APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1998, p. 4720–4728 0099-2240/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 64, No. 12
Molecular Cloning and Functional Expression in Lactobacillus plantarum 80 of xylT, Encoding the D-Xylose–H1 Symporter of Lactobacillus brevis ´ PHANE CHAILLOU,1,2 YEOU-CHERNG BOR,3 CARL A. BATT,3 STE PIETER W. POSTMA,1 AND PETER H. POUWELS1,2* EC Slater Institute, BioCentrum, University of Amsterdam, 1018 TV Amsterdam,1 and Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, 3700 AJ Zeist,2 The Netherlands, and Department of Food Science, Cornell University, Ithaca, New York 148533 Received 25 June 1998/Accepted 28 September 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–H1 symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the 1 1 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 mM, 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 mM. Lactobacillus brevis is a ubiquitous microorganism that can be isolated from various biotopes, such as milk, fermented vegetables, and the intestinal tracks of animals and that is often found as a spoilage contaminant in beer production (7, 11, 26). Fermented plant materials, one of the predominant ecological niches of L. brevis, are usually rich in hemicellulose fibers and therefore represent an abundant source of D-xylose, from which L. brevis can derive energy for growth. The fermentation of D-xylose is not a common property among Lactobacillus species. Besides L. brevis and Lactobacillus pentosus, most of the other lactobacilli are unable to utilize D-xylose as an energy source. The ability to ferment D-xylose, however, could improve the properties of heterofermentative lactic acid bacteria that are commonly used in fermented-food technology. Lactobacillus plantarum, for instance, is widely used to stimulate the fermentation of silage, sourdough, and diverse vegetables, such as the white cabbages used in the production of sauerkraut. L. plantarum cannot ferment D-xylose, although this property could potentially improve the competitive position of this or-
ganism in the fermentation of plant materials and would conform to the food-grade status of recombinant strains. The use of D-xylose metabolism as a food-grade selection marker for heterofermentative lactobacilli has already been proposed by Posno et al. (24). A plasmid, pLP3537-xyl, harboring the D-xylose catabolizing genes of L. pentosus, was used to complement the inability of Lactobacillus casei ATCC 393 to metabolize this pentose. However, the growth of the L. casei transformants carrying pLP3537-xyl was slow compared to that of L. pentosus, which naturally ferments D-xylose. In that study, the authors postulated that the transport of D-xylose in L. casei was the limiting function for growth on this compound. Indeed, pLP3537xyl lacked a specific transporter for D-xylose, the absence of which could limit its general use. Consequently, we aimed at the characterization and functional analysis of a D-xylose transporter from a Lactobacillus species that could be used to optimize the food-grade vector based on D-xylose fermentation. So far, no evidence indicating the presence of a specific transporter for D-xylose in L. pentosus has been obtained, but the situation could be different with L. brevis. The xylA gene (encoding D-xylose isomerase) of L. brevis has previously been cloned and sequenced (2). Sequencing of regions downstream of the xylA gene revealed the presence of another, albeit incomplete, gene: xylB (encoding D-xylulose kinase). The possibility that one or more genes specifying a
* Corresponding author. Mailing address: TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, P.O. Box 360, 3700 AJ Zeist, The Netherlands. Phone: 31-30-6944-462. Fax: 31-30-6944-466. E-mail:
[email protected] .nl. 4720
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D-XYLOSE–H
D-xylose transporter of L. brevis could be located in the surrounding of the xylA and xylB genes was investigated. We report here the cloning of the xylT gene encoding the proton motive force (PMF)-linked D-xylose transport system of L. brevis and its functional expression in L. plantarum 80 by using a Lactobacillus expression system suitable for developing a food-grade vector based on D-xylose fermentation. We also provide information on the arrangement of the xyl genes in L. brevis, an arrangement that is similar to that which is found in Bacillus megaterium (30) and Tetragenococcus halophila (33), except that a repressor gene, xylR, is lacking in front of xylA in L. brevis. We also show that the L. brevis D-xylose transporter is remarkably similar to XylE of Escherichia coli (8) and T. halophila and to XylT of B. megaterium in its primary sequence and substrate specificity.
MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. E. coli DH5a (supE44 DlacU169 (f80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used for the propagation of the Lactobacillus-E. coli shuttle vectors, and E. coli TG1 [supE hsdD5 thi D(lac-proAB) F9(traD36 proAB1 lacIq lacZDM15)] was used for the subcloning of the L. brevis xylABT locus. They were maintained on LuriaBertani broth, and ampicillin was added at a concentration of 100 mg/ml when necessary. Lactobacillus strains were cultivated in MRS medium (Difco Laboratories, Detroit, Mich.) or in M medium (19) supplemented with 0.5 or a 1% (wt/vol) concentration of the indicated sugar. Erythromycin (5 mg/ml) was used for the selection. Identification of L. plantarum 80(pLPA9) transformants was performed on M medium agar plates containing 25 mM D-glucose, 60 mg of 5-bromo-4-chloro-3-indolyl-b-D-glucuronide per ml (Sigma Chemicals Co., St. Louis, Mo.), and 100 mM potassium phosphate (pH 7.4). Inverse PCR. Genomic DNA was extracted from L. brevis as described previously (2). DNA was restricted with ApoI, BclI, EcoRV, and Sau3AI, and the restricted fragments were self-ligated with T4 DNA ligase. To amplify the xylT gene and the remainder of the xylB gene, the ligation mixtures were amplified by using Taq DNA polymerase (GIBCO BRL, Gathersburg, Md.) and two sets of primers: either Sen-2 (59-AGTCTTACGACCAGCGAT-39), specifying codons 93 to 99 of the xylB gene, and Ansen-4 (59-GGAGTAACTTAAGCCTTC-39), specifying anticodons 74 to 79 of the xylB gene, or Sen-3 (59-GGTGGTTGGT CAGGTTA-39), specifying codons 230 to 235 of the xylT gene, and Ansen-5 (59-CCACTTGGTCATGCTTGT-39), specifying codons 207 to 212 of the xylT gene. The purified PCR fragments were restricted with the appropriate restriction enzymes and ligated into pUC19, yielding plasmids pYCBS0.8 (Sau3AI), pYCBEV1.5 (EcoRV), and pYCBBc2.3 (BclI) or into pUC18, yielding plasmid pYCBA2.0 (ApoI) (see Fig. 1A). Several randomly selected clones were sequenced. Construction of the Lactobacillus xylT expression plasmid, pLPA9. The construction of plasmid pLPA9 was performed essentially as described previously for the construction of the L. pentosus xylP gene expression vector, pLPA6 (5). The xylT gene was amplified from L. brevis genomic DNA (by using the Expand high-fidelity system; Boehringer Mannheim) and cloned into the cloning vector pTUT-MCS2 (5). The forward primer (59-CCTTTGGTACCGAACGTCGTAA GGAGCG-39) created a KpnI site (underlined) and a stop codon (boldface) upstream of the xylT original ribosome binding site (RBS [italics]). The reverse primer (59-TTACCCATGGTGATCCCACCTCTTTCGTAATCG-39) starting 7 nucleotides downstream of the xylT gene stop codon, generated an NcoI site (underlined) overlapping the ATG codon (boldface) of the gusA gene from plasmid pTUT-MCS2. A putative RBS (italics) was also introduced eight nucleotides upstream of gusA and served as a potential translation start for this reporter gene. The resulting plasmid was digested by BglII and XhoI, and the cloning cassette was introduced between the BamHI and XhoI sites of the hybrid lactobacillus-E. coli shuttle expression vector pLP503(t) (25), yielding pLPA9(t). After NotI digestion and religation, yielding plasmid pLPA9, the stop codon introduced in the forward primer was inframe with the first few codons of the ldh gene (which is part of the ldh expression cassette) and permitted translation of the native XylT protein. Then, 5 mg of plasmid pLPA9 DNA was used to transform L. plantarum 80 as described previously (15). Enzyme assays. The conversion of D-xylose (200 mM) to D-xylulose by D-xylose isomerase was measured according to the sorbitol dehydrogenase method as described previously (3). D-Xylulose kinase activity was determined by measuring the appearance of ADP formed as a result of the D-xylulose kinase reaction (4). Beta-xylosidase activity was assayed with p-nitrophenyl-b-D-xylopyranoside (5 mM), as described previously for the measurement of a-xylosidase activity in L. pentosus (4). Enzyme activities were determined in cell extracts prepared from cells harvested during the logarithmic phase of growth as described earlier (4), except that cells (resuspended in 500 ml of 50 mM potassium phosphate buffer containing 0.5 mM EDTA and 1 mM dithiothreitol) were disrupted by shaking at full speed (IKA-VIBRAX-VXR; IKA-Labortechnik) for 2 h at 4°C with 100
SYMPORTER OF L. BREVIS
4721
