ABSTRACT
We have identified and characterized the d-xylose transport system of Lactobacillus pentosus. Uptake ofd-xylose was not driven by the proton motive force generated by malolactic fermentation and required d-xylose metabolism. The kinetics of d-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent Km of 8.5 mM and aVmax of 23 nmol min−1 mg of dry weight−1. In two mutants of L. pentosusdefective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on d-xylose was absent due to the lack ofd-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIBMan component of the EIIMancomplex. The EIIMan complex is also involved ind-xylose transport in L. casei ATCC 393 andL. plantarum 80. These two species could transport and metabolize d-xylose after transformation with plasmids which expressed the d-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-d-glucose were defective in EIIMan activity and were unable to transportd-xylose when transformed with plasmids containing thexylAB genes. Finally, transport of d-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. caseiATCC 393 containing plasmids coding for thed-xylose-catabolic enzymes, since the doubling time of these bacteria on d-xylose was proportional to the level of EIIMan activity.
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) andd-xylulose kinase (XylB). Until now, only two mechanisms ofd-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 ad-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 thermophilicBacillus 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 thed-xylose transport function could not be identified in thexyl gene cluster (2). This cluster comprises the xylPQ operon involved in the transport and metabolism of the disaccharide isoprimeverose [α-d-xylopyranosyl-(1,6)-d-glucopyranose]; the xylR gene, encoding the repressor of thexyl 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. pentosusremains to be characterized.
Recently, we have started a study of the phosphoenolpyruvate (PEP):d-mannose phosphotransferase system (PTS) ofL. pentosus. Several spontaneous 2-deoxy-d-glucose-resistant (2DGr) mutants defective in the activity of the EIIMan complex of the PTS were isolated. These mutants were impaired in growth ond-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 EIICBGlc have been isolated that allowd-glucose transport without phosphorylation (uncoupled transport) (25). Facilitated diffusion ofd-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 EIIMan 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
Bacterial strains, plasmids, and growth conditions.Table1 lists the Lactobacillusstrains and plasmids used. The plasmids were introduced into the various Lactobacillus species as previously described forL. pentosus (17), L. plantarum 80 (10), and L. casei ATCC 393 (4). Cells were grown on MCD medium (13) supplemented withl-aspartic and l-glutamic acids (50 mg of each per liter), which are essential amino acids for species related toL. plantarum (14). Carbohydrates were added to a final concentration of 1% (wt/vol), unless stated otherwise, and erythromycin (5 μg ml−1) 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.
Bacterial strains and plasmids used in this study
Isolation of 2DGr mutants.Spontaneous 2DGr 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) ord-gluconate (L. plantarum 80) on an M medium agar plate containing 25 mM sucrose (L. pentosus) or 50 mMd-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 mmol−1). 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 ofd-[U-14C]xylose ranged from 100 μM to 100 mM and its specific activity ranged from 0.05 to 0.5 μCi mmol−1. 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. plantarum80 (1). In all cases, transport was initiated by addition of 0.5 or 20 mM d-[U-14C]xylose (specific activity, 0.4 μCi mmol−1). Transport of 1 mM [U-14C]2DG (specific activity, 0.2 μCi mmol−1) 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.α-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-α-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 mmol−1),d-[U-14C]xylose (89 mCi mmol−1), and 2-deoxy-d-[U-14C]glucose (300 mCi mmol−1) were obtained from Amersham International, Amersham, United Kingdom.
RESULTS AND DISCUSSION
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 ind-xylose-grown cells of MD353/pLPA9 expressing thed-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. Whend-[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 ofd-[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.
(A) Uptake of 0.5 mMd-[U-14C]xylose at an extracellular pH of 4.5 (KPM buffer [see Materials and Methods]) byd-xylose-grown MD353 cells (●, with l-malate; ○, without l-malate) or MD353/pLPA9 cells (■, withl-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 mMd-[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 cyanidem-chlorophenylhydrazone added 30 s befored-[U-14C]xylose) ord-ribose-grown cells of MD353 (◊).
