INTRODUCTION

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The soil bacterium Sinorhizobium meliloti (Rhizobium meliloti) has the ability to produce the acidic exopolysaccharide (EPS) succinoglycan (EPS I), which is required for invasion of Medicago sativa root nodules by S. meliloti (2, 19, 30, 31, 32, 37, 54). Succinoglycan is composed of octasaccharide subunits, which consist of one galactose and seven glucose residues, joined by -glycosidic linkages (1). It can be modified by acetyl, succinyl, and pyruvyl groups (41). S. meliloti produces a high-molecular-weight (HMW) form and a low-molecular-weight (LMW) form of succinoglycan (30). LMW succinoglycan comprises monomers, dimers, and trimers of the octasaccharide subunit, and it has been shown that the trimer is the symbiotically active species (2, 20, 53). The production of succinoglycan is influenced by the osmolarity of the growth medium. An increase in osmotic pressure results in enhanced production of HMW succinoglycan at the expense of LMW succinoglycan (10).

The biosynthesis of succinoglycan is directed by 21 exo and exs genes, located in a 30-kb gene cluster on megaplasmid 2 (3-6, 8, 11, 17, 18, 36, 42). The octasaccharide repeating unit is synthesized on an undecaprenyl lipid carrier located in the cytoplasmic membrane (48). In a study of the roles of the various exo gene products in succinoglycan biosynthesis, membrane-associated proteins ExoP, ExoQ, and ExoT were determined to be involved in polymerization and secretion of succinoglycan (20). ExoQ was found to be required for production of HMW succinoglycan, and it was suggested that ExoT is involved in synthesis or secretion of the LMW succinoglycan dimers and trimers but not the monomers. A mutation in exoP blocked polymerization of succinoglycan octasaccharide subunits (20), indicating that ExoP plays an important role in the polymerization of succinoglycan.

Analysis of the membrane topology of the ExoP protein showed that this protein can be divided into an N-terminal domain located mainly in the periplasmic space and a C-terminal domain located in the cytoplasm (8). S. meliloti strains carrying a mutated exoP* gene, expressing only the N-terminal domain, produced a reduced amount of succinoglycan with an increased ratio of the LMW form to the HMW form. These exoP mutants were still able to invade root nodules (8). Specific amino acid substitutions in the proline-rich motif, which is located near the second transmembrane region in the N-terminal domain, also affected the ratio of HMW succinoglycan to LMW succinoglycan to the benefit of LMW succinoglycan (7). This led to the conclusion that the cytoplasmic C-terminal domain is not essential for production and export of succinoglycan but may have a regulatory function.

The ExoP protein has similarities to proteins involved in polysaccharide chain length determination (8). Related proteins involved in the biosynthesis of lipopolysaccharides (LPS), O-antigen polysaccharides, capsular polysaccharides (CPS), and EPS can be distinguished on the basis of their coiled-coil prediction profiles and other characteristics used for classification, such as size, type of polysaccharide synthesized, sequence similarity, location of transmembrane regions, and the presence of ATP-binding domains (35, 38). ExoP, with a periplasmic domain flanked by two transmembrane regions and an additional cytoplasmic domain, was placed in the PCP2a (polysaccharide copolymerase) family. Like the Ptk protein of Acinetobacter johnsonii or the Wzc protein of Escherichia coli K-12, the C-terminal domain of ExoP contains Walker A and B ATP-binding motifs (15, 24, 52). In several gram-positive bacteria (e.g., Staphylococcus aureus and Streptococcus pneumoniae) the cytoplasmic domain is encoded by a separate gene in CPS biosynthesis operons (34).

