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Originally published In Press as doi:10.1074/jbc.M305134200 on July 7, 2003

J. Biol. Chem., Vol. 278, Issue 41, 39323-39329, October 10, 2003
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Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase*

Christophe Grangeasse {ddagger} §, Brice Obadia {ddagger}, Ivan Mijakovic ¶, Josef Deutscher ¶, Alain J. Cozzone {ddagger} and Patricia Doublet {ddagger}

From the {ddagger}Institut de Biologie et Chimie des Protéines, CNRS, Université de Lyon, 69367 Lyon Cedex 07, France and Microbiologie et Génétique Moléculaire, CNRS, Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grinon, 78850 Thiverval-Grignon, France

Received for publication, May 15, 2003 , and in revised form, June 20, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein-tyrosine phosphorylation occurs in all living organisms and plays a central role in eukaryotes by regulating a wide variety of cellular processes (1–3). However, little is known about the physiological role of protein-tyrosine phosphorylation in bacteria. The first autophosphorylating bacterial protein-tyrosine kinases (PTKs)1 were detected in Acinetobacter (4, 5). Since then, autophosphorylating enzymes exhibiting significant similarity to these PTKs have been detected in a large number of Gram-negative and Gram-positive bacteria, and the recent progress in genome sequencing has revealed that this type of protein-modifying enzyme is, in fact, present in most bacteria (6–12). In addition, bacteria most often contain simultaneously phosphotyrosine-protein phosphatases (PTPs), which antagonize the activity of PTKs (13–16). The genes encoding PTPs are usually located immediately upstream of the genes encoding PTKs (17, 18). Moreover, the genes encoding both PTKs and PTPs are generally organized together with genes coding for enzymes involved in the biosynthesis or transport of capsular polysaccharides (cps) or exopolysaccharides (5, 6).

Such colocalization of the cps genes and the genes encoding the protein-tyrosine phosphorylating and dephosphorylating enzymes has suggested that reversible protein-tyrosine phosphorylation might regulate the biosynthesis of exopolysaccharides. Actually, PTKs and PTPs have been shown to play an important role in the production and/or processing of capsular polysaccharides and exopolysaccharides (19, 20). For example, in Escherichia coli the kinase Wzc and the phosphatase Wzb have been reported to participate in the biosynthesis of the exopolysaccharide colanic acid (21). Thus, when Wzc is phosphorylated, no colanic acid is synthesized by bacteria, whereas when Wzc is dephosphorylated by Wzb, colanic acid is produced. The same observation has been made in Streptococcus pneumoniae where autophosphorylation of the kinase CpsD decreases its activity and, consequently, lowers the production of capsular polysaccharides (12).

Although PTKs from Gram-negative and Gram-positive bacteria exhibit significant sequence similarity, they possess different domain organizations (Fig. 1). PTKs of Gram-negative bacteria are usually large proteins (~80 kDa) composed of an N-terminal transmembrane domain and a C-terminal cytosolic PTK domain (22) containing the active site Walker motifs A and B (23). When expressed separately, the soluble C-terminal domain of E. coli Wzc still exhibits autophosphorylating activity (24). By contrast, PTKs of Gram-positive bacteria are naturally separated into two distinct proteins, i.e. a transmembrane protein with limited similarity to the N-terminal domain and a soluble protein with significant similarity to the C-terminal domain of PTKs from Gram-negative bacteria. The soluble protein autophosphorylates at a tyrosine cluster located in its C terminus and also contains the Walker motifs A and B (11, 25, 26). The different domain organizations in PTKs from Gram-negative and Gram-positive bacteria have suggested that these enzymes might be regulated differently. Indeed, although intermolecular autophosphorylation of Wzc at the C-terminal tyrosine cluster (five tyrosines from position 708 to position 715) is stimulated by intramolecular autophosphorylation at Tyr569 (24), autophosphorylation of the S. pneumoniae kinase CpsD only occurs on the tyrosine cluster when the membrane protein modulator CpsC is also present (Fig. 1) (11, 12). Several studies have been performed to assess the biological role of phosphorylation of the tyrosine cluster located at the C-terminal end of PTKs. Thus, in E. coli K30 it has been shown that the overall level of phosphorylation in this region, rather than the phosphorylation of any specific residue(s), is important for the activity of Wzc in the capsule assembly (27). In the Gram-positive bacterium S. pneumoniae, the phosphorylation of only one of the different tyrosine residues in the tyrosine cluster has appeared to be sufficient to inactivate the CpsD kinase (25). In any case, whatever the mechanism used, it seems that phosphorylation is required to modulate PTKs activity in the metabolism of capsular polysaccharides.



