INTRODUCTION

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D-Xylose is abundant in nature, and many bacteria utilize it as a carbon source. D-Xylose must be taken up before it is metabolized by two intracellular enzymes: D-xylose isomerase (XylA) and D-xylulose kinase (XylB). Until now, only two mechanisms of D-xylose transport have been characterized in bacteria. One mechanism, identified in Escherichia coli (5, 6), Salmonella typhimurium (29), Bacillus megaterium (28), Lactobacillus brevis (1), Bacillus subtilis (12, 27), Tetragenococcus halophila (35), and some ruminal bacteria (31, 33), involves a D-xylose-H⁺ or -Na⁺ symporter. The second mechanism consists of a high-affinity D-xylose transport system involving a periplasmic binding protein and is driven by ATP. It has been characterized in E. coli (34) and is presumably present in a thermophilic Bacillus species (15) and several ruminal bacteria (32, 37).

L. pentosus is a bacterium which also can transport and metabolize D-xylose, although the gene(s) encoding the D-xylose transport function could not be identified in the xyl gene cluster (2). This cluster comprises the xylPQ operon involved in the transport and metabolism of the disaccharide isoprimeverose [-D-xylopyranosyl-(1,6)-D-glucopyranose]; the xylR gene, encoding the repressor of the xyl regulon; and the xylAB operon, encoding the D-xylose-catabolizing enzymes XylA and XylB. In this regulon, the xylP gene encodes an isoprimeverose cation symporter (3), which does not transport D-xylose. Thus, the protein(s) responsible for the transport of D-xylose in L. pentosus remains to be characterized.

Recently, we have started a study of the phosphoenolpyruvate (PEP):D-mannose phosphotransferase system (PTS) of L. pentosus. Several spontaneous 2-deoxy-D-glucose-resistant (2DG^r) mutants defective in the activity of the EII^Man complex of the PTS were isolated. These mutants were impaired in growth on D-xylose, suggesting a role of the D-mannose PTS in D-xylose utilization by L. pentosus.

The PTS catalyzes the concomitant transport and phosphorylation of various mono- and disaccharides at the expense of PEP (for a review, see reference 24). In a few cases, however, transport via the PTS can take place in the absence of phosphorylation. In E. coli, for instance, specific mutations in EIICB^Glc have been isolated that allow D-glucose transport without phosphorylation (uncoupled transport) (25). Facilitated diffusion of D-galactose via the D-glucose PTS in E. coli (11) or of D-galactose and trehalose via the D-mannose PTS in S. typhimurium (22, 23) was also demonstrated. Facilitated diffusion of carbohydrates is not widespread in bacteria or at least occurs much less frequently than in eukaryotic cells (30). Besides the few examples mentioned above, the best-characterized systems of facilitated diffusion in bacteria are the glycerol facilitator protein (GlpF) of E. coli (9, 40) and the glucose transporter (GlfZ) of Zymomonas mobilis (7, 19), two systems which are independent of the PTS.

In this report, we show that EII^Man of three species of facultative heterofermentative lactobacilli, L. pentosus, L. plantarum, and L. casei, can catalyze the facilitated diffusion of D-xylose. We also demonstrate that transport of D-xylose is the rate-controlling step in the metabolism and growth of L. pentosus on this compound.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, plasmids, and growth conditions. Table 1 lists the Lactobacillus strains and plasmids used. The plasmids were introduced into the various Lactobacillus species as previously described for L. pentosus (17), L. plantarum 80 (10), and L. casei ATCC 393 (4). Cells were grown on MCD medium (13) supplemented with L-aspartic and L-glutamic acids (50 mg of each per liter), which are essential amino acids for species related to L. plantarum (14). Carbohydrates were added to a final concentration of 1% (wt/vol), unless stated otherwise, and erythromycin (5 µg ml¹) was added when necessary. All incubations were carried out at 37°C in nonshaking tubes containing 25 ml (growth, phosphorylation, enzyme assays, and uptake studies) of MCD medium. Inoculations were performed by diluting an MCD culture (optical density at 600 nm [OD₆₀₀], 1.0; obtained after 8 to 24 h of incubation, depending on the energy source used) 1/100 into fresh medium.