mg of glass beads (ca. 0.1 to 0.3 mm in diameter; Pertorp Analytical). The protein concentration in these samples was determined by the method of Smith et al. (32). D-Xylose transport assays. L. plantarum 80 cells cultivated in M medium supplemented with 25 mM glucose and 75 mM L-malate were harvested by centrifugation (5,000 3 g, 4°C, 10 min), washed twice with uptake buffer (50 mM potassium phosphate buffer [pH 6.5 or 4.5] containing 2 mM MgSO4), and resuspended at a concentration of approximately 30 mg (dry weight)/ml. For the transport assay, cells were diluted to a concentration of 3 to 5 mg (dry weight)/ml in 800 ml of uptake buffer. Cells were preenergized at 30°C by incubation with either 50 mM L-malate (at an extracellular pH of 4.5) for 2 min or by incubation with 5 mM of glucose (at an extracellular pH of 6.5) for 5 min. Transport was initiated by the addition of D-[U-14C]xylose (specific activity, 0.4 mCi/mmol; Amersham) at a final concentration of 100 mM. At given time points, 100-ml samples were taken and diluted in 5 ml of ice-cold 0.1 M LiCl. The samples were rapidly filtered through glass fiber filters (Whatman GF/F) and washed with 2 ml of ice-cold 0.1 M LiCl. The radioactivity on the filter was determined by liquid scintillation analysis. To determine the initial rate of D-xylose uptake, the transport reaction was stopped after 20 s by quenching the whole mixture (total volume, 100 ml) in 5 ml of 0.1 M LiCl. Potential competitors or uncouplers were added 5 s before the initiation of the transport reaction, unless indicated otherwise. Accumulation levels of D-xylose in L. plantarum 80 were calculated by assuming an intracellular volume of 1.5 ml/mg (dry weight) (10). For the measurement of D-xylose transport in L. brevis, the cells were grown in M medium supplemented with 25 mM of the indicated sugar and 25 mM of L-arginine. Transport experiments were performed essentially as described above for L. plantarum 80, except that the cells were energized at pH 6.5, with 20 mM of L-arginine or 5 mM glucose, and added 2 and 5 min prior to the start of the transport reaction, respectively. For L. brevis, D-[U-14C]xylose (specific activity, 0.1 mCi/mmol) was used at a final concentration of 500 mM. DNA manipulation. All DNA manipulations, including Southern hybridization, ligation, and transformation of E. coli, were done according to standard procedures (28). All enzymes were used according to the specifications of the manufacturers. Double-stranded DNA sequencing was performed according to the method of Wang (34). The Blast comparisons and the amino acid Pileup comparisons were performed by using the Genetic Computer Group programs through the facilities of the CAOS/CAMM center, Nijmegen, The Netherlands. Nucleotide sequence accession number. The complete nucleotide sequence of the L. brevis xyl locus is available under GenBank accession number AF045552.
RESULTS Cloning of the D-xylulose kinase gene and flanking regions. The cloning strategy is summarized in Fig. 1A. Initially, a 2.5-kb NlaIII/EcoRI region from L. brevis was cloned using a PCR probe generated with primers deduced from the N-terminal sequence of the L. brevis D-xylose isomerase and a region conserved among virtually all bacterial D-xylose isomerases. Its reported sequence contained a 1,347-bp open reading frame (ORF) coding for XylA (2). Within the 2.5-kb NlaIII/EcoRI sequence, at 107 bp downstream from the xylA stop codon, an ATG codon at the beginning of an ORF was found. The deduced amino acid sequence of this ORF showed considerable homology to the N-terminal amino acid sequence of the E. coli and L. pentosus D-xylulose kinases (2). The sequence up to the NlaIII site was also determined and revealed a putative xylO operator site, starting 45 bp upstream of the xylA start codon, and showing similarity to xylO operator sites found near the two D-xylose-inducible promoters of the L. pentosus xylose regulon (4, 19). 235 and 210 promoter elements (TTGCAT and TATACT), spaced by 16 nucleotides, were present 81 and 59 nucleotides upstream of the xylA start codon, respectively (see Fig. 1B). In addition, a putative catabolite responsive element (cre), which is known to mediate CcpA-dependent catabolite repression in several gram-positive microorganisms (14, 35), was also found 90 bp upstream of the xylA start codon. The sequence of the 0.4-kb region upstream of the xylA gene did not contain any putative ORF in either orientation. To map the regions surrounding the xylA gene, a Southern hybridization analysis was performed on total genomic DNA by using a fragment from the L. brevis xylB gene as a probe. We found that a 4.2-kb HindIII fragment and a 5.8-kb EcoRI/SalI fragment located downstream of the xylA gene hybridized with
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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 210 and 235 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.
