Bacteriology Division, Department of Infectious Diseases and Immunology, Utrecht University, PO Box 80.165, 3508 TD Utrecht, The Netherlands1
Genencor International B. V., 2300 AE Leiden, The Netherlands2
National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands3
Author for correspondence: Jos P. M. van Putten. Tel: +31 30 2534888. Fax: +31 30 2540784. e-mail: j.vanputten{at}vet.uu.nl
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ABSTRACT |
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Keywords: capsular polysaccharide biosynthesis, glucosyltransferase
Abbreviations: CPS, capsular polysaccharide
The GenBank accession number for the sequence reported in this paper is AF402095.
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INTRODUCTION |
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Structurally, the capsular polysaccharide (CPS) of S. pneumoniae consists of a polymer of repeating oligosaccharide units. These basic building blocks are usually composed of two to eight monosaccharides that are linked via glycosidic bonds. Heterogeneity among capsules is largely established through variation in the type and linkage of the different monosaccharides that constitute the repeating unit, and their variable substitution with non-carbohydrate residues (Kamerling, 1999 ). Polymerization of the repeating units results in the high-molecular-mass capsule structure that surrounds the pneumococcus and protects the organism against hostile environments.
For a number of pneumococcal serotypes, the genes that encode the capsule biosynthesis machinery have been identified. The genes are clustered within the chromosome and in all but one serotype (serotype 37) are located between dexB and aliA (Llull et al., 1999 ). At this time the CPS synthesis (cps) loci of 15 serotypes (1, 2, 3, 4, 6B, 8, 14, 18C, 19F, 19A, 19B, 19C, 23F, 33F and 37) have been sequenced (Arrecubieta et al., 1995
; Dillard et al., 1995
; Iannelli et al., 1999
; Jiang et al., 2001
; Kolkman et al., 1997b
; Llull et al., 1998
, 1999
; Morona et al., 1997a
, b
, 1999a
, c
; Munoz et al., 1997
, 1999
; Ramirez & Tomasz, 1998
). Sequence analysis and function predictions suggest that the loci contain the complete repertoire of genes specifically required for capsule biosynthesis and its regulation. In a few cases, the function of a gene has been experimentally confirmed (Arrecubieta et al., 1995
, 1996
; Kolkman et al., 1996
, 1997a
, b
; Morona et al., 1997a
, 2000
; Munoz et al., 1997
).
Knowledge of the composition of the various cps loci and the function of the encoded proteins is important as it provides insight into the dynamics of the capsule biosynthesis pathway and the evolution of serotypes. Genetic exchange between cps loci occurs frequently and can lead to changes in serotype (Barnes et al., 1995 ; Coffey et al., 1991
, 1998a
, b
; Nesin et al., 1998
; Ramirez & Tomasz, 1999
). The recombination events can involve large pieces of DNA (1525 kb) with cross-over points often located outside the cps locus, as well as smaller fragments with exchange between regions of sequence similarity within the various loci (Morona et al., 1999b
). In the present study, we investigated the genetic organization of the cps locus of serotype 9V. This serotype is one out of 12 most commonly isolated serotypes worldwide (Butler, 1997
; Nielsen & Henrichsen, 1992
) and is included in the 7-valent conjugated vaccine (Hausdorff et al., 2000
; Shinefield & Black, 2000
) and the 23-valent polysaccharide vaccine (Robbins et al., 1983
). We have identified and sequenced the complete 9V locus and assigned putative functions to the genes. For one of the genes, its proposed function has been experimentally confirmed.
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METHODS |
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DNA techniques.
Chromosomal DNA was isolated as described by Ausubel et al. (1987) . Long-range PCR was performed with the Expand Long Template PCR system (Roche Diagnostics) using buffer 3, according to the manufacturers instructions. PCR involved a 2 min denaturation at 94 °C, followed by 10 cycles of 30 s denaturation, 30 s annealing with a touchdown from 60 to 55 or 50 °C (depending on the melting temperature of the primers) and 20 min extension at 68 °C, then 30 additional cycles consisting of 30 s denaturation, 30 s annealing at 55 or 50 °C and 20 min extension at 68 °C, lengthened by 20 s every cycle. The primers used to amplify the cps locus of serotype 9V were DEXBPR (5'-CCATGGGATGCTTTCTGTGTG-3') and ALIAST2 (5'-CAAATAGTTGAGGTTATCAGGGTCTGTCTC-3'). PCR products were purified from agarose gels with the Qiaquick gel extraction kit (Qiagen). Other DNA techniques were performed as described by Sambrook et al. (1989)
. Restriction endonucleases and T4 DNA ligase were purchased from Amersham Pharmacia Biotech.
