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Applied and Environmental Microbiology, August 2005, p. 4703-4712, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4703-4712.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities

Elisabete Silva, Ana Rita Marques, Arsénio Mendes Fialho,* Ana Teresa Granja, and Isabel Sá-Correia

Biological Sciences Research Group, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal

Received 27 October 2004/ Accepted 12 March 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-ß-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 µM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 µM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sphingomonas elodea (formerly Pseudomonas elodea and also referred to as Sphingomonas paucimobilis) ATCC 31461 synthesizes the exopolysaccharide (EPS) gellan, a gelling agent with applications in the food, pharmaceutical, and other industries (13, 41). This EPS is composed of a repeating linear tetrasaccharide unit consisting of D-glucose (Glc), D-glucuronic acid (GlcA), and L-rhamnose (Rha) in a 2:1:1 ratio, respectively, with glycerate and acetate substituents (22). The significant changes in rheology observed upon the deacylation of gellan are essentially due to the glycerate substituents (17).

The gellan repeat unit is formed by sequential transfer of the sugar nucleotides to a membrane-anchored lipid carrier by committed glycosyltransferases, followed by gellan polymerization and export (34). The pathway leading to the formation of the sugar nucleotides UDP-Glc, UDP-GlcA, and dTDP-L-Rha, donors of the monomers for assembling the gellan tetrasaccharidic repeat unit, was elucidated (27, 34). Eighteen genes, organized in the gel cluster (15, 34) and coding for enzymes presumably involved in the synthesis of dTDP-L-Rha, glucosyltransferases, and proteins required for gellan polymerization and export, were identified. The organization and the nucleotide sequence of the gel cluster are highly similar to those described for the gene cluster required for the synthesis of the sphingan S88 in Sphingomonas strain S88 (sps cluster) (15, 45).

The enzymes required for the synthesis of the sugar nucleotides UDP-Glc and UDP-GlcA from glucose-1-phosphate (G1P) are encoded by genes pgmG, ugpG, and ugdG, located outside the gel cluster (26, 34, 43).

The other activated sugar essential for the incorporation of L-rhamnose in the gellan repeat unit is dTDP-L-Rha. Rhamnose is a 6-deoxyhexose sugar widely distributed in gram-negative bacteria as part of the lipopolysaccharides (LPS), capsular polysaccharides (CPS), and EPSs. The nucleotide sugar dTDP-L-Rha is formed from G1P in a four-step reaction (21). This reaction involves the enzyme activities of glucose-1-phosphate thymidylyltransferase, dTDP-glucose-4,6-dehydratase, dTDP-4-keto-L-rhamnose-3,5-epimerase, and dTDP-L-rhamnose synthase, encoded by genes commonly designated rmlA, rmlB, rmlC, and rmlD, respectively (6). These genes are generally grouped together within the LPS, CPS, or EPS gene cluster (24, 34). The rml four-gene cluster has been identified for different gram-negative and gram-positive bacterial species; these clusters exhibit a remarkable sequence similarity, although the gene order of the operon-like structures may vary from species to species (11, 21, 24, 30). The two Sphingomonas strains S. elodea ATCC 31461 and Sphingomonas strain S88 exhibit a similar organization (ACBD->) and a high range of sequence conservation (87 to 96% amino acid identity) (15, 34).