The kinetic parameters of d-[U-14C]xylose uptake measured in d-xylose-grown MD353 cells suggested a system with a relatively low affinity for d-xylose (apparent Km of 8.5 ± 0.1 mM) and a Vmax of 23.2 ± 0.2 nmol min−1mg of dry weight−1.
Isolation of mutants unable to utilize d-xylose as a sole source of energy.To identify the low-affinityd-xylose transport system in L. pentosus, we studied several L. pentosus strains unable to utilized-xylose. We have isolated three 2DGr 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 andd-xylulose kinase activity and of growth ond-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. curvatusmannose PTS) into the three 2DGr 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 EIIBMan subunit of the L. pentosus EIIMan 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 ofL. 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.
In contrast, the Xyl− mutant MD358 was complemented for growth on xylose by plasmid pLP3537-xyl but not by plasmid pLPA9 (Table2). This result confirmed the lack ofd-xylose-catabolizing activity in this strain and stressed the importance of subsequent metabolism for the extensive uptake ofd-xylose. Moreover, it indicated that thed-xylose transport function was not defective in MD358.
Properties of wild-type and mutant L. pentosus strainsa
Transport and catabolism in the various Xyl− mutants and their respective Xyl+ transformants.The capacity to transport d-xylose, as well as to catabolized-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 α-xylosidase activity (encoded byxylQ) to investigate the effects of the various Xyl− mutations on the expression of thexylPQ 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 andd-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.
The data (Table 2) show clearly that 14C was not incorporated into cellular material, presumably due to the lack ofd-xylose transport in LPE5 and LPE8, caused by a defectived-mannose PTS, in agreement with the inability of these mutants to grow on d-xylose. The rates of ATP-dependent phosphorylation of the pentose and the α-xylosidase activity in the two mutants were approximately 10-fold lower than those of MD353. The question then arose of how the expression of xylQ andxylAB in these two 2DGr mutants can be induced if the inducer, d-xylose, is not transported into the cell. Considering the high d-xylose concentration used during the growth experiment (66 mM), a plausible explanation would be that under these conditions small amounts of d-xylose entered the cell and resulted in low-level expression of the xyl genes. In contrast, the xyl genes were fully induced in LPE5 and LPE8 transformed with pLAP9. The rate of ATP-dependent formation ofd-xylulose 5-phosphate and the α-xylosidase activity in strain LPE6 were similar to those in the wild-type strain, but the rate of incorporation of d-xylose in this strain was twofold lower than that in MD353, and this activity was significantly increased in LPE6/pMJ18. Therefore, the impairment of the ability of strain LPE6 to grow on d-xylose was due to inefficient transport ofd-xylose.
Finally, strain MD358, which lacked ATP-dependent formation ofd-xylulose 5-phosphate but was not defective in EIIMan activity, exhibited a low but significant rate of d-xylose transport. Expression of thed-xylose-catabolic enzymes XylA and XylB in MD358 from plasmid pLP3537-xyl did restore the rate of accumulation of14C-labelled material to the level observed in MD353, however. This result demonstrates that the continuous uptake ofd-xylose inside L. pentosus cells and the apparent accumulation of 14C label required subsequent metabolism, i.e., the trapping of compounds derived from xylose. This phenomenon is a typical feature of a facilitated-diffusion mechanism (30, 36). A similar phenomenon is observed in the MD353 parent strain. As mentioned in a previous section, ribose-grown MD353 cells did not take up much xylose, in contrast to xylose-grown cells, which contained the metabolic enzymes that converted xylose into xylulose 5-phosphate. EIIMan activity was almost similar, at 47 and 42 nmol min−1 mg of dry weight−1 in ribose- and xylose-grown cells, respectively. The overall data are strongly indicative of a facilitated-diffusion mechanism of d-xylose via the EIIMancomplex.
The involvement of the EIIMan complex ind-xylose uptake is further supported by the observation that uptake of 1 mM [U-14C]2DG (a substrate taken up and phosphorylated by EIIManin L. pentosus) in d-mannose-grown cells of MD353 was inhibited 40 and 65% by the addition of 20 and 40 mM unlabelled d-xylose, respectively. No inhibition was observed after the addition of 40 mM l-arabinose.