DNA sequencing and analysis.
DNA sequencing was performed on an ABI Prism 310 Genetic Analyser (Applied Biosystems) using the ABI Dye Terminator Cycle Sequencing Kit. DNA and protein data were analysed by using Lasergene software (DNAstar). The algorithm BLAST (Altschul et al., 1997 ) was used to compare sequences at the DNA and deduced amino acid levels to database sequences available at the National Center for Biotechnology Information (NCBI). Transmembrane segment predictions were done using the program DAS (http://au.expasy.org).
Membrane preparations.
Pneumococcal membranes for use in glycosyltransferase assays were prepared by the method of Osborn et al. (1972) with some modifications. Bacteria, grown in 10 ml ToddHewitt broth supplemented with 0·5% yeast extract in 15 ml tubes (15 h, 37 °C, no shaking), diluted 10-fold in 250 ml medium and re-grown to mid-exponential phase (OD600 0·3), were collected by centrifugation (5500 g, 15 min, 4 °C), resuspended in 10 ml Sol1 (0·7 M sucrose, 50 mM Tris/HCl, 1 mM EDTA, pH 8·0), centrifuged again and resuspended in 10 ml Sol1 supplemented with 20 mg lysozyme and 100 U mutanolysin. After 24 h incubation at 4 °C, 15 ml Sol1 was added and the protoplasts were collected by centrifugation and lysed in 20 ml Sol2 (20 mM Tris/HCl, 1 mM EDTA, 200 µM PMSF, pH 8·0). This suspension was sonicated (3x20 s) and stored overnight at -80 °C. Debris was removed by centrifugation (5500 g, 20 min, 4 °C) and membranes were collected by high-speed centrifugation (30000 g, 1 h, 4 °C). Membranes were washed once in 20 ml Sol3 (50 mM Tris/acetate, 1 mM EDTA, 200 µM PMSF, pH 8·3), centrifuged again and finally resuspended in 0·5 ml Sol3. Membrane preparations could be stored at -80 °C for a few months without apparent loss of enzyme activity.
Glycosyltransferase activity assays.
Glycosyltransferase activity was essentially determined as described previously (Kolkman et al., 1996 ). For each reaction, 40 µl membrane preparations was incubated (1 h, 10 °C) with 0·025 µCi uridine diphospho-[14C]glucose (Amersham Pharmacia) and 10 mM MgCl2 in a final volume of 50 µl. Reactions were stopped by the addition of 1 ml chloroform/methanol (2:1). The solution was extracted (1 min, 22 °C) with 0·2 ml PSUB (1·5 ml chloroform, 25 ml methanol, 23·5 ml water, 0·183 g KCl). The upper phase was discarded and the remaining solution was re-extracted twice. The incorporation of 14C-labelled glucose into the glycolipid fraction was measured in a Beckman LS3801 scintillation counter (Beckman Coulter).
Analysis of lipid-linked intermediates by TLC.
Lipid-linked intermediates were hydrolysed from the lipid carriers by mild acid hydrolysis. In this procedure one-fifth of the glycolipid fraction was dried in a Speed-Vac at 65 °C and was resuspended in 100 µl n-butanol. To this solution 100 µl 0·05 M trifluoroacetic acid was added and the solution was heated for 20 min at 90 °C. After drying (in a Speed-Vac at 65 °C), the pellet was resuspended in 40% 2-propanol containing 5 mg ml-1 each of unlabelled carrier glucose, galactose, N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc), and subjected to TLC using HPTLC silica gel plates (Merck). Constituents were separated with 1-butanol/ethanol/water (5:3:2), sprayed with En3hance (Perkin Elmer) and autoradiographed for 12 days. To visualize unlabelled sugar standards, dried TLC plates were sprayed with 5% H2SO4 in ethanol and heated to 100 °C for 10 min.
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RESULTS |
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Computer-assisted sequence analysis revealed that the locus contained 15 ORFs with a similar orientation as dexB and aliA (Fig. 1). These ORFs were designated cps9vA to cps9vO, according to their appearance. The G+C content of the ORFs varied between 42 and 29 mol% (Table 1
). A consensus
70 promoter (TAGACA-17 bp-TATAAT) was found 30 bp upstream of the first ORF (Guidolin et al., 1994
). All ORFs seemed to be preceded by a ribosome-binding site. Most of them were separated by small intergenic regions or showed a small overlap with the adjacent gene. On the opposite strand, one ORF (designated orf3) was present between cps9vM and cps9vN. Three additional ORFs (orf12 and orf4) were located on this strand directly adjacent to the dexB and aliA genes (Fig. 1
).
