Functional characterization of GumK, a membrane-associated ß-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis

Máximo Barreras, Patricia L. Abdian and Luis Ielpi1

Fundación Instituto Leloir, University of Buenos Aires and CONICET, Av. Patricias Argentinas 435, (C1405BWE)Buenos Aires, Argentina

Received on August 25, 2003; accepted on November 16, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Xanthomonas campestris is a Gram-negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely used in the industry as thickener and viscosifier. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components have not been examined and no data on glycosyltransferases have been reported. We describe the functional characterization of the gumK gene product, an essential protein for xanthan synthesis. Immunoblots and complementation studies showed that GumK is a 44-kDa protein associated to the membrane fraction. This value corresponds to the expected molecular mass for GumK encoded by an extended open reading frame than proposed from previous genetic data and in X. campestris published complete genome. The protein was expressed in Escherichia coli cells. The purified protein catalyzed the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-{alpha}-1,3-glucose-ß-1,4-glucose-P-P-polyisoprenyl with formation of a glucuronic acid-ß-mannose linkage. We examined the acceptor substrate specificity. GumK was unable to use the trisaccharide acceptor freed from the pyrophosphate lipid moiety. Replacement of the natural lipid moiety by phytanyl showed that the catalytic function could proceed with glucuronic acid transfer. These results suggest the enzyme does not show specificity for the lipidic portion of the acceptor. GumK showed diminished activity when tested with 6-O-acetyl-mannose-{alpha}-1,3-glucose-ß-1,4-glucose-P-P-polyisoprenyl, a putative intermediate in the synthesis of xanthan. This could indicate that acetylation of the internal mannose takes place after the formation of the GumK product.

Key words: glucuronosyltransferase / glycosyltransferase / polysaccharide / xanthan / Xanthomonas campestris


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Extracelular polysaccharides are major secreted products in many bacteria. There is a considerable interest in the molecular aspects of these compounds because they are involved in human and plant pathogenicity. In addition, some of them display unique physical properties useful for industrial applications (Sutherland and Tait, 1992Go). Most exopolysaccharides consist of polymerized oligosaccharide repeating units. Each repeating unit is assembled as glycoside-P-P-polyisoprenyl in a sequential series of reactions performed by specific glycosyltransferases (GTs).

The plant pathogen Xanthomonas campestris produces an exopolysaccharide known as xanthan gum. Xanthan is widely used as thickener or viscosifier in both food and nonfood industries, among many other applications (Becker et al., 1998Go; Harding et al., 1995Go). Xanthan is a branched acidic heteropolysaccharide composed of polymerized pentasaccharide repeating units containing glucose (Glc), mannose (Man), and glucuronic acid (GlcA) (see Scheme I). The internal and external mannoses of xanthan are substituted with variable proportions of 6-O-acetyl and 4,6-ketal pyruvate residues, respectively. A model for the biosynthesis of xanthan has been proposed. It mainly consists of the stepwise assembly and decoration of pentasaccharide units attached to a polyisoprenyl phosphate carrier, which are subsequently polymerized and exported (Ielpi et al., 1993Go). Pyruvic acid and acetal residues are transferred from phosphoenolpyruvate and acetyl–coenzyme A (Ac-CoA) before the polymerization of the repetitive unit. Classical genetic experiments indicated that a 14-kb X. campestris genome region named xpsI or gum encodes the proteins required for these processes (Harding et al., 1987Go; Vanderslice et al., 1988Go). This region is composed of 12 genes designated gumB to gumM (GenBank accession number U22511). Based on the analysis of the intermediate biosynthetic products produced by several gum mutants, it was possible to assign functions to several of the gum genes required for xanthan synthesis (Katzen et al., 1998Go; Vanderslice et al., 1988Go). Genes gumD, gumM, gumH, gumK, and gumI were suggested to encode specific GTs required for pentasaccharide unit assembly. However, to our knowledge no data on characterization of xanthan specific GTs are available.



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Scheme I. Structure of xanthan. Repetitive unit.

 
In this study we report the cloning and biochemical characterization of GumK from X. campestris (GenBank accession number AY170889). We provide direct evidence that GumK is the protein responsible for the transfer of a glucuronic acid residue from UDP-GlcA to Man-{alpha}-1,3-Glc-ß-1, 4-Glc-P-P-polyisoprenyl (Man-Cel-P-P-lipid), with inversion of the anomeric configuration. The reaction can be written as seen in Scheme II. We also show that this protein is encoded by an extended open reading frame (ORF) than was initially predicted and is located in X. campestris membrane fraction. Finally, we study the acceptor requirements of GumK, including the lipid moiety and the effect of 6-O-acetyl substitution on the mannose residue.



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Scheme II. Reaction catalyzed by GumK.

 

    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Analysis of gumK gene by functional complementation
Katzen et al. (1998)Go have shown that gumK mutant accumulates Man-Cel-P-P-lipid and is unable to produce glucuronic acid-ß-1,2-mannose-{alpha}-1,3-glucose-ß-1,4-glucose-P-P-polyisoprenyl (GlcA-Man-Cel-P-P-lipid). To confirm that the proposed GumK protein catalyzes the glucuronidation of Man-Cel-P-P-lipid, a complementation experiment with XcK mutant (gumK) carrying plasmid pBBRK-s (containing the postulated gumK ORF) was carried out. Xanthan biosynthesis in XcK strain is indicative of the restoration of the glucuronosyltransferase activity. Thus the exopolysaccharide was isolated and quantified from stationary-phase cultures of wild-type FC2 (as positive control) and XcK mutant carrying pBBRK-s. Surprisingly, XcK/pBBRK-s resulted in more than 95% decrease in the yield of xanthan compared to wild-type strain FC2 (Figure 1A), meaning that xanthan production was not restored in mutant cells carrying this plasmid.



