Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4350, USA1
Department of Infectious Diseases, Childrens National Medical Center, Washington DC 20010-2970, USA2
Institut für Organische Chemie, Technische Universität Darmstadt,D-64287 Darmstadt, Germany3
Author for correspondence: John Thompson. Tel: +1 301 496 4083. Fax: +1 301 402 0396. e-mail: jthompson{at}dir.nidcr.nih.gov
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ABSTRACT |
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Keywords: phospho--glucosidase, sucrose isomers, sucrose-6-phosphate hydrolase, Klebsiella pneumoniae, Escherichia coli O157:H7
Abbreviations: FK, fructokinase; G6P, glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; HK, hexokinase; 4-MU--G6P, 4-methylumbelliferyl
-D-glucopyranoside-6-phosphate; PEP:PTS, phosphoenolpyruvate-dependent sucrose:phosphotransferase system; pNP-
-G6P, p-nitrophenyl
-D-glucopyranoside 6-phosphate; pNP-ß-G6P, p-nitrophenyl ß-D-glucopyranoside 6-phosphate; S6PH, sucrose-6-phosphate hydrolase
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INTRODUCTION |
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The linkage between the two component sugars of sucrose, i.e. D-glucose and D-fructose, can be modified to yield five isomeric compounds (Fig. 1) that trivially are designated trehalulose, turanose, maltulose, leucrose and palatinose (Lichtenthaler & Rönninger, 1990
; Lichtenthaler et al., 1991
; Immel & Lichtenthaler, 1995
). Until recently (Thompson et al., 2001a
), there were no reports of the bacterial utilization of the five sucrose isomers. Indeed, the inability of mutans streptococci to metabolize these comparatively sweet disaccharides suggests their use as non-cariogenic substitutes for dietary sucrose (Ooshima et al., 1983
, 1991
; Ziesenitz et al., 1989
; Minami et al., 1990
; Peltroche-Llacsahuanga et al., 2001
). In light of these reports, we were surprised to discover that K. pneumoniae readily utilized all five
-D-glucosyl-D-fructoses as energy sources for growth (Thompson et al., 2001a
, b
), and that the enzymes encoded by the sucrose (scr) operon (Titgemeyer et al., 1996
) did not participate in dissimilation of these compounds. Remarkably, the sucrose isomers and structurally related
-glucosides (including maltose, isomaltose, maltitol and methyl
-D-glucoside) are first translocated by an
-glucoside-specific EII(CB) transport protein, and the accumulated 6-phospho-
-D-glucosides are hydrolysed by a metal-requiring, NAD+-dependent phosphoglucosyl hydrolase belonging to family 4 of the glycosylhydrolase superfamily (Henrissat, 1991
). In K. pneumoniae, the genes for the EII(CB) transport protein (aglA) and the phospho-
-glucosidase (aglB) lie adjacent, and are chromosomally encoded (Thompson et al., 2001b
).
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METHODS |
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Bacterial strains and culture media.
K. pneumoniae ATCC 23357, E. coli O157:H7 (EDL 933) and F. mortiferum ATCC 25557 were obtained from the American Type Culture Collection. K. pneumoniae and E. coli O157:H7 were grown in a medium of the following composition (per litre): Na2HPO4, 7·1 g; KH2PO4, 1·5 g; (NH4)2SO4, 3 g; MgSO4.7H2O, 0·1 g; FeSO4.7H2O, 5 mg. Filter-sterilized sugars were added to autoclaved media (pH 7·4) to a final concentration of 4 g per litre. Cells of K. pneumoniae were grown in standing cultures, but E. coli O157:H7 was grown with vigorous aeration on a rotary shaker (250 r.p.m.). E. coli PEP43(pCB4.11) was grown with aeration in LuriaBertani (LB) medium supplemented with 50 µg kanamycin ml-1. F. mortiferum was grown anaerobically (GasPak, BBL) in a medium comprising (per litre): Tryptone (Difco), 17 g; Protease Peptone (Difco), 3 g; Na2HPO4, 2·5 g; NaCl, 5 g; final pH 7·3.
DNA sequence analysis.
