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Introduction |
Streptococcus pneumoniae (pneumococcus) remains a major
cause of morbidity and mortality throughout the world
and its continuous increase in antimicrobial resistance is
rapidly becoming a leading cause of concern for public
health. Pneumococcal infection persists as the main causative agent of pneumonia, meningitis, and otitis media.
These diseases remain as the most prevalent infections in
many areas of the world, particularly in infants, the elderly, and in immunocompromised patients. As shown in a recent study (1), although a 68% reduction in total invasive
diseases due to Haemophilus influenzae type b was observed
in the United States after licensure of the conjugated vaccine, a concomitant increase of 74% in the number of cases
per 100,000 population for invasive pneumococcal diseases
was also found. Although S. pneumoniae produces several
virulence factors (for review see reference 2), as early as
1928 Griffith reported that unencapsulated pneumococcal variants were avirulent (3), and that loss of the capsule is accompanied by a 105-fold reduction of the virulence of S. pneumoniae. Unencapsulated pneumococci are readily phagocytized when added to a suspension of leukocytes in normal serum, whereas mucoid, capsulated organisms are resistant to phagocytosis and multiply rapidly. A quantitative relationship between the amount of type-specific polysaccharide and virulence has been found (4, 5), although
the chemical composition of the capsule (6) as well as the
cellular background in which the capsule is produced also
appear to play an important role in virulence (7).
90 different pneumococcal types have been described
(8). This remarkable phenotypic variability appears to be
present also at the genetic level (9), which has precluded until now the search for drugs capable of inhibiting
the synthesis of the capsular polysaccharides of S. pneumoniae. The chemical structure of the repeat unit of these
polysaccharides is known in more than half of the types
described, most of them contain glucose (Glc)1 and/or galactose (Gal) or various derivatives of them in addition to
other sugars (12). Early studies carried out by Mills and co-workers (for review see reference 13) showed that uridine diphosphoglucose (UDP-Glc) is a key component in
the biosynthetic pathway of pneumococcal capsular polysaccharides containing Glc, Gal, and/or UDP-glucuronic
(UDP-GlcA) or UDP-galacturonic (UDP-GalA) acids (Fig.
1). The enzyme UTP:glucose-1-phosphate uridylyltransferase (UDP-Glc pyrophosphorylase, UDPG:PP) (EC 2.7.7.9)
catalyzes the formation of UDP-Glc, which is the substrate
for the synthesis of UDP-GlcA. UDP-Glc is also required
for the interconversion of Gal and Glc by way of the Leloir
pathway (14), and consequently, mutants of Escherichia coli
deficient in UDPG:PP activity cannot ferment Gal and fail
to incorporate Glc and Gal into bacterial cell membranes,
resulting in the incomplete synthesis of lipopolysaccharide (15). The gene cap3C of S. pneumoniae, one of the three
genes located in the type 3-specific capsular operon, encodes
a UDPG:PP that has been shown to be dispensable for capsule production (16, 17). This result strongly suggests that
another different gene might also encode a UDPG:PP that,
in addition, might be common to all of the pneumococci. In
favor of this assumption is the fact that, apart from cap3C, a
gene putatively encoding a UDPG:PP has not been found in
any of the capsular clusters studied so far (9).
A partial (and still preliminary) nucleotide sequence of
the genome of a type 4 pneumococcus has been released
recently (ftp://ftp.tigr.org/pub/data/s_pneumoniae). This
has allowed the search for genes coding for proteins similar to the Cap3C pyrophosphorylase and we report here the
cloning, expression, and characterization of the galU gene
of S. pneumoniae. We show that the pneumococcal galU
mutants of types 1 and 3 did not synthesize detectable
amounts of capsular polysaccharide.
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Materials and Methods |
Bacterial Strains, Plasmids, and Growth Conditions.
The bacterial strains and the plasmids used in this study are shown in Table
1. Unless otherwise stated, S. pneumoniae was grown in liquid C
medium (18) containing 0.08% yeast extract (C + Y) without
shaking or on reconstituted tryptose blood agar base plates (Difco
Laboratories Inc., Detroit, MI) supplemented with 5% defibrinated sheep blood. E. coli cells were grown in Luria-Bertani medium (19). When required, ampicillin was added to the medium
at 100 µg/ml. Chromosomal DNA and plasmid purification, and
transformation of E. coli and laboratory strains of S. pneumoniae were carried out as described elsewhere (20). S. pneumoniae clones
obtained upon transformation with pUCEK2 or pMMG1 (Table 1) were scored on blood agar plates containing 0.7 µg of lincomycin (Ln) per ml. MacConkey agar plates (Difco Laboratories,
Inc.) containing 0.6% galactose were used for E. coli fermentation
tests.