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Fig. 1. (A) Relative xanthan production. The polymer formed was measured in X. campestris parental strain and in XcK mutant harboring pBBRK, pBBRK-s, or pBBRprom by the cetylpyridinium chloride method. Bars are ± the standard deviation from at least three independent experiments. (B) Partial map and genetic organization of the X. campestris gum operon. Nucleotide coordinates refer to the gum operon (GenBank accession number U22511). The arrows indicate the potential coding regions, including flanking ORFs gumJ and gumL as described in Katzen et al. (1998)Go. The 5' regions of gumK and gumK-s ORFs are presented showing the first translated amino acids. The potential ribosome binding sites are underscored. Both ORFs were amplified by PCR and cloned in pET29a(+), pET22b(+), and pBBRprom vectors generating pET29K, pET29K-s, pET22HisKC, pBBRK, and pBBRK-s.

 
By reexamining the published nucleotide sequence around gumK, it became evident that by using an earlier in frame ATG, gumK could be extended 315 bp at its 5' end. The name gumK was maintained for this extended ORF (GenBank accession number AY170889) and the original version of gumK will be referred to as gumK-s. A Shine-Dalgarno-like sequence was found eight nucleotides upstream the new putative initiation codon (Figure 1B).

The 1203-bp gumK gene was cloned in pBBRprom, and the resulting plasmid, pBBRK, was introduced into XcK. In this case we observed that xanthan synthesis was restored to wild-type levels (Figure 1A).

GumK is expressed in X. campestris from an extended ORF and is a membrane protein
The deduced amino acidic sequence derived from the original protein GumK-s is shorter than that of GumK protein (295 residues, 32.3 kDa versus 400 residues, 44.3 kDa). Western blot analysis using GumK-specific antiserum was used to establish the apparent molecular mass and the subcellular location of the expressed protein in the wild-type strain. A unique protein with an apparent molecular mass of 44 kDa was detected predominantly in the membrane fraction of wild-type strain FC2 carrying no plasmids (Figure 2, lane 1). This value corresponds to the predicted translation product of gumK gene. The protein was absent in the gum operon–deleted mutant Xc1231 and in the gene-specific mutant XcK (Figure 2, lanes 9–12), indicating that the 44-kDa protein detected corresponds to protein GumK. GumK was also mainly detected in membranes in Xc1231/pBBRK (Figure 2, lanes 3–4), as well as in XcK/pBBRK (Figure 2, lanes 5–6). Protein GumK-s was expressed from pBBRK-s as a ~32-kDa protein in XcK/pBBRK-s (Figure 2, lanes 7–8), showing a similar localization as that of GumK. These results clearly show that the 44-kDa protein is encoded from the 1203-bp gumK gene and shows substantial membrane localization, although this is not absolutely complete, a feature seen with other bacterial GTs (Videira et al., 2001Go).



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Fig. 2. A 1203-bp extended gene encodes GumK, a protein that is localized to the membrane. Western blot of proteins of membrane (M) and soluble (S) fractions from X. campestris strains. Lanes 1–2, FC2; 3–4, Xc1231/pBBRK; 5–6, XcK/pBBRK; 7–8 XcK/pBBRK-s; 9–10, Xc1231; and 11–12, XcK. Immunodetection was performed with mice anti-GumK polyclonal antibodies. The open arrows indicate the positions of the GumK and GumK-s proteins at ~44 kDa and ~32 kDa, respectively.

 
Production and purification of His-GumK
The 1203-bp gumK gene was expressed as His tag fusion protein. A prominent band of about 45 kDa for His-GumK was present in the isopropyl-ß-D-thiogalactopyranoside (IPTG)-induced Escherichia coli BL21(DE3)/pET22HisKC cell extracts (Figure 3, lanes 1–3). Subcellular localization of this recombinant protein by serial centrifugation revealed that His-GumK was found exclusively in 2,000 and 5,000 x g pellets and had no detectable enzymatic activity, suggesting the formation of inclusion bodies. Considering active protein is required for biochemical studies, we used milder induction conditions (IPTG 0.5 mM, 18°C) and extraction with detergent. The best optical density (OD600) for induction was 0.7, and the optimal expression was found at 15–18 h after IPTG addition. Escherichia coli BL21(DE3)/pET22HisKC disrupted cells were incubated with 1% Triton X-100, fractionated by centrifugation, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Recombinant GumK protein was mainly detected in the 13,000 x g supernatant (Figure 3, lanes 4–5), showing that a significant fraction of the protein was not forming inclusion bodies. His-GumK was purified by immobilized metal-affinity chromatography and gel filtration (Figure 3, lane 6).



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Fig. 3. SDS–PAGE analysis of proteins from recombinant E. coli showing purification of GumK. Lane 1: Soluble proteins from BL21(DE3)/pET22b after IPTG induction (negative control). Lane 2: Soluble proteins from uninduced BL21(DE3)/pET22HisKC. Lane 3: Soluble proteins from BL21(DE3)/pET22HisKC after IPTG induction. Lane 4: 13,000 x g pellet of BL21(DE3)/pET22HisKC disrupted cells after 1% Triton X-100 incubation. Lane 5: 13,000 x g supernatant of BL21(DE3)/pET22HisKC disrupted cells after 1% Triton X-100 incubation. Lane 6: Purified His-GumK protein eluted from the Superdex 200 size exclusion column. The open arrow indicates the position of the His-GumK protein at ~45 kDa.