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  		FIG. 1.
Sequence alignment and structural features of Gram-positive and Gram-negative PTKs. The PTK cytoplasmic domain of Wzc from E. coli K12 is presented in the middle section. The Walker A and B ATP-binding motifs are indicated as light gray shaded boxes. The Lys540 residue of Walker A, the intraphosphorylated Tyr569 residue, and the interphosphorylated tyrosine of the tyrosine cluster (Tyr708, Tyr710, Tyr711, Tyr713, and Tyr715) are indicated with one-letter code (Y708, Y710, Y711, Y713, and Y715, respectively). A structural comparison of Gram-negative (bottom section) and Gram-positive (top section) PTKs is presented. The two predicted transmembrane {alpha}-helices of both the Gram-negative PTKs and the Gram-positive transmembrane modulator, termed TM1 and TM2, are indicated by dark shaded boxes. The C-terminal cytosolic PTK domain is enlarged, and sequence alignment of PTKs from Gram-negative bacteria (E. coli K30 Wzc, E. coli K12 Wzc, Erwinia amylovora AmsA, Klebsiella pneumoniae Yco6, E. coli E2348/69 Etk, Acinetobacter johnsonii Ptk, and Rastonia solanacearum EpsB) and Gram-positive bacteria (S. pneumoniae Cps19fD, S. aureus Cap5B, and B. subtilis YwqD) was performed by using the CLUSTAL W program (31). Dashes indicate gaps introduced in the alignment process. Tyrosine residues of the tyrosine cluster and the Walker A and Walker B ATP-binding motifs are in gray boxes. Catalytic lysine of the Walker A motif is boldfaced. Tyr-569 is in a black box. GenBankTM accession numbers are AF104912 [GenBank] , U38473 [GenBank] , X77921 [GenBank] , D21242 [GenBank] , P38134 [GenBank] , Y15162 [GenBank] , U17898 [GenBank] , U09239 [GenBank] , U81973 [GenBank] and Z92952 [GenBank] , respectively.

 

UDP-glucose dehydrogenase is one of the enzymes catalyzing the formation of precursors for the synthesis of polysaccharides (19). In E. coli K12, the synthesis of colanic acid decreases when wzc or wzb is inactivated. From this observation, it seemed of interest to investigate whether the E. coli UDP-glucose dehydrogenase Ugd could be subject to protein-tyrosine phosphorylation.

In this work, we examined whether Ugd was modified by the PTK Wzc and if such a modification was able to modulate its UDP-glucose dehydrogenase activity. In addition, we analyzed the Wzb-catalyzed dephosphorylation of both Wzc and Ugd. Finally, in search of the mode of functioning of PTKs, we analyzed comparatively the phosphorylation mechanism of these enzymes in Gram-positive and Gram-negative bacteria.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, and Growth Conditions—Genomic DNA prepared from E. coli strain XL1-B K12 (28) was used as template for the PCR amplification of the ugd gene. This strain also served for the propagation of plasmids in cloning experiments and the overproduction of proteins. All strains were grown at 37 °C in Luria-Bertani (LB) or 2TY medium (16 g of Bacto-tryptone, 5 g of NaCl, and 10 g of Bacto-yeast extract per liter of water). When appropriate, media were supplemented with the antibiotics ampicillin (50 µg/ml) and tetracycline (15 µg/ml) at the concentrations given in parentheses. The plasmids used in this study are listed in Table I.


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  		TABLE I
Plasmids used in this study

 

DNA Manipulations—Plasmids were purified by using the Qiaprep purification kit (Qiagen). All restriction and DNA-modifying enzymes were used as recommended by the manufacturer (Promega). PCR amplifications were performed with Pfu polymerase (Promega). PCR products and plasmid-derived DNA fragments were purified using the QiaexII kit (Qiagen). The primers used in this study were synthesized by Sigma-Genosys Ltd. Transformation of E. coli cells was performed by following the method of Dagert and Ehrlich (29). DNA sequencing was carried out by Genome-Express Corp. to ensure error-free amplification. BLAST searches (30) and sequence alignments (31) were performed by using our server (NPS@) accessible via the World Wide Web (www.ibcp.fr).

Construction of a Vector for the Overproduction of His6-tagged Ugd— For ugd cloning, the sequences of the two primers were 5'-TATGGATCCAAAATCACCATTTCCGGTACTGGC-3' at the N terminus (the BamH1 site is italicized; the second codon of ugd is underlined) and 5'-TATAAGCTTTTAGTCGCTGCCAAAGAGATCG-3' at the C terminus (the HindIII site is italicized; the stop codon of ugd is underlined). The amplified DNA fragment (1164 bp) was restricted with BamHI and HindIII and ligated into pQE30 vector, which was opened with the same enzymes. The resulting plasmid was termed pQE30-U (Table I).

