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Applied and Environmental Microbiology, February 2002, p. 582-587, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.582-587.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

L-Glucitol Catabolism in Stenotrophomonas maltophilia Ac

Elke Brechtel, Alexander Huwig, and Friedrich Giffhorn*

Lehrstuhl für Angewandte Mikrobiologie, Universität des Saarlandes, D-66041 Saarbrücken, Germany

Received 18 June 2001/ Accepted 6 November 2001


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The carbohydrate catabolism of the bacterium Stenotrophomonas maltophilia Ac (previously named Pseudomonas sp. strain Ac), which is known to convert the unnatural polyol L-glucitol to D-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail. All enzymes operating in a pathway that channels L-glucitol via D-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified. D-Sorbose was converted by C-3 epimerization to D-tagatose, which, in turn, was isomerized to D-galactose. D-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation of D-galactose to D-galactonate, its dehydration to 2-keto-3-deoxy-D-galactonate, and its phosphorylation to 2-keto-3-deoxy-D-galactonate 6-phosphate. Finally, aldol cleavage yielded pyruvate and D-glycerate 3-phosphate as the central metabolic intermediates.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
In a previous study an obligate aerobic gram-negative rod with a polar flagellum was isolated from soil using a mineral medium and the unnatural polyol L-glucitol as the selective carbon source. On the basis of phenotypic characterizations the bacterium was assigned to the genus Pseudomonas and preliminarily designated Pseudomonas sp. strain Ac (30). Only recently, this strain was reclassified by the German Collection of Microorganisms and Cell Cultures as Stenotrophomonas maltophilia (DSM 14322). The genus Stenotrophomonas was proposed for Xanthomonas maltophilia (39) following a lengthy period of uncertainty in the taxonomic status of this bacterial species, which was formerly also known as Pseudomonas maltophilia (16, 42, 45). More recently, the distinction between S. maltophilia and members of the genus Xanthomonas was confirmed by restriction mapping (37) and sequence analyses of 16S rRNA genes (33). S. maltophilia is ubiquitous in a wide variety of environments and geographical regions, and strains of this organism were isolated from soil as well as from various nosocomial sources and clinical specimens (for a review see reference 7). Characteristic of S. maltophilia are unusual lipopolysaccharides containing relatively large amounts of uncommon fatty acids (35, 38) and O-specific polysaccharide chains with uncommon sugar moieties (8, 47-49). Strains of S. maltophilia isolated from natural environments revealed versatile biochemical and physiological characteristics, suggesting a potential for biotechnological exploitation of these organisms. These characteristics of S. maltophilia comprise activities against agricultural pests (3, 25, 26), expression of antifungal activities against human pathogenic fungi (22, 24), degradation of a variety of unusual substrates and recalcitrant compounds (5, 23, 36), and the production of enzymes with promising biotechnological potentials (18, 25, 28, 30, 31). On the other hand, strains of S. maltophilia isolated from nosocomial sources or clinical specimens were identified as multidrug-resistant opportunistic pathogens in humans which can cause severe disease, especially in immunocompromised individuals (for a review see reference 7). Because of both its biotechnological and clinical aspects, the bacterium S. maltophilia deserves to be studied more thoroughly (7). We contribute by characterizing a new pathway through which the organism is capable of utilizing rare and unnatural carbohydrate substrates.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Organism and growth conditions.
S. maltophilia Ac (DSM 14322) was grown at 30°C in 1-liter Erlenmeyer flasks with shaking (150 rpm) in a mineral medium, pH 6.8 (30), supplemented with L-glucitol or D-glucose (20 mM).

Preparation of cell extracts.
Wet cells (0.5 g) were washed in 50 mM potassium phosphate (pH 7.0), resuspended in 2.5 ml of the same buffer, and disintegrated by two or three passages through a French press at 14,000 N/cm2. Subsequently, the cell debris was removed by centrifugation at 4°C and 54,000 x g for 20 min. Protein concentrations were determined as described previously (13).