 
The gumK gene product is a glucuronosyltransferase
In vitro analyses were performed with purified GumK to unequivocally determine the activity of this protein. The enzymatic activity of recombinant His-GumK was investigated in a cell-free assay incubating it with its predicted substrates (Harding et al., 1993Go; Ielpi et al., 1993Go). As a source of glycolipid acceptor, we used X. campestris extract enriched in Man-Cel-P-P-lipid. Permeabilized X. campestris cells incubated with UDP-Glc and GDP-Man as sugar donors produce mainly Man-Cel-P-P-lipid. A detailed analysis of Man-Cel-P-P-lipid has been previously described (Ielpi et al., 1993Go).

Glycolipid acceptor and UDP-[14C]GlcA were incubated in the presence of GumK. The oligosaccharides were released from the polyisoprenyl anchor into the aqueous phase by mild acid hydrolysis, indicating that the sugars are bound to the lipid moiety by a pyrophosphate bridge (Couso et al., 1982Go). The released [14C]oligosaccharides were analyzed by thin-layer chromatography (TLC). A major radioactive product that comigrates with standard GlcA-Man-Cel was recovered (Figure 4A, lane 1), suggesting that the lipid-linked carbohydrate is a GlcA-containing tetrasaccharide. To confirm the identity of the molecule acting as acceptor in the glycolipid extract, [14C]Man-Cel-P-P-lipid was obtained and incubated with unlabeled UDP-GlcA and GumK as before. The radioactive material was processed and analyzed by TLC. In the absence of GumK, unmodified [14C]Man-Cel was detected (Figure 4A, lane 3). In contrast, a newly formed tetrasaccharide comigrating with standard GlcA-[14C]Man-Cel was found when GumK was present (Figure 4A, lane 2). It is worth mentioning that under the conditions of the experiment all the acceptor was converted into product. These results show that GumK catalyzes the transfer of a glucuronic acid residue from UDP-GlcA to Man-Cel-P-P-lipid to render GlcA-Man-Cel-P-P-lipid.



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Fig. 4. GumK is a ß-glucuronosyltransferase. (A) TLC analysis of oligosaccharides produced after in vitro incubations carried out as follows: lane 1, Man-Cel-P-P-lipid, UDP[14C]GlcA, and GumK; lane 2, [14C]Man-Cel-P-P-lipid, UDP-GlcA, and GumK; lane 3, [14C]Man-Cel-P-P-lipid, UDP-GlcA without GumK as a negative control. [14C]Man-labeled Man-Cel, GlcA-Man-Cel, and Man-GlcA-Man-Cel were used as standards (lane ST). (B) Anomeric configuration analysis. TLC analysis of released oligosaccharides after ß-glucuronidase treatment. GlcA-[14C]Man-Cel obtained employing GumK, before (lane 1) and after (lane 2) ß-glucuronidase treatment. [14C]GlcA-Man-Cel obtained employing GumK before (lane 3) and after (lane 4) ß-glucuronidase treatment. GlcA-[14C]Man-Cel obtained from FC2 cells before (lane 5) and after (lane 6) ß-glucuronidase treatment. [14C]GlcA-Man-Cel obtained from FC2 cells before (lane 7) and after (lane 8) ß-glucuronidase treatment. [14C]Man-labeled Man-Cel, GlcA-Man-Cel, and [14C]GlcA (lanes ST) were used as standards. TLCs were run in the direction indicated by the arrows.

 
To test whether GumK-s retained any enzymatic activity detectable in the cell-free assay, we overexpressed GumK-s and GumK in E. coli BL21 (DE3). Protein overexpression and activity assays were conducted as described in Materials and methods. We found no radioactivity incorporation in the organic phase when the 100,000 x g pellet containing GumK-s was incubated with UDP-[14C]GlcA and glycolipid acceptor, indicating that this protein has no detectable glucuronosyltransferase activity. On the other hand, the 100,000 x g pellet containing GumK showed glucuronosyltransferase activity with the formation of GlcA-Man-Cel-P-P-lipid as detected by TLC (data not shown). These results agree with our previous studies on functional complementation and protein expression, confirming that the glucuronosyltransferase activity is carried out by the 400-amino-acid protein. For all further experimentation, purified His-GumK was used.

Characterization of the glucuronosyl linkage
We have used liver ß-glucuronidase to determine the stereochemistry of the GlcA linkage formed by GumK. The tetrasaccharide obtained using either a radioactive donor or a radioactive acceptor was digested with liver ß-glucuronidase. As shown in Figure 4B the [14C]Man-labeled GlcA-Man-Cel released a [14C]Man-trisaccharide comigrating with standard [14C]Man-Cel (lanes 1–2), whereas the [14C]GlcA-labeled GlcA-Man-Cel released free [14C]GlcA (lanes 3–4). The authentic GlcA-Man-Cel-P-P-lipid obtained from wild-type FC2 is also sensitive to liver ß-glucuronidase (Figure 4B, lanes 5–8). These results are consistent with a ß-glucuronosyl-glycosidic linkage. Taken together these results indicate that GumK is a ß-glucuronosyltransferase.

Acceptor specificity of GumK
The specificity of GumK was investigated to elucidate the structural requirements for the acceptor. Data are shown in Figure 5. First we analyzed whether GumK could achieve the transfer of a glucuronic acid residue on a P-P-lipid free acceptor. [14C]Man-Cel obtained by mild acid hydrolysis treatment was incubated with GumK. No product formation was detected by TLC (Figure 5A, lane 1), suggesting that the pyrophosphate bridge and/or the lipid moiety is indispensable for the glucuronic acid transfer reaction.