Overproduction and Purification of His6-tagged Proteins—Cells of E. coli strain XL1-B were transformed with pQE30-derived vectors expressing Ugd or the cytoplasmic PTK domain of Wzc (with or without the C-terminal tyrosine cluster) (Table I). 100 ml of 2TY medium containing ampicillin and tetracycline was inoculated with an overnight cell culture and incubated at 37 °C under shaking until it reached an A600 of 0.5. Isopropyl-1-thio-{beta}-D-galactopyranoside (IPTG) was subsequently added at a final concentration of 0.5 mM, and growth was continued for 3 h at 37 °C under shaking. Cells were harvested by centrifugation at 3,000 x g for 10 min, resuspended in buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 10 mM {beta}-mercaptoethanol), and centrifuged again. The resulting cell pellet was resuspended in buffer A containing 5 µg/ml DNase I and RNase A. Cells were disrupted in a French pressure cell at 16,000 p.s.i. The resulting cell extract was centrifuged at 4 °C for 30 min at 30,000 x g. The supernatant was added to Ni2+-NTA-agarose suspension, and batch binding was allowed to proceed for 1 h at 4 °C under gentle shaking. The cell lysate/Ni2+-NTA-agarose mixture was filled in a column that was washed with buffer A and subsequently with buffer A containing 20 mM imidazole until the absorption at A280 remained stable. Elution of His6-tagged proteins was carried out with buffer B (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 200 mM imidazole, 10% glycerol, and 10 mM {beta}-mercaptoethanol). Aliquots of the collected fractions were analyzed by SDS-PAGE, and those containing purified His6-tagged Ugd were pooled, applied to a HiTrap desalting column (Amersham Biosciences) and stored at –20 °C in 50 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM DTT. The purification procedures for His6-tagged mutant PTK domains of Wzc, His6-Wzb, His6-YwqD, GST-YwqC and GST-YwqC-NCter have been described elsewhere (6, 24, 26).

Phosphorylation Assay—An in vitro phosphorylation assay was carried out by incubating the reaction mixtures (20 µl) containing ~2 µg of the various purified wild-type or mutant proteins and 25 mM Tris-HCl, pH 7.0, 1 mM DTT, 5 mM MgCl2, 1 mM EDTA, and 10 µM [{gamma}-32P]ATP (4 µCi) for 10 min at 37 °C. The reaction was stopped by either adding an equal volume of 2x sample buffer and heating for 5 min at 100 °C or by precipitating the P-proteins with 5 volumes of acetone. One-dimensional gel electrophoresis was performed as described by Laemmli (32), whereas two-dimensional gel electrophoresis was carried out as described by O'Farrell (33). After electrophoresis, gels were soaked in 16% trichloroacetic acid for 10 min at 90 °C. They were stained with Coomassie Blue, and radioactive proteins were visualized by autoradiography using x-ray films (Kodak BIOMAX-MR).

Protein Dephosphorylation Assay—Purified wild-type or mutant His6-tagged proteins were phosphorylated in the presence of [{gamma}-32P]ATP as described above and subsequently dialyzed overnight against a buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1 mM DTT. The dephosphorylation reaction was carried out by adding 1 µg of purified Wzb to the P-proteins and diluting the assay mixtures 2-fold with a buffer containing 100 mM sodium citrate, pH 6.5, 1 mM EDTA, and 0.1% {beta}-mercaptoethanol. The reaction mixture was incubated at 37 °C and, at various time intervals (from 1 to 30 min) aliquots were withdrawn and analyzed by gel electrophoresis and autoradiography.

Phosphotransferase Assay—The purified PTK domain of Wzc (His6-Wzc-(Ser447–Ala704)) was autophosphorylated with [{gamma}-32P]ATP before it was repurified on a Ni2+-NTA-agarose column as described above. The capacity of the 32P-labeled cytoplasmic His6-Wzc domain to transfer its phosphoryl group bound to Tyr569 to Ugd was tested by incubating the two proteins in phosphorylation buffer in the absence or presence of 50 µM non-radioactive ATP.

UDP-Glucose Dehydrogenase Assay—Ugd purified from E. coli was tested for UDP-glucose dehydrogenase activity by using a spectrophotometric assay described by Pagni et al. (34). The absorbance at 340 nm was measured for 10 min at 37 °C by using a thermostated spectrophotometer. UDP-glucose dehydrogenase activity was also determined after phosphorylating or dephosphorylating purified Ugd. For this purpose, 2 µg of Ugd was either phosphorylated with Wzc as described above or dephosphorylated with calf intestinal alkaline phosphatase by incubating Ugd solutions (20 µl) for 10 min at 37 °C before 230 µl of 100 mM Tris-HCl buffer, pH 9.0, containing 100 mM NaCl and 2 mM DTT and 300 µl of the same buffer also containing 10 mM NAD+ and 10 mM UDP-glucose (UDPG) were added.

Phosphoamino Acid Analysis—The method used to detect acid-stable phosphoamino acids was described in detail in (35). Briefly, Ugd was phosphorylated by Wzc in the presence of [{gamma}-32P]ATP. Proteins were subsequently separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane, and labeled proteins were detected by autoradiography. The radioactive band corresponding to 32P-labeled Ugd was excised, and the protein was hydrolyzed in 6 M HCl for 1 h at 110 °C. The acid-stable phosphoamino acids thus liberated were lyophilized and dissolved in water containing Ser(P), Thr(P), and Tyr(P) standards. This mixture was separated by electrophoresis at pH 1.9 (800 V x h) in a buffer containing 7.8% acetic acid and 2.5% formic acid (first dimension) followed by ascending chromatography in 2-methyl-1-propanol/formic acid/water (8:3:4) (v/v/v) (second dimension). After migration, the plates were dried and stained with ninhydrin, and radioactive molecules were detected by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Tyrosine Phosphorylation of Ugd Is Catalyzed by the Cytoplasmic PTK Domain of Wzc—Two recent reports suggested that E. coli Ugd might be subject to phosphorylation at tyrosine. (i) The two UDP-glucose dehydrogenases of another bacterium, Bacillus subtilis, were found to be phosphorylated by the endogenous kinase YwqD (26); and (ii) colanic acid synthesis in E. coli was decreased when disrupting the PTK- and PTP-encoding genes wzc and wzb, respectively (21). Because the E. coli UDP-glucose dehydrogenase Ugd not only exhibits ~30% sequence identity with the B. subtilis enzymes but also catalyzes the formation of UDP-glucuronic acid, a precursor of colanic acid synthesis (19), one could expect that Ugd might be phosphorylated by Wzc.