Enzyme assays.
Cell extracts of S. maltophilia Ac were used to determine the following enzyme activities. (i) L-Glucitol dehydrogenase (LGDH; EC 1.1.1.-) activity was determined at 30°C in the presence of 100 mM L-glucitol by monitoring the reduction of NAD at 340 nm (30, 40). (ii) D-Tagatose 3-epimerase (EC 5.1.3.-) activity was determined as the rate of D-tagatose formation from D-sorbose. The reaction mixture contained in a final volume of 0.6 ml at 30°C the following: 14 µmol of Tris-HCl (pH 8.0), 1 µmol of MgCl2, 12 µmol of D-sorbose, and 0.2 ml of cell extract (2.5 mg of protein). Samples of 0.1 ml were taken at 0, 15, 30, and 50 min and assayed by high-performance liquid chromatography (HPLC; see below) following heat treatment of the samples and removal of the precipitated protein by centrifugation. (iii) D-Galactose isomerase (EC 5.3.1.-) activity was qualitatively determined by analyzing the formation of D-tagatose from D-galactose by HPLC and thin-layer chromatography (TLC; see below). The reaction mixture was similar to that for D-tagatose 3-epimerase. (iv) D-Galactose dehydrogenase (GalDH; EC 1.1.1.48) activity was determined at 30°C by monitoring the reduction of NAD at 340 nm (4). (v) D-Galactonate dehydratase (EC 4.2.1.6) activity was measured as the rate of 2-keto-3-deoxy-D-galactonate (KDGalA) formation from D-galactonate using the thiobarbiturate assay (43, 46). (vi) KDGalA kinase (EC 2.7.1.58) and KDGalA 6-phosphate aldolase (EC 4.1.2.21) activities were determined by a coupled assay using KDGalA as the substrate (see below), which, in the presence of ATP, is converted into pyruvate and D-glyceraldehyde 3-phosphate (GAP). The reaction mixture contained in a final volume of 1.0 ml at 30°C the following: 76 µmol of Tris-HCl (pH 8.0), 0.5 µmol of MgCl2, 2.5 µmol of KDGalA, 0.2 µmol of ATP, and 100 µl of cell extract. After 15 min of incubation the mixture was heated for 1 min at 100°C, and the precipitated protein was removed by centrifugation. In the supernatant pyruvate (27) and GAP (32) levels were determined. D-Gluconate 6-phosphate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-D-glucose 6-phosphate aldolase (EC 4.1.2.14) were assayed as described in item vi above by using 2.5 µmol of D-glucose 6-phosphate as the substrate. D-Fructose 6-phosphate kinase (EC 2.7.1.11), galactokinase (EC 2.7.1.6), and galactose 1-phosphate uridylyltransferase (EC 2.7.7.12) activities were assayed as described in references 4, 15, and 9, respectively, and D-glyceraldehyde activity was assayed as described in reference 14.

Preparation of D-tagatose.
To demonstrate the reaction product of the D-sorbose conversion by D-tagatose 3-epimerase, a reaction mixture similar to that used for D-tagatose 3-epimerase was used; the reaction was stopped after 40 min by heat treatment when a maximum level of about 4 mM D-tagatose, which corresponded to 20% of the initial D-sorbose concentration, was reached. D-Tagatose was isolated from the supernatant of the reaction mixture by collecting the sugar at its specific retention time (11 to 12 min) after several runs of HPLC separations. The combined fractions were concentrated by evaporation and used for TLC and nuclear magnetic resonance (NMR) spectroscopic analyses (see below).

Preparation of KDGalA.
The preparative conversion of D-galactonate to KDGalA was performed in a reaction mixture which contained in a final volume of 5.0 ml at 30°C the following: 95 µmol of Tris-HCl (pH 8.0), 2.5 µmol of MgCl2, 40 µmol of D-galactonic acid, and 2 ml of cell extract (8.2 mg of protein/ml). Samples of 0.1 ml were taken after 0, 0.5, 1, 4, 6, 8, and 10 h and assayed for KDGalA production by the thiobarbiturate assay (see item vi of "Enzyme assays"). After 10 h the KDGalA concentration reached 6.9 mM; on the basis of an initial concentration of 8 mM D-galactonic acid, the conversion was 86%. Then the reaction was stopped by the addition of 0.2 ml of 20% trichloroacetic acid, and the precipitated proteins were removed by centrifugation. The supernatant was adjusted with water to a concentration of 5 mM KDGalA for use in the enzyme assay (as described in item vi of "Enzyme assays").

HPLC.
Substrates and products of carbohydrate conversions were determined by HPLC on the basis of comparisons with authentic sugars. Prior to analysis proteins were removed by heat treatment and centrifugation. A Benson-100 carbohydrate column (Benson Polymeric Inc., Reno, Nev.) linked with a refractive index detector (model 156; Beckman, Frankfurt, Germany) was used, and water was the mobile phase at a column temperature of 85°C and a flow rate of 0.85 ml/min (17).