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Fig. 5. Acceptor specificity analysis of GumK. (A, B) Contribution of pyrophosphate bridge and lipid moiety to the reaction. TLC analysis of oligosaccharides produced after incubation with (A) P-P-polyprenyl-free acceptor or (B) Man-Cel-P-P-phytanyl. (A) Incubations were carried out using: lane 1, [14C]Man-Cel, UDP-GlcA, and GumK; lane 2, [14C]Man-Cel, UDP-GlcA without GumK as a negative control; lane 3, [14C]Man-Cel-P-P-lipid, UDP-GlcA, and GumK as a positive control. (B) Incubation was carried out using Man-Cel-P-P-phytanyl, UDP-[14C]GlcA, and GumK (lane 1). (C) Effect of the O-acetylation on GumK activity. (Ac)Man-Cel-P-P-lipid acceptor was prepared and incubated as described in Materials and methods. Lane 1, labeled mixture of (Ac)[14C]Man-Cel-P-P-lipid and [14C]Man-Cel-P-P-lipid, UDP-GlcA, and GumK; lane 2, [14C]Man glycolipid mixture used as acceptor in lanes 1 and 3; lane 3, labeled mixture of (Ac)[14C]Man-Cel-P-P-lipid and [14C]Man-Cel-P-P-lipid, UDP-GlcA incubated in the absence of GumK as negative control. Lane 4, [14C]Man-Cel-P-P-lipid, UDP-GlcA, and GumK, as a positive control. Lane 5, unlabeled mixture of (Ac)Man-Cel-P-P-lipid and Man-Cel-P-P-lipid, UDP-[14C]GlcA, and GumK. [14C]Man-labeled Man-Cel, GlcA-Man-Cel, (Ac)Man-Cel, and GlcA-(Ac)Man-Cel were used as standards (lane ST). TLCs were run in the direction indicated by the arrows.

 
To further elucidate the contribution of the lipid moiety to the recognition process and based on the reported results that phytanyl can satisfactory replace the natural polyisoprenyl portion (Lellouch et al., 2000Go; Watt et al., 1997Go), we studied the capability of GumK to transfer glucuronic acid to Man-Cel-P-P-phytanyl. Man-Cel-P-P-phytanyl was prepared by transferring mannose from GDP-Man to Cel-P-P-phytanyl in a reaction catalyzed by the mannosyltransferase AceA from Acetobacter xylinum (Lellouch et al., 2000Go) (Materials and methods). Figure 5B shows that the phytanyl derivative can function as acceptor of a GlcA residue as detected by TLC of the reaction products.

The internal mannose residue of xanthan repeating units is substituted with O-acetyl residues. We have previously described that Ac-CoA is the donor and Man-Cel-P-P-lipid as well as mannose-ß-1,4-glucuronic acid-ß-1,2-mannose-{alpha}-1, 3-glucose-ß-1,4-glucose-P-P-polyisoprenyl (Man-GlcA-Man-Cel-P-P-lipid) function as acceptors forming (Ac)Man-Cel-P-P-lipid and Man-GlcA-(Ac)Man-Cel-P-P-lipid, respectively (Ielpi et al., 1983Go; Katzen et al., 1998Go). One point to clarify was whether GumK could accept (Ac)Man-Cel-P-P-lipid as substrate. (Ac)Man-Cel-P-P-lipid was prepared adding Ac-CoA in addition to UDP-Glc and GDP-Man to the incubation mixture. About 60% of Man-Cel-P-P-lipid acceptor produced was transformed into the (Ac)Man-Cel-P-P-lipid acceptor as determined by densitometry (Figure 5C, lane 2). A mixture of (Ac)Man-Cel-P-P-lipid plus Man-Cel-P-P-lipid was incubated with UDP-GlcA and GumK with either the acceptor mixture or the sugar nucleotide [14C]-labeled. The incubation carried out with [14C]Man-labeled mixture showed that ~10% of (Ac)Man-Cel-P-P-lipid was transformed to GlcA-(Ac)Man-Cel-P-P-lipid, and ~60% of Man-Cel-P-P-lipid is transformed to GlcA-Man-Cel-P-P-lipid (Figure 5C, lane 1). This result is clearly different from that regularly observed, where 100% of Man-Cel-P-P-lipid is transformed to GlcA-Man-Cel-P-P-lipid (Figure 5C, lane 4; see also Figure 4A). The incubation carried out in the presence of UDP-[14C]GlcA produced 10% of GlcA-(Ac)Man-Cel-P-P-lipid (Figure 5C, lane 5). These findings show that (Ac)Man-Cel-P-P-lipid is slowing the rate of reaction and, on the other hand, there clearly exists a marked preference of GumK for the nonacetylated acceptor.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
GumK is the first GT from the X. campestris gum operon whose biochemical properties have been characterized in cell-free assays. According to our previous report (Katzen et al., 1998Go), the putative protein responsible for the ß-glucuronosyltransferase activity in the biosynthesis of xanthan repetitive unit was coded by an 888-bp ORF, giving a 295-amino-acid protein named GumK. The results shown here indicate that the 400-amino-acid extended protein carries out this activity. It is worth mentioning that the ORF coding for the extended protein is present in the closely related Xylella fastidiosa and Xanthomonas axonopodis pv. citri, judging from its published genomic sequences (Da Silva et al., 2002Go; Simpson et al., 2000Go).