The present work focuses on the first gene of the S. elodea ATCC 31461 rml four-gene cluster, the rmlA gene. The heterologous overexpression of rmlA in Escherichia coli, its purification as a fusion protein, and the biochemical characterization of the encoded enzyme were carried out. The protein RmlA was demonstrated to be a glucose-1-phosphate thymidylyltransferase (EC 2.7.7.24) involved in the reversible conversion of G1P and dTTP into dTDP-glucose (dTDP-Glc) and diphosphate. Besides this dTDP-glucose pyrophosphorylase (TGP) activity, RmlA also exhibits a UDP-glucose pyrophosphorylase (UGP) activity (EC 2.7.7.9). In the present study, we also demonstrated that the S. elodea UgpG protein, identified before as the enzyme required for the synthesis of UDP-glucose in the same bacterial strain (26), also recognizes both deoxythymidine and deoxyuridine nucleotides as substrates, and the kinetic parameters of these two proteins were compared. Since the ugpG gene maps outside the gel cluster (26), the adjacent chromosomal regions were sequenced to gain more insight into its physiological function.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, plasmids, and growth conditions.
Strains and plasmids used in this study are described in Table 1. E. coli strains were grown in Lennox broth (Sigma Chemical Co., St. Louis, Mo.) or in Superbroth medium (36) at 37°C with orbital agitation. When required, the culture medium was supplemented with ampicillin (150 mg/liter) or kanamycin (100 mg/liter). The oligonucleotides were purchased from MWG Biotech (Ebersberg, Germany). Restriction enzymes were from GibcoBRL-Life Technologies (Portugal), and DNA polymerases were from Boehringer GmbH (Mannheim, Germany). UDP-Glc, dTDP-Glc, NADP+, glucose-6-phosphate dehydrogenase, phosphoglucomutase (PGM), glucose-6-P-dehydrogenase (ZWF), and inorganic pyrophosphate (PPi) were obtained from Sigma.


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  		TABLE 1. Bacterial strains and plasmids used in this work

 
DNA manipulation.
All standard procedures, including DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, DNA ligation, and transformation of E. coli strains, were performed by conventional methods (36). DNA was recovered from gel slices by using a Concert gel kit (GibcoBRL-Life Technologies). PCR amplifications were performed using Pwo polymerase obtained from Roche Diagnostics (Mannheim, Germany) and 200 ng of template DNA.

Sequence analysis.
Previous studies have shown the presence of the gellan gene cluster in the p96 cosmid of the genomic library of S. elodea ATCC 31461 (44). To confirm the presence of the rmlA gene in the p96 insert, an internal region of this gene was amplified by PCR with the synthetic oligonucleotides AU (5'-GCAGCTGCTTCCCGTCTATGA-3') and AL (5'-CGAGCCGGGTGATGTGGAGGTC-3'), designed on the basis of the nucleotide sequence of the homologous gene of Sphingomonas strain S88, given that the organization of the sphingan S88 gene cluster is identical to that of the gellan biosynthetic cluster (45). The PCR product was recovered from a 1% (wt/vol) low-melting-point agarose gel (FMC Bioproducts, Rockland, ME), purified with a Concert gel kit (GibcoBRL), and sequenced with the oligonucleotide primers AU and AL to confirm the identity. The complete sequence of rmlA was obtained by directly sequencing the p96 insert with primers designed on the basis of the sequenced internal regions of this gene.

DNA sequencing of the ugpG gene-adjacent regions was carried out by using cosmid pC22 as the template (26) and specific primers based on the ugpG sequence. Sequence similarity searches were performed using BLAST 2.0 with default settings from the National Center for Biotechnology Information (NCBI), Bethesda, Md. A phylogenetic analysis of the TGP (RmlA) and UGP proteins compared in this study was performed. The manually edited 20-sequence alignment performed with the CLUSTAL X program (16) served to calculate the distance matrix by the TREE-PUZZLE method (39). The phylogenetic tree was constructed by the neighbor-joining method (35) and supported by 1,000 bootstrap steps (12).

Construction of plasmids.
To overexpress the rmlA gene, the complete sequence of this gene was amplified by PCR with the oligonucleotides TGP1 (5'-AAAGGATCCATGAAGGGCATCAT-3') and TGP2 (5'-AAAAAGCTTTCATGCGGCAGCCA-3') and with cosmid p96 as the template DNA. The PCR product (897 bp) was digested with BamHI and HindIII (underlined for the primers used) and cloned into compatible sites of pWH844, generating pRmlA. This recombinant plasmid carries the rmlA gene preceded by a sequence coding for six histidine residues present in the pWH844 vector. The insert cloned in pRmlA was then sequenced to confirm the fidelity of DNA amplification.

Protein overexpression and purification.
Expression and purification of histidine-tagged proteins from plasmids pRmlA (this study) and pUgpG (26) were carried out as previously described (26).

