Glycobiology Advance Access originally published online on January 3, 2007
Glycobiology 2007 17(4):433-443; doi:10.1093/glycob/cwl084
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The dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene is part of the surface layer glycoprotein glycosylation gene cluster of Geobacillus tepidamans GS5-97T
3 Zentrum für NanoBiotechnologie
4 Department für Chemie, Universität für Bodenkultur Wien, A-1190 Wien, Austria
1 To whom correspondence should be addressed; Tel: +43-1-47654-2202; Fax: +43-1-4789112; e-mail: paul.messner{at}boku.ac.at
Received on November 13, 2006; revised on December 22, 2006; accepted on December 24, 2006
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Abstract |
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 Top Abstract IntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The glycan chain of the S-layer protein of Geobacillus tepidamans GS5-97T consists of disaccharide repeating units composed of L-rhamnose and D-fucose, the latter being a rare constituent of prokaryotic glycoconjugates. Although biosynthesis of nucleotide-activated L-rhamnose is well established, D-fucose biosynthesis is less investigated. The conversion of
-D-glucose-1-phosphate into thymidine diphosphate (dTDP)-4-dehydro-6-deoxyglucose by the sequential action of RmlA (glucose-1-phosphate thymidylyltransferase) and RmlB (dTDP-glucose-4,6-dehydratase) is shared between the dTDP-D-fucose and the dTDP-L-rhamnose biosynthesis pathway. This key intermediate is processed by the dTDP-4-dehydro-6-deoxyglucose reductase Fcd to form dTDP--D-fucose. We identified the fcd gene in G. tepidamans GS5-97T by chromosome walking and performed functional characterization of the recombinant 308-amino acid enzyme. The in vitro activity of the enzymatic cascade (RmlB and Fcd) was monitored by high-performance liquid chromatography and the reaction product was confirmed by 1H and 13C nuclear magnetic resonance spectroscopy. This is the first characterization of the dTDP--D-fucopyranose biosynthesis pathway in a Gram-positive organism. fcd was identified as 1 of 20 open reading frames contained in a 17471-bp S-layer glycosylation (slg) gene cluster on the chromosome of G. tepidamans GS5-97T. The sgtA structural gene is located immediately upstream of the slg gene cluster with an intergenic region of 247 nucleotides. By comparison of the SgtA amino acid sequence with the known glycosylation pattern of the S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a, two out of the proposed three glycosylation sites on SgtA could be identified by electrospray ionization quadrupole-time-of-flight mass spectrometry to be at positions Ser-792 and Thr-583. Key words: surface layer glycoprotein / fcd / dTDP-4-dehydro-6-deoxyglucose reductase / glycosylation gene cluster / Geobacillus tepidamans
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Introduction |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Many prokaryotic cells of the domains Bacteria and Archaea are completely covered by regularly arrayed cell surface layers (S-layers) (Sleytr et al. 1993). Glycosylation is a common posttranslational modification of these S-layer proteins (Messner and Schäffer 2003
). The glycan structures of bacterial S-layer glycoproteins resemble the architecture of lipopolysaccharide (LPS) O-antigens of Gram-negative bacteria (Raetz and Whitfield 2002). They possess a tripartite structure with a variable carbohydrate chain, usually composed of repeating units, a core oligosaccharide, and the glycosidic linkage of the glycan to the S-layer polypeptide. In Gram-positive bacteria, the common target sites of glycosylation are serine, threonine, or tyrosine residues of particular S-layer proteins (Schäffer and Messner 2004). The genetic information of the enzymes involved in S-layer glycan biosynthesis is organized in S-layer glycosylation (slg) gene clusters (Novotny, Pfoestl et al. 2004). Polycistronic slg gene clusters generally comprise nucleotide sugar pathway genes that are often consecutively arranged, glycosyltransferase genes, glycan processing genes, transporter genes, and a ligase. The recently affiliated Geobacillus tepidamans GS5-97T is a Gram-positive, moderately thermophilic organism that possesses a S-layer glycoprotein with disaccharide repeating units (Figure 1) consisting of L-rhamnopyranose and D-fucopyranose residues (Kählig et al. 2005).