the L. brevis xylB probe. In Bacillus spp. and other gram-positive bacteria, the xylAB operon was shown to be negatively regulated at the level of transcription by a repressor protein, designated XylR (16, 18, 27, 29, 31). Since a putative xylO operator site (XylR binding site) was found in the promoter region of the L. brevis xylA gene, another Southern hybridiza-
tion analysis to locate a xylR homologue was performed with the L. pentosus xylR gene as a probe. A 2.9-kb EcoRI/SalI fragment hybridized to this latter probe. Based upon a restriction map of the L. brevis xyl locus and the nearest possible 2.9-kb EcoRI/SalI fragment, the xylR gene must be located more than 2 kb upstream or 5 kb downstream of the xylA gene. To clone
VOL. 64, 1998
the entire xylB gene, a minilibrary was constructed in E. coli TG1 by using size-selected HindIII fragments. Although colony hybridization revealed several positively hybridizing clones, restriction analyses revealed that these recombinants carried inserts which were much smaller than the expected 4.2 kb and appeared to have lost most of the sequences downstream of the xylB gene. The deletions were confirmed by DNA sequencing. The cause of these deletions is unknown, however, and this dictated the use of another approach to clone xylB. Since inverse PCR was successfully applied to clone the 432-bp xylA 59 region (2), it was also employed to amplify the flanking region of xylB. Results of Southern hybridization were used to select restriction enzymes which generated fragments in the 0.8- to 2.5-kb range. Size-selected restriction fragments, including 2.3-kb BclI, 1.5-kb EcoRV, and 0.8-kb Sau3AI fragments, were self-ligated under conditions favoring the formation of monomeric circles and amplified by using primers Sen-2 and Ansen-4 to yield the xylB gene and the flanking region. The sequence of the fragments obtained was determined. Based upon this new sequence information, two additional inverse PCR primers (Sen-3 and Ansen-5) from the region downstream of xylB were synthesized and then used to amplify a 2-kb fragment from a religated ApoI size-selected population. This fragment was cloned into pUC18, and its sequence was determined. Nucleotide sequence of the xylB and xylT genes. The xylB ORF (1,506 nucleotides long) starts with an ATG codon (located 96 nucleotides after the xylA stop codon) and terminates with a TAG codon. The xylB gene codes for a protein of 502 amino acids and showed a high degree of identical amino acids (66%) when compared to the product of the L. pentosus xylB gene (data not shown). Another ORF (1,371 nucleotides long), xylT, begins 222 bp downstream of the xylB stop codon with an ATG codon and terminates with a TAA codon. Both the xylB and the xylT ORFs are preceded by putative Shine-Dalgarno sequences. No obvious promoter motifs were found immediately upstream of xylB. However, starting 110 bp upstream of the xylT start codon, a xylO sequence was observed, which showed similarity to xylO found in the upstream region of the L. brevis and L. pentosus xylA genes and in the upstream region of the L. pentosus xylP gene (Fig. 1C). 235 and 210 promoter elements (TTTCAA and TATGAT), spaced by 17 nucleotides, were found 145 and 128 bp upstream of the xylT start codon. Moreover, a putative cre site overlapping the 235 element was also identified. Potential Rho-independent transcriptional terminator sequences were found within the noncoding regions after xylA (DG8 5 217.1 kcal mol21), xylB (DG8 5 214.6 kcal mol21), and xylT (DG8 5 218.4 kcal mol21). Sequence homology of XylT with sugar transporters. The Blast computer program (1) was used to search entries in the Swiss-Prot protein database showing similarity to the deduced amino acid sequence of xylT from L. brevis. XylT demonstrated strong similarity throughout the entire sequence to other bacterial monosaccharide transporters of the “major facilitator superfamily” (MFS) (13, 20, 22), especially to the L-arabinose– H1 symporter of E. coli, AraE (60%); the D-galactose–H1 symporter of E. coli, GalP (59.5%); the glucose facilitator of Zymomonas mobilis, GlfZ (57%); the D xylose–H1 symporters of B. megaterium, XylT (58%) and of E. coli, XylE (57%); and the D-xylose–Na1 symporter of T. halophila, XylE (57%). An alignment is presented in Fig. 2. Surprisingly, the similarity score shared between XylT of L. brevis and the three bacterial D-xylose–cation symporters was significantly lower than the score shared between XylE of E. coli and T. halophila and XylT of B. megaterium (73 to 81%). Moreover, XylT of L. brevis
1
D-XYLOSE–H
SYMPORTER OF L. BREVIS
4723