Transformants of E. coli BL21(DE3) harboring plasmid pRmlA or pUgpG or the respective cloning vector, pWH844 or pET29b (controls), were grown in 200 ml of Superbroth and supplemented with the appropriate antibiotic at 37°C until the optical density at 640 nm of the culture reached 0.5 ± 0.1. The transcription of the rmlA and ugpG genes was then induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG), followed by an additional 3-h period of cultivation. Cells were harvested by centrifugation, washed with 0.9% (wt/vol) NaCl, and resuspended in 3 ml of cold start buffer (1 mM phosphate buffer [pH 7.4], 50 mM NaCl, and 10 mM imidazole). Cells were sonicated on ice (VibraCell; Sonics Material, Danbury, Conn.) and centrifuged at 18,000 x g for 1 h at 4°C to remove cell debris and insoluble proteins. The overexpressed RmlA and UgpG proteins were purified under native conditions by following the one-step procedure for His-tagged proteins as described by the column manufacturer (Amersham Pharmacia Biotech). After the equilibration of the column with 10 ml of the start buffer, the induced cell crude extract was loaded on the column. The columns were then washed in several steps with the same start buffer containing increasing imidazole concentrations, and the His6 recombinant protein, RmlA or UgpG, was eluted with a buffer containing 200 mM or 300 mM imidazole, respectively.

The proteins present in the cell crude extracts or in the purified fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% acrylamide-bisacrylamide gels). Protein bands were revealed by staining with Coomassie brilliant blue, and the molecular masses of RmlA and UgpG were estimated with low-molecular-weight standards (Amersham Pharmacia Biotech). Protein concentrations were determined by the method of Bradford (7), with bovine serum albumin fraction V (Sigma) as the standard.

Enzyme assays.
dTDP- and UDP-glucose pyrophosphorylase activities were measured by coupled reaction with the enzymes PGM and ZWF, leading to the formation of NADPH. Enzyme reactions were performed at 30°C in a final volume of 1 ml containing the following activity assay buffer: 100 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 2 mM dTDP-D-glucose or UDP-D-glucose, 1 U PGM, 1 U ZWF, and 1 mM NADP+. The reaction was initiated by the addition of the cell crude extracts (1 mg) or the purified proteins (6 µg) and inorganic PPi (2 mM). The rate of formation of NADPH was followed at 340 nm with a double-beam spectrophotometer (model V-200; Hitachi Ltd., Tokyo, Japan). Control assays were performed with enzyme reaction mixtures lacking the extracts or the substrate. Under the assay conditions used, 1 U of enzyme activity was defined as the amount of enzyme needed to reduce 1µmol of NADP per min. TGP and UGP activities are the means of the values of at least three enzyme assays. The kinetic constants Km and Vmax for dTDP-Glc or UDP-Glc, corresponding to the TGP or UGP activities of purified RmlA and UgpG (ranges of 20 to 2,000 µM for RmlA and 3.3 to 25 µM for UgpG), were determined by using the coupled enzyme reaction mixtures mentioned above, in which PGM- and ZWF-catalyzed reaction mixtures were largely saturated. The adequacy of the coupling-enzyme system was verified by the fact that the apparent activity of an enzyme assay containing 30 U of pyrophosphorylase/mg protein, at the upper end of the range of data analyzed in Results, was unaffected by a doubling of the coupling-enzyme concentration. The calculation of the kinetic constants was based directly on the Michaelis-Menten equation; the rectangular hyperbolic function was solved by using iterative procedures with the Solver computer program (from Microsoft Excel). The kcat values (min–1) were calculated on the basis of a molecular mass of 31.5 kDa for RmlA and 31.2 kDa for UgpG. The catalytic efficiency was based on the kcat/Km ratio. All assays were performed at 30°C with 6 µg of purified RmlA or UgpG in the presence of 2 mM PPi.