To check this hypothesis, phosphorylation experiments were carried out with His6-Ugd and His6-Wzc-(Ser447–Ala704), the catalytic PTK domain of Wzc missing the C-terminal tyrosine cluster. When these two purified proteins were incubated together with [{gamma}-32P]ATP, phosphorylation of Ugd was indeed detected after SDS/PAGE. Although no radioactive protein was observed when His6-Ugd was incubated with [{gamma}-32P]ATP (data not shown), two labeled bands appeared when His6-Wzc-(Ser447–Ala704) was also present in the incubation mixture (Fig. 2A). The faster migrating radioactive band with an apparent molecular mass of ~31 kDa corresponded to His6-Wzc-(Ser447–Ala704) autophosphorylated at Tyr569, whereas the slower radioactive band (~44 kDa) migrated to the same position as His6-Ugd. Phosphorylation of Ugd appeared to be dependent on a functional Walker motif A of Wzc. Indeed, when Lys540 in the Wzc Walker A motif (Fig. 1) was replaced with methionine, not only autophosphorylation of Wzc but also phosphorylation of Ugd was abolished (data not shown).



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  		FIG. 2.
Phosphorylation of Ugd protein. His6-Ugd was overproduced, purified on Ni2+-immobilized matrix, and assayed for in vitro phosphorylation in the presence of the Wzc cytoplasmic fragment His6-Wzc-(Ser447–Ala704) and [{gamma}-32P]ATP. Proteins were analyzed by SDS-PAGE (A) and two-dimensional electrophoresis (B), and radioactive bands were revealed by autoradiography. Phosphorylated proteins are indicated by arrows. Coomassie Blue staining of unphosphorylated Ugd is shown as a dashed circle on the two-dimensional electrophoresis autoradiogram. The phosphoamino acid content of Ugd (C) was analyzed, after acid hydrolysis by electrophoresis in the first dimension (1D) and ascending chromatography in the second dimension (2D). After migration, radioactive molecules were revealed by autoradiography. Authentic phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) were run in parallel and visualized by staining with ninhydrin. Radioactive spots on both the left and right sides of the diagram correspond to unhydrolyzed phosphopeptides and inorganic phosphate, respectively (53).

 

Ugd seemed to be monophosphorylated, because a single radioactive spot was detected after His6-Wzc-(Ser447–Ala704)-catalyzed phosphorylation with [{gamma}-32P]ATP and subsequent two-dimensional gel electrophoresis (Fig. 2B). As expected, radiolabeled [32P]Ugd exhibited a slightly lower pI than unmodified Ugd (dotted circle in Fig. 2B). No radioactive spot corresponding to autophosphorylated His6-Wzc-(Ser447–Ala704) was detected on the two-dimensional gel. His6-Wzc-(Ser447–Ala704), which has a theoretical pI of 9.25, probably ran out of the gel. To determine whether phosphorylation of Ugd by Wzc, similarly to autophosphorylation of Wzc, would also occur at a tyrosine residue, total acid hydrolysis of 32P-labeled Ugd previously separated on SDS/polyacrylamide gel was carried out. Two-dimensional electrophoresis/chromatography of the corresponding radioactive hydrolysis products revealed that only radioactive phosphotyrosine was present, whereas no phosphoserine or phosphothreonine could be detected (Fig. 2C).

Autophosphorylation of Wzc at Tyr569 Stimulates Ugd Phosphorylation—As already mentioned, intramolecular autophosphorylation at Tyr569 is known to stimulate the intermolecular phosphorylation of His6-Wzc-(Ser447–Lys720) (24) at its C-terminal tyrosine cluster. Thus, when Wzc is mutated on tyrosine 569 it still exhibits tyrosine kinase activity on the C-terminal tyrosine cluster but to a much lower extent than non-mutated Wzc. To test whether autophosphorylation at Tyr569 would also affect Ugd modification, phosphorylation experiments were carried out with the PTK domain of Wzc in which Tyr569 had been replaced with phenylalanine. The results in Fig. 3A (lanes 1 and 2) showed that the extent of phosphorylation of Ugd by the mutated kinase (His6-Wzc-(Ser447–Ala704)Y569F) was much lower than that observed for the non-mutated molecule (His6-Wzc-(Ser447–Ala704)). These results strongly suggested that autophosphorylation of Wzc at Tyr569 stimulates the phosphorylation of Ugd.