We have previously shown that the transcription of the gum genes is essentially directed by a single promoter mapped upstream of the gumB gene. A second alternative promoter was also found upstream of gumK-s ORF (Katzen et al., 1996Go). This second promoter is located inside the gumK gene, specifically in its 5' region. To address the possibility that this alternative promoter could play a role in regulating the synthesis of xanthan, we decided to express GumK and GumK-s in X. campestris wild-type strain FC2. Strains bearing the truncated or the complete gumK gene produced xanthan at the same level as the wild-type parental strain (data not shown). Although this result suggests that under our conditions the presence of GumK-s does not interfere with a function essential for xanthan production, a regulating role for this promoter cannot be ruled out.

GTs are extremely diverse in terms of functionality and sequence (Breton et al., 1999). A classification for GTs was created (Coutinho and Henrissat, 1999Go) based on sequence homology, donor/product stereochemistry and donor sugar identity. The number of identified and characterized GTs has increased over the past 5 years. This has resulted in an increment of the number of different GTs families. This classification currently comprises 65 families, and the number keeps growing (Coutinho and Henrissat, 1999Go). Surprisingly, from structural data it has turned out that GTs belong to only two different structural folds, named GT-A and GT-B (Bourne and Henrissat, 2001Go; Unligil and Rini, 2000Go). Both folds include retaining and inverting enzymes, forming four clans (Coutinho et al., 2003Go). In terms of comparison, GT-A enzymes show a conserved DXD motif involved in divalent cation binding essential for catalysis. GT-B enzymes do not display the DXD motif; neither do they depend on metal ions for catalysis (Hu and Walker, 2002Go). GumK is a GT that can transfer a sugar moiety in the absence of divalent cations (unpublished data from our laboratory). Given this, we could presume that it belongs to the GT-B fold. In any case, if we take into account the sequence homology to other GTs, GumK does not belong to any family in CAZy's classification. Based on the biochemical characterization reported in this work and the fact that this protein does not show significant sequence homology to any other GT, we propose that GumK should be included in CAZy's classification as a nonclassified GT.

In this study we have used western blot analysis to determine the subcellular location of GumK. Our results show that this protein is predominantly located in the membrane fraction of X. campestris. Many bacterial GTs are found to be integral membrane proteins; others, like GumK, are membrane associated despite the lack of predicted transmembrane regions. For these, the nature of the membrane–protein interaction is not clearly understood. It has been suggested that a patch of residues in the N-domain of MurG protein may be involved in electrostatic as well as hydrophobic interactions with the membrane (Ha et al., 2000Go), or else C-terminal paired basic residues may interact with phospholipids in the inner membrane, as described for the GTs responsible for the biosynthesis of Neisseria meningitidis lipooligosaccharides (Wakarchuk et al., 1998Go). Another possibility is the formation of putative complexes among membrane-bound and cytoplasmic proteins involved in the biosynthesis of polysaccharides, as has previously been suggested (Hodson et al., 2000Go; Rigg et al., 1998Go; Whitfield and Roberts, 1999Go). Our results suggest that association of GumK to the membrane seems not to require the presence of the rest of the gum operon proteins. This is shown by the fact that GumK is located to membranes in the gum operon–deleted strain Xc1231 expressing GumK protein (Figure 2).

We observed that GumK is incapable of transferring a GlcA residue to a polyisoprenyl-pyrophosphate free acceptor. This result is in agreement with earlier reports on other GTs (Geremia et al., 1999Go; Lellouch and Geremia, 1999Go). The structural bases for this requirement are not clearly understood. In the case of MurG (a GT-B fold protein) it has been proposed that two rich glycine loops at the N-terminal, variants of the consensus sequence GXGXXG found in dinucleotide binding proteins, would be responsible for the recognition of the pyrophosphate in the P-P-lipid moiety of the acceptor substrate (Ha et al., 2000Go). The amino acidic sequence for these rich G-loops or its variants are not present in GumK; neither are they present in another characterized bacterial glucuronosyltransferase, GelK from Sphingomonas paucimobilis (Videira et al., 2001Go). The question of how phosphates and the lipid portion of the acceptor are recognized by GumK is still unanswered. To address this and other questions, structural data on GumK alone and in complex with substrates will be of great value. We are currently carrying out crystallization assays to reach this goal.

Many exopolysaccharides from Gram-negative bacteria are assembled from polyisoprenyl-pyrophosphate-linked units (Sutherland and Tait, 1992Go). In the few cases analyzed, the lipid portion turned out to be a polyisoprenol, specifically undecaprenol, a C55 molecule. In the case of xanthan the exact chain length of the polyisoprenyl unit is not known. Interestingly, the chain length of the lipid moiety does not seem to be a stringent feature for recognition. The substitution of the natural lipid moiety for (C20) phytanyl to give Man-Cel-P-P-phytanyl renders an acceptor recognized by the enzyme. Furthermore, kinetic analyses using different lipid chain lengths for E. coli GT MurG acceptor substrate showed that the enzyme recognizes a lipid chain as short as 10 carbon atoms (Chen et al., 2002Go). The minimum chain length for the lipid portion to be recognized, as well as the requirement of a pyrophosphate bridge in the acceptor, awaits further studies.

