Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5,D-91058 Erlangen, Germany1
Author for correspondence: Tel: +49 9131 8528818. Fax: +49 9131 8528082. e-mail: jstuelke{at}biologie.uni-erlangen.de
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ABSTRACT |
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Keywords: phosphorylation, Walker A box, mutagenesis, catabolite repression
Abbreviations: PTS, phosphoenolpyruvate:sugar phosphotransferase system; HPr, histidine-containing phosphocarrier protein of the PTS; HPrK/P, HPr kinase/phosphatase; FBP, fructose 1,6-bisphosphate
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INTRODUCTION |
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We are interested in the control of carbon catabolism in the human pathogen Mycoplasma pneumoniae. This bacterium contains a small genome of about 816 kb (Himmelreich et al., 1996 ). The metabolic capacities of M. pneumoniae are rather limited. There are enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) required for the transport of glucose, fructose and mannitol and all the enzymes of the glycolytic pathway. Glycerol and glycerol 3-phosphate may be transported by the glycerol facilitator and an ABC-type transporter, respectively, and are further catabolized by glycolysis. In contrast, the enzyme complement for the pentose-phosphate pathway is incomplete. Since M. pneumoniae lacks enzymes for the tricarboxylic acid cycle, quinones and cytochromes, ATP generation is restricted to substrate-level phosphorylation (Razin et al., 1998
; Himmelreich et al., 1996
; Miles, 1992
). In addition to carbohydrates, M. pneumoniae can probably catabolize arginine, yielding ammonia and ATP (Himmelreich et al., 1996
).
Knowledge of the regulation of carbon catabolism in M. pneumoniae and other mollicutes is very limited. The complete genome of M. pneumoniae encodes only a few regulatory proteins. Two-component systems or alternative sigma factors commonly found in other bacteria are absent (Himmelreich et al., 1996 ; Himmelreich et al., 1997
). M. pneumoniae and Mycoplasma genitalium encode an HPr kinase/phosphatase (HPrK/P), the key regulatory enzyme of carbon catabolism in low-GC Gram-positive bacteria (Himmelreich et al., 1996
; Fraser et al., 1995
). Moreover, HPrK/P activity is also present in mollicutes such as Mycoplasma capricolum and Acholeplasma laidlawii (Hoischen et al., 1993
; Zhu et al., 1997
). In Bacillus subtilis, HPrK/P senses the metabolic state of the cell and reversibly phosphorylates HPr of the PTS and an HPr homologue, Crh, at seryl residues (Galinier et al., 1997
, 1998
; Reizer et al., 1998
). HPr-Ser-P and Crh-Ser-P serve as cofactors for the transcriptional regulator, CcpA. HPr-Ser-P is a poor substrate for phosphorylation by Enzyme I of the PTS (Galinier et al., 1997
; Reizer et al., 1998
; Deutscher et al., 1995
). The regulatory consequences of HPr phosphorylation by HPrK/P have been reviewed in detail (Stülke & Hillen, 1999
). The presence of an hprK gene in M. pneumoniae and other mollicutes suggests that carbon metabolism in these bacteria may be governed by this master regulator. The assumed involvement of HPrK/P in regulation of carbon and energy metabolism is reinforced by the accumulation of fructose 1,6-bisphosphate (FBP), a trigger of HPr kinase activity in B. subtilis, in glycolytically active cells of Mycoplasma gallisepticum (Reizer et al., 1998
; Egan et al., 1986
; Mason et al., 1981
).
We have studied the activity of HPrK/P isolated from M. pneumoniae. Our results demonstrate that the protein has both kinase and phosphatase activities. In contrast to HPrK/P of B. subtilis which is active as a phosphatase in the absence of any metabolic intermediates, the M. pneumoniae enzyme exhibits kinase activity. The activity of the protein in vivo, and thus the phosphorylation state of HPr, is adjusted by the ratio of ATP, FBP and Pi. Mutagenesis experiments indicate that a conserved Walker A box nucleotide-binding motif and the conserved HPr kinase signature sequence (Reizer et al., 1998 ) are required for both activities. Several mutations in the nucleotide-binding motif of HPrK/P completely eliminate phosphatase activity without affecting the kinase activity.
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METHODS |
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DNA manipulation and plasmid constructions.