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So far, D-fucose has rarely been described as a constituent of prokaryotic glycans. An example is Actinobacillus actinomycetemcomitans Y4 serotype b that has a capsular polysaccharide-like antigen with the same glycan components as G. tepidamans GS5-97T, namely L-rhamnopyranose and D-fucopyranose (Yoshida et al. 1998
, 1999). In Escherichia coli O52, the O-antigen constituents are D-fucofuranose and 6-deoxy-D-manno-heptopyranose (Feng et al. 2004). Exopolysaccharides of Caulobacter crescentus contain D-fucose amongst other monosaccharides (Ravenscroft et al. 1991). In addition to these Gram-negative organisms, the Gram-positive bacterium Eubacterium saburreum T15 has an antigenic octasaccharide with D-fucofuranose as constituent (Sato et al. 2003). Some antibiotic substances contain D-fucose and methyl ethers of D-fucose (Flowers 1981); for instance, the oligosaccharide antibiotic avilamycin A produced by Streptomyces viridochromogenes Tü57 possesses a heptasaccharide side chain with a 4-O-methyl-D-fucose moiety (Flowers 1981; Weitnauer et al. 2004). Apart from prokaryotes, D-fucose has been isolated from a number of cardiac glycosides in plants (Flowers 1981). An example of an animal source of D-fucose is the spider venom of Tegenaria agrestis (Taggi et al. 2004).
Although the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, is well established in various bacterial systems (Giraud and Naismith 2000), the biosynthesis of dTDP-D-fucose has been characterized only in A. actinomycetemcomitans Y4, serotype b (Yoshida et al. 1999). Nucleotide sugar anabolism for both sugars follows successive activation reactions, with the first steps, catalyzed by glucose-1-phosphate thymidylyltransferase RmlA and thymidine diphosphate (dTDP)-glucose 4,6-dehydratase RmlB, in common. Subsequently, the biosynthesis of the precursors branches off. While the pathway for dTDP-L-rhamnose biosynthesis is completed by the reactions catalyzed by the dTDP-4-dehydrorhamnose 3,5-epimerase RmlC and the dTDP-4-dehydrorhamnose reductase RmlD, the final step of dTDP-D-fucose biosynthesis is catalyzed by the dTDP-4-dehydro-6-deoxyglucose reductase Fcd (Figure 2 and Table I; Shibaev 1986; Yoshida et al. 1999).
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Because of the rare occurrence of D-fucose in prokaryotic glycans and the inconsistency of information available in data bases concerning the dTDP-4-dehydro-6-deoxyglucose reductase Fcd, it was necessary to identify the fcd gene and functionally characterize the encoded reductase in G. tepidamans GS5-97T as a prerequisite to perform carbohydrate engineering based on the S-layer glycan of that organism (Sleytr et al. 1999
; Schäffer and Messner 2004). In the present study, the fcd gene was identified as part of the slg gene cluster of that organism. Cloning, overexpression, and functional analysis of the enzyme provides the first confirmation of the dTDP-4-dehydro-6-deoxyglucose reductase Fcd in a Gram-positive organism. This finding indicates that the biosynthesis of dTDP-D-fucose follows a conserved route in different organisms. |
Results and discussion |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Identification of the S-layer structural gene and its glycosylation cluster
On the basis of the known S-layer glycan structure of G. tepidamans GS5-97T (Kählig et al. 2005
) and on the assumption that the rml genes, encoding the four enzymes responsible for dTDP-L-rhamnose biosynthesis, are part of the chromosomal slg gene cluster of this organism, the alignment of several rmlB genes was chosen for the design of primers. These considerations resulted in the wobble primer pair wRmlB for (5'-GTNACNGGNGGNGCNGGNTTYATHGG-3'), derived from the amino acid sequence VTGGAGFIG, including the conserved Wierenga motif at the N-terminal part of the enzyme, and wRmlB_rev (5'-TGHTANGGNCCRTARTTRTT-3'), derived from the amino acid sequence NNYGPY. The 500 bp fragment of rmlB obtained after polymerase chain reaction (PCR) was sequenced and used for further up- and down-stream sequencing by chromosome walking. This yielded a part of the slg gene cluster of about 9600 bp, containing the rml genes in the order rmlACBD.