The availability of heterologous expressed GumK gave us the opportunity to examine in detail the incorporation of acetyl groups in xanthan biosynthesis. The synthesis of (Ac)Man-Cel-P-P-lipid in permeabilized cells suggested that it could be a putative acceptor substrate for GumK (Katzen et al., 1998Go). The product of this reaction GlcA-(Ac)Man-Cel-P-P-lipid would be transformed in Man-GlcA-(Ac)Man-Cel-P-P-lipid. However, we found that in vitro GumK is barely capable of using (Ac)Man-Cel-P-P-lipid as an acceptor. Figure 5C shows that the amount of GlcA-(Ac)-Man-Cel-P-P-lipid produced represents only 10% of the amount of acetylated acceptor present in the reaction. This is far lower than the rate of product formation we observed when we used nonacetylated acceptor. Moreover, the presence of the acetylated derivative seems to exert a negative effect on GumK activity, where only 60% of Man-Cel-P-P-lipid is transformed to GlcA-Man-Cel-P-P-lipid.

The study of the kinetics of GumK to understand the preference toward the nonacetylated acceptor is in progress. Considering that xanthan production is a very efficient process, this finding suggests either that the reactions catalyzed by GumK and GumF (the enzyme responsible for the addition of the acetyl group to mannose) take place in different compartments, GumK and GumF belongs to different protein complexes, or else the activity of GumF employing Man-Cel-P-P-lipid as substrate is negatively regulated by an unknown mechanism.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials and general procedures
UDP-Glc, GDP-Man, UDP-GlcA, Ac-CoA, and bovine liver ß-glucuronidase were purchased from Sigma (St. Louis, MO). GDP-[U-14C]Man (280 Ci/mol) and UDP-[U-14C]GlcA (310 Ci/mol) were prepared as previously described (Ielpi et al., 1993Go). Proteins were analyzed by SDS–PAGE in 10% gels followed either by Coomassie blue staining or western blot (Sambrook et al., 1989Go). Anti-GumK polyclonal antibodies were obtained from mice immunized with GumK inclusion bodies. Mice immunization was carried out according to established protocols (Harlow and Lane, 1988Go). Cross-reactive antibodies were removed by absorption with E. coli BL21(DE3)/pET29a(+) and Xc1231 cell extracts. Anti-mouse IgG alkaline phosphatase–conjugated antibodies (Sigma) were used as secondary antibody, and detection was performed with BCIP/NBT color development substrate (Promega, Madison, WI).

Bacterial strains, media, and plasmids
E. coli DH5{alpha} was used for DNA subcloning and E. coli BL21(DE3) for protein expression. E. coli cells were grown in Luria broth medium (Sambrook et al., 1989Go) in a shaker at 200 rpm, 37°C, unless otherwise indicated. X. campestris FC2 (wild type) (Katzen et al., 1998Go)], XcK (gumK) (Katzen et al., 1998Go) and Xc1231 ({Delta}gum) (Capage et al., 1987Go) cells were grown in TY medium (tryptone 5 g/L, yeast extract 3 g/L, and CaCl2 0.7 g/L) or in modified XOL medium (K2HPO4 4 mM, KH2PO4 1.5 mM, (NH4)2SO4 1 g/L, FeSO4 0.01 g/L, MnCl2 5 mM, MgCl2 0.5 mM, triptone 1.25 g/L, yeast extract 1.25 g/L, glucose 4 g/L) in a shaker at 200 rpm, 28°C. Antibiotics were used at the following concentrations when required: for E. coli: kanamicyn, 30 µg/ml; gentamicin, 10 µg/ml; ampicillin 200 µg/ml; for X. campestris: kanamicyn, 50 µg/ml; gentamicin, 30 µg/ml.

Plasmids were constructed as follows: (i) pBBRK-s. The original version of gumK gene (accession number U22511), referred to in this paper as gumK-s, was amplified by polymerase chain reaction (PCR) from pCHC3 (Harding et al., 1987Go) using specific oligonucleotides FGKCS (5'-GGAAGACCATATGTTCCGCTGGTATG-3' [NdeI site underscored]) and BGKC (5'-CGCGGATCCCTCCTCAATGTGAGAGCGCTGCC-3' [BamHI site underscored]), and digested with NdeI and BamHI. The 902-bp fragment was ligated to the corresponding sites of the broad host-range vector pBBRprom (pBBR1MCS-5 [Kovach et al., 1995Go] with gum operon promoter region inserted into MCS) (Ielmini, unpublished data) to yield plasmid pBBRK-s. (ii) pBBRK. The extended ORF for gumK (accession number AY170889), for which we keep the name gumK, extending 315 bp upstream +1 position of gumK-s, was amplified from pCHC3 using specific oligonucleotides FGKLS (5'-GACACGGCATATGAGCGTCTCTC-3' [NdeI site underscored]) and BGKC. The 1217-bp PCR product was digested with NdeI and BamHI and ligated to the corresponding sites of pBBRprom to yield plasmid pBBRK. (iii) pET22HisKC. gumK ORF was amplified from pCHC3 using specific oligonucleotides FGKL (5'-CACGGCCCATGGGCGTCTCTC-3' [NcoI site underscored]) and BHisKC (5'-CCGCTCGAGATGTGAGAGCGCTGC-3' [XhoI site underscored]). The 1216-bp PCR product was digested with NcoI and XhoI and ligated to the corresponding sites of pET22b(+) (Novagen) expression vector to yield plasmid pET22HisKC. This plasmid encodes a GumK fusion protein carrying a LE(H)6 C-terminal sequence. (iv) pET29K and pET29K-s. The NdeI-BamHI amplified DNA fragments for gumK-s and gumK ORFs were cloned in pET29a(+) (Novagen, Madison, WI) to obtain pET29K-s and pET29K, respectively. All cloned fragments were sequenced to ensure fidelity in DNA sequences encoding GumK.