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  		FIG. 3.
Wzc phosphorylates Ugd by using a Tyr569 autophosphorylation activating kinase activity. A, role of Wzc-Tyr569 autophosphorylation in Ugd phosphorylation. Autoradiography of the SDS/polyacrylamide gels on which reaction mixtures containing [{gamma}-32P]ATP and the following proteins were separated: His6-Ugd and His6-Wzc-(Ser447–Ala704) (designated His6-Wzc-(S447-A704) here) (lane 1); and His6-Ugd and His6-Wzc-(Ser447–Ala704)Y569F (lane 2). B, Wzc phosphorylates Ugd by using a kinase activity rather than a phosphotransferase activity. Autoradiography of the SDS/polyacrylamide gels on which phosphorylation reaction mixtures and the following proteins were separated: re-purified 32P-Tyr569-labeled His6-Wzc-(Ser447–Ala704 (lane 1); re-purified 32P-Tyr569-labeled His6-Wzc-(Ser447–Ala704) and [{gamma}-32P]ATP (lane 2); re-purified 32P-Tyr569-labeled His6-Wzc-(Ser447–Ala704), His6-Ugd, and [{gamma}-32P]ATP (lane 3); re-purified 32P-Tyr569-labeled His6-Wzc-(Ser447–Ala704) and His6-Ugd (lane 4); and re-purified 32P-Tyr569-labeled His6-Wzc-(Ser447–Ala704), His6-Ugd, and non-radioactive ATP (lane 5).

 

To exert its stimulating effect on Ugd phosphorylation, autophosphorylation at Tyr569 of Wzc could either enhance its ATP-dependent kinase activity, or the phosphoryl group bound to Tyr569 might itself be transferred to Ugd. The latter reaction could either be ATP-independent or require the presence of ATP. To distinguish between these possibilities, His6-Wzc-(Ser447–Ala704) was first autophosphorylated with [{gamma}-32P]ATP at Tyr569 and subsequently separated from the radioactive nucleotide. The purified, partially modified His6-Wzc-(Ser447–Ala704)-Tyr(P)-569 (Fig. 3B, lane 1) was active, because it could use [{gamma}-32P]ATP to autophosphorylate (Fig. 3B, lane 2) or phosphorylate Ugd (Fig. 3B, lane 3). To detect a possible phosphoryl group transfer from radioactive His6-Wzc-(Ser447–Ala704)-Tyr(P)-569 to His6-Ugd, the two proteins were incubated in the absence of ATP. In these conditions, no phosphorylation of Ugd was detected (Fig. 3B, lane 4). To analyze whether a possible phospho transfer from His6-Wzc-(Ser447–Ala704)-Tyr(P)-569 to Ugd might require ATP binding or hydrolysis, the two proteins were incubated in the presence of unlabeled ATP. Again, no radioactive labeling of Ugd was observed (Fig. 3B, lane 5).

Different Mechanisms Regulate PTKs of Gram-positive and Gram-negative Bacteria—Because the PTK domain of Wzc was capable of phosphorylating the UDP-glucose dehydrogenase YwqF from B. subtilis (26), we tested whether the B. subtilis PTK YwqD could phosphorylate E. coli Ugd. As mentioned above, the amino acid sequences of PTKs of Gram-positive and Gram-negative organisms are similar, but they exhibit a different domain organization (Fig. 1). In addition, the soluble PTKs of Gram-positive bacteria have no counterpart to Tyr569 and need to interact with a membrane protein to autophosphorylate (11, 12). Phosphorylation of B. subtilis UDP-glucose dehydrogenase YwqF is more extensive when YwqD is allowed to interact with YwqC, the membrane protein modulator (26). By contrast, in our experiments the N-terminal transmembrane domain of Wzc exerted no stimulation on the phosphorylation of Ugd by the Wzc cytoplasmic domain (Fig. 4, lanes 1 and 2). B. subtilis PTK YwqD, alone or together with the N-terminal transmembrane domain of Wzc, was also unable to phosphorylate E. coli Ugd (Fig. 4, lanes 5 and 6). However, the presence of the entire YwqC or only its N- and C-terminal parts (first 15 and last 50 amino acids) fused to GST (GST-YwqC and GST-YwqC-NCter, respectively) (26) allowed YwqD to phosphorylate E. coli Ugd (Fig. 4, lanes 8 and 9). These data suggested that Gram-positive and Gram-negative PTKs are activated through a different mechanism to phosphorylate UDP-glucose dehydrogenases.



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  		FIG. 4.
Different mode of action of Gram-positive and Gram-negative protein-tyrosine kinases to promote Ugd phosphorylation. Autoradiography of the SDS/polyacrylamide gels on which reaction mixtures containing [{gamma}-32P]ATP and the following proteins were separated: His6-Ugd and His6-Wzc-(Ser447–Ala704) (designated His6-Wzc(S447-A704) here) (lane 1); His6-Ugd, His6-Wzc-(Thr2-Gly452) and His6-Wzc-(Ser447–Ala704) (lane 2); His6-YwqD (lane 3); His6-Wzc-(Thr2-Gly452) and His6-YwqD (lane 4); His6-Ugd and His6-YwqD (lane 5); His6-Ugd, His6-Wzc-(Thr2-Gly452), and His6-YwqD (lane 6); GST-YwqC and His6-YwqD (lane 7); GST-YwqC, His6-Ugd, and His6-YwqD (lane 8); and GST-YwqC-NCterm, His6-Ugd, and His6-YwqD (lane 9).