Isolation of 2DG^r mutants. Spontaneous 2DG^r mutants were isolated by plating 100 µl of a late-exponential-phase culture grown in M medium (17) containing 1% (wt/vol) sucrose (L. pentosus) or D-gluconate (L. plantarum 80) on an M medium agar plate containing 25 mM sucrose (L. pentosus) or 50 mM D-gluconate (L. plantarum 80) and 10 mM 2DG. Resistant clones were purified by streaking two times on the same selective medium.

Preparation of permeabilized cells. Bacterial cultures were grown as described above, washed two times with ice-cold 50 mM potassium phosphate buffer (pH 6.5) containing 2 mM MgSO₄ (KPM buffer), resuspended in 1/100 culture volume of KPM containing 20% (wt/vol) glycerol, rapidly frozen in liquid nitrogen, and kept at 80°C. After thawing, cells were washed once with KPM buffer and resuspended to an OD₆₀₀ of 50 in 50 mM potassium phosphate buffer (pH 6.5) containing 12.5 mM NaF, 5 mM MgCl₂, and 2.5 mM dithiothreitol. Cells were permeabilized as follows. First, 2.5 µl of toluene-acetone (1:9, vol/vol) was added per 250 µl of cell suspension and the mixture was vortexed for 5 min at 4°C. Cells were centrifuged (150 × g at 4°C for 2 min), and the supernatant was discarded. This step was necessary to remove the large cytoplasmic PEP pool of L. pentosus. The cell pellet was resuspended in the same buffer (OD₆₀₀, 50) and treated with toluene-acetone as described above. After 5 min of vortexing, permeabilized cells were kept on ice.

PEP- and ATP-dependent ¹⁴C-labelled carbohydrate phosphorylation assay. For each assay, 2.5-, 5-, and 10-µl volumes of permeabilized cells (OD₆₀₀, 50) were incubated in a final volume of 100 µl of 50 mM potassium phosphate buffer (pH 6.5) containing 12.5 mM NaF, 5 mM MgCl₂, 2.5 mM dithiothreitol, 10 mM PEP or ATP, and 10 mM ¹⁴C-labelled carbohydrate (specific activity, 0.1 µCi mmol¹). After incubation for 15 to 30 min at 37°C, the phosphorylated carbohydrate was separated from the unreacted carbohydrate (21) on Dowex AG 1-X2 columns and the radioactivity was determined. The rate of PEP- or ATP-dependent phosphorylation was calculated by applying a line through the three values and subtracting the rate of phosphorylation obtained in permeabilized cells without the phosphoryl donor (background).

Transport assays. L. pentosus cells were cultivated in MCD medium supplemented with 1% (wt/vol) D-xylose, unless indicated otherwise. D-[U-¹⁴C]xylose transport was performed in KPM buffer (pH 6.5) as previously described (2). For the kinetic studies, the concentration of D-[U-¹⁴C]xylose ranged from 100 µM to 100 mM and its specific activity ranged from 0.05 to 0.5 µCi mmol¹. D-Xylose transport rates were determined after 2 min of uptake. Transport of D-xylose under proton motive force (PMF)-generating conditions was assayed with KPM buffer (pH 4.5) as previously described for L. plantarum 80 (1). In all cases, transport was initiated by addition of 0.5 or 20 mM D-[U-¹⁴C]xylose (specific activity, 0.4 µCi mmol¹). Transport of 1 mM [U-¹⁴C]2DG (specific activity, 0.2 µCi mmol¹) was performed under conditions identical to those described for the transport of D-xylose (pH 6.5), except that cells were grown on D-mannose.

Enzymatic determinations. -Xylosidase activity was determined at 37°C in 750 µl of KPM buffer containing 10 to 20 µl of permeabilized cells (OD₆₀₀, 50) and 5 mM p-nitrophenyl--D-xylopyranoside. The reaction was stopped by adding 250 µl of 1 M Na₂CO₃, and the OD₄₁₀ was measured.