shared the highest similarity score with the L-arabinose–H1 symporter, AraE, of E. coli. Regulation of D-xylose uptake and D-xylose catabolism in L. brevis. It is not known whether D-xylose is required for the expression of the L. brevis xyl genes. We could, however, identify putative xylO sequences in the regions upstream of the xylA and xylT genes, suggesting that their expression would be induced by D-xylose. Moreover, the presence of putative cre sites upstream of the xylA and of the xylT genes suggested a possible negative regulation by glucose and other carbohydrates. Therefore, the activity of D-xylose isomerase and D-xylulose kinase were measured in cell extracts of L. brevis grown in the presence of D-xylose, D-ribose, L-arabinose, D-glucose, maltose, and a mixture of D-glucose and D-xylose. Active D-xylose transport was also measured in L. brevis cells in the presence of an exogenous energy source, L-arginine or D-glucose (Table 1). D-Xylose isomerase and D-xylulose kinase activity and the transport of D-xylose were only detected when cells were grown on D-xylose. Moreover, the addition of D-glucose to cells growing on D-xylose decreased the total activity of D-xylose isomerase and D-xylulose kinase by fourfold, and the D-xylose transport activity was decreased by sixfold. In addition, it has been demonstrated that the repressor gene, xylR, of the Bacillus subtilis and L. pentosus xyl regulons is involved in the negative control of a b-xylosidase and of an a-xylosidase encoding gene, respectively (4, 12, 17). In L. brevis, we could identify a xylR repressor gene homologue, although it was not found within the xylABT locus. This finding prompted us to investigate the presence of a- or b-xylosidase activities in L. brevis. No a-xylosidase activity could be detected, but a low level of b-xylosidase activity was detected in cell extracts with most of the growth substrates (see Table 1). However, this activity was increased about 10-fold in the presence of D-xylose, suggesting a mechanism of induction. The b-xylosidase activity was reduced about twofold when both D-glucose and D-xylose were added to the growth medium compared to that of cells grown on Dxylose only. Functional expression of the D-xylose transport gene in L. plantarum 80. The L. brevis strain studied here could not be transformed with plasmid DNA when standard Lactobacillus electrotransformation procedures were used (unpublished observations). Therefore, the xylT gene could not be inactivated by plasmid integration to determine its role in D-xylose uptake in L. brevis. However, we have recently developed a Lactobacillus expression system which was used to characterize the a-xyloside transporter of L. pentosus, XylP (5). Consequently, an xylT expression vector was constructed, pLPA9 (Fig. 3) and was used to transform L. plantarum 80, a strain lacking PMFlinked D-xylose transport activity. To demonstrate that the Dxylose transport gene of L. brevis was functionally expressed in L. plantarum 80, D-xylose transport activity with D-[U-14C]xylose was assayed under conditions in which a PMF was generated from either malolactic fermentation (21) or glucose fermentation. Under both conditions, L. plantarum 80 harboring plasmid pLPA9 could transport and efficiently accumulate significant amounts of D-xylose, whereas the parental wild-type strain could not (Fig. 4). The initial rates of uptake in L. plantarum 80(pLPA9) were approximately 9 and 12 nmol/min per mg (dry weight) when L-malate and D-glucose were the source of the PMF, respectively. The accumulation level of D-xylose ([D-xylose]in/[D-xylose]out) after 2 min was around 60 when the PMF was generated by L-malate uptake and metabolism. The accumulation level was 30-fold when 5 mM glucose was the source of metabolic energy. In addition, increasing the glucose concentration to 20 mM resulted in a slower rate of uptake (ca. 3 nmol/min per mg [dry weight]), and in a fourfold decrease of
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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.
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TABLE 1.
D-Xylose
Maltose Fructose Glucose Xylose Glucose-xylose Arabinose Ribose
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transport and D-xylose catabolic enzyme activities in L. brevis grown on different carbohydratesa Initial D-xylose transport rateb (nmol/min/mg [dry wt]) with:
Energy source
SYMPORTER OF L. BREVIS
Enzyme activity (nmol/min/mg of total protein) with:
No addition
20 mM of L-arginine
5 mM of D-glucose
,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1
,0.1 ,0.1 ,0.1 9.1 6 0.4 1.9 6 0.1 0.4 6 0.1 ,0.1
,0.1 ,0.1 ,0.1 18.1 6 0.5 3.1 6 0.1 1.4 6 0.1 ,0.1
D-Xylose
isomerase
0 0 0 516 6 45 116 6 11 0 0
D-Xylulose
kinase
0 0 0 3,210 6 70 861 6 27 0 0
b-Xylosidase
5.8 6 0.3 4.8 6 0.1 5.5 6 0.9 68 6 4.0 32 6 1.0 9.7 6 1.6 6.8 6 1.7
a Cells were grown in M medium supplemented with 0.5% (wt/vol) of the corresponding energy source. In all cases, inoculations were performed by diluting an overnight M medium culture (OD600, ;4.0) 1/100 into fresh medium. Pregrowth was conducted in the presence of the same energy source used for the assay, except for the mixture of D-glucose plus D-xylose, for which cells were pregrown on D-xylose. Each assay was conducted in triplicate with individual L. brevis cultures. b Initial rates of D-xylose transport were measured after 20 s. Transport was carried out at 30°C and at pH 6.5 in the presence of 0.5 mM of D-[U-14C]xylose. L. brevis cells were energized with either 20 mM of L-arginine 2 min prior to start the transport reaction or with 5 mM of D-glucose 5 min prior to start the transport reaction.