Nucleotide sequence accession numbers.
The nucleotide sequences of the S. elodea ATCC 31461 rmlA gene and the UgpG genomic region were deposited in the GenBank database under the accession numbers AY247402 and AY873993, respectively.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence analysis of S. elodea rmlACBD biosynthetic cluster.
A 35-kb chromosomal region of S. elodea ATCC 31461, encompassing a cluster of genes involved in gellan synthesis, was cloned and partially sequenced in a previous study (34, 44). We started the present study by sequencing the first gene (rmlA) of the putative rml operon, located within the gel cluster. More recently, the nucleotide sequence of this four-gene rml cluster was deposited in GenBank by Harding et al. (15) and used in this work to deduce the amino acid sequences of the RmlB, RmlC, and RmlD proteins. These sequences were compared with the corresponding sequences from seven gram-negative and two gram-positive organisms (Table 2). The S. elodea ATCC 31461 RmlA, -B, -C, and -D proteins showed the highest identities with the four proteins of Sphingomonas strain S88 (96, 94, 94, and 87%, respectively) (Table 2). The comparison of the rml gene products from the nine bacterial species examined indicated that the rmlA gene product exhibited the highest identity values, in particular with the proteins from gram-positive bacteria (Table 2). Based on these sequence homology studies, the following putative functions were assigned to the Rml proteins of S. elodea ATCC 31461: RmlA is the glucose-1-phosphate thymidylyltransferase, RmlB the dTDP-D-glucose-4,6-dehydratase, RmlC the dTDP-4-keto-L-rhamnose-3,5-epimerase, and RmlD the dTDP-L-rhamnose synthase. According to what has been described in the literature for other rml-like clusters, it is likely that this S. elodea rml cluster is involved in dTDP-L-rhamnose synthesis.


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  		TABLE 2. Comparison of amino acid sequences of RmlA, RmlB, RmlC, and RmlD from S. elodea ATCC 31461 with other dTDP-L-rhamnose biosynthetic enzymes

 
Significant differences in the genetic organizations of the rml clusters were observed among the gram-negative strains examined (Fig. 1). The two Sphingomonas strains, S. elodea ATCC 31461 and Sphingomonas strain S88, exhibit a similar organization of the rml genes (ACBD->). However, this organization diverges from the gene order registered for all of the other gram-negative species examined and coincides with that registered for the two gram-positive species (Lactococcus lactis and Streptococcus pneumoniae) (Fig. 1).



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  		FIG. 1. Diagrammatic representation of putative or demonstrated operonic organization of dTDP-L-rhamnose-synthesizing genes from different bacterial species. Genes are represented by arrows. rmlA, rhsA, and rfbA, glucose-1-phosphate thymidylyltransferase; rmlB, rhsB, and rfbB, dTDP-glucose-4,6-dehydratase; rmlC, rhsC, and rfbC, dTDP-4-keto-L-rhamnose-3,5-epimerase; and rmlD, rhsD, and rfbD, dTDP-L-rhamnose synthase.

 
The four gel genes of the S. elodea ATCC 31461 rml cluster are contiguous, and sequence analysis suggests an operon-like structure, like those documented for the equivalent clusters of L. lactis (6) and Salmonella enterica (23). Putative ribosome-binding site sequences that precede the translational start codons of the four genes were identified (Table 3). The rml genes have overlapping stop/start codons (rmlA and rmlC [4 bp], rmlB and rmlD [1 bp]) (Table 3), reinforcing the idea that they may be cistronically linked. A putative rho-independent transcription terminator, located downstream of the last gene of the operon, the rmlD gene, was also identified. The G+C contents of the four genes, as well as information on the deduced polypeptides, are shown in Table 3. The rmlACBD block of genes has a mean G+C content of 65.8 mol%, consistent with the high values described for the Sphingomonas genes already characterized (26, 43, 44, 45)


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  		TABLE 3. Characteristics of rml genes from S. elodea ATCC 31461

