Laboratoire de Génétique des Microorganismes, INRA-CNRS URA 1925, F-78850 Thiverval-Grignon, France1
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan2
Author for correspondence: Josef Deutscher. Tel: +33 1 30 81 54 47. Fax: +33 1 30 81 54 57. e-mail: jdeu{at}platon.grignon.inra.fr
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ABSTRACT |
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Keywords: Thermus flavus, glycerol metabolism, glycerol kinase, PEP:sugar phosphotransferase system
Abbreviations: HPr, histidine-containing protein; MTP, multiphosphoryl transfer protein; PEP, phosphoenolpyruvate; PTS, phosphoenolpyruvate:sugar phosphotransferase system
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INTRODUCTION |
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Recently, glpK, the gene encoding glycerol kinase, and a fragment of glpF, which encodes the first 156 amino acids of the glycerol facilitator of Thermus flavus, have been cloned and sequenced (Huang et al., 1998a ). Surprisingly, the thermostable glycerol kinase from T. flavus, which belongs to the Thermus/Deinococcus group, showed an unusually high degree of sequence identity (80·6%) when compared to glycerol kinase of B. subtilis and 64·0% when compared to glycerol kinase of Ent. casseliflavus. An equivalent of His-232, the site of PEP-dependent phosphorylation in glycerol kinase from Ent. casseliflavus, is present in glycerol kinase of T. flavus and the surrounding sequence is well-conserved. We therefore wanted to study whether T. flavus is capable of transporting glycerol and whether glucose exerts a repressive effect on glycerol transport and metabolism. We also wanted to test whether T. flavus possesses the general PTS components enzyme I and HPr allowing PEP-dependent phosphorylation of purified T. flavus glycerol kinase, whether purified enzyme I and HPr from B. subtilis can phosphorylate T. flavus glycerol kinase and whether this PEP-dependent phosphorylation would have an influence on T. flavus glycerol kinase activity.
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METHODS |
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Protein purification.
Recombinant T. flavus glycerol kinase was produced in the Esc. coli strain DH5 transformed with plasmid pGYK12 and purified by sequential chromatography on DEAE-Toyopearl and Toyopearl HW650 columns as previously described (Huang et al., 1997
, 1998a
). The Esc. coli strain M15 carrying the plasmid pREP4 (Qiagen) and transformed with plasmids pAG2 or pAG3 was used for the overproduction of B. subtilis His-tagged HPr and His-tagged enzyme I, respectively (Galinier et al., 1997
).
Glycerol-transport assay.
A 150 ml culture of T. flavus TE3420 cells was grown to mid-exponential phase and cells were harvested by centrifugation at 4 °C, washed twice with MM medium (Anagnostopoulos & Spizizen, 1961 ) and resuspended in the same medium at a concentration corresponding to an OD600 of 2. A peptone solution was added (1% final concentration) and the mixture was prewarmed at 50 °C for 10 min before [1,3-14C]glycerol [ICN; 0·5 mM final concentration, specific activity, 0·5 mCi (18·5 MBq) mmol-1] was added. Aliquots (120 µl) were withdrawn at different time intervals (0·5, 1·5, 3, 5, 10, 15 and 20 min), filtered through a 0·45 µm pore-size filter and washed twice with 5 ml cold Tris/maleate buffer, pH 7·4. The filters were dried and the radioactivity retained was determined by liquid scintillation counting.
Protein-phosphorylation assays.
Phosphorylation of purified T. flavus glycerol kinase with crude extracts prepared from T. flavus cells was carried out with [32P]PEP, which was prepared from [-32P]ATP via the pyruvate kinase exchange reaction at equilibrium (Roossien et al., 1983
). T. flavus cells were grown in 150 ml 2x YT medium in the absence of a carbohydrate or in the presence of 1% glycerol, 1% glucose or 1% fructose to OD600 between 0·65 and 0·70. Cells were centrifuged, washed three times with 50 mM Tris/HCl buffer, pH 7·4, resuspended in 2 ml of the same buffer and broken by sonication. Cell debris was removed by centrifugation and the supernatant was dialysed overnight against 50 mM Tris/HCl, pH 7·4, containing 0·1 mM PMSF and 0·1 mM DTT. Phosphorylation of glycerol kinase was carried out in a reaction mixture of 30 µl containing 4 µg T. flavus glycerol kinase, 10 mM MgCl2, 10 µM [32P]PEP (0·1 µCi; 3·7 kBq), 50 mM Tris/HCl buffer, pH 7·4, and 6 µl T. flavus crude extracts. The assay mixture was incubated for 20 min at 37 °C. The phosphorylation reaction was stopped by adding an equal volume of sample buffer (Laemmli, 1970
) to the assay mixtures. Phosphorylation assays with T. flavus glycerol kinase and purified B. subtilis enzyme I(His)6 and HPr(His)6 were also carried out in a total volume of 30 µl and incubated for 20 min at 37 °C. They contained 4 µg T. flavus glycerol kinase, 0·5 µg B. subtilis enzyme I(His)6, 0·5 µg B. subtilis HPr(His)6, 10 mM MgCl2, 10 mM PEP or 10 µM [32P]PEP (0·1 µCi; 3·7 kBq) and 50 mM Tris/HCl buffer, pH 7·4. Proteins were separated by PAGE performed either under denaturing (1% SDS) or non-denaturing conditions. Gels were dried and exposed to x-ray film (Biomax MR, Kodak) for experiments carried out with [32P]PEP or stained with Coomassie brilliant blue R for experiments with non-radioactive PEP.