Identification of the S-layer structural gene sgtA on the chromosome of G. tepidamans GS5-97T was achieved by alignment of peptide sequences, derived from tandem mass spectrometry analyses of peptides that originated from different proteolytic digests of the S-layer protein SgtA of G. tepidamans GS5-97T, with the sequence of the homologous S-layer protein SgsE of G. stearothermophilus NRS 2004/3a (GenBank accession number AF328862
[GenBank]
). Several conserved regions have been identified (Steiner 2006). These peptide sequences were used for designing the degenerate primers wsgtA_for2 (5'-CAYACNGTNACNAAYACNGG-3' - MS peptide: HTVTNTG) and wsgtA_rev7 (5'-GCRTCNARRTTYTGNGTRAANG-3' - MS peptide: TFTQNDLA), which were then applied in a wobble primer-based PCR experiment. To complete the sgtA sequence, further sequencing steps were carried out by chromosome walking. The resulting sequence of the sgtA precursor gene including the promoter region comprising 3541 bp was verified with a second sequencing step and deposited at GenBank with the accession number AY883421
[GenBank]
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At that stage the sequence of the still incomplete slg gene cluster sequence of G. tepidamans GS5-97T showed high homology to the slg gene cluster of G. stearothermophilus NRS 2004/3a. Furthermore, the slg gene cluster of the latter organism is located directly downstream of the S-layer gene sgsE on the chromosome (Novotny, Pfoestl et al. 2004; Novotny, Schäffer et al. 2004). On the basis of the assumed analogy of slg gene cluster organization of the two Geobacillus species, we used a forward primer in the sequence of the S-layer gene sgtA (sgtAseq_for4 5'-CTATGCTTATTGGAAAGGGG-3') and a reverse primer (GS5_revA 5'-TCCATCTCTCAACTCCTGTTC-3') in the slg sequence upstream of the rmlA gene to complete the sequence of the slg gene cluster of G. tepidamans GS5-97T. The PCR fragment obtained by using the Expand Long Template PCR System was approximately 8000 bp long and closed the gap between sgtA and the known part of the slg gene cluster. The final sequence comprises 21167 bp including the S-layer structural gene sgtA and 20 open reading frames (ORFs) encoding proteins, presumably involved in S-layer glycan biosynthesis (this sequence has been deposited at GenBank with the accession number AY883421
[GenBank]
). We named the genes of the slg gene cluster ws** followed by a species-specific third letter to confirm the designation to the bacterial polysaccharide gene nomenclature (Reeves et al. 1996; BPGD database, http://www.microbio.usyd.edu.au/BPGD). These results are summarized in Table II.
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The average G + C content of the genome of G. tepidamans GS5-97T is 43.2%, the G + C content of the S-layer protein SgtA is 38.0% and the average G + C content of the slg gene cluster yields approximately 33.9%. This observation may be taken as an indication for lateral gene transfer of the slg gene cluster from an organism with lower G + C content into G. tepidamans GS5-97T (Keenleyside and Whitfield 1999
).
Identification of the gene coding for the dTDP-4-dehydro-6-deoxyglucose reductase
The conserved domain search of all 20 translated ORFs in the slg gene cluster of G. tepidamans GS5-97T revealed that there is only one protein encoded (WsbK) besides the RmlACBD enzymes, which is supposed to be part of the NAD(P)H dependent epimerase/dehydratase family (pfam01370) (Marchler-Bauer and Bryant 2004). This protein family utilises NAD(P)H as a cofactor and uses nucleotide-sugar substrates for a variety of chemical reactions. Basic local alignment search tool (BLAST)p alignment (Altschul et al. 1997) of WsbK showed homology (35% identities/53% positives) with Fcf1, a putative dTDP-4-dehydro-6-deoxyglucose reductase of E. coli (Feng et al. 2004) (GenBank accession number AAS99161
[GenBank]
.1). Unexpectedly, no hit could be observed with either ORF14 of A. actinomycetemcomitans Y4, serotype b (Yoshida et al. 1999), which is the only dTDP-4-dehydro-6-deoxyglucose reductase encoding gene (GenBank accession number of protein BAA19641
[GenBank]
.1) with characterized function, deposited so far, or with the GenBank entry annotated fcd (GenBank accession number of protein AAG49410
[GenBank]
.1), which is part of a gene cluster of A. actinomycetemcomitans CU1000, serotype f (Kaplan et al. 2001). The latter organism, however, does not contain D-fucose in the glycan chain.
The ORF wsbK codes for a protein of 308 amino acids. As part of the SDR (short-chain dehydrogenases/reductases) family the CDS translation of wsbK shows motifs described for other reductases also involved in the biosynthesis of nucleotide activated 6-deoxy sugars, such as dTDP-4-dehydrorhamnose reductase RmlD (Graninger et al. 1999) or GDP-6-deoxy-D-lyxo-4-hexulose reductase Rmd (Kneidinger, Graninger, Adam et al. 2001). These motifs are a slightly modified Wierenga motif G8XGXXG for coenzyme binding, motif 2 S114AGTVY and the extended motif 3 PXXXY139XXXK143XXXE, where the serine residue of motif 2 and the tyrosine and lysine of motif 3 are likely to build up the catalytic triad (Figure 3). The function of the catalytic triad is the protonation or deprotonation of the substrate's carbonyl or hydroxyl group (Blankenfeldt et al. 2002).