 
A complete multiple-amino-acid alignment with the dTDP-glucose pyrophosphorylases (encoded by the A genes of the respective clusters) from prokaryotic organisms available in databases was performed using the CLUSTAL X alignment program (16). From all of the sequences examined in this work, only the RmlA proteins from Pseudomonas aeruginosa (29) and Lactococcus lactis (6) and the RfbA proteins from E. coli (46) and Salmonella enterica (25) were biochemically characterized. The amino acid sequences of three bacterial UGPs were also included in this study. Two regions (domains I and II) were found to be highly conserved throughout the sequences compared (Fig. 2). The N-terminal region (domain I) of these proteins exhibits the motif G-X-G-T-R-X2-P-X-T. Domain II, found among the bacterial UGPs and TGPs, is also present in S. elodea ATCC 31461 RmlA; this domain has a conserved lysine residue (K163), located within the E-E-K-P motif, which has been proven to be essential for glucose-1-phosphate binding in P. aeruginosa RmlA (5) (Fig. 2). This motif, or a closely related sequence (A-E-K-P, K-E-K-P, Q-E-K-P, I-E-K-P, or V-E-K-P), is found in many bacterial enzymes which share the ability of catalyzing the synthesis of nucleoside diphosphate sugars from sugar-1-phosphates and nucleoside triphosphates (5, 42).



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  		FIG. 2. Multiple-amino-acid alignment of the two characteristic consensus sequences, including the activator binding region (domain I) and the sugar binding region (domain II), of known bacterial TGPs and UGPs. Each consensus sequence delineates the conserved N-terminal peptide motif found in the large family of XDP-sugar pyrophosphorylase enzymes and a lysine residue located within the X-E-K-P motif. Numbers of intervening amino acids are given in parentheses. CLUSTAL X software (16) was used to generate this multiple-sequence alignment. Organisms include Mesorhizobium loti, Caulobacter crescentus, Azotobacter vinelandii, Burkholderia pseudomallei, Pseudomonas syringae, Pseudomonas fluorescens, and Zymomonas mobilis.

 
The phylogenetic tree prepared with the 17 RmlA proteins (putative or confirmed TGPs) and the three putative or confirmed UGP proteins reveals that RmlA and UGP proteins form two divergent groups (Fig. 3). Within the RmlA group, it is interesting that the RmlA proteins of S. elodea and Sphingomonas strain S88 are a branch apart from others, including Novosphingobium aromaticivorans, that are phylogenetically close to S. elodea. This atypical feature, which is not coincident with the phylogenetic distance between the species, may indicate that the corresponding S. elodea and Sphingomonas strain S88 rmlA genes were introduced to their current positions more recently as the result of recombination events.



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  		FIG. 3. Phylogenetic tree of the dTDP-glucose pyrophosphorylase (RmlA) and UDP-glucose pyrophosphorylase (Ugp) proteins listed in Fig. 2. The distance matrix was calculated by the TREE-PUZZLE method (39), based on the manual alignment of the complete protein sequences performed with the CLUSTAL X program. The tree was constructed by the neighbor-joining method (35). The numbers for the interior branches are bootstrap percentages. Percentages less than 50 are not shown. A branch length of 0.5 substitution/site is given for phylogenetic distances. Organisms include Mesorhizobium loti, Pseudomonas syringae, P. fluorescens, Caulobacter crescentus, Burkholderia pseudomallei, Azotobacter vinelandii, and Zymomonas mobilis.

 
Overproduction in E. coli and purification of heterologous S. elodea rmlA.
To overproduce the S. elodea ATCC 31461 RmlA protein for biochemical characterization, the rmlA gene was overexpressed from plasmid pRmlA in E. coli BL21(DE3) cells, and the dTDP-glucose pyrophosphorylase activity was assayed in cell crude extracts and compared with that present in the cell crude extract prepared from E. coli BL21(DE3) with the cloning vector pWH844. This extract, prepared from the control cells, exhibited a low level of dTDP-glucose pyrophosphorylase activity (21 mU/mg), while those from E. coli BL21(DE3) overproducing RmlA protein from plasmid pRmlA exhibited increased levels of dTDP-glucose pyrophosphorylase activity following IPTG induction (73 mU/mg). The fused His6-RmlA protein was purified to homogeneity from this cell extract by using a His tag purification kit. Under denaturing conditions, the purified fusion protein migrated as a single polypeptide with an estimated molecular mass of approximately 32.8 kDa (31.5 kDa from the native protein, plus 1.3 kDa corresponding to the His6 tag) (results not shown). Enzyme assays performed with the purified RmlA protein (2,952 mU/mg) reinforced the idea that RmlA is a dTDP-glucose pyrophosphorylase (Fig. 4).