 

Phosphorylated Ugd Is a Poor Substrate for the PTP Wzb—Wzb, a well established E. coli PTP (6), is encoded by the gene located immediately upstream of wzc in the cps gene cluster. We have previously shown that Wzb is able to dephosphorylate autophosphorylated Wzc, both in vivo and in vitro (21). However, after incubation with Wzb, a significant amount of Wzc still remains phosphorylated (21). We suspected that such Wzb-resistant phosphorylation might correspond to autophosphorylation at Tyr569. We therefore compared the Wzb-catalyzed dephosphorylation of Wzc previously phosphorylated only at Tyr569 with that of Wzc phosphorylated only at the C-terminal tyrosine cluster. 32P-labeled, phosphorylated His6-Wzc-(Ser447–Ala704) and His6-Wzc-(Ser447–Lys720)Y569F were prepared and incubated with Wzb for various time periods. Although His6-Wzc-(Ser447–Lys720)Y569F, phosphorylated at the tyrosine cluster, was rapidly and extensively dephosphorylated in the presence of Wzb, His6-Wzc-(Ser447–Ala704), phosphorylated exclusively at Tyr-569, was not, or only slowly, dephosphorylated (Fig. 5). We also determined whether Wzb was capable of dephosphorylating phosphorylated Ugd. 32P-labeled phosphorylated His6-Ugd was only slowly dephosphorylated by Wzb, and a detectable level of phosphorylation persisted even after 30 min incubation (Fig. 5, lane labeled 30').



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  		FIG. 5.
Wzc and Ugd dephosphorylation by the phosphotyrosine-protein phosphatase Wzb. Reaction mixtures containing either the Wzc cytoplasmic fragments His6-Wzc-(Ser447–Ala704) (designated His6-Wzc(S447-A704) here) or His6-Wzc-(Ser447–Lys720)Y569F (designated His6-Wzc(S447-K720)Y569F here) or both His6-Ugd and His6-Wzc-(Ser447–Ala704) were incubated with [{gamma}-P]ATP and dialyzed, and then aliquots were incubated for various times (0 to 30 min) with the phosphotyrosine-protein phosphatase Wzb. As control, experiments were carried out in the absence of Wzb. Separation by SDS-PAGE was performed, and Ugd and Wzc dephosphorylation was determined by autoradiography.

 

Phosphorylation of Ugd Enhances Its UDP-Glucose Dehydrogenase Activity—Ugd is an UDP-glucose dehydrogenase catalyzing the NAD+-dependent transformation of UDP-glucose into UDP-glucuronic acid (34). Because Ugd is efficiently phosphorylated by Wzc, we tested whether phosphorylation of Ugd would affect its UDP-glucose dehydrogenase activity. When His6-Ugd was purified from E. coli, it exhibited weak UDP-glucose dehydrogenase activity; but this activity was up to 6-fold higher when Ugd was incubated previously with ATP and His6-Wzc-(Ser447–Ala704) (Fig. 6). By contrast, incubation of His6-Ugd with alkaline phosphatase led to a nearly complete loss of its enzymatic activity. It therefore appeared that phosphorylated Ugd was the active form of the enzyme and that His6-Ugd purified from E. coli contained a small fraction of phosphorylated enzyme responsible for the observed weak UDP-glucose dehydrogenase activity.



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  		FIG. 6.
Activation of the UDP-glucose dehydrogenase activity of Ugd by tyrosine phosphorylation. Ugd UDP-glucose dehydrogenase activity was monitored at 340 nm for 10 min by measuring NADH formation by His6-Ugd ({diamondsuit}), His6-Ugd previously treated by calf intestinal alkaline phosphatase (PhoA) (•), His6-Ugd previously phosphorylated by 0.5 µg of His6-Wzc-(Ser447–Ala704) (designated Wzc(S447-A704) here) ({blacksquare}), or 1 µg of His6-Wzc-(Ser447–Ala704) ({blacktriangleup}).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The finding that the UDP-glucose dehydrogenases of the model organisms for Gram-positive and Gram-negative bacteria, B. subtilis and E. coli, are phosphorylated by PTKs, suggests that UDP-glucose dehydrogenases are frequent substrates for PTKs in bacteria. In B. subtilis, the genes encoding the YwqD kinase, the transmembrane modulator YwqC, and the UDP-glucose dehydrogenase YwqF, respectively, are organized within the same operon (26), whereas in E. coli the wzc gene encoding the kinase and the ugd gene encoding the UDP-glucose dehydrogenase are not located in the same operon, even though they are rather close to each other in the genome. From data base analysis in GenBankTM, a similar close localization of PTK- and UDP-glucose dehydrogenase-encoding genes can be found in several other bacteria, including epsB and epsD in Ralstonia solanacearum, cpsD and cpsK in S. pneumoniae, the Oceanobacillus iheyensis genes OB2884 (ugd) and OB2900 (ptk), and the Novosphingobium aromaticovorans genes ZP 95590.1 (ugd) and ZP 95561.1 (ptk), further supporting the concept that PTK-catalyzed phosphorylation of UDP-glucose dehydrogenases is widely distributed in bacteria. Predicted UDP-glucose dehydrogenases and PTKs are present in many other bacteria including Salmonella typhimurium (Ugd and Wzc), Staphylococcus aureus (CapL and CapB), and Vibrio vulnificus (genes vv10781 and vv10774). However, it is likely that UDP-glucose dehydrogenases are not the only substrates for bacterial PTKs, because some organisms, such as Lactobacillus plantarum or Lactobacillus gasseri, contain one or more PTKs but do not possess proteins with significant similarity to E. coli and B. subtilis UDP-glucose dehydrogenases. It would be interesting to identify the substrates of the PTKs in these organisms.