TLC.
TLC was performed on silica gel plates (Merck, Darmstadt, Germany) using acetone-butanol-water (4:5:1) as the mobile phase. Spots were detected with dinitrophenylhydrazine reagent (17).

NMR spectroscopy.
NMR spectra were recorded in D2O on a Bruker AMX 500 spectrometer at 500.14 MHz for 1H and 125.76 MHz for 13C at 300°K. Chemical shifts were referenced to internal acetone ({delta} = 2.030 and 30.50 ppm) (11, 12).

Chemicals.
L-Glucitol was prepared by chemical hydrogenation of D-gulono-1,4-lactone from Aldrich (Deisenhofen, Germany) (30). D-Sorbose was prepared from L-glucitol by bioconversion with S. maltophilia Ac (formerly Pseudomonas sp. strain Ac) (18). D-Tagatose was prepared chemoenzymatically with pyranose oxidase as described previously (11). D-Galactonic acid and all other chemicals were reagent grade from Sigma (Deisenhofen, Germany). Biochemicals were purchased from Roche Molecular Biochemicals (Mannheim, Germany).


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
L-Glucitol catabolism in S. maltophilia.
In a previous study it was shown that bacterium S. maltophilia Ac grew readily in a mineral medium containing the unnatural polyol L-glucitol as the sole carbon source and that a new enzyme, LGDH, catalyzes the initial conversion of L-glucitol into the rare sugar D-sorbose (30). This finding posed the question of how D-sorbose is further metabolized and channeled into the central metabolic pathways of S. maltophilia Ac. Of several possible conversions of D-sorbose we considered aldol cleavage, isomerization into one of the epimeric rare hexoaldoses D-gulose and D-idose, and C-3 epimerization to D-tagatose. First, aldol cleavage was studied by enzymatic analysis, but we were unable to detect either of the possible cleavage products, D-glyceraldehyde (14) and D-glyceraldehyde 3-phosphate (32), upon incubation of cell extracts of S. maltophilia Ac with 20 mM D-sorbose in the absence and presence of ATP plus MgCl2 (both 0.4 mM). However, when the cell extracts were assayed by HPLC (see Materials and Methods), two peaks were detected, one of which was identified as D-sorbose with a retention time at 8.6 min (Table 1) and the other of which was considered the putative conversion product of D-sorbose with the retention time of authentic D-tagatose, 11.6 min. Since D-gulose had a similar retention time of 11.3 min (Table 1), a clear distinction between D-gulose and D-tagatose by HPLC was not possible. Therefore the putative conversion product was isolated from a similar reaction mixture (see Materials and Methods) and characterized as tagatose by comparison with authentic D-tagatose using 1H- and 13C-NMR spectroscopy. The recorded spectra (Fig. 1) are in accordance with the NMR data obtained for D-tagatose in our recent studies (11, 12, 19). The results showed that in cell extracts of S. maltophilia Ac D-sorbose was converted to D-tagatose by C-3 epimerization and excluded D-gulose as a possible conversion product. It was likely that the interconversion of D-sorbose into D-tagatose in cell extracts of S. maltophilia Ac was catalyzed by an enzyme similar to D-tagatose 3-epimerase of Pseudomonas cichorii ST-24 (20, 21). Accordingly, the activity of the D-tagatose 3-epimerase in cell extracts of S. maltophilia Ac was determined by HPLC as the rate of D-tagatose formation from D-sorbose (21). The specific activity of D-tagatose 3-epimerase (0.03 U per mg of protein) was calculated on the basis of initial rate measurements during which D-tagatose formation was linear with time. Figure 2 shows the course of D-tagatose formation during the incubation of a cell extract of S. maltophilia Ac in the presence of 20 mM D-sorbose (see Materials and Methods). Within a period of 30 min, the formation of D-tagatose increased linearly with time, reaching a maximum that amounted about 20% of the initial D-sorbose concentration, and then the formation of D-tagatose slowed down, suggesting that the epimerase reaction was striving for an equilibrium. For the D-tagatose 3-epimerase of P. cichorii ST-24 the equilibrium of the D-sorbose-D-tagatose interconversion was shown to be far in favor of D-sorbose (21). In view of the fact that D-sorbose was converted to D-tagatose, the question of how D-tagatose was further metabolized arose. It was reasonable to assume that D-tagatose