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  		FIG. 4. Saturation curves of purified His6-RmlA and His6-UgpG with various concentrations of TGP or UGP, conforming to Michaelis-Menten kinetics. The apparent Michaelis parameters (Table 4) were calculated directly based on the Michaelis-Menten equation.

 
S. elodea RmlA and UgpG exhibit both TGP and UGP activities.
The kinetic analysis of the TGP activity of the RmlA protein for the pyrophosphorolysis reaction showed a typical Michaelis-Menten saturation pattern (Fig. 4). Compared with the other characterized prokaryotic glucose-1-phosphate thymidylyltransferase proteins, the estimated apparent Km (Table 4) showed the highest values for dTDP-Glc (12.0 µM), compared with 36.0 µM for Pseudomonas aeruginosa RmlA (29), 83.0 µM for Salmonella enterica serovar Typhimurium RfbA (25), and 95.0 µM for Escherichia coli RfbA (46).


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  		TABLE 4. Kinetic parameters of RmlA and UgpG from S. elodea ATCC 31461

 
With respect to UGP activity, the estimated apparent Km for S. elodea RmlA was 229.0 µM, 19-fold higher than the Km value for TGP activity. On the other hand, the Vmax value for UDP-Glc (37.05 µmol min–1 mg–1) was approximately 15-fold higher than the corresponding value for dTDP-Glc (Table 4). These results, obtained for the pyrophosphorolysis reactions catalyzed by RmlA in vitro, suggest that for limited concentrations of dTDP-Glc and UDP-Glc, the RmlA enzyme may act preferentially as a glucose-1-phosphate thymidylyltransferase, as suggested before for the P. aeruginosa RmlA enzyme (29).

The S. elodea UgpG protein was previously characterized in our laboratory as a UGP (or glucose-1-phosphate uridylyltransferase) and was hypothesized to be involved in the synthesis of UDP-glucose, one of the activated precursors for gellan biosynthesis (26). In the present work, we further demonstrate that, besides being a glucose-1-phosphate uridylyltransferase, UgpG also exhibits glucose-1-phosphate thymidylyltransferase activity (Fig. 4). The affinity of the two enzyme activities associated with UgpG (Km values for UDP-glucose and TDP-glucose are, respectively, 7.5 and 9.4 µM) is higher than the affinity estimated for RlmA (Km values for UDP-glucose and TDP-glucose are, respectively, 229.0 and 12.0 µM), but the Vmax values associated with UgpG are lower (Table 4).

Analysis of the nucleotide sequence of the ugpG gene-adjacent regions.
The apparent redundancy of UgpG and RmlA proteins suggested by the results of the enzyme assays carried out in vitro led us to consider the inspection of the chromosomal region surrounding this putative gellan gene, since the insertional inactivation of the ugpG gene in ATCC 31461 was repeatedly tried before, without success (26).

A 4,638-bp region containing adjacent genes upstream and downstream from the ugpG gene was thus sequenced to gain insight into the physiological function of the encoded protein that might (also) be involved. This analysis revealed the presence of four open reading frames (ORFs) surrounding the ugpG gene (Fig. 5). Results of the comparison of the deduced amino acid sequences of these ORFs with the primary structures of proteins available in databases are shown in Table 5. The deduced amino acid sequence of the partially sequenced region of ORF 1 (362 bp) showed homology with proteins with UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase activity, ORF 2 showed strong homology with proteins with UDP-N-acetyl-D-glucosamine-2-epimerase activity, ORF 3 showed homology with proteins belonging to the UDP-glucose/GDP-mannose dehydrogenase family, and the sequenced region of ORF 4 (623 bp) showed homology with proteins with UDP-glucuronic acid epimerase activity.



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  		FIG. 5. Genetic organization of the ugpG flanking regions in the S. elodea ATCC 31461 chromosome (GenBank accession no. AY873993). The nucleotide sequences of ORF 1 and ORF 4 were not completely determined.