Several members of the PCP2a family have been shown to be autophosphorylating protein tyrosine kinases. In the case of Ptk of A. johnsonii, the ATP-binding motif is required for this activity (15). Morona et al. (34) obtained evidence that protein tyrosine phosphorylation negatively regulates CPS production in S. pneumoniae. A similar finding for E. coli was recently described by Vincent et al. (51), although these authors hypothesized that the processes of protein autophosphorylation are different in gram-positive and gram-negative bacteria. The role of autophosphorylation in EPS biosynthesis has not been determined yet. In this context we focused on the biochemical activity of ExoP, particularly with regard to the C-terminal cytoplasmic domain, and demonstrated that ATPase activity and tyrosine phosphorylation occur. The possible role of the ATP-binding motif and individual tyrosine residues in biosynthesis of succinoglycan was investigated.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.

Culture media and growth conditions. E. coli strains were grown in Pennassay broth or in Luria-Bertani broth (43) at 37°C. For overexpression the strains were grown in Superbroth (43) at 37 or 30°C. S. meliloti strains were grown in TY (9) or in Luria-Bertani medium. For succinoglycan production S. meliloti strains were grown at 30°C in glutamate-D-mannitol-salts (GMS) medium (pH 7.0) supplemented with 0.24 M sodium chloride, biotin, thiamine, and trace elements (55).

DNA and protein biochemistry. Preparation of plasmid DNA, DNA restriction, agarose gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), cloning procedures, and transformations of E. coli cells were carried out by using previously described protocols (28, 43). Southern hybridizations were performed as described by Kessler (25). Total DNA was isolated from rhizobia as described by Meade et al. (33).

DNA sequencing. DNA sequencing to verify new plasmid constructs or mutations was carried out by the Institut für Innovationstransfer an der Universität Bielefeld (IIT Biotech) with an ABI PRISM 377 DNA sequencer (Perkin-Elmer). Sequence data were obtained and processed by using the ABI software according to the manufacturer's instructions.

Construction of the exoP expression plasmid. Plasmid pExoP (6, 7) (exoP sequence EMBL/GenBank/DDBJ accession number Z22636) served as the template for PCR amplification of the exoP_C gene flanked by a BamHI restriction site and a SmaI restriction site. The sequences of the two primers were 5'-GCC CGG ATC CTT GCC TTC CTC GAA TTC CGC G-3' at the N terminus and 5'-GCA ACC CGG GTC GAT CGC CGC AAG GCT TGA C-3' at the C terminus. The BamHI-SmaI fragment comprising 925 bp of the 3' portion of exoP and 70 bp downstream of the exoP coding region was ligated into vector pGEX-5x-1 (Pharmacia Biotech), resulting in plasmid pGEX-exoP_C. The sequence of the PCR fragment containing exoP_C was verified by DNA sequencing.

Site-directed mutagenesis. Site-directed mutagenesis was carried out by using a Chameleon double-stranded site-directed mutagenesis kit from Stratagene according to the manufacturer's protocol. The target mutagenic primers and the selective primer which were used to generate site-specific mutations are shown in Table 2. The mutations were introduced into plasmid pHIP1-EB, which resulted from insertion of the 1.77-kb EcoRI-BamHI fragment of the exoP-thi gene into the vector pHIP1. All mutations were verified by sequencing.

Purification of the ExoP_C protein. E. coli BL21 cells were transformed with plasmid pGEX-exoP_C. Cells from an overnight culture of this strain were used to inoculate 1 liter of Superbroth supplemented with ampicillin. Cultures were incubated at 37°C with shaking until the A₆₀₀ was 0.6. Then isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. Incubation was continued at 30°C for 2.5 h with shaking. Cells were harvested by centrifugation at 4,200 × g for 10 min at 4°C and suspended in 40 ml of buffer 1 (pH 7.4) (10 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 10% glycerol) containing 20 µg of RNase A per ml and 10 µg of DNase 1 per ml. The cells were disrupted with a French pressure cell at 20,000 lb/in² two or three times. The state of the cells was checked by light microscopy. Each cell suspension was supplemented with Triton X-100 at a final concentration of 1% and centrifuged at 12,500 × g for 10 min at 4°C.