Automated DNA sequencing incorporating Big Dye terminators was used to sequence malB, and an adjacent upstream gene (malR), directly from genomic DNA of F. mortiferum. From data previously reported by Bouma et al. (1997) , a reverse primer 1R1 (5'-AACTCTCTCTAACTTGTGGTACTGAAAGTC-3') was designed to obtain initial sequence information. Subsequent data were obtained by the primer synthesis and the chromosomal walking technique. PCR primers were designed for sequencing of the second strand, and for amplification (from genomic DNA) of the fragment encoding the two genes by use of Taq DNA polymerase. The amplicon was cloned into the TOPO TA cloning vector (Invitrogen). All sequencing was performed by BioServe Biotechnologies (Laurel, MD, USA), and the MacVector 7.0 sequence analysis package (Genetics Computer Group, Madison, WI, USA) was used to assemble and analyse the data.
Metabolism of sugars by washed cells of F. mortiferum.
To maintain anaerobic conditions, centrifuge tubes were flushed with a gas mixture (5% CO2, 5% H2, 90% N2, by vol.) prior to harvesting of the sucrose-grown cells (5000 g for 10 min at 5 °C). The supernatant fluid was discarded, and the cell pellet was resuspended as quickly as possible in 30 ml anaerobically prepared wash solution [50 mM potassium phosphate buffer (pH 7) containing 1 mM MgCl2]. After centrifugation, the washed cells were resuspended in 5 ml wash buffer, and the mixture was maintained at 0 °C under anaerobic conditions until required. For studies of disaccharide utilization, the washed cells (equivalent to 30 mg total cell protein) were added to 10 ml of wash buffer containing the desired sugar (sucrose or isomer) at a final concentration of 10 mM. The cell suspensions were incubated at 37 °C in 100 ml serum bottles filled with anaerobic gas and, at intervals, 1 ml samples were withdrawn by insertion of a gas-flushed tuberculin syringe through the butyl rubber cap. The cells were removed by filtration through Millex-GS filter units (0·22 µm pore size; Millipore) and filtrates were collected. Samples were heated in 1 M HCl for 1 h at 100 °C, cooled, and neutralized with 1 M KOH. Glucose (equivalent to disaccharide remaining) was determined by the ATPG6PDH/HKNADP+ coupled enzyme assay.
Preparation and analysis of F. mortiferum extracts.
Cells of F. mortiferum grown on the different sugars were harvested from 400 ml anaerobic culture. The cell pellets (12 g wet weight) were resuspended with 3 vols 25 mM Tris/HCl buffer (pH 7·5) containing 0·1 mM NAD+ and 1 mM MnCl2 (designated TNM buffer). The cells were disrupted by sonication at 0 °C (6x15 s bursts in a Branson instrument, model 185), and centrifuged at 14000 r.p.m. for 20 min at 5 °C in an Eppendorf bench-top instrument. The clarified supernatants were assayed for S6PH, FK and phospho-
-glucosidase activities.
Enzyme assays.
The activities of S6PH, FK and phospho--glucosidase (with disaccharide phosphate substrates) were determined from the formation of glucose, fructose 6-phosphate and G6P, respectively, in the appropriate reaction mixture. Production of the three metabolites was coupled to the enzymic reduction of NADP+ (measured as A340), and rates were determined in a Beckman DU 640 recording spectrophotometer. In all calculations, a molar absorption coefficient (
) of 6220 M-1 cm-1 was assumed for NADPH.
S6PH.
This enzyme catalyses the hydrolysis of both sucrose 6-phosphate (to G6P and fructose) and sucrose (to glucose and fructose), albeit with significantly different Km for the two compounds (0·1 mM and 100 mM, respectively; see Thompson et al., 1992
). Because of the limited supply of sucrose 6-phosphate, sucrose was used as substrate for the spectrophotometric assay of S6PH in cell extracts. The standard 1 ml assay contained: 0·1 M potassium phosphate buffer (pH 7·2); 50 mM sucrose; 5 mM ATP; 10 mM MgCl2; 1 mM NADP+,
3 U each of G6PDH/HK, and cell extract.
FK.
Activity was determined in a similar mixture to that used for S6PH, containing 10 mM fructose as substrate, 3 U G6PDH and 5 U phosphoglucose isomerase.
