Characterization of the galU Gene of Streptococcus pneumoniae Encoding a Uridine Diphosphoglucose Pyrophosphorylase: A Gene Essential for Capsular Polysaccharide Biosynthesis

The galU gene of Streptococcus pneumoniae has been cloned and sequenced. Escherichia coli cells harboring the recombinant plasmid pMMG2 (galU) overproduced a protein that has been shown to correspond to a uridine 5′-triphosphate:glucose-1-phosphate uridylyltransferase (uridine diphosphoglucose [UDP-Glc] pyrophosphorylase) responsible for the synthesis of UDP-Glc, a key compound in the biosynthesis of polysaccharides. A gene very similar to the S. pneumoniae galU has been found in a partial nucleotide sequence of the Streptococcus pyogenes genome. Knockout galU mutants of type 1 pneumococci are unable to synthesize a detectable capsule. An identical result was found in type 3 S. pneumoniae cells in spite of the fact that these bacteria contain a type-specific gene (cap3C) that also encodes a UDP-Glc pyrophosphorylase. Since eukaryotic UDP-Glc pyrophosphorylases appear to be completely unrelated to their prokaryotic counterparts, we postulate that GalU may be an appropriate target for the search of new drugs to control the pathogenicity of bacteria like pneumococcus and S. pyogenes.

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Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.

Journal of Experimental Medicine ◽

10.1084/jem.181.3.973 ◽

1995 ◽

Vol 181 (3) ◽

pp. 973-983 ◽

Cited By ~ 106

Author(s):

J P Dillard ◽

M W Vandersea ◽

J Yother

Keyword(s):

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Gene Cassette ◽

Polysaccharide Biosynthesis ◽

Hybridization Data ◽

Major Virulence Factor ◽

The Right ◽

Type 3

The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types.

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Type 3-specific synthase of Streptococcus pneumoniae (Cap3B) directs type 3 polysaccharide biosynthesis in Escherichia coli and in pneumococcal strains of different serotypes.

Journal of Experimental Medicine ◽

10.1084/jem.184.2.449 ◽

1996 ◽

Vol 184 (2) ◽

pp. 449-455 ◽

Cited By ~ 41

Author(s):

C Arrecubieta ◽

R López ◽

E García

Keyword(s):

Escherichia Coli ◽

Streptococcus Pneumoniae ◽

High Molecular Mass ◽

Cell Fractionation ◽

Periplasmic Space ◽

Polysaccharide Biosynthesis ◽

E Coli ◽

Escherichia Coli Cells ◽

Immunological Tests ◽

Type 3

The cap3B gene, which is involved in the formation of the capsule of Streptococcus pneumoniae type 3, encodes a 49-kD protein that has been identified as a polysaccharide synthase. Escherichia coli cells harboring the recombinant plasmid pTBP3 (cap3B) produced pneumococcal type 3 polysaccharide, as demonstrated by immunological tests. Biochemical and cell fractionation analyses revealed that this polysaccharide had a high molecular mass and was localized in substantial amounts in the periplasmic space of E. coli. Unencapsulated (S-2), laboratory pneumococcal strains synthesized type 3 polysaccharide by transformation with plasmid pLSE3B harboring cap3B. In addition, encapsulated pneumococci of types 1, 2, 5, or 8 transformed with pLSE3B can direct the synthesis of pneumococcal type 3 polysaccharide, leading to the formation of strains that display binary type of capsule.

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The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

Biochemical Journal ◽

10.1042/bj20050217 ◽

2005 ◽

Vol 389 (1) ◽

pp. 63-72 ◽

Cited By ~ 10

Author(s):

Nehmé SAKSOUK ◽

Ludovic PELOSI ◽

Pierre COLIN-MOREL ◽

Manel BOUMEDIENNE ◽

Patricia L. ABDIAN ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Capsular Polysaccharide ◽

Biochemical Characterization ◽

Repeat Unit ◽

Sugar Residue ◽

Functional Identification ◽

Polysaccharide Biosynthesis ◽

Sugar Addition ◽

Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

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Characterization of the Type 8 Capsular Gene Cluster of Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.181.19.6214-6219.1999 ◽

1999 ◽

Vol 181 (19) ◽

pp. 6214-6219 ◽

Cited By ~ 23

Author(s):

Rosario Muñoz ◽

Marta Mollerach ◽

Rubens López ◽

Ernesto García

Keyword(s):

Streptococcus Pneumoniae ◽

Nucleotide Sequence ◽

Gene Cluster ◽

Complete Nucleotide Sequence ◽

Capsular Polysaccharide ◽

Open Reading Frames ◽

Dna Fragment ◽

Type 3 ◽

Reading Frames

ABSTRACT The complete nucleotide sequence of the capsular gene cluster (cap8) responsible for the biosynthesis of the capsular polysaccharide of Streptococcus pneumoniae type 8 has been determined. The cap8 gene cluster, located between the genes dexB and aliA, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the cap8genes was sufficient to transform an unencapsulated type 3 S. pneumoniae strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.

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