Although PTKs of Gram-positive and Gram-negative bacteria exhibit significant sequence similarity, their domain organization is quite different. This difference in domain organization reflects their different modes of regulation. Although the soluble PTKs of Gram-positive bacteria need to interact with a transmembrane protein to be active, the PTK domain in Gram-negative bacteria is fused to a transmembrane polypeptide, and stimulation of in vitro intermolecular autophosphorylation at the C-terminal tyrosine cluster occurs via intramolecular autophosphorylation at Tyr569 (Fig. 1) (24). The autophosphorylation state of Wzc seems to regulate the assembly of capsular polysaccharides. However, despite the clearcut effect of Tyr569 phosphorylation observed in vitro, a mutant with the Y569F replacement of E. coli K30 still functions in vivo in capsular polysaccharide assembly (27). Interestingly, autophosphorylation at Tyr569 also regulated the protein-tyrosine kinase activity of E. coli Wzc. Replacing Tyr569 in the PTK domain of Wzc with phenylalanine strongly diminished phosphorylation of Ugd, the first substrate of a PTK identified to date in Gram-negative organisms. We showed that the phosphoryl group bound to Tyr569 is not transferred to Ugd, thus excluding the possibility that Wzc might function as a phosphoryl transferase. Phosphorylation at Tyr569, therefore, rather stimulates the PTK activity of Wzc. Any counterpart of Tyr569 of Wzc is absent from all known PTKs in Gram-positive bacteria, but this residue is conserved in the PTKs of many {gamma}-proteobacteria such as Salmonella, Erwinia, Pseudomonas, Acinetobacter, and Klebsiella, further supporting the concept that regulation of PTKs in these two types of bacteria is controlled by different mechanisms. In fact, regulation of PTKs of {gamma}-proteobacteria resembles that of eukaryotic receptor tyrosine kinases, which are transmembrane proteins with a hydrophilic PTK domain, the activity of which is often stimulated by autophosphorylation at a tyrosyl residue (36). Protein ExoP of the {alpha}-proteobacterium Sinorhizobium meliloti, which controls succinoglycan production, might be regulated by a similar mechanism (9). Although ExoP does not possess a conserved Tyr569, it has been shown to be phosphorylated on Tyr505, and replacement of Tyr505 by a serine was found to drastically enhance the production of low molecular weight succinoglycan at the expense of high molecular weight succinoglycan (9). However, it remains to be confirmed whether this amino acid plays an important role in enzyme regulation similar to that of Tyr569 of Wzc.

Autophosphorylated Wzc has been shown previously to be rapidly dephosphorylated in vivo and in vitro by Wzb, a low molecular weight PTP, although demodification of Wzc by Wzb was not complete (6). By carrying out experiments with soluble PTK domains of Wzc missing either the C-terminal tyrosine cluster or the phosphorylatable Tyr569, we could demonstrate that Wzb rapidly and exhaustively dephosphorylated Wzc previously phosphorylated at the C terminus but that the modification at Tyr569 was not, or only very slowly, removed by Wzb. This result can explain the failure of Wzb to completely dephosphorylate autophosphorylated wild-type Wzc. Interestingly, Wzb was found to hardly dephosphorylate tyrosine-phosphorylated Ugd, and this reaction was relatively slow compared with the dephosphorylation of Wzc phosphorylated at the C-terminal tyrosine cluster. The question therefore arises whether E. coli possesses additional PTPs able to efficiently demodify phosphorylated Ugd and/or Wzc phosphorylated at Tyr569. The E. coli proteins PrpA and PrpB (37, 38), which belong to the PPP family of protein phosphatases, could be such candidates. Although members of this family were first described as phosphoserine/phosphothreonine protein phosphatases, some of them, including PrpA and PrpB, have been found to also dephosphorylate phosphotyrosine (39).