Quantification of xanthan production
X. campestris wild type and mutant strains were inoculated in XOL modified medium and incubated for 72 h. Xanthan was precipitated by the cetylpyridinium chloride polysaccharide precipitation method (Scott, 1965Go) with modifications. Briefly, 20 µl of culture supernatants containing xanthan and xanthan standard aqueous solutions of 0.75%, 1.5%, 2.25%, 3%, and 3.75% were mixed with 3 ml deionized water. Three milliliters of 0.36% cetylpyridinium chloride solution were added, and the mixture was incubated at 30°C for 1 h. This mixture was centrifuged at 5000 x g to settle the cetylpyridinium chloride/xanthan precipitate. The 260 nm absorbance of supernatants from xanthan standards were used to construct a calibration curve. The amount of polysaccharide produced in the samples was estimated from its A260 referring to the calibration curve.

Cell fractionation and protein detection
X. campestris cells were grown in TY medium to stationary phase. Cells were collected by centrifugation, washed twice with water, and resuspended at 50 OD equivalents/ml in resuspension buffer (Tris–HCl 70 mM pH 8.2, MgCl2 10 mM). Cells were broken by three passages through a French pressure cell at 20,000 psi. Unbroken cells were removed by centrifugation at 2000 x g, 4°C, for 10 min. The lysate was subjected to ultracentrifugation at 100,000 x g, 4°C, for 2 h to separate total membrane fraction from soluble fraction. Total membrane fraction was solubilized in resuspension buffer plus SDS 4% in an equivalent volume to the soluble fraction. Both fractions were solubilized with 2x denaturing buffer (urea 10 M, SDS 3%, dithiothreitol 0.4 M, Tris–HCl 100 mM, pH 8.8), subjected to SDS–PAGE, and transferred to polivinildifluoride membranes for western blot.

Production of GumK-s and GumK
For expression of GumK and GumK-s, 200 ml Luria broth media were inoculated with E. coli BL21(DE3) carrying pET29K or pET29K-s and incubated in a rotary shaker at 150 rpm, 37°C. At an OD600 of 0.8, 10 µM IPTG was added, and incubations were continued at 18°C for additional 15–18 h. Cells were centrifuged; washed twice in Tris–HCl 70 mM, pH 8.2; and resuspended in the same buffer at 50 OD equivalents/ml. Resuspended cells were disrupted by two passages in a French pressure cell at 20,000 psi. Cell extracts were subsequently centrifuged at 2000 and 5000 x g to remove unbroken cells and cellular debris and finally at 100,000 x g to separate total membranes from the soluble fraction. Each centrifugation pellet was resuspended in Tris–HCl 70 mM, pH 8.2. The 100,000 x g pellet was used as the source of enzyme for activity assays.

Purification of His-GumK
For purification of His-GumK, 1 L of E. coli BL21(DE3)/pET22HisKC cells at an OD600 of 0.7 were induced with 0.5 mM IPTG and incubated at 18°C for additional 15–18 h. Cells were harvested; washed twice in Tris–HCl 70 mM, pH 8.2; resuspended in the same buffer at 50 OD equivalents/ml; and disrupted by two passages in a French pressure cell at 20,000 psi. Solubilization of GumK from membranes and subsequently purification steps were performed according to P.L. Abdian (unpublished data). Briefly, disrupted cells were diluted with buffer Tris–HCl 70 mM, pH 8.2, Triton X-100 1% to a protein concentration of 5 mg/ml, incubated at 4°C for 2 h, and centrifuged at 13,000 x g for 10 min. The supernatant was recovered and dialyzed against column buffer (Tris–HCl 20 mM pH 8, NaCl 0,5 M, imidazole 20 mM, Triton X-100 0,05 %) and filtered through a 0.22 µm filter. The filtrate was applied to Ni-NTA columns (Pharmacia, Uppsala, Sweden) equilibrated with column buffer. Following elution with an imidazole gradient, the protein was concentrated by ultrafiltration and applied to a Superdex 200 size exclusion column. SDS–PAGE profiles of the eluted protein show a single band around 45 kDa (see Figure 3, lane 6), the expected molecular weight of the protein. Purity was estimated to be greater than 95% and the yield of enzyme approximately 7 mg/L of bacterial culture. For activity and acceptor specificity assays, the Superdex 200 eluates were used.

Preparation of glycolipid acceptors
Because the acceptor Man-Cel-P-P-lipid is not commercially available, it was was obtained by incubation of permeabilized X. campestris FC2 cells with UDP-Glc and GDP-Man (or GDP-[14C]Man for radiolabeled acceptor) (acceptor incubation mixture) as previously reported (Ielpi et al., 1993Go). (Ac)Man-Cel-P-P-lipid acceptor was prepared adding 0.7 mM Ac-CoA to the glycolipid acceptor incubation mixture. Under these conditions ~60% of Man-Cel-P-P-lipid is transformed into the acetylated form. The extracts containing the glycolipid acceptor were reduced under air stream to a volume of ~15 µl and used in GumK activity assays.

Man-Cel-P-P-phytanyl was obtained by incubation of Cel-P-P-phytanyl and GDP-Man with A. xylinum mannosyltransferase AceA as reported (Lellouch et al., 2000Go). Briefly, the glycolipid incubation mixture to prepare Man-Cel-P-P-phytanyl contained 0.4 µg of purified AceA (Abdian et al., 2000Go; Geremia et al., 1999Go), 1% Triton X-100, 6 mM MgCl2, 20 µM synthetic acceptor Cel-P-P-phytanyl and 0.25 mM of GDP-Man in a final volume of 100 µl. After incubation at 37°C for 10 min, the reaction was stopped by addition of 200 µl chloroform:methanol (1:1) and 120 µl water. The glycolipid acceptor was recovered in the organic phase after centrifugation at 6000 x g for 5 min and reduced under air stream to ~15 µl (Abdian et al., 2000Go).