Immunoblot analysis. Samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by using a semidry electrophoretic transfer cell (Trans-Blot SD-Dry Transfer Cell; Bio-Rad) and the procedure of Towbin et al. (49).

ATPase activity. For in situ demonstration of ATPase activity by detection of released inorganic phosphate (Pi), the affinity-purified fusion proteins were separated on nondenaturing acrylamide gel as described by Koronakis et al. (26, 27). The gels were either stained with Coomassie brilliant blue or incubated with ATP buffer (40 mM Tris-HCl, 4 mM ATP, 4 mM MgCl₂, 5% glycerol) for 20 min at 37°C. After incubation the reaction was stopped with color reagent (0.034% malachite green, 0.1% Triton X-100, and 10.5 g of ammonium molybdate per liter in 1 M HCl) and 34% citric acid (29).

Production of succinoglycan. S. meliloti strains were grown at 30°C for 10 days in GMS medium as described by Zevenhuizen and van Neerven (55). Cells were removed by centrifugation (11,200 × g, 1 h, 10°C), and the clear culture supernatants, containing the secreted EPS, were lyophilized. After suspension in water (20% of the primary volume), carbohydrates were precipitated with 10 volumes of ethanol and pelleted by centrifugation. The carbohydrates were resuspended and desalted by dialysis (Spectra/Por membrane; molecular weight cutoff, 1,000; Roth) against water for 4 days, and this was followed by concentration of EPS by lyophilization.

Analysis of extracellular carbohydrates by high-performance liquid chromatography (HPLC)-gel permeation chromatography. Succinoglycan fractions were separated by gel permeation chromatography on Nucleogel columns (2× GFC 4000-8, 1× GFC 300-8; 300 by 7.7 mm; Macherey-Nagel, Düren, Germany) by using a flow rate of 0.8 ml min¹ as described by Becker and Pühler (7); the eluent was 200 mM sodium chloride-200 mM sodium phosphate buffer (pH 7.0).

Analysis of extracellular carbohydrates by gel filtration chromatography and HPAEC-PAD. Cyclic glucans and HMW and LMW succinoglycan fractions were separated by gel filtration chromatography (Bio-Gel P6, fine mesh; Bio-Rad) by using the procedure of Wang et al. (53); the size of the column (Merck) was 1.6 by 120 cm, 1 ml was loaded, the flow rate was 0.2 ml min¹ and the buffer was pyridine-0.1 M acetic acid (pH 5.0). Ninety 1.5-ml fractions were collected and analyzed for total carbohydrates by the HCl-L-cysteine method (14).

1

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The C-terminal domain of ExoP fused to GST is phosphorylated on tyrosine. To investigate the function of the C-terminal domain of ExoP independent from the N-terminal domain, an exoP gene lacking the coding region for the N-terminal domain was synthesized by PCR and inserted into vector pGEX-5x-1. The resulting plasmid, termed pGEX-exoP_C, expressed a 57-kDa fusion protein consisting of the C-terminal domain of ExoP with the GST at its N terminus.

GST-ExoP_C fusion protein possesses ATPase activity. The ExoP protein contains two conserved sequences in the cytoplasmic domain, S⁵⁸²ALPDEGKS⁵⁹⁰ and V⁶⁹¹VVD⁶⁹⁴, which are similar to the Walker A motif ([AG]X₄GK[ST]) and the Walker B motif ([hhhD]), respectively (X indicates any amino acid; h indicates a hydrophobic amino acid; and alternative residues are enclosed in brackets). These motifs were also identified in ExoP homologues like Ptk from A. johnsonii and CpsD from S. pneumoniae (15, 34). Doublet et al. showed that these conserved features are involved in binding of ATP by the Ptk protein kinase of A. johnsonii. Hence, the affinity-purified GST-ExoP_C protein was assayed for ATPase activity in nondenaturing polyacrylamide gels. When ATP was provided, the GST-ExoP_C protein produced free inorganic phosphate, while GST alone did not (Fig. 1). If ATP was not added to the incubation buffer, free inorganic phosphate was not observed. This result confirmed that the cytoplasmic domain of ExoP is able to hydrolyze ATP.