Besides the synthesis of colanic acid in E. coli, UDP-glucose dehydrogenases catalyze the formation of glucuronic acid, which serves as building block for the synthesis of specific exopolysaccharides in a variety of bacterial species. Thus, The UDP-glucose dehydrogenase RkpK of S. meliloti is involved in the synthesis of capsular polysaccharides (40), whereas similar proteins encoded by the udgH gene of Xanthomonas campestris or hasB of Streptococcus pyogenes are required for the biosynthesis of xanthan gum (41) and hyaluronic acid (42), respectively. Similarly, the synthesis of alginate, a polysaccharide produced by Pseudomonas aeruginosa and Azotobacter vinelandii, depends on the UDP-glucose dehydrogenase AlgD (43, 44), and the ugd gene product of Acinetobacter lwoffii is involved in emulsan biosynthesis (20). Also, mutations in the UDP-glucose dehydrogenase-encoding cap3A gene of S. pneumoniae are responsible for a change from the encapsulated to unencapsulated phenotype (45). It is noteworthy that most of the organisms mentioned above contain a PTK homologue of Wzc (ExoP in S. meliloti, CpsD in S. pneumoniae, EpsB in P. aeruginosa, and Ptk in A. lwoffii), making it likely that the diverse functions of these UDP-glucose dehydrogenases are regulated by tyrosine phosphorylation. E. coli Ugd as well as the two B. subtilis UDP-glucose dehydrogenases YwqF and TuaD (26) are active only when they are tyrosyl-phosphorylated. Ugd purified from E. coli is probably slightly phosphorylated, because it exhibited weak UDP-glucose dehydrogenase activity. This activity disappeared when Ugd was incubated with alkaline phosphatase, whereas incubation with ATP and the PTK domain of Wzc increased the UDP-glucose dehydrogenase activity ~6-fold.

Recently, ugd was also reported to be controlled by the RcsC-YojN-RcsB phospho relay and the RcsA protein (46). This result is of special interest, because expression of the wzb- and wzc-containing cps operon of E. coli is also regulated by RcsA, RcsB, and RcsC (47–49). Expression of the genes coding for the tyrosine kinase Wzc, the phosphotyrosine phosphatase Wzb, and their substrate Ugd, seems therefore to be controlled by the same transcription regulatory system. Interestingly, Ugd of E. coli and S. typhimurium also catalyzes the first step of a metabolic pathway leading to resistance toward the peptide antibiotic polymixin B and several cationic antimicrobial peptides of the innate immune system (50, 51). Through the action of ArnA and ArnB, the UDP-glucuronic acid formed by Ugd is converted into UDP-4-amino-4-deoxy-L-arabinose (UDP-L-Ara4N) (52). If the L-Ara4N moiety of this compound is attached to the phosphate groups of lipid A, the resulting modified lipopolysaccharide is known to render the organisms resistant to polymyxin B.

By combining previous observations of Wzb/Wzc functions and Ugd activities in E. coli with the results reported here, we can propose a model (Fig. 7) that predicts that Wzc would regulate both polysaccharide production and resistance to antimicrobial peptides. The protein kinase activity of Wzc is first enhanced by autophosphorylation on Tyr569. The resulting activation of Wzc stimulates autophosphorylation at the C-terminal tyrosine cluster and phosphorylation of Ugd. The latter phosphorylation reaction increases the UDP-glucose dehydrogenase activity of Ugd leading to elevated synthesis of UDP-glucuronic acid, a building block for colanic acid synthesis and a precursor of UDP-L-Ara4N. Wzc also plays a role in colanic acid polymerization and/or transport, but it is not yet clear whether these activities require unphosphorylated Wzc or Wzc phosphorylated at specific tyrosine(s) in the C-terminal tyrosine cluster. Because polysaccharides are important for bacterial virulence, a more detailed analysis of PTKs is expected to allow deeper insights into the regulatory network controlling bacterial pathogenicity.



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  		FIG. 7.
Schematic model for Wzc intraphosphorylation, interphosphorylation, and Ugd phosphorylation as control elements of bacterial metabolic pathways. Wzc is sensitive to an activator of its autophosphorylation on Tyr569 (Y569-P). Its protein-tyrosine kinase activity is then enhanced, which induces Wzc interphosphorylation on the five C-terminal tyrosines Tyr708, Tyr710, Tyr711, Tyr713, and Tyr715 (YC). Wzc dephosphorylation by Wzb would thus control the polymerization or transport of colanic acid. Simultaneously, phosphorylation of Ugd by Wzc enhances its UDP-glucose dehydrogenase activity to produce UDP-glucuronic acid. This compound is then used in colanic acid unit production and/or UDP-4-amino-4-deoxy-L-arabinose (UDP-L-Ara4N) synthesis to possibly modify lipopolysaccharide (LPS) and promote polymyxin resistance.

 


   FOOTNOTES
 
* This work was supported by Ministère de la Recherche Grant FNS 2000 (Microbiologie), Région Rhône-Alpes Grant Emergence 97.027, Société Ezus-Lyon 1 Grant 482.022, and a grant from the Institut Universitaire de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Institut de Biologie et Chimie des Protéines, 7, passage du Vercors, 69367 Lyon Cedex 07, France. Tel.: 33-4-72-72-26-79; Fax: 33-4-72-72-26-01; E-mail: c.grangeasse{at}ibcp.fr.

1 The abbreviations used are: PTK, protein-tyrosine kinase; PTP, phosphotyrosine-protein phosphatase; Cps, capsular polysaccharide; Ni2+-NTA, nickel-nitrilotriacetic acid; Ugd, UDP-glucose dehydrogenase; DTT, dithiothreitol; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We gratefully thank Jean-Michel Jault for helpful discussion.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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