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Cps9vE, F, G, J and L are putative glycosyltransferases
Five ORFs of the 9V locus showed similarity with putative glycosyltransferases (Table 1). Cps9vE was homologous (>83% similarity) to proteins in several other S. pneumoniae serotypes, including Cps14E which we previously demonstrated to function as a UDP-glucosyl-1-phosphate transferase (Kolkman et al., 1996
, 1997a
). Cps9vF resembled (>90% similarity) serogroup 19 ß-1,4-N-acetylmannosaminyltransferases, which transfer UDP-ManNAc in a ß-1,4-glycosidic linkage to a lipid-linked glucose. Cps9vG showed similarity to several putative glycosyltransferases, including WciL of serotype 4 (47%) which (as serotype 9V; Fig. 2
) carries a galactose attached in an
-1,3-glycosidic linkage to a ManNAc residue. Cps9vJ and Cps9vL were most similar to putative glycosyltransferases in other species, although Cps9vL showed some similarity (37%) with WciU of S. pneumoniae serotype 18C.
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Cps9vI and Cps9vK are involved in capsule assembly
On the basis of similarities to an O-antigen polymerase of Pseudomonas putida (43%) (Rodriguez-Herva et al., 1999 ) and a repeating unit polymerase of Streptococcus agalactiae (42%) (GenBank accession no. AF337958), Cps9vI was considered to be the CPS repeating unit polymerase. This idea was supported by transmembrane segment analysis using the program DAS which predicted that Cps9vI contained 12 transmembrane domains and showed a similar DAS profile to other putative repeating unit polymerases (data not shown).
The sequence of Cps9vK was most similar to polysaccharide export proteins and O-antigen repeating unit transporters. This suggests that this protein acts as the CPS repeating unit transporter that transports the repeating oligosaccharide unit across the cell wall.
Cps9vM and Cps9vO are putative O-acetyltransferases
Cps9vM and Cps9vO both showed similarity at the amino acid level to putative O-acetyltransferases. Cps9vM was 53% similar to Cap1F of S. pneumoniae serotype 1, which is a putative O-acetyltransferase (Munoz et al., 1997 ). Cps9vO showed 41% similarity to WciX of S. pneumoniae serotype 18C. WciX is a protein of unknown function, but since serotype 18C contains one O-acetyl group in its structure (Jiang et al., 2001
) and no other gene in the type 18C locus was predicted to encode an O-acetyltransferase, this protein may likely act as the O-acetyltransferase. Transmembrane segment predictions suggest that Cps9vO has various transmembrane segments, whileCps9vM was predicted to be cytosolic.
Insertion sequences
Four sequences were found in the cps9V region that were similar to known insertion sequences (Table 2). An insertion sequence similar to RUPA, but with a deletion of 19 nt was found in front of the locus. orf1 (582 bp) and orf2 (783 bp), located between the genes cps9vO and aliA on the opposite strand, were similar to insertion sequence IS1167 with a frameshift in the ORF. This insertion element has also been found in other cps loci and at other positions within the S. pneumoniae chromosome (Coffey et al., 1998a
; Kolkman et al., 1997b
; Munoz et al., 1997
; Tettelin et al., 2001
). At the protein level, ORF3 (1044 bp) showed 52 % similarity to a putative transposase of Mycobacterium tuberculosis (Cole et al., 1998
). ORF4 (348 bp) is almost identical (99%) to the first part of the insertion sequence IS630-Spn1, which is also found in several other pneumococcal cps loci and elsewhere on the chromosome (Coffey et al., 1998a
; Iannelli et al., 1999
; Llull et al., 1999
; Munoz et al., 1997
, 1999
; Tettelin et al., 2001
). We cannot exclude that the entire transposon is present in serotype 9V as the region between dexB and cps9vA was not fully sequenced.
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cps9vE encodes a UDP-glucosyl-1-phosphate transferase
To confirm the proposed function of Cps9vE as the glycosyltransferase that confers the first step in capsule biosynthesis, i.e. the transfer of glucose to the lipid carrier, we performed a direct functional enzyme assay. Membrane preparations of serotype 9V, the 9Vcps9vE mutant, and of the serotypes 14 and 4 as positive and negative controls, respectively, were incubated with 14C-labelled UDP-glucose (Kolkman et al., 1996
, 1997a
). The synthesis of labelled intermediates was determined after extraction of the glycolipid fraction. Liquid scintillation counting of the obtained fractions showed that the incorporation of glucose catalysed by serotype 14- and serotype 9V-derived membranes was much higher than by the mutant 9V
cps9vE and serotype 4 fractions (Fig. 3
).