Transformation of E. coli and plasmid DNA extraction were performed using standard procedures (Sambrook et al., 1989 ). Restriction enzymes, T4 DNA ligase and DNA polymerase were used as recommended by the manufacturers. DNA fragments were purified from agarose gels using a Nucleotrap Gel Extraction kit (Macherey & Nagel). DNA sequences were determined using the dideoxy chain-termination method (Sambrook et al., 1989
).
To overexpress the wild-type HPrK/P protein as well as the HPr protein fused to a hexahistidine sequence at the N terminus, plasmids were constructed as follows. DNA fragments corresponding to the hprK and ptsH ORFs, respectively, were amplified by PCR using the cosmids G07 and D09 bearing the M. pneumoniae hprK and ptsH sequences (Himmelreich et al., 1996 ). The primer pair used for amplification of the hprK gene was KS9 (5'-AAAGTCGACATGAAAAAGTTATTAGTCAAGGAG-3') and KS10 (5'-ATTAAGCTTGGTCTGCTACTAACACTAGGATTCAT CTTTTTTACG-3'). The primers used for amplification of the ptsH gene were KS 34 (5'-AAAGTCGACATGAAGAAGATTCAAGTAGTCGTTAAAGAC-3') and KS 35 (5'-AAAAAGCTTTTAAATAACTTGGTGTTTTTCTAAAACTGC-3'). The PCR products were digested with SalI and HindIII, and the resulting fragments were cloned into the expression vector pWH844 (Schirmer et al., 1997
) cut with the same enzymes. The resulting plasmids were pGP204 (for hprK) and pGP217 (for ptsH).
Site-directed mutagenesis of the M. pneumoniae hprK gene was performed by a two-step PCR approach as described previously (Hanson et al., 2002 ). The mutant alleles were cloned into pWH844 as described for the wild-type.
Protein purification.
E. coli DH5 was used as host for the overexpression of recombinant proteins. Expression was induced by the addition of IPTG (final concentration 1 mM) to exponentially growing cultures (OD600 0·8). Cells were lysed using sonication (8x30 s, 4 °C, 50 W) or a French press cell (2000 p.s.i.=13·8 MPa). After lysis the crude extracts were centrifuged at 15000 g for 30 min. For purification of the HPrK/P proteins the resulting supernatants were passed over a Ni2+ HiTrap chelating column (Pharmacia) followed by elution with an imidazole gradient (in a buffer containing 10 mM Tris/HCl, pH 7·5, 200 mM NaCl).
For the recombinant HPr protein the overproduced protein was detected in the pellet fraction of the lysate. Therefore, after centrifugation as described above, the supernatant was discarded and the pellet was resuspended using 6 M urea. After an additional centrifugation of the resuspended pellet at 15000 g for 30 min, the resulting supernatant containing the solubilized HPr protein was passed over the Ni2+ HiTrap chelating column (Pharmacia). The protein was renatured while attached to the column by using an extended wash-out, followed by elution via an imidazole gradient. Renaturation of the HPr was assayed by using it as a substrate for in vitro phosphorylation by Enzyme I and HPrK/P. Complete phosphorylation was taken as an indication that the renatured protein was present in a native form.
After elution the fractions were tested for the desired protein using 12·5% SDS-PAGE gels for HPrK/P and 10% Tris/Tricine gels (Schägger & von Jagow, 1987 ) for HPr. The relevant fractions were combined and dialysed overnight.
Purified proteins were concentrated using Microsep Microconcentrators with a molecular mass cut-off of 3 and 10 kDa for HPr and HPrK/P, respectively (Pall Filtron). Protein concentration was determined according to the method of Bradford (1976) using the Bio-Rad dye-binding assay where bovine serum albumin served as the standard.
(His6)HPr and (His6)HPrK/P of B. subtilis were purified as described previously (Hanson et al., 2002 ).
Activity assays of HPrK/P.
Activity assays were carried out with purified HPrK/P in assay buffer (10 mM MgCl2, 25 mM Tris/HCl, pH 7·6, 1 mM dithiothreitol) using purified (His6)HPr or (His6)HPr-Ser-P. ATP, potassium phosphate and FBP were added as indicated. The assays were carried out at 37 °C for 15 min followed by thermal inactivation of the enzyme (4 min at 95 °C). The assay mixtures were analysed on 10% native polyacrylamide gels as described previously (Hanson et al., 2002 ). Proteins were visualized by Coomassie staining.