Radiochemicals. D-[U-¹⁴C]mannose (286 mCi mmol¹), D-[U-¹⁴C]xylose (89 mCi mmol¹), and 2-deoxy-D-[U-¹⁴C]glucose (300 mCi mmol¹) were obtained from Amersham International, Amersham, United Kingdom.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Characteristics of D-xylose transport in L. pentosus MD353. D-[U-¹⁴C]xylose uptake was measured in starved, D-xylose-grown MD353 cells (Fig. 1A and B). The results showed that the rate of D-[U-¹⁴C]xylose uptake was not stimulated by a PMF-generating system (malolactic fermentation). To verify that addition of 50 mM L-malate to L. pentosus cells can generate a PMF under these conditions, uptake of D-[U-¹⁴C]xylose was also measured in D-xylose-grown cells of MD353/pLPA9 expressing the D-xylose-H⁺ symporter of L. brevis. MD353/pLPA9 cells could accumulate D-xylose under PMF-generating conditions at a rate about 15-fold higher than that found in the absence of a PMF. When D-[U-¹⁴C]xylose uptake was measured in the absence of a PMF-generating system for a longer period (Fig. 1B), MD353 cells accumulated ¹⁴C-labelled material as a result of D-[¹⁴C]xylose transport, followed by its subsequent metabolism and incorporation into metabolic intermediates and cellular material. The rate of ¹⁴C label accumulation was not decreased by addition of the protonophore carbonyl cyanide-m-chlorophenylhydrazone to 1 µM, a result suggesting that D-xylose uptake is not dependent on the PMF. Moreover, cells of MD353 grown on D-ribose did not take up much D-xylose, indicating that D-xylose incorporation requires a function inducible by D-xylose, most likely the enzymes required for xylose metabolism.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   (A) Uptake of 0.5 mM D-[U-14C]xylose at an extracellular pH of 4.5 (KPM buffer [see Materials and Methods]) by D-xylose-grown MD353 cells (, with L-malate; , without L-malate) or MD353/pLPA9 cells (, with L-malate; , without L-malate). Cells were pre-energized at 37°C by incubation with 50 mM L-malate for 2 min. (B) Uptake of 0.5 mM D-[U-14C]xylose at an extracellular pH of 6.5 (KPM buffer) by D-xylose-grown cells of MD353 (, no addition; , in the presence of 1 µM carbonyl cyanide m-chlorophenylhydrazone added 30 s before D-[U-14C]xylose) or D-ribose-grown cells of MD353 ().

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Isolation of mutants unable to utilize D-xylose as a sole source of energy. To identify the low-affinity D-xylose transport system in L. pentosus, we studied several L. pentosus strains unable to utilize D-xylose. We have isolated three 2DG^r mutants which lacked PEP-dependent phosphorylation of D-mannose (see below). Two of these mutants, LPE5 and LPE8, had a Xyl phenotype, whereas the growth of the third mutant (LPE6) on D-xylose was impaired but not completely abolished. Another strain, MD358, was a natural Xyl isolate from a cucumber fermentation. An insertion of about 8 kb has been identified within the xylA gene of this strain, which resulted in the absence of D-xylose isomerase and D-xylulose kinase activity and of growth on D-xylose (data not shown).

Complementation of the inability of the Xyl mutants to grow on D-xylose. Introduction of plasmid pMJ18 (expressing the manB gene of the L. curvatus mannose PTS) into the three 2DG^r mutants resulted in restoration of PEP-dependent D-mannose phosphorylation in LPE6 but not in LPE5 and LPE8. This result suggested that the mutation in LPE6 affected the activity of the EIIB^Man subunit of the L. pentosus EII^Man system (38). To characterize the defective function of mutants LPE5 and LPE8, the two strains were transformed with either plasmid pLP3537-xyl (expressing the D-xylose-catabolizing enzymes of L. pentosus MD353, XylA and XylB) or plasmid pLPA9 (expressing the D-xylose-H⁺ symporter of L. brevis). Introduction of pLPA9, but not of pLP3537-xyl, restored the ability of these mutants to grow on D-xylose. These observations indicated that mutants LPE5 and LPE8 were defective in the D-xylose transport function.