is converted by an isomerase to the corresponding aldose D-galactose, a substrate for which catabolic routes are well known (see below). However, analysis of the forward reaction (D-tagatose->D-galactose) by HPLC was hampered, because D-sorbose was produced from D-tagatose by cell extracts of S. maltophilia Ac and both D-sorbose and the expected isomerase product, D-galactose, exhibited the same retention time of 8.6 min (Table 1). In fact, HPLC analysis of cell extracts of S. maltophilia Ac incubated with 20 mM D-tagatose for 30 min revealed only the D-tagatose peak at 11.6 min and the peak at 8.6 min, which did not resolve D-sorbose and D-galactose. Therefore, samples of the above conversion were analyzed by TLC, which allowed a clear distinction between D-galactose (Rf = 0.31) and D-sorbose (Rf = 0.39). However, TLC analysis revealed only the confluent spots of D-tagatose (Rf = 0.41) and D-sorbose and no perceptible spot of D-galactose. Since the analysis of the forward reaction did not lead to a clear result, the reverse reaction (D-galactose->D-tagatose) was also analyzed. In this case, D-galactose served as a substrate for the isomerase only and both the substrate and the product of the isomerase could be clearly distinguished by HPLC and TLC. Actually, the formation of D-tagatose by cell extracts incubated with 20 mM D-galactose for 30 min could be detected both by HPLC (Table 1) and TLC (Fig. 3), thus demonstrating the presence of a D-galactose isomerase in cell extracts of S. maltophilia Ac. This conversion was not quantified (noted in Table 2), because in cell extracts there was no linear relationship between D-galactose formation and the reaction time. Our inability to demonstrate the forward reaction (D-tagatose->D-galactose) is not yet clear; it may be due to the fact that two enzymes compete for D-tagatose and that D-tagatose 3-epimerase may have a much higher affinity for this substrate than D-galactose isomerase. Since to our knowledge D-galactose isomerase has not yet been described in the literature, we can only refer to similar aldose ketol-isomerases, which typically exhibit poor substrate affinities (1, 34, 51). A possible phosphorylation of D-tagatose and subsequent isomerization to D-galactose 6-phosphate as in Lactococcus lactis (44) were excluded, because we were able to demonstrate the complete catabolic pathway for D-galactose (see below). Since the key enzyme of this pathway, GalDH, catalyzes the irreversible oxidation of D-galactose to D-galactonate, it was reasonable to assume that this reaction pulls the sugars into the catabolic pathway. So far, it can be concluded from the results that S. maltophila Ac is capable of utilizing the unnatural polyol L-glucitol by producing a set of unique enzymes which convert L-glucitol into the more common substrate D-galactose (Fig. 4, reactions 1 to 3). For comparison, the pattern of enzymes which were produced by S. maltophilia Ac grown on D-glucose was also analyzed. Table 2 shows that D-glucose-grown cells produced only LGDH, not D-tagatose 3-epimerase and D-galactose isomerase. In addition, the presence of D-gluconate-6-P dehydratase and 2-keto-3-deoxy-D-gluconate 6-phosphate aldolase (see Materials and Methods) and the absence of D-fructose 6-phosphate kinase (4) indicated that in S. maltophilia Ac D-glucose is metabolized via the Entner-Doudoroff pathway (10). LGDH was also produced in cells grown on D-galactose, ribitol, xylitol, galactitol, and D-glucitol (sorbitol), and the LGDH activities were 0.2 to 1.6 U/mg of protein, depending on the substrate. The activities of LGDH were still present after several transfers of the cultures to fresh media. The finding that the activity of LGDH was retained whereas those of D-tagatose 3-epimerase and D-galactose isomerase were absent in S. maltophilia Ac grown on substrates other than L-glucitol (e.g., D-glucose) led to the development of an efficient bioconversion process for the production of the rare sugar D-sorbose from L-glucitol (18).


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  		TABLE 1. HPLC and TLC characteristics of various sugarsa

 


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  		FIG. 1. 1H-NMR spectrum (A) and 13C-NMR spectrum (B) of the conversion product of D-sorbose, which was identified as tagatose (see text).