For the radioactive assay, HPrK/P activity was determined in a mixture containing 10 mM MgCl2, 25 mM Tris/HCl, pH 7·6, 1 mM dithiothreitol, 0·1 mM [32-P]ATP (0·8 µCi nmol-1), 20 mM FBP and purified (His6)HPr from M. pneumoniae or HPr from Bacillus megaterium in a volume of 20 µl. Incubation was performed for 15 min at 37 °C. The reaction was stopped by adding 5 µl SDS quenching buffer (Reizer et al., 1998
) followed by boiling for 3 min. Proteins were separated by SDS-PAGE on a 15% gel. HPr-Ser-32P was analysed by autoradiography using the TINA software of a Phosphoimager (Fujifilm BAS 1500).
(His6)HPr-Ser-P of M. pneumoniae was prepared as follows. After incubation of purified (His6)HPr with HPr kinase in assay buffer in the presence of 10 mM ATP and 20 mM FBP the enzyme was thermally inactivated. The reaction led to complete phosphorylation of (His6)HPr which was confirmed on a 10% native gel. To separate the phosphorylated protein from low-molecular-mass effector molecules used in the phosphorylation reaction, the assay mixture was passed over a HiTrap Desalting column (Sephadex G25, Pharmacia). (His6)HPr-Ser-P of B. subtilis was prepared as described previously (Hanson et al., 2002 ).
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RESULTS |
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His6-HPr was phosphorylated by Enzyme I and HPrK/P in a phosphoenolpyruvate or ATP-dependent manner, respectively (data not shown). His6-HPr was also recognized by polyclonal antibodies raised against HPr from B. megaterium (data not shown). The purified His6-HPrK/P in vitro phosphorylated HPr from B. megaterium and M. pneumoniae; however, the efficiency of phosphorylation was about 20-fold higher for the cognate HPr (data not shown).
Regulation of kinase activity of M. pneumoniae HPrK/P
The effect of increasing concentrations of ATP on kinase activity of M. pneumoniae HPrK/P was tested. All HPr in the reaction was phosphorylated at ATP concentrations of 25 µM and above. An ATP concentration as low as 1 µM did yield kinase activity (note that ATP was the limiting component in this reaction) (Fig. 1a).
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Both activities of HPrK/P may be controlled by the concentration of Pi, as shown for the enzyme from Enterococcus faecalis (Kravanja et al., 1999 ). The addition of 1 mM Pi to the M. pneumoniae kinase reaction in the presence of 50 µM ATP resulted in partial inhibition of kinase activity (data not shown). At 5 mM Pi, kinase activity was completely inhibited (Fig. 1b
). We investigated whether FBP as a major glycolytic intermediate might counteract the inhibitory effect of Pi. Indeed, FBP prevented kinase inhibition by Pi (see Fig. 1b
).
Regulation of phosphatase activity of M. pneumoniae HPrK/P
Phosphatase activity of His6-HPrK/P from M. pneumoniae was analysed using His6-HPr-Ser-P from M. pneumoniae as a substrate. Significant phosphatase activity was detected only if the Pi concentration in the reaction mixture exceeded 1 mM (Fig. 2). The observed antagonistic effects between FBP and Pi regarding kinase activity of HPrK/P from other bacteria prompted us to investigate the influence of FBP on phosphatase activity as well. In the presence of 5 mM Pi, no effect of FBP on phosphatase activity was observed (data not shown). However, if ATP (50 µM) was included in the assay mix, FBP shifted the activity towards the kinase reaction.
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In addition to the three conserved glycine residues in the Walker A box motif, another universally conserved glycine was analysed by site-directed mutagenesis. This mutation, G140A, had no effect on kinase activity; however, the phosphatase activity was observed even at low concentrations of Pi (1 mM).
Two mutations of the signature sequence were introduced and their effects studied. An R204K mutation affected only phosphatase activity, which was strongly reduced as compared to the wild-type protein. The G207A mutation completely abolished all activities of HPrK/P.