Transport and catabolism in the various Xyl mutants and their respective Xyl⁺ transformants. The capacity to transport D-xylose, as well as to catabolize D-xylose (ATP-dependent formation of D-xylulose 5-phosphate from D-xylose as a result of XylA and XylB activities), was determined in the various strains mentioned above. In addition, we measured the level of -xylosidase activity (encoded by xylQ) to investigate the effects of the various Xyl mutations on the expression of the xylPQ operon. Finally, PEP-dependent phosphorylation of D-mannose was determined to study the correlation between D-xylose transport and the activity of the D-mannose PTS. All of these measurements were performed with cells grown in a mixture of glycerol and D-xylose and then permeabilized with toluene. Glycerol is a neutral energy source in L. pentosus which does not mediate catabolite repression of the xyl genes.

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Mechanism of D-xylose transport via the EII^Man complex. The phenotype of mutant LPE6 suggested that some, but not all, mutations that affected EII^Man activity in the 2DG^r mutants resulted in a lack of D-xylose uptake. To explain such a phenotype, it may be important to understand the role of the different components of the EII^Man complex in the process of facilitated diffusion of D-xylose. EII complexes of the mannose class studied to date consist of two integral membrane proteins, EIIC^Man and EIID^Man, and of two cytoplasmic phosphoryl transfer domains, EIIA^Man and EIIB^Man (which may be separate proteins or a single polypeptide, as in the case of the E. coli EII^Man complex). In E. coli, EIIC^Man and EIID^Man are not phosphorylated. The cytosolic EIIB^Man domain is supposed to be the phosphoryl donor to the carbohydrate (8). This implies that the membrane-bound subunits EIICD^Man of the EII^Man complex should bind and translocate the PTS carbohydrate to the cytosolic side in order to allow phosphorylation by the phosphorylated EIIB^Man domain. Therefore, facilitated diffusion of a sugar may occur in the absence of phosphorylation (22, 23, 25). It should be mentioned that PEP-dependent phosphorylation of D-xylose could not be detected in permeabilized cells of MD353; the activity was less than 0.5 nmol of xylose phosphorylated per min per mg of dry weight in xylose-grown cells. Given these data, it seems plausible to assume that D-xylose, the pyranoside ring of which is similar to that of D-glucose, might be recognized by the EIIC/D^Man domains and transported inside the cell by facilitated diffusion. The other components, EIIB^Man, EIIA^Man, HPr, and enzyme I, of the PTS would not be required for such a process. Indeed, a Xyl⁺ phenotype is observed in mutant LPE6, which is presumably impaired in EIIB^Man activity. However, EIIB^Man is apparently required for maximal D-xylose transport activity via the EIIC/D^Man components. This observation suggests that EIIB^Man controls the active conformation of EIIC/D^Man even if it is not functioning in phosphoryl transfer to D-xylose. We do not know in which component of the EII^Man complex the mutation of LPE5 and LPE8 is localized, but it seems likely that these two mutants at least lack EIIC/D^Man activity or have altered EIIC/D^Man activity.