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On the basis of this theoretical approach, it is conceivable to assume that WsbK is the dTDP-4-dehydro-6-deoxyglucose reductase Fcd. WsbK and additionally RmlB, as enzyme for the conversion of dTDP-D-glucose into dTDP-4-dehydro-6-deoxy-D-glucose which is the key intermediate in the biosynthesis of dTDP-L-rhamnose and dTDP-D-fucose, were cloned, and heterologously overexpressed in E. coli. The recombinant enzymes were purified by application of Ni2+ affinity chromatography recovering the proteins with 40% elution buffer. The in vitro enzyme assay was performed with dTDP-D-glucose as substrate and NAD+ and NADH as cofactors for the enzymes RmlB and WsbK, respectively. The conversion of dTDP-D-glucose to dTDP-D-fucose was monitored by HPLC (Figure 4A). Because there is no dTDP-D-fucose standard substance commercially available, we established an alternative method for analyzing the reaction product prior to nuclear magnetic resonance (NMR) spectroscopy. After hydrolysis of the product with 2.2 M trifluoroacetic acid (TFA) at 110 °C for 4 h the released monosaccharide was analyzed by high-performance anion-exchange chromatography (HPAEC). Thus, fucose could be determined unambiguously (Figure 4B). The product of the enzyme assay was desalted and its structure was verified by NMR analysis. Ten milligram of dTDP-D-glucose were converted into 7.2 mg of dTDP-D-fucose, which corresponds to a yield of approximately 70%.
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NMR results
The 300 MHz 1H NMR spectrum of the sample indicated the presence of a homogeneous single component in addition to residual Tris-buffer (Figure 5), whereas the 31P signals at – 10.13 and – 11.61 ppm were consistent with the assignment of the sugar diphospho nucleoside. On the basis of homo- and hetero-nuclear experiments [1H/13C heteronuclear multiple-quantum coherence (HMQC)- and 1H/13C heteronuclear multiple-bond coherence (HMBC)-measurements] all proton and carbon signals could be unambiguously assigned (Table III). Furthermore, the signals of the 2'-deoxythymidine could be readily distinguished from those of the sugar moiety and were in full accordance with the literature data (Naundorf and Klaffke 1996
). The structural assignment of the reaction product as nucleotide-activated D-fucopyranose was deduced from the 1H and 13C NMR signals of the 6-deoxy unit (1.21 and 16.25 ppm, respectively), whereas the galacto-configuration of the pyranose ring was evident from the small homonuclear coupling constant J4,5 and J4,5 (less than 1.0 and 3.3 Hz, respectively) and the unchanged -anomeric configuration was established from the coupling constant J1,2 (3.7 Hz).
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slg Gene cluster comparison
The comparison of the slg gene clusters of G. tepidamans GS5-97T and G. stearothermophilus NRS 2004/3a (Novotny, Pfoestl et al. 2004; Novotny, Schäffer et al. 2004) revealed that the clusters are organized in a similar way (Figure 6). The first three ORFs downstream of the S-layer gene include highly homologous genes coding for a putative ligase and a putative rhamnosyltransferase and are followed by the ABC-2 transporter encoding genes wzm and wzt. The subsequent sequence segments are different. In G. tepidamans GS5-97T, there are five ORFs including four putative glycosyltransferase genes and the dTDP-4-dehydro-6-deoxyglucose reductase gene fcd. This segment is terminated by three transposases and one small ORF with unknown function. In G. stearothermophilus NRS 2004/3a, only two ORFs in that region of the gene cluster are found, a putative methyltransferase gene and a putative glycosyltransferase gene. Downstream of these variable parts of the slg gene clusters, the rmlACBD genes, a putative rhamnosyltransferase gene and the putative UDP-galactose lipid carrier transferase gene, are located. These parts of both gene clusters have the highest homology (up to 96% identity).