View larger version (53K): [in this window] [in a new window]   FIG. 1.   In situ demonstration of GST-ExoP ATPase activity. Affinity-purified GST-ExoP, GST-ExoP. K589I, and control GST protein were separated on a nondenaturing acrylamide gel and either stained with Coomassie brilliant blue (A) or incubated with ATP. A dark green precipitate was formed in the gel upon reaction of released Pi with malachite green (B).

Substitution of amino acids in the Walker A ATP-binding motif blocks ATPase activity and phosphorylation of tyrosine residues. To assess the relevance of the ATP-binding motif in ExoP in general and with respect to protein tyrosine kinase activity, GST-ExoP_C fusion proteins characterized by specific amino acid substitutions in the conserved segment were constructed by site-directed mutagenesis of the exoP_C gene.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Presence of the 57-kDa GST-ExoP wild-type and mutant proteins, overexpressed in E. coli BL21. Western immunoblots were probed with the ExoP peptide antibody (A) and with anti-phosphotyrosine antibody PT-66 (B).

Replacement of specific tyrosine residues results in a reduced phosphorylation state in the ExoP protein. To investigate the role of tyrosine phosphorylation in ExoP, single tyrosine residues were replaced in the ExoP_C protein. Site-directed mutagenesis was performed as described above. Grangeasse et al. (21) suggested that there are three putative autophosphorylation tyrosine sites in ExoP. The sequence flanking these tyrosine residues often includes arginine or lysine residues, like the consensus autophosphorylation motifs present in various eukaryotic kinases (39). On the basis of this information, we replaced residue Y⁴⁷⁷, which is situated in the transmembrane region and is followed by an arginine residue at position +7 (R⁴⁸⁴). The second tyrosine residue that was replaced was Y⁵⁰⁵, which is located between the second transmembrane region and the amphiphilic helix at the C terminus of the cytoplasmic domain. This tyrosine was also followed by an arginine residue at position +7 (R⁵¹²).

Mutations in the ATP-binding motif result in much lower levels of succinoglycan which consists only of monomers of the repeating unit. To investigate the phenotypic effect of mutations on the biosynthesis of succinoglycan, S. meliloti Rm2011 mutant strains expressing the exoP mutant genes were constructed. The 665-bp EcoRI-StuI fragment of pHIP1-EB carrying the mutations was inserted into plasmid pExoP, replacing the wild-type fragment. The exoP mutant genes were expressed in S. meliloti exoP deletion mutant RmPII15 in order to eliminate interference from ExoP proteins encoded by the endogenous gene and ExoP proteins encoded by the exoP mutant genes, as described by Becker and Pühler (7). Integration of wild-type and mutant pExoP plasmids into the genome of RmPII15 by homologous recombination occurred upstream of the deletion site, thereby restoring the native genomic structure, which was verified by Southern hybridization.

View larger version (8K): [in this window] [in a new window]   FIG. 3.   Chromatographic separation of LMW succinoglycan. (A) Wild type; (B) tyrosine mutant RmPII15.pExoP-Y505S; (C) ATP-binding motif mutant RmPII15.pExoP-K589I. The monomer fraction eluted after 14 min, the dimer fraction eluted after 19 min, and the trimer fraction eluted after 22 min.