GumK activity assays
GumK activity was measured by the transfer of a GlcA residue from UDP-GlcA either into Man-Cel-P-P-lipid or Man-Cel-P-P-phytanyl. The reaction mixture contained 15 µl glycolipid acceptor, 0.5 mM MgCl2, 3% Triton X-100, and 0.15 µCi UDP-[14C]GlcA, 70 mM Tris–HCl, pH 8.2, in a final volume of 100 µl. The reaction was started with the addition of 10 µg His-GumK or 50 µg of 100,000 x g pellet containing either GumK or GumK-s. Reactions were carried out at 20°C for 30 min and stopped by the addition of 200 µl chloroform:methanol (1:1) and 120 µl water. Newly formed lipid-bound oligosaccharides were isolated by organic solvent extraction as previously described (Geremia et al., 1999Go; Lellouch and Geremia, 1999Go). The oligosaccharide moiety was released from the polyisoprenol by mild acid hydrolysis (0.01 N HCl for 10 min at 100°C). Under this conditions, only the phosphate linkages are split, releasing the labeled oligosaccharide from the polyisoprenol. This lipid fraction was extracted with 0.6 ml chloroform:methanol (2:1). The aqueous phase containing the oligosaccharides was completely air-dried and resuspended in 0.5 ml pyridine. Salts were removed by centrifugation, and the supernatant containing the labeled compounds was recovered. A 10% fraction of the pyridine supernatant was counted in a liquid scintillation counter, and the rest was air-dried, resuspended in water, and spotted on a TLC plate. TLC plates were developed twice in 1-propanol:nitromethane:water (5:2:2). Radioactive spots corresponding to the oligosaccharides were visualized and quantified using ImageQuant software in a Storm 820 scanner (Molecular Dynamics).

Preparation of labeled oligosaccharides used as standards
Labeled Man-Cel, (Ac)Man-Cel, and Man-GlcA-Man-Cel were prepared by mild acid treatment of the corresponding polyprenyl-phosphosugars, obtained with X. campestris FC2 ethylenediamine tetra-acetic acid–permeabilized cells as previously described (Ielpi et al., 1993Go). Labeled GlcA-Man-Cel and its acetylated form (GlcA-[Ac]Man-Cel) were prepared by a similar procedure, replacing the wild-type strain with X. campestris XcI mutant. This strain is unable to transfer the external ß-1,4-mannose to the tetrasaccharide intermediate (Katzen et al., 1998Go).

ß-Glucuronidase treatments
Treatments with ß-glucuronidase were performed as described previously (Ielpi et al., 1993Go). Briefly, the enzyme reaction mixture contained 100 mM sodium citrate pH 5.0, 30,000 cpm labeled oligosaccharide, and 0.4 U of bovine liver ß-glucuronidase in a final volume of 50 µl. Incubations were carried out for 8 h at 37°C, and reactions were stopped by adding 1 vol ethanol. Supernatants were desalted as indicated for GumK activity assays and analyzed by TLC using butanol:acetic acid:water (2:1:1) as the mobile phase.


    Acknowledgements
 
We are grateful to Susana Raffo for the preparation of GDP-[14C]Man and GDP-[14C]GlcA, Marta Bravo for technical assistance with high-pressure liquid chromatography, and Jimena Ortega for technical assistance with DNA sequencing. We thank Dr. R. A. Geremia (CERMO, CNRS, Université Joseph Fourier, Grenoble, France) for providing Cel-P-P-phytanyl and M. V. Ielmini (Fundación Instituto Leloir) for plasmid pBBRprom. This work was supported by grant X647-2003 from University of Buenos Aires. P.L.A. is the recipient of a doctoral fellowship from Fundación Antorchas.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: lielpi{at}leloir.org.ar Back


    Abbreviations
 
Ac, acetyl; CoA, coenzyme A; (Ac) Man-Cel, 6-O-acetyl-mannose-{alpha}-1,3-glucose-ß-1,4-glucose; Man-Cel-P-P-lipid, 6-O-acetyl-mannose -{alpha}-1,3-glucose-ß-1,4-glucose-P-P-polyisoprenyl; GlcA-Man-Cel, glucuronic acid-ß-1,2-mannose-{alpha}-1, 3-glucose-ß-1,4-glucose; GT, glycosyltransferase; IPTG, isopropyl-ß-D-thiogalactopyranoside; Man-GlcA-Man-Cel, mannose-ß-1,4-glucuronic acid-ß-1,2-mannose-{alpha}-1, 3-glucose-ß-1,4-glucose; Man-GlcA-Man-Cel-P-P-lipid, mannose-ß-1,4-glucuronic acid-ß-1,2-mannose-{alpha}-1,3-glucose-ß-1,4-glucose-P-P-polyisoprenyl; Man-Cel, mannose-{alpha}-1, 3-glucose-ß-1,4-glucose; Man-Cel-P-P-lipid, mannose-{alpha}-1,3-glucose-ß-1,4-glucose-P-P-polyisoprenyl; OD, optical density; ORF, open reading frame; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrilamide gel electrophoresis; TLC, thin-layer chromatography


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
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