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The glycoproteins of both organisms possess an extended tripartite structure consisting of a similar core and linkage region and a variable glycan chain (Schäffer and Messner 2004
). It seems that the variable part in the centre of the slg gene clusters is responsible for the biosynthesis of the individual repeating units and terminating elements, whereas the region with higher homology codes for proteins involved in assembling the core region, transport of the glycan to the cell surface, and its ligation to the S-layer protein. This resembles the organization of O-antigen gene clusters in Gram-negative organisms, where the variability of O-antigens is considered as recombination events in the central region of the O-antigen gene clusters (Wang et al. 1992; Kaplan et al. 2001).
S-layer glycoprotein SgtA and its glycosylation sites
The ORF sgtA codes for a protein of 901 amino acids including a signal peptide of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 92 302 Da and a theoretical isoelectric point of 5.38. The observation of four distinct bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) can be explained by the presence of different glycoforms in addition to nonglycosylated S-layer protein SgtA (Kählig et al. 2005). For determination of the accurate mass of the three S-layer glycoprotein species of G. tepidamans GS5-97T, the isolated S-layer protein was analyzed by infrared matrix-assisted laser desorption/ionization orthogonal-time-of-flight mass spectrometry (IR-MALDI-oTOF MS). Four well resolved peaks could be obtained (data not shown). The molecular masses of the peaks were determined to be 94.1, 101.3, 108.5, and 115.7 kDa, by using the positively charged molecular ions. The average mass differences between two neighbouring peaks of the singly charged ions were calculated to be 7.2 kDa, which corresponds to the average mass of a glycan chain composed of 21 repeating units, implicating the presence of three putative glycosylation sites. The first peak (94.1 kDa) would originate from nonglycosylated S-layer protein. On different S-layer protomers one, two, or all of the three proposed glycosylation sites are randomly glycosylated resulting in the second (101.3 kDa), third (108.5 kDa), and fourth peak (115.7 kDa), respectively. This corresponds to monoglycosylated, diglycosylated, and triglycosylated SgtA, respectively.
The target amino acids of glycosylation are threonine and serine residues of the S-layer protein SgtA. In a recent NMR analysis of the glycan chain structure of the S-layer glycoprotein of G. tepidamans GS5-97T, three short peptide sequences of putative protein glycosylation sites (SAD, TQN and TA) have been determined (Kählig et al. 2005). Only the SAD site is present once throughout the sequence of SgtA, TQN occurs twice in the sequence of SgtA, and TA even 8 times. Four of the putative TA-glycosylation sites could be proven to be nonglycosylated in the material of the highest mass band, where all glycosylation sites are supposed to be glycosylated. The glycosylation site Ser-792 of SgtA corresponds to Ser-794 in SgsE (Schäffer et al. 2002). The neighbouring amino acid sequence is conserved between the two proteins. Additionally, one of the putative TQN glycosylation sites (Thr-583) of SgtA is located in a region, which is highly homologous to the sequence of SgsE containing the glycosylation site Thr-590 (Steiner et al. 2006; Figure 7). The sequence in vicinity of Thr-620 of SgsE is not as conserved as for the other glycosylation sites and none of the putative glycosylation sites of SgtA could be aligned to this sequence. The exact positions of the threonine glycosylation sites (especially the TA sites) could not be identified, because no glycopeptides with longer peptide sequences were available at the time of the analysis.
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Conclusions |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The ORF named wsbK codes for the dTDP-4-dehydro-6-deoxyglucose reductase Fcd in the slg gene cluster of G. tepidamans GS5-97T. The rmlACBD genes for the biosynthesis of dTDP-D-rhamnose are found in the downstream region (G + C content, 38.6%) of the cluster, whereas the fcd gene is located in the central region, displaying the lowest G + C content (29.5%) within the gene cluster. The G + C content of the organism is 43.2%. Unique to the slg gene cluster of G. tepidamans GS5-97T, the fcd gene and the other four ORFs wsbG-wsbJ of the central region are supposed to have arisen from one or more gene transfer events. The assumption is substantiated by the fact that the central region of the slg gene cluster is separated from the rml genes by three transposases (Figure 6A).