Enzyme-activity assays.
Glycerol kinase activity was determined by using a coupled spectrophotometric assay carried out in 100 mM glycine/hydrazine buffer, pH 8·8 (Deutscher & Sauerwald, 1986 ). Enzyme activities are expressed as nmol product formed min-1 and (µg protein)-1. To measure the effect of phosphorylation on the enzyme activity, glycerol kinase (6·4 µg) was first incubated for 20 min at 37 °C together with 10 mM PEP, 10 mM MgCl2, 1 µg enzyme I(His)6 and 1 µg HPr(His)6 in a total volume of 60 µl. An aliquot of 7·5 µl of the reaction mixture (corresponding to 0·5 µg glycerol kinase) was used for the glycerol kinase assay. To determine to what extent glycerol kinase used in these enzymic tests was phosphorylated, the proteins present in a 30 µl aliquot of the glycerol kinase phosphorylation mixture were separated on a denaturing 10% polyacrylamide gel.
The presence of enzyme I and HPr in crude extracts of T. flavus has been demonstrated with the Staphylococcus aureus mutant complementation assay by using crude extracts prepared from the mutants S710A (ptsI lacR) and S797A (ptsH lacR) (Hengstenberg et al., 1969 ).
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RESULTS |
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Effect of phosphorylation of T. flavus glycerol kinase on its enzyme activity.
PEP-dependent, enzyme I- and HPr-catalysed phosphorylation of enterococcal glycerol kinases caused a 10-fold increase in enzyme activity (Deutscher, 1985 ; Charrier et al., 1997
). We therefore wanted to test whether phosphorylation of T. flavus glycerol kinase would lead to a similar stimulation with regard to its enzyme activity. T. flavus glycerol kinase was phosphorylated with PEP, enzyme I and HPr as described in Methods, and glycerol kinase assays were carried out using saturating concentrations of glycerol (5 mM) and ATP (5 mM). The Km values of T. flavus glycerol kinase for glycerol and ATP have been reported to be 38 and 162 µM, respectively (Huang et al., 1997
). Under the above conditions, very similar activities were found for unphosphorylated (4·4 units) and phosphorylated glycerol kinase (5·6 units). In contrast to enterococcal glycerol kinases, phosphorylation of T. flavus glycerol kinase does not seem to increase the Vmax of this enzyme. The observed absence of a stimulatory effect of PEP-dependent phosphorylation of glycerol kinase on its enzyme activity cannot be due to incomplete phosphorylation of T. flavus glycerol kinase under the reaction conditions employed. When an aliquot of the phosphorylated T. flavus glycerol kinase used for the activity assays was separated on SDS polyacrylamide gels, more than 90% of the glycerol kinase was found to be present in the phosphorylated form (data not shown).