Tyrosine mutants produce modified ratios of LMW and HMW succinoglycans. Gel filtration chromatography indicated that some of the tyrosine mutants differed from the wild type in terms of the ratio of HMW succinoglycan to LMW succinoglycan. This observation was verified by gel permeation HPLC on Nucleogel columns. Mutant RmPII15.pExoP-Y477G produced the same peak areas for LMW and HMW succinoglycans as the wild type. This was not surprising because the mutation at position 477 was located in the putative transmembrane region and therefore was probably not phosphorylated. Mutant RmPII15.pExoP-Y505S produced a completely different result (Table 4). The mutation of this mutant resulted in drastically enhanced production of LMW succinoglycan at the expense of HMW succinoglycan. A ratio of HMW succinoglycan to LMW succinoglycan of 10:90 was obtained. The total amount of EPS was less than 50% of the amount of wild-type EPS produced. Mutant RmPII15.pExoP-Y775S acted like the wild type. Strain RmPII15.pExoP-Y505S/Y775S carrying the double mutation also produced more LMW succinoglycan than the wild type, but the double mutation did not result in as drastic an alteration as that observed with mutant RmPII15.pExoP-Y505S. The ratio of HMW succinoglycan to LMW succinoglycan determined for the double mutant was 24:76. Finally, the mutation at position 758 (RmPII15.pExoP-Y758S) resulted in a ratio of HMW succinoglycan to LMW succinoglycan of 30:70 and slightly decreased production of total EPS (Table 4).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study the C-terminal cytoplasmic domain of ExoP from S. meliloti was shown to influence the polymerization and export of succinoglycan. The function of ExoP is affected by its ATPase activity and the phosphorylation state of its tyrosine residues in the cytoplasmic domain.

Doublet et al. (15) showed that the ATP-binding motif in the C-terminal domain of A. johnsonii Ptk was required for phosphorylation activity. The ATP molecule which serves as the phosphoryl donor binds to highly conserved Walker A and B motif protein sites. The presence of Walker motifs A and B has been described for a wide variety of prokaryotic and eukaryotic ATP-or GTP-binding proteins (22), and these motifs were also found in the protein sequence of the cytoplasmic ExoP domain. Previously (8), we reported a potential ATP- and GTP-binding motif in ExoP and local homology to prokaryotic ATPases. In this study we verified the ATPase activity of ExoP. On the basis of strong structural similarities between Ptk of A. johnsonii (21) and ExoP, it seems very likely that tyrosine phosphorylation in ExoP also occurs due to an autophosphorylating activity, such as that described for Ptk.

In accordance with this assumption, the ExoP_C proteins of the ATP-binding motif mutants were not phosphorylated on tyrosine. This finding might be an indication that ATP is the phosphoryl donor, as in Ptk. It also supports the observation of Doublet et al. (15) that binding and hydrolysis of ATP occur at a site different from the phosphorylation site. We found that binding and/or hydrolysis of ATP is essential for the function of ExoP, as the phenotypes of the ATP-binding mutants did not differ from those of exoP deletion mutants with regard to succinoglycan biosynthesis.

Phosphorylation of the CpsD protein in the gram-positive bacterium S. pneumoniae required the presence of another protein, CpsC, which enabled ATP to bind to CpsD (34). However, in contrast to CpsD, no additional protein was necessary to catalyze phosphorylation of Wzc, a protein tyrosine kinase in E. coli comprising a C-terminal cytoplasmic domain and an N-terminal periplasmic domain (51). CpsD corresponds to the C-terminal domain and CpsC is similar to the N-terminal domain of proteins belonging to the PCP2a family, like Wzc and ExoP. However, Vincent et al. (51) reported that even the C-terminal domain of Wzc alone could be phosphorylated. These results supported our hypothesis that the tyrosine kinase activity and a regulatory function in biosynthesis of succinoglycan should be assigned to the C-terminal domain of the ExoP protein of S. meliloti.

In contrast to tyrosine phosphorylation in eukaryotic cells, in which a high number of protein tyrosine kinases and the essential roles of these molecules in the control of various cellular functions, including signal transduction, growth control, and metabolism, are well known (16, 23), tyrosine phosphorylation in prokaryotic organisms is still poorly understood. IIan et al. (24) observed that protein tyrosine kinases in bacterial pathogens are commonly associated with production of EPS and virulence. A direct influence of tyrosine phosphorylation on a phenotype was demonstrated only for the CpsD protein. Morona et al. (34) reported that tyrosine phosphorylation negatively regulated CPS biosynthesis. Recently, Vincent et al. (51) demonstrated that synthesis of the CPS colanic acid in E. coli is modulated by reversible tyrosine phosphorylation of protein Wzc, which has to be dephosphorylated to be active in colanic acid synthesis.