Phospho--glucosidase (MalH).
Activity was determined by two methods in which either chromogenic analogues or phosphorylated disaccharides served as substrates. Cofactors NAD+ and Mn2+ were included in both reaction mixtures. Throughout the purification of MalH, enzyme activity was determined in a discontinuous assay with pNP--G6P and pNP-ß-G6P as substrates. The 2 ml reaction mixture (at 37 °C) contained: 50 mM Tris/HCl buffer (pH 7·5); 0·5 mM NAD+; 1 mM MnCl2; and 0·5 mM of the chromogenic substrate. After enzyme addition, samples (0·25 ml) were removed at intervals throughout a 3 min period of incubation, and were immediately added to 0·75 ml 0·5 M Na2CO3 solution containing 0·1 M EDTA to stop the reaction. The A400 was measured, and the amount of pNP formed (substrate hydrolysed) was calculated from the molar absorption coefficient of the yellow p-nitrophenolate anion,
=18300 M-1 cm-1. A continuous NADP+-coupled assay was used to measure G6P formed by MalH-catalysed hydrolysis of phosphorylated disaccharides. The assay mixture contained in 1 ml: 0·1 M HEPES buffer (pH 7·5); 1 mM MgCl2; 1 mM MnCl2; 1 mM NAD+; 1 mM NADP+; 2 mM disaccharide phosphate, 3 U G6PDH and purified MalH (35 µg protein).
Purification of MalH from E. coli PEP43(pCB4.11).
A 2·2 kb Sau3AI chromosomal DNA fragment of F. mortiferum that includes the malH gene and its promoter has previously been cloned and the enzyme has been expressed from plasmid pCB4.11 (Bouma et al., 1997 ). This plasmid was transferred by electroporation to E. coli PEP43
cel
(bglpho) leu metA or B his rpsL lacZ
4680 lacY+ arbT+ Tn10::bglA dTn10cam::ebgA5100 ebgR+ L532 (B. G. Hall, Biology Department, University of Rochester, NY, USA, laboratory collection). E. coli PEP43 expresses no phospho-ß-glucosidases because the cel and bglGFB operons have been deleted, bglA is disrupted by Tn10 and the asc operon is cryptic.
Cells of E. coli PEP43(pCB4.11) (25 g wet weight) were resuspended with 40 ml TNM buffer, and the organisms were disrupted by 2x1·5 min sonication with a Branson model 350 instrument. The preparation was clarified by ultracentrifugation (180000 g for 2 h at 5 °C), and the supernatant fluid was dialysed against 4 litres of TNM buffer. The dialysed material was transferred (
0·6 ml min-1) to a column of TrisAcryl M-DEAE (2·6x10 cm) that had been equilibrated with TNM buffer. The column was washed to elute non-adsorbed material, and then MalH activity was eluted with 500 ml of a linear, increasing concentration gradient of NaCl (0150 mM) in TNM buffer. Fractions of 5 ml were collected, and those containing highest MalH activity (5465 inclusive) were pooled and concentrated in an Amicon pressure cell to
3 ml. The concentrated sample was transferred (0·15 ml min-1) to an Ultrogel AcA-44 gel filtration column (1·6x94 cm; linear fractionation range, 10130 kDa) previously equilibrated with TNM buffer containing 0·1 M NaCl. Fractions of 2 ml were collected, and tetrameric MalH (
200 kDa) was eluted at the void volume of the column. Fractions that contained a single protein by SDS-PAGE (4750, inclusive) were pooled, and concentrated to yield
5 mg purified MalH [specific activity 2·9 µmol pNP-
-G6P hydrolysed min-1 (mg protein)-1].
Analytical methods.