the D-xylose level of accumulation. These findings suggest that the metabolism of glucose can negatively affect the activity of XylT and/or the maintenance of D-xylose inside the cell. Without L-malate or glucose as source of the PMF, D-xylose could not be transported or accumulated in L. plantarum 80(pLPA9). These results clearly indicate that the xylT gene of L. brevis encodes a D-xylose transporter. Substrate specificity and kinetic parameters of XylT. The kinetic parameters of XylT in L. plantarum 80(pLPA9) were determined with an L-malate-generated PMF. The Km and Vmax for D-xylose transport were 215 6 15 mM and 35 6 2 nmol/min per mg (dry weight), respectively. The effects of various sugars or sugar analogs on the initial rate of uptake of D-xylose were also tested (Table 2). An excess of the test substrate (50-fold) was added 5 s before the addition of D-[U14 C]xylose, and the initial velocity of D-xylose transport was measured (20 s). D-Xylose transport by XylT was poorly inhibited (20%) by a 50-fold excess of L-arabinose and methyl-a-Dxylose, but no inhibition could be detected with methyl-b-Dxylose, D-ribose, D-fucose, and D-galactose. 6-Deoxy-D-glucose and D-glucose inhibited the transport of D-xylose. The inhibition of D-xylose transport by 6-deoxy-D-glucose was found to be competitive (Fig. 5), with a Ki of 220 6 3 mM. As described above, the metabolism of D-glucose in L. plantarum 80 affected both the initial rate of uptake and the accumulation level of D-xylose. Since the metabolic inhibition of D-xylose was different with two different concentrations of D-glucose, the role of D-glucose as a potential competitive inhibitor of D-xylose uptake by XylT could not be assessed in our assay conditions. Role of the PMF components on D-xylose uptake. At pH 4.5, the PMF generated by L-malate transport and metabolism ('160 mV) is composed of an electrochemical membrane potential, Dc ('70 mV) and of an electrochemical proton gradient, ZDpH ('90 mV), where Z equals 2.3 (RT/F) and R, T, and F have their usual meanings (21). To determine the role of these components in the transport of D-xylose by XylT, the effects of uncoupling agents on the initial rate of uptake and on the accumulation level of D-xylose were studied (Table 3). The ionophore nigericin (H1/K1 antiporter), which dissipates the DpH, decreased the initial rate of uptake and lowered the accumulation level about 80% when used at a concentration of 0.5 mM or higher. A similar but stronger effect was obtained with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which collapses the total PMF.
DISCUSSION The aim of this study was twofold. First, we wanted to characterize a D-xylose transporter from a Lactobacillus species and, second, we wanted to construct a system enabling efficient expression of the transporter. The expression system should be of value in the development of food-grade vectors for heterofermentative lactobacilli based on D-xylose fermentation. Similarity between the L. brevis xylABT locus, the xylABT operon of B. megaterium, and the xylABE operon of T. halophila. The cloning and sequencing of the region downstream from the xylA and xylB genes of L. brevis revealed the presence of a potential xyl gene, xylT, encoding a putative membraneembedded protein. On the basis of homology between the primary structure of XylT from L. brevis (further referred to as XylTLb) and some members of the MFS, including the lowaffinity D-xylose–cation symporters XylE of E. coli (further
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 (Pldh from L. casei ATCC 393), the xylT gene, the E. coli b-glucuronidase gene (gusA) used as a marker of gene expression, and the terminator sequence of the L. plantarum 80 cbh gene (Tcbh [6]). The two terminator sequences (Tldh from L. casei ATCC 393 [25]) downstream from the strong Pldh promoter are used to circumvent instability of the expression vector in E. coli. These two Tldh 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.
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APPL. ENVIRON. MICROBIOL. TABLE 2. Substrate specificity of the L. brevis XylT transporter Addition of sugars or sugar analogs (5 mM, final concn)
Relative uptake of 14 D-[U- C]xylose (%)a
None ........................................................................................... D-Xylose...................................................................................... 6-Deoxy-D-glucose..................................................................... D-Glucose ................................................................................... methyl-a-D-Xylose...................................................................... L-Arabinose ................................................................................ D-Galactose ................................................................................ methyl-b-D-Xylose...................................................................... D-Ribose ..................................................................................... D-Fucose .....................................................................................
100 15 10 60 80 80 90 100 100 100
a Initial rates of D-xylose transport in L. plantarum 80(pLPA9) were measured after 20 s at pH 4.5 in the presence of 50 mM of L-malate as a PMF-generating system and with 100 mM of D-[U-14C]xylose. The control rate (100%) was 9 nmol/min per mg (dry weight). The potential competitors were added 5 s before the initiation of the transport reaction. All values are the average of two separate experiments.