It can be assumed that a putative dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene is part of a nucleotide sugar biosynthesis cluster in various organisms. The O-antigen gene cluster of E. coli O52 (GenBank accession number AY528413
[GenBank]
) contains a putative dTDP-D-fucofuranose cluster starting at the proximal (5'-) end of the cluster including the successively arranged genes rmlB (putative RmlB, AAS99159
[GenBank]
.1), rmlA (putative RmlA, AAS160.1), fcf1 (putative Fcd, AAS99161
[GenBank]
.1), and fcf2, coding for a putative mutase (AAS99162
[GenBank]
.1) (Feng et al., 2004). Genes required for the biosynthesis of the antibiotic avilamycin A produced by Streptomyces viridochromogenes Tü57 are clustered in a avi biosynthetic gene cluster (GenBank accession number AF333038
[GenBank]
). In the middle of the gene cluster, four genes are successively arranged, namely aviD (putative RmlA, AAK83195
[GenBank]
.1), aviE1 (putative RmlB, AAK83196
[GenBank]
.1), aviQ2 (putative Fcd, AAK83179
[GenBank]
.1), and aviG that is responsible for the methylation of the hydroxyl group at position 4 of the D-fucose moiety (proved function, AAK83180
[GenBank]
.1), which, at the current state of knowledge, we interpret as a putative dTDP-4-O-methyl-D-fucose cluster within the avi biosynthetic gene cluster (Weitnauer et al. 2004).
The GenBank entries concerning the dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene show some inconsistencies. The only annotated dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene (CDS AF213680
[GenBank]
.2: nt 8425-8817), which is part of a gene cluster of A. actinomycetemcomitans CU1000, serotype f, is described as a protein with two different functions. In addition to the putative reductase activity this small protein (130 amino acids) shows similarity to the GtrA-like protein family, but the accurate function remains unknown. It is unlikely that this protein possesses reductase activity, because all required SDR protein family motifs are missing and the polysaccharide antigen of that organism does not contain D-fucose. Therefore, we suggest withdrawing the annotation dTDP-4-dehydro-6-deoxyglucose reductase. ORF14 of A. actinomycetemcomitans Y4, serotype b (Yoshida et al. 1999), which was the first dTDP-4-dehydro-6-deoxyglucose reductase encoding gene with characterized function (CDS AB002668
[GenBank]
.1: nt 15271-15963), is still designated as unnamed protein product. We suggest renaming ORF14 of that organism as dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene.
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Materials and methods |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Materials
dTDP-D-glucose, NAD+ and NADH were purchased from Sigma (Vienna, Austria), isopropyl-1-thio-ß-D-galactopyranoside from Fermentas (St. Leon-Rot, Germany). HisTrapRHP ready-to-use and Sephadex G-10 columns were obtained from GE Healthcare (Vienna, Austria). All primers were synthesized by Invitrogen (Lofer, Austria).
Bacterial strains and culture conditions
G. tepidamans GS5-97T (ATCC BAA-942T, DSM 16325T) was grown in SVIII culture medium at 57 °C and 150 rpm (Kählig et al. 2005). E. coli DH5 (K12 F–
80d lacZ
M15 endA1 recA1 hsdR17 (rK–mK–) supE44 thi-1 gyrA96relA1 (lacZYA-argF) U169) (Invitrogen) was used for cloning. Enzyme overexpression was done in E. coli BL21(DE3) [F– ompT hsdSB (rB–mB–) gal dcm (DE3)]. For selective growth Luria–Bertani (LB) medium was supplemented with ampicillin (Sigma) or kanamycin (Invitrogen) at a concentration of 50 µg/mL each, when appropriate.
DNA manipulation, PCR, and DNA sequencing
All standard DNA manipulation and transformation procedures were performed according either to Sambrook and Russell (2001) or the manufacturer's protocols. The identification of the S-layer gene and the slg gene cluster was accomplished by both, PCR using wobble primers including the following steps: initial dissociation (94 °C for 120 s), 30 amplification cycles (94 °C for 30 s, 47 °C for 45 s. and 72 °C for 120 s), final extension (72 °C for 420 s) and chromosome walking as described elsewhere (Kneidinger, Graninger, Messner 2001). Taq DNA polymerase, Pwo DNA polymerase and the Expand Long Template PCR System (Roche, Mannheim, Germany) were used for PCR. PCR was performed in a PCR Sprint thermocycler from Hybaid (Ashford, UK). PCR products were eluted from agarose gels using the Min Elute Kit (Qiagen, Hilden, Germany) and DNA sequencing was done by AGOWA (Berlin, Germany). The resulting nucleotide sequences were managed with OMIGAR (Accelrys, Cambridge, UK). Homology searches, conserved domain searches and sequence alignments were done with the BLAST tools (Altschul et al. 1997) and CD-search (Marchler-Bauer and Bryant 2004) at NCBI and Multalin (Corpet 1988), respectively.