DNA Techniques and Plasmid Construction.
Restriction endonucleases, T4 DNA ligase, and the Klenow (large) fragment of
DNA polymerase I were obtained commercially and used according to the recommendations of the suppliers. Gel electrophoresis of plasmids, restriction fragments, and PCR products was carried out in agarose gels as described (19). DNA was recovered from gel slices with the Gene Clean Kit (Bio 101, La Jolla, CA).
S. pneumoniae DNA digested with either SmaI, SacII, or ApaI
was analyzed by pulsed-field gel electrophoresis (PFGE) using a
contour-clamped homogeneous electric field DRII apparatus
(Bio-Rad, Hercules, CA) as previously described (21).
PCR amplifications were performed using 2 U of AmpliTaqTM
DNA polymerase (Perkin-Elmer Applied Biosystems, Norwalk,
CT), 1 µg of chromosomal (or plasmid) DNA, 1 µM of each synthetic oligonucleotide primer, 200 µM of each deoxynucleoside
triphosphate, and 2.5 mM of MgCl2 in the buffer recommended by
the manufacturer. Conditions for amplification were chosen according to the G + C content of the corresponding oligonucleotide.
The oligonucleotides used were: (624), 5'-TTGGTAccTGAAACAACTGGCATGC-3' (primer OGalU1); (1139/c) 5'-CCCAACGTCGTAACGAGcTCCTG-3' (primer OGalU2); (1464/c),
5'-GAGCAaTTGGTGGCGCATTTCTAGC-3' (primer OGalU3);
(323), 5'-TGAGTcGaCTTAACCCTCTATAGAAAG-3' (primer OGalU4); (659/c), 5'-GAAATGAAGGCGCATGCCAGTTG-3'
(primer IGalU1); (773), 5'-CGAGAAGGCCGTTCCTTTGAC-3'
(primer IGalU2). Numbers indicate the position of the first nucleotide of the primer in the sequence reported in this paper (see below), and /c means that the corresponding sequence corresponds
to the complementary strand. Lowercase letters indicate nucleotides introduced to construct appropriate restriction sites. These
are shown underlined. For inverse PCR, DNA prepared from
strain 406 was digested with ClaI, and the resulting fragments were diluted 10-fold and self-ligated. Afterwards, the DNA was concentrated by ethanol precipitation and amplified by PCR using oligonucleotides IGalU1 and IGalU2.
To construct pMMG2 (Table 1), DNA prepared from the type
3 strain 406 was amplified with oligonucleotides OGalU3 and
OGalU4 and the 1.1-kb DNA fragment was purified, digested
with MunI and SalI, and ligated to EcoRI/SalI-digested pUC19.
Plasmid pMMG1, which contains a 0.5-kb internal fragment of
the gene galU (Table 1), was constructed as follows: DNA prepared from strain 406 was amplified with primers OGalU1 and
OGalU2 and the 0.5-kb DNA fragment was purified, digested
with KpnI and ClaI, and ligated to pUCE191 previously digested
with KpnI plus AccI.
NEBlotTM PhototopeTM Kit (Millipore Corp., Bedford, MA)
was used to construct biotin-labeled probes and PhototopeTM 6K
Detection Kit (Millipore Corp.) for the chemiluminescent detection. Southern blots, dot blots, and hybridizations were carried
out according to the manufacturer's instructions.
Nucleotide Sequence and Data Analysis.
DNA sequencing was
carried out by using an Abi Prism 377TM DNA sequencer (Applied Biosystems Inc., Foster City, CA). DNA and protein sequences were analyzed with the Genetics Computer Group software package (version 9.0) (22). Sequence similarity searches were done by using the EMBL/GenBank, SWISS-PROT, and
PIR databases.
SDS-PAGE and NH2-terminal Amino Acid Sequence Analysis.
SDS-PAGE was carried out as described by Laemmli using 10%
gels (23). After SDS-PAGE, proteins were blotted onto a polyvinylidene difluoride membrane (Immobilon-PSQ, Millipore Corp.),
and stained briefly with Amido black (Sigma Chemical Co., St.
Louis, MO). Subsequently, the desired band was cut and the
NH2-terminal amino acid sequence was determined as described
elsewhere (24).
Miscellaneous Techniques.