Protein concentrations were determined by the BCA protein assay (Pierce). The Novex X-Cell system was used for both native (nonreducing) and SDS-PAGE. For SDS-PAGE experiments, precast NuPage (412%) Bistris gels and MES-SDS running buffer (pH 7·3) were used with Novex Mark 12 protein size standards. Proteins were stained with Coomassie brilliant blue R-250. Electrophoresis of cell extracts under nonreducing conditions was carried out at 10 °C in Tris/glycine (420%) precast gels from Novex, with Tris/glycine (pH 8·3) supplemented with 1 mM MnCl2 and 0·1 mM NAD+ as the running buffer. For detection of phospho--glucosidase activity, the gel was immersed in 30 ml of a solution that contained 25 mM Tris/HCl buffer (pH 7·5); 1 mM MnCl2; 0·1 mM NAD+ and 0·1 mM 4MU-
-G6P. After
5 min incubation, the gel was photographed under long-wave UV light with Ektopan Kodak film (2 min exposure with a green filter). For Western blot analysis, proteins in the cell extracts together with pre-stained markers (SeeBlue from Novex), were first separated by SDS-PAGE and then transferred to a nitrocellulose membrane. Immunodetection of phospho-
-glucosidase was performed with polyclonal antibody to MalH from F. mortiferum as described previously (Thompson et al., 1995
). Molecular dynamics simulations and procedures for the determination of solvent-accessible surfaces have been described previously (Immel & Lichtenthaler, 1995
; Thompson et al., 2001b
).
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RESULTS |
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Growth studies with F. mortiferum, K. pneumoniae and E. coli O157:H7
Growth studies were performed to determine if possession of the putative operons would permit growth of the three organisms on sucrose isomers and other -glucosides. Both K. pneumoniae and F. mortiferum showed excellent growth on all sugars tested, including glucose, fructose, methyl
-glucoside, maltose, maltitol, sucrose and all five
-D-glucosyl-D-fructoses. E. coli O157:H7 grew well on glucose, fructose, maltose and sucrose, but the pathogen was unable to grow on methyl
-glucoside, maltitol or any of the sucrose isomers.
Enzyme expression during growth of F. mortiferum on sucrose and its isomers
S6PH and ATP-dependent fructokinase (FK) are induced by growth of F. mortiferum on sucrose (Robrish et al., 1991 ; Thompson et al., 1992
). Because sucrose and its isomers comprise the same hexose moieties, it was of interest to determine whether sucrose-grown cells of F. mortiferum would also metabolize the isomeric compounds. As expected, sucrose-grown cells readily fermented sucrose, but there was no detectable metabolism of the five isomers, including palatinose and turanose (Fig. 3
). Furthermore, whereas sucrose-grown cells of F. mortiferum contained high levels of S6PH and FK activity (Table 1
), after growth on the isomers, the activities of the two enzymes were not significantly greater than the constitutive levels found in glucose- or fructose-grown cells. These findings contrast markedly with the high levels of S6PH and FK that are expressed during growth of K. pneumoniae on the isomeric compounds (Table 2
).
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DISCUSSION |
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Proteins encoded by the scr operons of K. pneumoniae and F. mortiferum are expressed during growth of both organisms on sucrose (Sprenger & Lengeler, 1988 ; Thompson et al., 1992
). Hydrolysis of sucrose 6-phosphate by S6PH yields G6P and fructose, and for K. pneumoniae, fructose is believed to be the inducer of the scr operon (Jahreis & Lengeler, 1993
). Hydrolysis of the phosphorylated isomers by AglB of K. pneumoniae also yields G6P and fructose, and formation of the latter ketohexose is consistent with the high levels of S6PH and FK present in cells grown on the isomers (Table 2
). Surprisingly, similar studies with F. mortiferum showed that, for this organism, growth on the isomeric compounds did not induce significant expression of either S6PH or FK (Table 1
). These findings explain why these cells were unable to metabolize sucrose, and additionally, the data point to sucrose 6-phosphate (rather than fructose) as the likely inducer of the scr operon in F. mortiferum.