Mechanism of d-xylose transport via the EIIMan complex.The phenotype of mutant LPE6 suggested that some, but not all, mutations that affected EIIMan activity in the 2DGr 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 EIIMan 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, EIICMan and EIIDMan, and of two cytoplasmic phosphoryl transfer domains, EIIAManand EIIBMan (which may be separate proteins or a single polypeptide, as in the case of the E. coliEIIMan complex). In E. coli, EIICMan and EIIDMan are not phosphorylated. The cytosolic EIIBMan domain is supposed to be the phosphoryl donor to the carbohydrate (8). This implies that the membrane-bound subunits EIICDManof the EIIMan complex should bind and translocate the PTS carbohydrate to the cytosolic side in order to allow phosphorylation by the phosphorylated EIIBMandomain. 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/DMan domains and transported inside the cell by facilitated diffusion. The other components, EIIBMan, EIIAMan, 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 EIIBMan activity. However, EIIBMan is apparently required for maximal d-xylose transport activity via the EIIC/DMan components. This observation suggests that EIIBMan controls the active conformation of EIIC/DMan even if it is not functioning in phosphoryl transfer to d-xylose. We do not know in which component of the EIIMan complex the mutation of LPE5 and LPE8 is localized, but it seems likely that these two mutants at least lack EIIC/DMan activity or have altered EIIC/DMan activity.
Role of EIIMan in the transport ofd-xylose in other facultative heterofermentative lactobacilli.It has been previously demonstrated that introduction of plasmid pLP3537-xyl into L. casei andL. 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 EIIMancomplex could be responsible for the facilitated diffusion ofd-xylose in these species. An L. caseiEIIMan-defective mutant (BL23D) has been described (39), but no EIIMan-defective mutant of L. plantarum was available. Therefore, we isolated several spontaneous L. plantarum 80 2DGr 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 min−1 mg of dry weight−1 for LPL1 versus 48 ± 4 nmol min−1 mg of dry weight−1for LPL80). This suggested that LPL1 was impaired in EIIMan 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 thexylAB operon in L. casei; see reference16). 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 ofd-gluconate (an energy source to support the growth of Xyl− transformants) and d-xylose. The results are shown in Table 3. In bothLactobacillus species transformed with plasmids pLP3537-xyl and pLP3537-xyl*, the inability to grow on d-xylose correlated with a low EIIMan activity. Moreover, the lack of d-xylose transport in 2DGr 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 EIIMan is also responsible for the facilitated diffusion of d-xylose inL. casei and L. plantarum whend-xylose catabolic activity is provided to the cell.
Properties of wild-type and mutant L. caseiATCC 393 and L. plantarum 80a
Role of d-mannose in the growth rate of L. pentosus, L. casei, and L. plantarum ond-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 EIIMan (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 Lactobacillusstrains 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 ofd-mannose in permeabilized cells (Fig. 3). Indeed, a linear relationship could be found between the doubling times of the variousL. pentosus strains on d-xylose and the levels of PEP-dependent phosphorylation of d-mannose (level of EIIMan 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.
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 (○).
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.
In conclusion, facilitated diffusion of d-xylose via the EIIMan complex in L. pentosus increases the number of examples of sugar transport via the PTS which occurs in the absence of phosphorylation. Such a phenomenon might prove to be widespread, and similar examples could exist for many other bacteria.
ACKNOWLEDGMENTS
We are grateful to Gaspar Pérez-Martı́nez (Instituto de Agroquı́mica y Technologı́a de Alimentos, Valencia, Spain) for kindly providing plasmid pMJ18 and L. casei ATCC 393 strains BL23 and BL23D and to M. Daeschel (Department of Food Science, North Carolina State University) for the gift of L. pentosus MD358.
This work was supported by a grant from the EU (BIO2-CT92-0137).
FOOTNOTES
- Received 4 November 1998.
- Accepted 10 June 1999.
- Copyright © 1999 American Society for Microbiology