Plasmid construction
The ORFs of rmlB (coding for dTDP-D-glucose 4,6-dehydratase, RmlB) and wsbK (coding for dTDP-4-dehydro-6-deoxyglucose reductase, Fcd) were amplified by PCR. The oligonucleotide primers were designed with attB1 and attB2 sites for the recombination of the PCR product into the donor vector pDONR221 of the GatewayR Technology (Invitrogen). The following primers were used for wsbK: gORFgt110_fwd3 5'-attb1-gcgaaggagatagaacc ATGAAAAGAATTCTTATACTAGGC-3'; ORFgt110_rev3 5'-attb2-tttaCATCATATTCTCTCCGTTCC-3'; and rmlB: grmlBgt_for1 5'-attb1-gcgaaggagatagaaccATGCAGATGAAAGTATTGATTAC-3'; grmlBgt_rev1 5'-attb2-tttaACCTAACTGCCCATTTG-3'. After recombination into the donor vector pDONR221 and transformation into E. coli DH5, entry clones were obtained. To generate expression clones, the genes were transferred in a second recombination step from the donor vector pDONR221 into the destination vector pDEST17, which provides a hexahistidine tag for N-terminal fusion to the protein. The corresponding plasmids gORFgt110_5 and gRMLB_6 were used for overexpression of the hexahistidine tagged proteins in E. coli BL21(DE3). Obtained plasmid constructs were confirmed by sequencing (AGOWA).
Overexpression and purification of recombinant proteins
After inoculation of 400 mL of LB medium, containing 50 µg/mL ampicillin, with 2 mL of an overnight culture of E. coli BL21(DE3) carrying the plasmids gORFgt110_5 or gRMLB_6, cells were grown at 37 °C to an OD600 of 0.6. Overexpression of the recombinant proteins was induced with 1 mM IPTG for 3 h. The biomass was collected by centrifugation at 5000 rpm at 4 °C for 20 min.
The cells were resuspended in the binding buffer for Ni2+ affinity chromatography (20 mM sodium phosphate, 0.5 M NaCl, and 5 mM imidazole pH 7.4) and disrupted on ice by ultrasonication. Cell debris was removed by ultracentrifugation at 60 000 rpm at 4 °C for 40 min using a Ti 70.1 rotor in a Beckman LE-80 ultracentrifuge. The supernatant was immediately applied to a HisTrapR HP column for performing Ni2+ affinity chromatography. Elution was done by a stepwise gradient with 10, 20, 40, and 60% elution buffer (20 mM sodium phosphate, 0.5 M NaCl, and 0.5 M imidazole pH 7.4). The fractions containing the desired proteins were dialyzed against 50 mM Tris/HCl pH 7.5, followed by a concentration step with centrifugal filter devices (cutoff 10 000 Da; Millipore, Vienna, Austria). Proteins were stored at a concentration of 5.3 mg/mL (RmlB) and 0.9 mg/mL (Fcd). Storage conditions were 4 °C, with addition of 1 mM 1,4-dithio-DL-threitol, and –20 °C, with addition of 50% glycerol, respectively. Protein purity was checked by SDS-PAGE and protein concentration was determined by the Bio-Rad protein assay (Bio-Rad, Richmond, CA).
Enzyme assay
The conversion of dTDP-D-glucose into dTDP-D-fucose using the purified enzymes RmlB and Fcd was assayed using the following conditions: 150 µL of reaction mixture containing 50 mM Tris/HCl (pH 7.0), 10 mM MgCl2, 100 nmol of dTDP-D-glucose as substrate and 100 nmol NAD+ and NADH as cofactors were incubated at 37 °C for 2 h. Enzymes (1–10 µg per assay) were added either simultaneously or successively.
For confirmation of the reaction product by NMR analysis, the assay was scaled up, using 10 mg of dTDP-D-glucose, the product was desalted on a Sephadex G10 column (100 x 1 cm) and the pooled fractions were lyophilized.
Analytical techniques—HPLC
To identify the nucleotide-activated monosaccharides dTDP-D-glucose, dTDP-4-dehydro-6-deoxy-D-glucose, and dTDP-D-fucose, the reverse phase-HPLC method of Martin et al. (1989) was used with slight modifications. Briefly, the method comprises an isocratic elution on a reverse phase column (Nucleosil 120-3 C18, Macherey-Nagl, Düren, Germany) with 0.4 M KH2PO4, pH 6.0 as elution buffer, a flow rate of 0.6 mL/min, and UV detection at 267 nm and was performed on a Summit HPLC System (Dionex, Sunnyvale, CA). To identify the sugar moiety of the reaction product, hydrolysis with 2.2 M TFA at 110 °C for 4 h was performed and the released monosaccharide was analyzed by HPAEC (Dionex, Sunnyvale, CA; Lee 1990).