Substrate specificity and hydrolysis of chromogenic substrates by MalH
MalH is an oligomeric protein comprising four identical subunits (molecular mass 50 kDa) that, by sequence-based alignment, is assigned to family 4 of the glycosylhydrolase superfamily. As reported for other members of this unusual family, MalH is inherently unstable and Mn2+ and NAD+ are prerequisite cofactors for activity (Nagao et al., 1988
; Thompson et al., 1998
, 1999
; Raasch et al., 2000
). Whether the nucleotide and metal ion fulfil catalytic or structural functions has not been ascertained for any member of family 4. Phosphorylation at O-6 of the glucosyl moiety of the isomers is necessary for substrate cleavage, and MalH is unable to hydrolyse the corresponding non-phosphorylated compounds. MalH is also exacting with respect to the
-O linkage of its PTS-derived substrates (see below) and, because there is no detectable hydrolysis of ß-O-linked stereomers such as cellobiose 6'-phosphate and gentiobiose 6'-phosphate (Table 4
), the enzyme may reasonably be classified as a phospho-
-glucosidase. In this context, it is not clear why MalH should hydrolyse both pNP-
-G6P and pNP-ß-G6P with comparable efficiency (Table 3
). Co-purification of MalH with a phospho-ß-glucosidase resident in the host (E. coli PEP43) can be discounted because of gene inactivation or crypticity, and analysis of the final preparation by SDS-PAGE provided evidence for only a single polypeptide. That the same cofactors should also be required for catalysis is further evidence that the same enzyme hydrolyses both
- and ß-forms of the chromogenic compound(s). Unlike the phosphorylated sucrose isomers (where G6P is linked to a fructose moiety), the essential G6P moiety of the chromogenic substrates is attached to p-nitrophenol. Perhaps the aromatic aglycone exerts an effect (electron-withdrawing?) upon the O-linkage such that
/ß conformation is no longer a determinant of substrate specificity. It is of comparative interest to note that cellobiose-6-phosphate hydrolase (CelF) from E. coli is also a member of glycosylhydrolase family 4 (Thompson et al., 1999
). In contrast to MalH, this NAD+- and metal-dependent phospho-ß-glucosidase hydrolyses only pNP-ß-G6P.
Molecular basis for substrate recognition by MalH
Sucrose 6-phosphate and its phosphorylated isomers are not commercially available, but all of these derivatives were recently prepared in our laboratory (Thompson et al., 2001a ). Studies of substrate specificity showed that whereas MalH hydrolysed all of the phosphorylated isomers, the enzyme failed to hydrolyse sucrose 6-phosphate itself. Insight into the molecular basis for this remarkable discrimination among potential substrates was gained by molecular dynamics simulations, which revealed the probable solution-state geometries of the various disaccharide phosphates (Thompson et al., 2001b
). Molecular dynamics simulations and determination of solvent-accessible surfaces indicate pronounced conformational differences between sucrose 6-phosphate and its five isomeric 6'-phosphates. By virtue of an interresidue water bridge between Glc-2-O
H2O
O-1-Fru, both sucrose and sucrose 6-phosphate assume a compact, globular shape in solution (Immel & Lichtenthaler, 1995
; Thompson et al., 2001b
). This water bridge is not present in the 6'-phosphoglucosyl-fructoses and, in consequence, the phosphorylated isomers adopt a more linear (extended) molecular geometry. The specificity of MalH for the isomeric phosphates presumably reflects recognition by the enzymes binding domain of both the shape and the molecular lipophilicity potential of the contact surfaces of these particular molecules (Thompson et al., 2001b
).
Conclusions
Both F. mortiferum and K. pneumoniae readily metabolize the five isomers of sucrose. In contrast, E. coli O157:H7 (which grows well on sucrose) failed to grow on any of the isomeric compounds. These results were surprising, because this enterohaemorrhagic strain has three genes (yidP, glvA and glvG) whose organization and deduced amino acid sequences are virtually identical to those of aglR, aglA and aglB, respectively, in K. pneumoniae. Although contrary to expectation, the results obtained for E. coli O157:H7 were nevertheless important. First, the data established for E. coli O157:H7 (as for the other species), that the sucrose-PTS/S6PH pathway is neither induced by, nor does it provide a route for dissimilation of, sucrose isomers. Secondly, the data indicate that possession of genes encoding -glucoside-specific EII(CB) and phospho-
-glucosidase (while necessary), may not be entirely sufficient for dissimilation of
-D-glucosyl-D-fructoses by micro-organisms.
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ACKNOWLEDGEMENTS |
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Received 3 September 2001;
revised 5 November 2001;
accepted 8 November 2001.