The test for carbohydrate fermentation by pneumococcal strains was carried out in Heart infusion
broth (Difco Laboratories, Inc.) with the appropriate carbohydrate added at 1% (final concentration) as previously described
(25). For immunoagglutination assays (26), S. pneumoniae cells
were incubated on C medium containing 0.1% BSA and different
amounts of anti-R antiserum. Anti-R (antisomatic) antiserum
contains group-specific agglutinins that, at a convenient dilution,
agglutinate only rough pneumococci, and was raised in rabbits as
previously described (26). Purification of capsular polysaccharides
and double-diffusion experiments were performed as described
previously (27). To estimate the amount of polysaccharide present in a sample, 5 µl of serial dilutions of the extract and known amounts of purified polysaccharide were analyzed by immunodiffusion against 5 µl of the same batch of antiserum.
Nucleotide Sequence Accession Number.
The DNA sequence described here is deposited with the EMBL database under accession
No. AJ004869.
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Results |
Identification, Cloning, Sequencing, and Mapping of a Pneumococcal Gene Similar to cap3C.
The deduced amino acid
sequence of the cap3C gene was compared to the translated
version of the nucleotide sequence of a type 4 pneumococcal strain that has been released recently. A gene (hereafter
designated galU) encoding a protein 77% identical to Cap3C
was located in the 12,128-bp contig No. 4225. Analysis of this gene and its surrounding regions (Fig. 2) revealed that
contig No. 4225 does not contain the type 4-specific capsular cluster which appears to be localized in the contig
No. 4108 (34,280 bp). The putative galU gene is preceded
by a gene whose product showed strong similarity to the
GpsA NAD(P)H-dependent dihydroxyacetone-phosphate reductase of Bacillus subtilis (28) (Table 2). The galU and the
gpsA genes are only 21 bp apart. Other genes surrounding
gpsA-galU were preliminarily identified on the basis of sequence similarities with the exception of orf5 and orf6,
which did not show any significant similarities to those
available in the data banks.

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Fig. 2.
Genetic organization of two S. pneumoniae strain (types 3 and
4) DNAs containing the galU gene. The corresponding region of the S. pyogenes DNA is also shown. Thick and thin arrows represent complete or
interrupted orfs, respectively. Identical shading represents DNA regions
coding for the same putative protein. Relevant restriction sites are indicated (C, ClaI; K, KpnI; M, MunI; Sa, SalI). An asterisk indicates that the
corresponding restriction site has been introduced using a synthetic oligonucleotide. Filled key represents a putative transcription terminator.
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Table 2
The galU Genes of S. pneumoniae and S. pyogenes and Their Surrounding orfs: Properties and Similarities in the Database
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Preliminary efforts to amplify by PCR the galU gene using DNA prepared from the S. pneumoniae type 3 strain 406 were unsuccessful when using oligonucleotide primers designed from the nucleotide sequence of genes gpsA and orf5
from type 4 pneumococci. However, successful amplification was achieved with OGalU3 and OGalU4, which correspond to the 5' end of orf6 and to the 3' end of gpsA, respectively. The 1.1-kb PCR fragment was cloned into
pUC19 to create pMMG2 (Table 1). Moreover, inverse
PCR using oligonucleotides IGalU1 and IGalU2 (designed
from the nucleotide sequence of the SalI-MunI insert of
pMMG2) produced a 2.3-kb DNA fragment that was partially sequenced. Determination of the sequence of 1,464 bp revealed that the orf5 gene was not present in 406 DNA,
which accounts for the amplification failures discussed
above. It should be noted that the nucleotide sequence of
the galU406 gene (900 bp) was nearly identical (89%) to that
of the type 4 isolate (not shown).
The absence of orf5 in strain 406 prompted us to analyze
whether other important differences exist among various
pneumococcal isolates. Southern blot analysis of HindIII-digested chromosomal DNA prepared from a variety of
pneumococcal strains using a 0.7-kb SalI-ClaI fragment of
pMMG2 showed that all the strains tested contain at least
one copy of the galU gene (Fig. 3). In the case of strains
6028/95 (S8+) and SSIP 33A/1 (S33A+), the pattern of hybridization might suggest the existence of a second copy of
galU in their DNAs, although we favor the hypothesis that
the 5.5-kb DNA band was incompletely cleaved in those
two strains. Nevertheless, this point was not further investigated.

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Fig. 3.
Identification of the galU gene of S. pneumoniae by DNA-
DNA hybridization. Chromosomal DNA from 15 different pneumococcal strains of various types was digested with HindIII, electrophoresed,
blotted, and hybridized with a 0.7-kb SalI-ClaI fragment of pMMG2 (see
Table 1). As size standards, we used a HindIII digest of DNA.