regulation by XylR and CcpA homologues. However, the transcriptional regulation of the L. brevis xyl locus is not yet known, and the role of these transcriptional regulators remains to be demonstrated. Characteristics of XylTLb. The L. brevis xylT gene was functionally expressed in L. plantarum 80 by using a recently described Lactobacillus expression system, which confirmed XylTLb to be a D-xylose–H1 symporter and enabled the determination of some properties of this transporter. It is interesting to note that throughout the whole sequence, XylTLb appeared to be more closely related to the E. coli L-arabinose–H1 transport protein, AraE, than to the bacterial D-xylose–cation symporters, XylEEc, XylETh, and XylTBm. This difference extended to the large gap found between the third and the fourth putative transmembrane region in the sequence of XylTLb, AraE, and GalP introduced to optimize the alignment with
FIG. 4. D-Xylose uptake by cells of L. plantarum 80(pLPA9) (E; 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 (F), 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%.
referred to as XylEEc), XylE of T. halophila (further referred to as XylETh) and XylT of B. megaterium (further referred to as XylTBm), a D-xylose–H1 symport activity has been assigned to XylTLb. The organization of the L. brevis xylABT locus showed similarity to that of the B. megaterium xylABT operon (30) and the T. halophila xylABE operon. No tightly linked xylR gene could be found upstream of xylA in L. brevis. This finding does not exclude, however, that the xylR gene in L. brevis may be located in another operon, perhaps serving to regulate a distinct set of genes. Preliminary results have indicated that the expression of the xylABT genes in L. brevis is inducible by D-xylose and is repressed by D-glucose. Putative xylO sequences (XylR binding sites) and cre-like elements (CcpA binding sites) upstream of xylAB and xylT genes strongly suggest a negative
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 (E) or with 0.5 mM (}) or 1 mM (F) 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.
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D-XYLOSE–H
TABLE 3. Effect of ionophores and of the F0F1-ATPase inhibitor CCCP on D-xylose uptake by the L. brevis XylT transportera Addition
Concn (nM)
None
Initial rate of uptakea (nmol/min/mg [dry wt])
Level of accumulation [xylosein]/[xyloseout])
9
60
CCCP
50 200 500 1,000
6 1 0.5 0.5
40 4 2 1
Nigericin
50 200 500 1,000
9 5.5 2 1
52 30 10 2
a The transport assays were carried out with L. plantarum 80(pLPA9) cells at pH 4.5 and in the presence of 100 mM D-[U-14C]xylose and 50 mM L-malate as the PMF-generating system. Initial rates were measured after 20 s, and the accumulation levels were calculated after an uptake of 2 min. The potential ionophores CCCP and nigericin were added 5 s before the initiation of the transport reaction. All values are the average of two separate experiments.
XylEEc, XylETh, GlfZ, and XylTBm. Even though such a homology would suggest a higher affinity of XylTLb for L-arabinose than for D-xylose, L-arabinose proved to be a very poor competitor of D-xylose uptake by XylTLb. This finding suggests that L-arabinose is presumably not a physiological substrate for the XylTLb protein. In fact, the pattern of inhibition of D-xylose transport by several sugars or sugar analogs indicated that the properties of the XylEEc and XylTLb transport proteins are very similar. In contrast to the AraE protein of E. coli, for which L-arabinose, D-fucose, and D-xylose are substrates (13), transport of D-xylose by XylTLb is only inhibited by 6-deoxyLb D-glucose (6-methyl-D-xylose) and possibly D-glucose. XylT can also discriminate between D-xylose and D-xylose analogs with a methyl substitution on the C1 of the pyranoside ring, since neither methyl-a-D-xylose nor methyl-b-D-xylose were efficient competitors of D-xylose transport. A similar specificity was previously found for XylEEc (13). These results indicate that the primary sequence homology between members of the MFS is clearly not sufficient to predict their substrate specificity. Substrate recognition by these proteins may reside in the positions of specific charged residues (mostly histidine, glutamic acid, or aspartic acid) located in the hydrophilic regions, which may help to bind the substrates (for a review, see reference 23). The alignment shown in Fig. 3, however, did not reveal such conserved residues in XylTLb, XylTBm, XylEEc, and XylETh, which could discriminate the D-xylose–cation symporters from the other monosaccharide-cation symporters. Nevertheless, the apparent affinity constant of the XylTLb protein for D-xylose (215 mM) is substantially higher than the apparent affinity constant of XylTBm (100 mM) and of XylEEc (60 mM) for D-xylose (36). Finally, the susceptibility of D-xylose transport to the protonophore CCCP, which collapses the PMF, and to the ionophore nigericin, which discharges the DpH, indicates that D-xylose transport by XylTLb proceeds most likely in symport with a proton. The metabolism of D-glucose in L. plantarum 80, resulted in inhibition of D-xylose transport via XylTLb. A similar inhibition of D-xylose transport could be observed in L. brevis when a high concentration of D-glucose (.20 mM) was used to energize the cells for transport (data not shown). Addition of D-glucose decreased the initial rate of uptake and the overall accumulation level, suggesting a mechanism which may inactivate the activity of XylTLb. A similar mechanism of inhibition, called inducer exclusion, has already been suggested to regulate