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DISCUSSION |
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HPrK/P from B. subtilis is active as a phosphatase in the absence of any metabolites if Pi is present. Kinase activity occurs only if the concentration of ATP is high (>0·2 mM) and glycolytic intermediates such as FBP are present (Jault et al., 2000 ). Similarly, the enzymes from other low-GC Gram-positive bacteria are active as a kinase only at high ATP concentrations that indicate a good supply of nutrients (Kravanja et al., 1999
; Brochu & Vadeboncoeur, 1999
; Dossonnet et al., 2000
; Huynh et al., 2000
). In contrast, M. pneumoniae HPrK/P exhibits an inverse mode of regulation: this enzyme needs Pi for phosphatase activity, whereas the kinase activity occurs at ATP concentrations as low as 1 µM. Thus, this protein is by default a kinase rather than a phosphatase. M. pneumoniae HPrK/P phosphorylates HPr at Ser-46 as the standard reaction (see Figs 3
and 4
). This may result in permanent carbon catabolite repression of genes required for the utilization of secondary carbon sources, and in controlled sugar uptake. This correlates well with the nutrient-rich environment of M. pneumoniae, in which there is no limitation of preferred sources of carbon and energy. On the other hand, the standard phosphatase activity of the B. subtilis HPrK/P results in absence of catabolite repression as a standard regulatory mechanism in poor environments.
In the course of this study it turned out that M. pneumoniae HPrK/P phosphorylated the cognate HPr much more efficiently than its counterpart from B. megaterium. This finding is in good agreement with the identification of specificity determinants required for the interaction between HPrK/P and HPr of M. capricolum. Residues 48, 49 and 5153 of HPr are important for kinase-HPr recognition (Zhu et al., 1998 ). While the latter three residues are shared by the HPr sequences of M. pneumoniae and B. megaterium (Himmelreich et al., 1996
; Wagner et al., 2000
), the former are not conserved and may be responsible for the weak phosphorylation of B. megaterium HPr by M. pneumoniae HPrK/P.
There are two strongly conserved sequence motifs in HPrK/P: the potential ATP-binding site and the signature sequence. A mutational analysis of these sequences revealed that both motifs contain residues that are indispensable for the enzymic function of HPrK/P. The recent elucidation of the three-dimensional structure of L. casei HPrK/P indicated that both motifs are located in close proximity (Fieulaine et al., 2001 ). The GKSE cluster in the ATP-binding site is most important for both kinase and phosphatase activities. Even conservative substitutions (G159A, K160R) result in complete inactivation. Similarly, a replacement at the corresponding position in B. subtilis and L. casei HPrK/P (G158A and G160S, respectively) results in a strong decrease of kinase and abolition of phosphatase activity (Monedero et al., 2001
; Hanson et al., 2002
). The mutational separation of the enzymic activities is also observed with M. pneumoniae HPrK/P. The E162D substitution results in complete loss of phosphatase activity without affecting kinase activity. A corresponding mutation of L. casei HPrK/P may have similar consequences, since the mutant strain exhibits constitutive catabolite repression indicative of constitutive phosphorylation of HPr and loss of dephosphorylation (Monedero et al., 2001
). In HPrK/P of L. casei, amino acids of the KSE motif interact with Pi (Fieulaine et al., 2001
). Moreover, the phosphatase reaction was shown to be enzymically distinct from the kinase reaction for HPrK/P of L. casei (Monedero et al., 2001
). Thus, the specific effect of mutations in this region on phosphatase activity may result from loss of Pi binding. In contrast, the G140A and S156A mutant proteins exhibit normal kinase activity, but their phosphatase activity is strongly enhanced as compared to the wild-type protein. The fully conserved G207 residue of the signature sequence is essential for HPrK/P function in M. pneumoniae. The corresponding mutant of the B. subtilis enzyme exhibits very weak kinase and no phosphatase activity (Hanson et al., 2002
). Of the mutations studied so far, no substitutions that reduce kinase activity without affecting phosphatase activity have been identified, whereas mutations abolishing phosphatase activity but allowing kinase activity have been found in M. pneumoniae, B. subtilis and L. casei (Monedero et al., 2001
; Hanson et al., 2002
). Interestingly, a mutational analysis of another bifunctional protein kinase/phosphatase from E. coli, isocitrate dehydrogenase kinase/phosphatase (IDH K/P), yielded results similar to those reported here: mutations of the ATP-binding site inactivate both kinase and phosphatase activities (Stueland et al., 1989
). In addition, mutations selectively abolishing phosphatase activity were found (Ikeda et al., 1992
).
To the best of our knowledge, HPrK/P is the first bifunctional enzyme for which opposing basal activities have been found in orthologues from different organisms. It will be interesting to analyse the structural basis of this phenomenon. The protein has been crystallized (Steinhauer et al., 2002 ) and analysis of the structure is under way.
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ACKNOWLEDGEMENTS |
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Received 12 March 2002;
revised 27 June 2002;
accepted 5 July 2002.