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Fig. 4 shows a multiple alignment of the amino acid sequence of the galU406 gene product with the proteins available in the databases. HasC, a UDPG:PP from the has operon required for expression of the hyaluronic acid capsule
of Streptococcus pyogenes (29), showed the highest similarity
to the pneumococcal GalU (>85% identical amino acids
and 90.9% similarity). About 87% sequence similarity (77%
identity) was found between the pneumococcal GalU and
Cap3C proteins. Although not yet included in the data
banks, a search of a partial nucleotide sequence of the S. pyogenes genome (http://www.genome.ou.edu/strep.html) for genes similar to galU (and hasC) showed that, as in S. pneumoniae, group A streptococci (GAS) also contain a galU
homologue located in the contig No. 234 (8061 bp) (Fig.
2). The similarity between the GalU proteins of S. pneumoniae and S. pyogenes was even higher than that found between the former and the type 3-specific Cap3C UDPG:PP
of S. pneumoniae (Fig. 4). Lower but significant similarities were also found between the pneumococcal GalU and
UDPG:PPs from B. subtilis (30) and E. coli (31) as well as
with the GalF protein of E. coli that modulates the activity
of the GalU enzyme (32).

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Fig. 4.
Computer-generated alignment (PILEUP) of the GalU protein
of S. pneumoniae (SpnGalU) with other similar proteins included in the
databases. The following UDPG:PP were aligned: S. pneumoniae Cap3C
(Spn_Cap3C) (reference 16), S. pyogenes HasC (SpyHasC) (reference 29), B. subtilis GtaB (BsuGtaB) (reference 30), and E. coli GalU (EcoGalU) (reference 31). Other proteins used were GalF of E. coli (EcoGalF) (reference 32)
and the putative UDPG:PP of S. pyogenes, the GalU protein described in
this paper (SpyGalU). The inset shows the percentages of identical/similar amino acid residues resulting from pairwise comparisons (BESTFIT).
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To determine the location of galU in the pneumococcal
genome, chromosomal DNA of strain M24, a descendant
of the classical laboratory strain R6 (33), was digested with
SmaI, ApaI, or SacII, subjected to PFGE, blotted, and hybridized with the 0.7-kb SalI-ClaI fragment of pMMG2
(see above). The results shown in Fig. 5 indicated that the
galU gene resides in fragments 5 (SmaI, 235 kb), 4 (ApaI,
235 kb), and 13 (SacII, 54 kb) of the physical map of the
pneumococcal genome (34).

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Fig. 5.
PFGE of the DNA
prepared from strain M24 of S. pneumoniae digested with ApaI,
SacI, or SmaI and blotted and
hybridized with the 0.7-kb SalI-ClaI fragment of pMMG2. The
sizes (in kb) of the SmaI fragments (reference 34) are indicated at the right.
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Overproduction and Characterization of the S. pneumoniae
GalU Protein.
E. coli DH5
cells harboring pMMG2
were incubated overnight at 37°C with shaking in LB medium containing ampicillin (100 µg/ml). Crude extracts
obtained by sonication were analyzed by 10% SDS-PAGE and a prominent protein band of ~37 kD was observed
(Fig. 6, lane 2). This band was absent in crude extract prepared from E. coli DH5
cells containing the vector plasmid pUC19 (Fig. 6, lane 3). The molecular mass of the 37-kD overproduced protein was in fair agreement with that
deduced from the nucleotide sequence of the galU gene
(33,213 D). It should be mentioned that an apparent 38,000 Mr has been reported for the purified E. coli GalU
whereas a molecular mass of 32,291 D was predicted from
the sequence of the galU gene (31). The NH2-terminal
amino acid sequence of the protein was determined, yielding Met-Thr-Ser-Lys-Val-Arg-Lys-Ala-Val-Ile, which confirmed the sequence deduced from the nucleotide sequence of the gene (Fig. 4).

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Fig. 6.
Expression of the
GalU protein of S. pneumoniae by
E. coli containing the recombinant plasmid pMMG2. E. coli
DH5 cells containing either
pMMG2 or pUC19 vector alone
were incubated overnight on LB
medium, and the crude sonicated
extracts were analyzed by 10%
SDS-PAGE. Lane 2, extracts from
bacteria containing pMMG2.
Lane 3, extracts from bacteria
harboring pUC19. The molecular masses of the standards (lane
1) are indicated on the left.
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To ascertain that the pneumococcal galU gene codes for
a UDPG:PP, the pGMM2 plasmid was introduced by
transformation into the galU E. coli mutant strain FF4001.