Role of EII^Man in the transport of D-xylose in other facultative heterofermentative lactobacilli. It has been previously demonstrated that introduction of plasmid pLP3537-xyl into L. casei and L. plantarum, two Lactobacillus species that do not naturally ferment D-xylose, resulted in a Xyl⁺ phenotype (20). Since these species did not possess PMF-dependent D-xylose transport activity (our unpublished results), this finding suggested that a non-energy-dependent D-xylose transporter might be present. The results obtained with L. pentosus prompted us to investigate whether the presence of a similar EII^Man complex could be responsible for the facilitated diffusion of D-xylose in these species. An L. casei EII^Man-defective mutant (BL23D) has been described (39), but no EII^Man-defective mutant of L. plantarum was available. Therefore, we isolated several spontaneous L. plantarum 80 2DG^r mutants. One of these mutants, named LPL1, was further characterized and showed a severely reduced rate of PEP-dependent phosphorylation of D-mannose (2.5 ± 0.7 nmol min¹ mg of dry weight¹ for LPL1 versus 48 ± 4 nmol min¹ mg of dry weight¹ for LPL80). This suggested that LPL1 was impaired in EII^Man activity. L. plantarum LPL1 and LPL80 were transformed with plasmid pLP3537-xyl, and L. casei BL23D and its parent wild-type strain (BL23) were transformed with plasmid pLP3537-xyl* (a pLP3537-xyl derivative with a promoter-up mutation required for high expression of the xylAB operon in L. casei; see reference 16). The transformants LPL1/pLP3537-xyl and BL23D/pLP3537-xyl* were not able to utilize D-xylose as a sole source of energy, whereas the respective wild-type strains LPL80/pLP3537-xyl and BL23/pLP3537-xyl* were. D-Xylose-catabolic capacity, i.e., formation of xylulose 5-phosphate from xylose, and D-xylose uptake activity were also determined in the transformants grown in a mixture of D-gluconate (an energy source to support the growth of Xyl transformants) and D-xylose. The results are shown in Table 3. In both Lactobacillus species transformed with plasmids pLP3537-xyl and pLP3537-xyl*, the inability to grow on D-xylose correlated with a low EII^Man activity. Moreover, the lack of D-xylose transport in 2DG^r mutants LPL1/pLP3537-xyl and BL23D/pLP3537-xyl* resulted in a 15-fold-lower rate of ATP-dependent formation of D-xylulose 5-phosphate, as was shown for L. pentosus (Table 2). From these results, we conclude that EII^Man is also responsible for the facilitated diffusion of D-xylose in L. casei and L. plantarum when D-xylose catabolic activity is provided to the cell.

Role of D-mannose in the growth rate of L. pentosus, L. casei, and L. plantarum on D-xylose. We have investigated the influence of a low concentration of D-mannose (0.5 mM) in the growth medium on the growth of MD353 on D-xylose (Fig. 2). MD353 grew approximately 1.9-fold faster when D-mannose was present (generation time of 240 min in the presence of mannose versus 455 min in its absence), whereas 0.5 mM D-mannose in the absence of D-xylose was not sufficient to promote the growth of L. pentosus (Fig. 2). This increased growth rate in medium containing mannose was paralleled by an increased rate of PEP-dependent mannose phosphorylation via EII^Man (Fig. 3). This observation suggested that transport of D-xylose could be the rate-limiting step in the growth of MD353 on D-xylose. Hence, we have measured the doubling times of the various other Lactobacillus strains used in this study on D-xylose in the presence of 0.5 mM D-mannose in the growth medium and, for each case, we determined the rate of PEP-dependent phosphorylation of D-mannose in permeabilized cells (Fig. 3). Indeed, a linear relationship could be found between the doubling times of the various L. pentosus strains on D-xylose and the levels of PEP-dependent phosphorylation of D-mannose (level of EII^Man expression). Interestingly, the values obtained with LPL80/pLP3537-xyl and BL23/pLP3537-xyl* fit the line, indicating that transport of D-xylose in these two species is also the limiting step in growth.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Growth of L. pentosus MD353 on MCD medium containing 1% (wt/vol) D-xylose (), 0.01% (wt/vol) D-mannose (), or 1% (wt/vol) D-xylose plus 0.01% (wt/vol) D-mannose ().

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Relationship between the PEP-dependent phosphorylation rates of D-[U-14C]mannose and the doubling times of various L. pentosus strains. Shown are results for MD353 () and LPE6/pMJ18 () grown on 1% (wt/vol) D-xylose and MD353 (), LPE6/pMJ18 (), MD358/pLP3537-xyl (), LPL80/pLP3537-xyl (), and BL23/pLP3537-xyl* () grown on 1% (wt/vol) D-xylose plus 0.01% (wt/vol) D-mannose.