 


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  		FIG. 2. Time course of D-tagatose production from 20 mM D-sorbose by cell extracts of S. maltophilia Ac. The reaction mixture is described in Materials and Methods. Samples were taken at the times indicated and were assayed by HPLC.

 


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  		FIG. 3. TLC analysis of a cell extract of S. maltophilia Ac (lane 4) incubated with 20 mM D-galactose at 30°C. For further details see Materials and Methods. Lanes 1 to 3, authentic sugars D-sorbose (lane 1), D-tagatose (lane 2), and D-galactose (lane 3) used as references.

 

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  		TABLE 2. Activities of various enzymes in cell extracts of S. maltophilia Ac grown in mineral medium with L-glucitol or D-glucose

 


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  		FIG. 4. Enzymatic conversions involved in L-glucitol catabolism in S. maltophilia Ac. Reactions: 1, LGDH; 2, D-tagatose 3-epimerase; 3, D-tagatose isomerase; 4 to 7, DD pathway (6); 4, GalDH; 5, D-galactonate dehydratase; 6, KDGalA kinase; 7, KDGalA 6-phosphate aldolase.

 
D-Galactose catabolism in S. maltophilia via the DD pathway.
Many microorganisms can utilize D-galactose as a substrate for growth by virtue of two alternative metabolic routes. Most organisms convert D-galactose to D-glucose-1-P by the Leloir pathway (29), whose key enzymes, galactokinase (15) and galactose 1-phosphate uridylyltransferase (9), could not be detected in cell extracts of S. maltophilia Ac (Table 2). Alternatively, D-galactose can be metabolized by the so-called De Ley-Doudoroff (DD) pathway, consisting of enzymes similar to those of the Entner-Doudoroff pathway (6). The DD pathway has been demonstrated so far in only a few bacterial species; it was first discovered in Pseudomonas saccharophila (6) and later was discovered in Rhizobium meliloti L5-30 (2) and in Azotobacter vinelandii (50). The key enzyme of the DD pathway is GalDH, which catalyzes the oxidation of D-galactose to D-galactonate. This enzyme was detected at considerable activities in cell extracts of S. maltophilia Ac. In addition, all other enzymes involved in the DD pathway were demonstrated, and their activities are summarized in Table 2 and illustrated in Fig. 4 (reactions 4 to 7). D-Galactonate dehydratase was present in sufficient amounts to prepare the reaction product KDGalA from D-galactonate, as illustrated in Fig. 5. KDGalA (2.5 µmol) was then used as a substrate to demonstrate the presence of KDGalA kinase and KDGalA 6-phosphate aldolase in a coupled assay (see Materials and Methods), yielding pyruvate (0.60 µmol) and D-glyceraldehyde 3-phosphate (0.64 µmol) in almost equimolar amounts after 15 min. The study shows that L-glucitol catabolism in S. maltophilia Ac is mediated by two consecutive but independently operating reaction routes. To our knowledge, the first reaction sequence has yet not been demonstrated to occur in microorganisms. It converts the unnatural polyol L-glucitol (not known as a natural compound) into the common substrate D-galactose, which, in turn, induces the DD pathway in S. maltophilia Ac. For broader substrate profiles, as shown for LGDH (30) and D-tagatose 3-epimerase from P. cichorii ST-24 (20, 21), the enzymes involved in the first reaction sequence may contribute to the synthesis of rare and unusual sugars found in the lipopolysaccharides of S. maltophilia (see the introduction). The origin of LGDH in S. maltophilia Ac is not yet clear. Since the organism was selected on L-glucitol (30), it may represent an acquired (gain-of-function) capability (41).



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  		FIG. 5. Time course of KDGalA production from 8 mM D-galactonate by cell extracts of S. maltophilia Ac. The reaction mixture is described in Materials and Methods. Samples were taken at the times indicated and subjected to the colorimetric thiobarbiturate assay.

 


   ACKNOWLEDGMENTS
 
This work was supported by the Bundesminister für Forschung und Technologie (grant no. 0319515A).


   FOOTNOTES
 
* Corresponding author. Mailing address: Lehrstuhl für Angewandte Mikrobiologie, Universität des Saarlandes, Postfach 15 11 50, D-66041 Saarbrücken, Germany. Phone: 49-(0)681-302-2704. Fax: 49-(0)681-302-4360. E-mail: giffhorn{at}mx.uni-saarland.de. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, February 2002, p. 582-587, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.582-587.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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