Analytical techniques—mass spectrometry
S-layer glycoprotein SgtA was isolated and purified according to standard procedures (Messner et al. 1995; Kählig et al. 2005). IR-MALDI-oTOF MS of S-layer glycoproteins was performed as described elsewhere (Steiner et al. 2006).
For sequencing of the S-layer protein routinely, 200 pmol of S-layer protein were treated with 0.2 µg of trypsin, chymotrypsin, endoproteinase Asp-N, or pepsin (Roche) in a final volume of 20 µL of 10 mM NH4HCO3 (trypsin and chymotrypsin) or 50 mM NaH2PO4 (AspN) or 10 mM NH4Ac (pH 3.8, pepsin). Digestion was carried out at 37 °C for 18–36 h. Peptides were desalted prior to mass spectrometric analysis on ZipTip clean-up columns (Millipore, Eschborn, Germany) according to the manufacturer's instructions. For tryptic and chymotryptic degradation also in-capillary digestion was performed (Pohlentz et al. 2005). Positive ion mode electrospray-ionization quadrupole TOF (ESI-QTOF) MS of the peptides was carried out using an ESI-QTOF instrument (Micromass, Manchester, UK). Capillary voltage applied on an internal wire electrode was 1100 V, the cone voltage used for peptide analysis was 40 V. Peptide sequencing was conducted using argon as collision gas. The applied collision energy was 15–40 V. The samples were dissolved in water yielding a concentration of the stock solution of 20 pmol/µL. Four microlitre of that solution were mixed with 5 µL of methanol and 1 µL of 10% formic acid. The final concentration of peptides was 8 pmol/µL.
Analytical techniques—NMR spectroscopy
Approximately 2 mg of the purified reaction product was lyophilized and taken up in D2O prior to NMR analysis. Spectra were recorded at 300 K at 300.13 MHz for 1H, at 75.47 MHz for 13C and at 121.49 MHz for 31P with a Bruker AVANCE 300 spectrometer equipped with a 5 mm quadrupole nuclear probehead with z-gradients. Data acquisition and processing were performed with the standard XWINNMR software (Bruker, Rheinstetten, Germany). 1H spectra were referenced internally to 2,2-dimethyl-2-silapentane-5-sulfonic acid (
= 0), 13C spectra were referenced externally to 1,4-dioxane ( = 67.40), and 31P spectra were referenced externally to phosphoric acid ( = 0). 1H/13C HMQC- and 1H/13C HMBC-spectra were recorded in the phase-sensitive mode using time-proportional phase increments and pulsed field gradients for coherence selection.
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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We would like to thank Dr René Novotny (Austrian Research Center, Seibersdorf, Austria) for discussion of the molecular biology work and Dr Gottfried Pohlentz and Dr Klaus Dreisewerd (Institute of Medical Physics and Biophysics, University of Münster, Germany) for help with the MS analysis. Financial support came from the Austrian Science Fund, projects P18013 [GenBank] -B10 (to P.M.) and P19047 [GenBank] -B12 (to C.S.) and the Federal Ministry of Science and Research.
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Footnotes |
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2 Present address: Sonn & Partner, Patent attorneys, Riemergasse 14, A-1010 Wien, Austria
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Abbreviations |
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BLAST, basic local alignment search tool; dTDP, thymidine diphosphate; ESI-QTOF, electrospray-ionization quadrupole time-of-flight; HMBC, heteronuclear multiple bond coherence; HMQC, heteronuclear multiple-quantum coherence; HPAEC, high-performance anion-exchange chromatography; HPLC, high performance liquid chromatography; IR-MALDI-oTOF MS, infrared matrix-assisted laser desorption/ionization orthogonal-TOF mass spectrometry; LB, Luria-Bertani; LPS, lipopolysaccharide; NAD, nicotinamide adenine dinucleotide; NMR, nuclear magnetic resonance; ORF, open reading frame; PCR, polymerase chain reaction; SDR, short-chain dehydrogenases/reductases.; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; slg, S-layer, glycosylation; TFA, trifluoroacetic acid.
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References |
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