 

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  		TABLE 5. Comparison of ORFs in UgpG vicinity with primary structures of proteins available in databases

 
The investigation of the neighboring regions of genes encoding UDP-glucose pyrophosphorylase proteins in other bacterial genomes (Novosphingobium aromaticivorans, Sinorhizobium meliloti, Agrobacterium tumefaciens, Pseudomonas aeruginosa, Xanthomonas campestris, Escherichia coli, Ralstonia solanacearum, Staphylococcus aureus, and Vibrio cholerae) indicated that the gene organization observed in the referred chromosomal region of the gellan-producing strain is distinct. However, orthologues of S. elodea ATCC 31461 ORF 1 and ORF 2 appear together in different bacterial species (e.g., Escherichia coli, Ralstonia solanacearum, Staphylococcus aureus, Vibrio cholerae, and Bacteroides fragilis) (Fig. 6A). These two genes are involved in the synthesis of UDP-N-acetyl-D-mannosaminuronic acid from UDP-N-acetylglucosamine, required for the incorporation of this activated sugar in the CPS of S. aureus (33, 37) and Bacteroides fragilis (9) and also for the synthesis of the enterobacterial common antigen (2, 28).



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  		FIG. 6. Genetic organization of homologues of S. elodea ATCC 31461 ORF 1 and ORF 2 (A) and ORF 3 and ORF 4 (B) in diverse genomes. Accession numbers are given in parentheses below the strain/plasmid names. nt, nucleotide; str. or str, strain.

 
The deduced amino acid sequence of ORF 3 showed homology with proteins belonging to the UDP-glucose/GDP-mannose dehydrogenase family, and the sequenced region of ORF 4 (623 bp) showed homology with proteins with UDP-glucuronic acid epimerase activity. The presence of ORF 3 close to ORF 4 suggests that the products of these genes may be necessary for the synthesis of UDP-glucuronic acid or GDP-mannuronic acid. In Sinorhizobium meliloti strain 1021, Agrobacterium tumefaciens strain C58, and E. coli serotype O113, the UDP-glucose-6 dehydrogenase and UDP-glucuronic acid epimerase proteins are also encoded by two adjacent genes (rkpK and lpsL, AGR_L_1413 and AGR_L_1415, or ugd and wbnF, respectively) (Fig. 6B). In S. meliloti, these two proteins are involved in the synthesis of UDP-galacturonic acid for the incorporation of galacturonic acid in the CPS of this species (20). In E. coli, WbnF may be involved in the synthesis of UDP-galacturonic acid or UDP-N-acetyl galactosamine, necessary for the incorporation of the respective sugars in the oligosaccharide repeat unit of the E. coli O113 O antigen (32).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we carried out a functional analysis of the first gene of the four-gene cluster of S. elodea ATCC 31461 with homology to other genes demonstrated to be involved in the synthesis of L-rhamnose for incorporation into bacterial exopolysaccharides (6, 21). As predicted, the encoded RmlA protein was demonstrated to exhibit TDP-glucose pyrophosphorylase (or glucose-1-phosphate thymidylyltransferase) activity, thus being involved in the reversible conversion of G1P and dTTP into dTDP-Glc and PPi. S. elodea RmlA was found to recognize both deoxythymidine and deoxyuridine nucleotides as substrates, as described before for RmlA homologues (25, 31, 40). These results are in accordance with previously described results for other RmlA enzymes, which were found to catalyze both the formation of dTDP-Glc and UDP-Glc and pyrophosphorolysis (25, 29, 46). For practical reasons, in the present study, the kinetic constants were determined for the pyrophosphorolysis reaction.

The three-dimensional structures of different enzymes with glucose-1-phosphate thymidylyltransferase activity and of the complexes they form with their substrate(s), products, and inhibitors were determined for Pseudomonas aeruginosa RmlA (5), Escherichia coli RmlA (46), and Salmonella enterica LT2 RmlA (3). Studies with P. aeruginosa RmlA revealed that the arginine residue in conserved domain I, G-X-G-T-R*-X2-P-X-T (R*13 in the RmlA sequence of S. elodea ATCC 31461), is involved in binding the triphosphate group of dTTP (5). It was postulated that the referred conserved domain constitutes at least part of the activator binding site (5, 8, 42), representing a prototypic structure characteristic of the large family of bacterial sugar nucleotidyl transferases (8). The overall conservation of particular residues that were proven to be catalytically important for P. aeruginosa RmlA, specifically, K25, D110, and D225 (5), suggests a common overall three-dimensional structure for dTDP- and UDP-glucose pyrophosphorylase enzymes. According to Blankenfeldt et al. (5), there are three other important conserved residues in the P. aeruginosa RmlA protein (G10, Q82, and G87), all of which form hydrogen bonds with the pyrimidine ring (corresponding to G8, Q80, and G85 in S. elodea RmlA) (the last two residues are not shown in Fig. 2).

