylose by L. plantarum 80(pLPA9) without PMF-generating conditions is shown in both panels (•), and the effect of 20 mM glucose on the accumulation of d-xylose by L. plantarum 80(pLPA9) is indicated (⧫) in panel B. Each experiment was performed at least in triplicate, and the standard deviation never exceeded 10%.

FIG. 5

Eadie-Hofstee plot of the d-[U-¹⁴C]xylose uptake rate by cells of L. plantarum 80(pLPA9) as a function of the d-xylose concentration, without inhibitor (○) or with 0.5 mM (⧫) or 1 mM (•) 6-deoxy-d-glucose. Cells were preenergized at 30°C by incubation with 50 mM l-malate for 2 min at an extracellular pH of 4.5. Rates were calculated after an uptake of 20 s.