The presence of proteins which catalyze dephosphorylation of proteins at tyrosine residues has also been demonstrated for A. johnsonii and E. coli (51, 52). In these organisms phosphotyrosine protein phosphatases have been identified which are encoded by genes genetically linked to protein tyrosine kinase-encoding genes ptk of A. johnsonii and wzc and etk of E. coli. Interestingly, a phosphatase-encoding gene was not found to be linked to exoP in S. meliloti. The gene cluster involved in biosynthesis of EPS does not encode a gene product that is similar to phosphotyrosine protein phosphatases. A Blast search of the genome of S. meliloti (http://sequence.toulouse.inra.fr/rhime /complete/doc/complete.html) revealed only two potential phosphatases. One of these phosphatases, encoded on pSymA, exhibited 30% identity and 40% similarity to the phosphatase encoded by wzb in E. coli. The other phosphatase, identified an arsenate reductase, is encoded on the chromosome and exhibited 30% identity and 43% similarity to the wzb-encoded enzyme.

Our results also imply that the composition of succinoglycan in S. meliloti is influenced by the phosphorylation state of tyrosine residues in ExoP_C. The levels of succinoglycan produced by the tyrosine substitution mutants differed significantly, although the mutant proteins still displayed ATPase activity, indicating that the mutation did not eliminate the function of the proteins. In particular, the tyrosine substitution at amino acid position 505 resulted in a significant decrease in the amount of HMW succinoglycan. When the phosphotyrosine immunoblot analysis procedure was used, the signal intensity did not necessarily indicate that the succinoglycan composition was modified. The signal of the double mutant indicated that we did not replace every tyrosine residue which could be phosphorylated in the cytoplasmic domain. Our results led to the inference that phosphorylation of one tyrosine residue might be influenced by the phosphorylation state of other tyrosine residues. It may be that one phosphorylated tyrosine residue promotes phosphorylation of other residues, independent of its direct contribution to the function of the ExoP protein. This would explain the presence of the intense, almost wild-type-like protein band of mutant RmPII15.pExoP-Y758S on the phosphotyrosine immunoblot and the strongly modified phenotype of this mutant.

Mutant RmPII15.pExoP-Y505S exhibited phenotypic similarities to mutant RmP*1, which expressed a truncated ExoP protein lacking the whole C-terminal domain (8). The LMW succinoglycan fraction of RmPII15.pExoP-Y505S contained normal amounts of mono-, di-, and trimers, which was not surprising because like all tyrosine mutants, RmPII15.pExoP-Y505S was still able to produce at least small amounts of HMW succinoglycan. It is thought that ExoP is required for production of dimers of succinoglycan (20). Since mutant RmPII15.pExoP-Y505S produced dimers of the succinoglycan repeating unit, the substitution at position 505 did not destroy this possible function of ExoP. However, the data indicated that an ExoP protein not phosphorylated at position 505 influences the biosynthetic pathway. As the ExoQ protein of S. meliloti is thought to be necessary for production of HMW succinoglycan (20), the mutated ExoP protein might have negative regulatory effects on ExoQ. Otherwise, it is assumed that the ExoT protein is involved in the synthesis of LMW oligosaccharides of succinoglycan (20). An ExoP protein not phosphorylated at position 505 might also be a positive regulator of ExoT activity. Both possibilities are consistent with the hypothetical model which states that ExoP regulates the degree of succinoglycan polymerization by controlling polymerization activities of other proteins, most likely ExoQ and ExoT (20). Whether these proteins also contribute to the phosphorylation process and how the mechanism of tyrosine phosphorylation itself is involved remain to be investigated.