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DISCUSSION |
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The genetic organization of the cps9V locus shows strong resemblance to cps loci of other serotypes (Jiang et al., 2001; Paton & Morona, 2000 ). The locus is located between dexB and aliA. It contains the regulatory genes at the 5' end, the genes that confer the sequential steps in the oligosaccharide biosynthesis and the assembly in the central part and the genes encoding modifying enzymes and/or enzymes for the synthesis of activated monosaccharide precursors at the 3' end of the locus. Furthermore, the locus has a conserved
70 promoter site just in front of the first gene. The locus carries several genes with a relatively low G+C content. The conserved nature of the 9V locus compared with sequenced loci from other serotypes feeds the concept that the 9V locus is part of a gene pool that contributes to the evolution of capsule diversity. Genetic exchange of capsule genes resulting in altered antigenic properties of the capsule have been reported both at the level of (nearly) complete loci and of individual genes (Barnes et al., 1995
; Coffey et al., 1991
, 1998a
, b
; Nesin et al., 1998
; Ramirez & Tomasz, 1999
). The low G+C content of some of the genes, about 2934 mol%, compared to the mean for pneumococcal genes, 39·7 mol% (Tettelin et al., 2001
), suggests that some cps genes may have originally been acquired from other species. Knowledge of the cps9V sequence may help to define the extent of the pneumococcal capsule gene repertoire and, ultimately, perhaps reconstruction of the evolution of the capsule serotypes.
At one point, the cps9V locus differs in organization from other known loci. In the 9V locus, the cps9vH gene, involved in nucleotide sugar biosynthesis, is located in the central part of the locus between the serotype-specific glycosyltransferase and assembly genes. In serotypes 4 and 19 with a cps9vH homologue, the gene is located at the end of the locus, adjacent to other nucleotide sugar biosynthesis genes. A second notable difference with other cps loci is the presence of a putative transposase-encoding gene on the opposite strand between two cps genes. The functional relevance of this gene (if any) remains to be determined.
The cps9V locus contains 15 ORFs. On the basis of their putative functions and current knowledge about capsule biosynthesis, the following sequence of reactions can be proposed for the biosynthesis of the 9V CPS (Fig. 4). First, a glucose is attached to the lipid carrier, possibly undecaprenyl phosphate (Paton & Morona, 2000
; Whitfield & Roberts, 1999
). This reaction is catalysed by Cps9vE as confirmed by the TLC results of the enzymic assay. The repeating unit of serotype 9V CPS contains two glucose residues, but based on the structural homology between serotypes 9V and 14 (which contains only one glucose residue), this reaction likely involves the glucose residue that will be linked to the ManNAc residue. After formation of the glucose-lipid intermediate, the four additional glycosyltransferases, Cps9vF, Cps9vG, Cps9vJ and Cps9vL, sequentially add monosaccharides to the growing chain. On the basis of sequence similarities and the observation that genes in the cps loci of S. pneumoniae seem to be arranged in the order that the assembly of the repeating unit is expected to occur, we anticipate that Cps9vG, Cps9vJ and Cps9vL exhibit
-1,3-galactosyltransferase,
-1,3-glucuronic acid transferase and
-1,4-glucosyltransferase activities, respectively. Some of the substrates used in these reactions are available in the pneumococcus via the activity of the GlcNAc-1-phosphate uridyltransferase (GlmU) (Sulzenbacher et al., 2001
), UTP-glucose-1-phosphate uridylyltransferase (GalU) (Mollerach et al., 1998
) and UDP-glucose-4-epimerase (GalE) (Tettelin et al., 2001
) that are located outside the 9V locus. The substrates UPD-ManNAc and UDP-glucuronic acid, which do not seem to be part of 9V housekeeping, are synthesized with the help of the Cps9vH and Cps9vN proteins encoded by the 9V locus. To the completed oligosaccharide unit, additional O-acetyl groups may be variably attached to the ManNAc, glucuronic acid and glucose residues (Fig. 2
) (Perry et al., 1981
; Rutherford et al., 1991
) by the putative O-acetyltransferases Cps9vM and Cps9vO. After transport across the membrane facilitated by Cps9vK, the repeating units are likely polymerized via the Cps9vI polymerase. Finally, the CPS is linked to the peptidoglycan. The mechanism by which this step is established has not yet been determined.
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ACKNOWLEDGEMENTS |
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Received 23 November 2001;
revised 29 January 2002;
accepted 5 February 2002.