In the present work, we also found that UgpG, the previously characterized UDP-glucose pyrophosphorylase involved in UDP-glucose formation (reference 26 and our more recent results), also exhibits in vitro dTDP-glucose pyrophosphorylase activity. Similarly, the UGP and RmlA enzymes from E. coli (4, 40), S. enterica serovar Typhimurium (31), and S. enterica (25) also exhibit these two activities. Interestingly, the affinities of UgpG for both substrates are similar (Km of 7.5 µM for UDP-Glc and Km of 9.4 µM for dTDP-Glc), and the Vmax values are also of the same level. The affinities of UgpG for both substrates are also above the affinities estimated for other UGP and RmlA enzymes previously characterized (4, 25, 31, 40). Taken together, the results of the in vitro pyrophosphorolysis assays indicate that these two enzymes, RmlA and UgpG, putatively involved in the gellan biosynthetic pathway, can use both UDP-Glc and dTDP-Glc, at least in vitro. Nevertheless, when plasmid pRmlA was introduced into the UDP-glucose pyrophosphorylase (GalU) mutant strain E. coli FF4001, the ability of the mutant to grow on galactose was not restored (results not shown), which was different from earlier conclusions made when UgpG was expressed from plasmid pUgpG in the same genetic background with the galU mutation (26). This test monitored the capacity of the recombinant cells to ferment galactose, which requires the expression of UGP activity. These observations suggest that, despite the similarities of the biochemical characteristics examined by the in vitro pyrophosphorolysis assays, RmlA and UgpG proteins are not redundant in vivo.

In order to obtain further insight into the role of UgpG in S. elodea, we sequenced the genes in the vicinity of the ugpG gene. Interestingly, we found that the genetic organization of the overall region has no homologous regions in other prokaryotes, not even in N. aromaticivorans, which is phylogenetically close to S. elodea. Nevertheless, the four genes in the chromosomal region surrounding the ugpG gene were found in other organisms, organized in genetic clusters involved in the synthesis of capsular polysaccharides (9) or teichuronic acids (1, 10, 14) in gram-positive bacteria and lipopolysaccharides (2, 28, 33, 37) in gram-negative bacteria. This observation raised the question of the putative involvement of this genomic region in the biosynthesis of a cell wall polysaccharide in S. elodea. Little is known about the structure of the cell wall of bacteria belonging to the genus Sphingomonas, but it was demonstrated that it does not contain the LPS characteristic of gram-negative bacteria in the outer membrane. Interestingly, this LPS is replaced by two types of glycosphingolipids composed of glucuronic acid (GLS-1) or glucuronic acid, galactosamine, galactose, and mannose in the glycosyl portion (GLS-4) (18, 19). However, no evidence was obtained in this work to support the hypothetical involvement of the chromosomal region where ugpG maps in the synthesis of these or other cell glycans.


   ACKNOWLEDGMENTS
 
This work was supported by FEDER and Fundação para a Ciência e a Tecnologia (FCT), Portugal (grants PRAXIS/P/BIO/12020/1998, POCTI/BIO/35733/2000, POCTI/BME/44441/2002, POCTI/BIO/58401/2004, and Ph.D. and postdoctoral grants to E. Silva, A. T. Granja, and A. R. Marques).


   FOOTNOTES
 
* Corresponding author. Mailing address: Biological Sciences Research Group, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal. Phone: 351-21-8417684. Fax: 351-21-8419199. E-mail: afialho{at}ist.utl.pt. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, August 2005, p. 4703-4712, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4703-4712.2005
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