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methyl-b-D-thiogalactopyranoside accumulation in L. brevis (36). Whether a similar mechanism is active in L. plantarum 80 and whether it is responsible for the inhibition of D-xylose uptake by a high concentration of D-glucose in L. brevis remains to be demonstrated. Development of a D-xylose food-grade vector. In this study, we have shown that the Lactobacillus expression system, pLPA9, allowed the functional expression of a D-xylose transporter in L. plantarum 80. This plasmid could serve as the basis for the construction of a food-grade vector based on the metabolism of D-xylose by cloning of the xylA and xylB genes downstream of the xylT gene. Such a vector, which makes use of a strong and constitutive Lactobacillus promoter (Pldh from L. casei ATCC 393) may represent a useful tool for improving the catabolic capacity of heterofermentative lactic acid bacteria that are important in the fermentation of plant materials and in the production of fermented food. ACKNOWLEDGMENT This work was supported by a grant from the EC BIOTECH program (contract BIO2-CT92-0137). REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. 2. Bor, Y.-C., S.-P. Moraes, S.-P. Lee, W. L. Crosby, A. J. Sinskey, and C. A. Batt. 1992. Cloning and sequencing the Lactobacillus brevis gene encoding xylose isomerase. Gene 114:127–131. 3. Callens, M., H. Kerstens-Hildersion, O. van Opstal, and C. K. de Bruine. 1986. Catalytic properties of D-xylose isomerase from Streptomyces violaceoruber. Enzyme Microb. Technol. 8:696–700. 4. Chaillou, S., B. C. Lokman, R. J. Leer, C. Posthuma, P. W. Postma, and P. H. Pouwels. 1998. Cloning, sequence analysis and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus. J. Bacteriol. 180:2312–2320. 5. Chaillou, S., P. W. Postma, and P. H. Pouwels. 1998. Functional expression in Lactobacillus plantarum 80 of xylP encoding the isoprimeverose transporter of Lactobacillus pentosus. J. Bacteriol. 180:4011–4014. 6. Christiaens, H., R. J. Leer, P. H. Pouwels, and W. Verstraete. 1992. Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay. Appl. Environ. Microbiol. 58:3792– 3798. 7. Daeschel, M. A., R. E. Anderson, and H. P. Fleming. 1987. Microbial ecology of fermenting plant material. FEMS Microbiol. Rev. 46:357–367. 8. Davis, E. O., and P. J. F. Henderson. 1987. The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli. J. Biol. Chem. 262:13928–13932. 9. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive sequence analysis program for the VAX. Nucleic Acids Res. 12:387–395. 10. Glaasker, E., F. S. B. Tjan, P. F. ter Steeg, W. N. Konings, and B. Poolman. 1998. The physiological response of Lactobacillus plantarum to salt and nonelectrolyte stress. J. Bacteriol. 180:4718–4723. 11. Hammes, W. P., and R. F. Vogel. 1995. The genus Lactobacillus, p. 18–54. In B. J. B. Wood and W. H. Holzapfel (ed.), The genera of lactic acid bacteria, vol. 1. Blackie Academic & Professional, Glasgow, Scotland. 12. Hastrup, S. 1988. Analysis of the Bacillus subtilis xylose regulon, p. 79–84. In A. T. Cramson and J. A. Hoch (ed.), Genetics and biotechnology of bacilli, vol. 2. Academic Press, Inc., New York, N.Y. 13. Henderson, P. J. F., and M. C. J. Maiden. 1990. Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes. Philos. Trans. R. Soc. Lond. 326:391–410. 14. Hueck, C. J., W. Hillen, and J. Saier, Jr. 1994. Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria. Res. Microbiol. 145:418–503. 15. Josson, K., T. Scheirlinck, F. Michiels, C. Platteeuw, P. Stanssens, H. Joos, P. Dhaese, M. Zabeau, and J. Mahillon. 1989. Characteristics of a grampositive broad host range plasmid isolated from Lactobacillus hilgardii. Plasmid 11:9–20. 16. Kreuzer, P., D. Ga ¨rtner, R. Allmansberger, and W. Hillen. 1989. Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J. Bacteriol. 171:3840–3845. 17. Lindner, C., J. Stu ¨lke, and M. Hecker. 1994. Regulation of xylanolytic enzymes in Bacillus subtilis. Microbiology 140:753–757. 18. Lokman, B. C., M. Heerikhuisen, R. J. Leer, A. van den Broek, Y. Borsboom, S. Chaillou, P. Postma, and P. H. Pouwels. 1997. Regulation of expression of the Lactobacillus pentosus xylAB-operon. J. Bacteriol. 179:5391–5397.
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