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DISCUSSION |
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Most glycerol kinases from Gram-positive bacteria, but not glycerol kinases from Gram-negative bacteria, seem to be regulated by a mechanism that implicates stimulation of glycerol kinase activity by PEP-dependent phosphorylation at a histidyl residue catalysed by enzyme I and HPr of the PTS (Deutscher & Sauerwald, 1986 ; Charrier et al., 1997
). As a consequence, ptsH or ptsI mutants of Gram-positive bacteria, in which glycerol kinase cannot be activated by PEP-dependent phosphorylation, were unable to grow on glycerol as the sole carbon source (Reizer et al., 1984
; Romano et al., 1990
; Gonzy-Treboul et al., 1991
; Beijer & Rutberg, 1992
). Interestingly, the phosphorylatable histidyl residue was also found to be present in glycerol kinase from T. flavus. The sequences flanking this histidyl residue are well-conserved when compared to the corresponding sequences of glycerol kinase from B. subtilis (Fig. 2
) or other Gram-positive bacteria. In the presence of [32P]PEP, purified T. flavus glycerol kinase was found to be phosphorylated by crude extracts of T. flavus cells (Fig. 3a
), suggesting that this organism contains the general PTS proteins enzyme I and HPr. This assumption was confirmed by the observation that crude extracts prepared from T. flavus cells were capable of reconstituting the lactose-specific PTS activity in mutant complementation assays with S. aureus ptsI and ptsH mutants (Hengstenberg et al., 1969
). Deinococcus radiodurans, a close relative of T. flavus, was found to possess a multiphosphoryl transfer protein (MTP) similar to Rhodobacter capsulatus (Wu et al., 1990
) and Xanthomonas campestris (de Crécy-Lagard et al., 1995
). The MTP is part of the fructose-specific PTS and contains an enzyme IIAFru domain followed by an HPr and an enzyme I domain. The sequence around the phosphorylatable histidyl residue in the HPr domain of D. radiodurans was found to exhibit strong similarity to the corresponding sequence in B. subtilis HPr (data not shown). However, the experiments presented in Fig. 3a
suggest that T. flavus does not possess an MTP, but that enzyme I and HPr of this organism are probably distinct proteins, as radioactive bands migrating to the approximate positions of enzyme I and HPr from B. subtilis were detected when crude extracts of T. flavus were incubated with [32P]PEP and separated by SDS-PAGE. Even in fructose-grown cells (Fig. 3a
, lanes 8 and 9), no labelled band migrating to the position corresponding to the molecular mass of the MTP (90 kDa) was observed. However, similar to the HPr domain of D. radiodurans MTP, HPr of T. flavus probably possesses an active centre strongly resembling the active centre of HPr in Gram-positive bacteria, which would explain why HPr present in crude extracts prepared from T. flavus cells is functional in the S. aureus mutant-complementation assay with the ptsH mutant S797A and why P-His-HPr from both B. subtilis and T. flavus can efficiently phosphorylate T. flavus glycerol kinase.
Although phosphorylation of glycerol kinase changes the electrophoretic mobility of T. flavus glycerol kinase in a similar way to that which has been observed for glycerol kinase from Ent. faecalis (Deutscher & Sauerwald, 1986 ; Charrier et al., 1997
), only a very weak stimulation of glycerol kinase activity was found to accompany phosphorylation of T. flavus glycerol kinase. The physiological function of the PEP-dependent PTS-catalysed phosphorylation of T. flavus glycerol kinase therefore remains unknown. It is interesting to note that glycerol kinase from another thermophilic organism, Bacillus stearothermophilus, exhibited a very similar behaviour to T. flavus glycerol kinase. It was also phosphorylated by PEP, enzyme I and HPr, and phosphorylation caused changes in electrophoretic mobility identical to those found with T. flavus glycerol kinase (Reizer & Peterkofsky, 1987
). In addition, no increase in enzyme activity was observed when B. stearothermophilus glycerol kinase became phosphorylated. It is possible that phosphorylation of glycerol kinase in the two thermophilic organisms T. flavus and B. stearothermophilus causes an increase in enzyme activity only at higher temperatures. Since no thermostable glycerol-3-phosphate dehydrogenase was available, the enzyme assays reported in this paper were carried out at 37 °C and not at the preferred temperature of T. flavus, i.e. 60 °C. It is interesting to note that when phosphorylated glycerol kinase from T. flavus was kept at 60 °C in Tris/HCl buffer, pH 7·4, about 70% of the protein was dephosphorylated within 10 min, as was observed with other proteins phosphorylated at the N-3 position of a histidyl residue (Waygood et al., 1986
). However, the remaining 30% of the protein was found to remain phosphorylated even after 1 h incubation at 60 °C (data not shown). It is possible that under physiological conditions, most of the T. flavus glycerol kinase is converted into this phosphorylated, heat-resistant form, which might exhibit elevated enzyme activity.
After this paper was accepted, we noticed when carrying out BLAST searches with the amino acid sequence of T. flavus glycerol kinase in microbial databases that D. radiodurans, a close relative of T. flavus, contains two glycerol kinases: one without the conserved phosphorylatable histidyl residue of Gram-positive bacteria, rather resembling Esc. coli glycerol kinase (58% sequence identity), the other possessing an equivalent of His-232 and exhibiting an unexpectedly high sequence identity of 88% to glycerol kinase from Streptococcus pyogenes. D. radiodurans possesses an MTP, which is composed of a fructose-specific EIIA, an HPr and an enzyme I domain (Wu et al., 1990 ). It is therefore likely that the His-232-containing glycerol kinase of this organism is also phosphorylated in vivo with PEP.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 June 1999;
revised 19 July 1999;
accepted 23 July 1999.
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