This mutant contains a T to C mutation causing the substitution of a proline residue by a serine one in the predicted
amino acid sequence of the GalU protein and virtually
lacks UDPG:PP activity (31). Ampicillin-resistant transformants scored on MacConkey-galactose plates grew as red colonies indicating that they were able to ferment the sugar
(not shown). This result confirmed that the galU gene of S. pneumoniae encodes a UDPG:PP.
Construction and Characterization of galU Mutants of Pneumococcus.
To construct galU mutants of S. pneumoniae
lacking UDPG:PP, insertion-duplication mutagenesis was
carried out using plasmid pMMG1 that contains an internal
fragment of the galU gene cloned into pUCE191 (Table 1).
A Ln-resistant transformant (designated as M24g) was isolated upon transformation of the pneumococcal strain M24
with pMMG1 isolated from E. coli TG1. Carbohydrate-fermentation tests showed that M24g fermented Glc but
was unable to use Gal as a carbon source (not shown).
Chromosomal DNA prepared from strain M24g was
used to transform the encapsulated strains M23 (S3+) and
M25 (S1+) and Ln-resistant clones were picked for further
study. As found for M24g, the transformant strains M23g
and M25g were also unable to ferment Gal whereas M23c
did ferment either Glc or Gal. Furthermore, when the
M23g strain was grown in blood agar plates, small, rough-like colonies were formed, in sharp contrast with the big,
smooth colonies typical of type 3 strains of S. pneumoniae
(Fig. 7). As reported before (16), no major differences were
observed between the colonies of M23 and M23c strains.
Since type 1 pneumococcal strains form small colonies, no
significant differences were found on the morphology of
the colonies of M25 and M25g (not shown). Encapsulated pneumococci grow typically in suspension when incubated
in broth, whereas unencapsulated mutants show a tendency
to aggregate at the bottom of the tube. The pneumococcal
galU mutants, but not the cap3C mutants (M24c), grew as
true unencapsulated strains (Fig. 7) and were agglutinated
with anti-R serum (not shown). Moreover, immunodiffusion analysis of cell extracts using either type 1- or type
3-specific antisera demonstrated that M23g and M25g did
not synthesize any detectable capsular polysaccharide (Fig.
7). In contrast, M23c produced type 3 polysaccharide in amounts comparable to the M23 strain, which confirmed
previous results (16). To further confirm that GalU is essential for the biosynthesis of capsular polysaccharides in S. pneumoniae, strains M23g and M25g were repeatedly subcultured in Ln-free C medium containing 0.1% BSA and
anti-R antiserum (see Materials and Methods). After several
passages, putatively encapsulated revertants that were not
agglutinated by the serum and thus grew in suspension
were isolated. They appeared to be true galU+ revertants as
judged from the findings that were no longer Ln-resistant and that the size (1.1 kb) of the PCR fragments obtained
by using oligonucleotide primers OGalU3 and OGalU4
and DNA isolated from every revertant tested corresponded to that of the intact galU gene (see above). As expected, those revertants were able to synthesize a capsular
polysaccharide corresponding to the original, encapsulated parental strain (type 1 or type 3) as demonstrated immunologically (data not shown).

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Fig. 7.
Unencapsulated phenotype of the pneumococcal galU mutants. (Top) Colony morphology on blood-agar plates. Bar, 1 mm. (Middle) Growth characteristics after overnight incubation at 37°C in C + Y
medium. (Bottom) Double immunodifussion in agarose. The center wells
received type 3 (a) or type 1 (b) antisera. The pneumococcal strains analyzed were: M23 (A); M23c (Cap3C) (B); M23g (GalU) (C); M25 (D);
M25g (GalU) (E). Purified pneumococcal polysaccharides from type 1 (1) and type 3 (3) were used as positive controls.
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Discussion |
Duplicated genes appear to be rather unusual in bacteria
and consequently type 3 pneumococci and S. pyogenes are
somehow exceptional in having two genes coding for the
same enzyme, namely, a UDPG:PP. The capsular polysaccharides of both bacteria contain glucuronic acid, and the
genes responsible for their biosynthesis are organized in a
similar fashion (16, 35), i.e., each operon contains three
genes, the gene responsible for the synthesis of UDP-Glc (cap3C and hasC, in S. pneumoniae and GAS, respectively),
the gene encoding a UDP-Glc dehydrogenase (cap3A and
hasB), and the gene coding for the enzyme responsible for
the synthesis of the type 3 polysaccharide in S. pneumoniae
(cap3B) or hyaluronic acid in S. pyogenes (hasA). As shown
for type 3 pneumococci where cap3C is not required for
capsule formation, only HasA and HasB appear to be required for hyaluronic acid capsule production both in GAS
and in heterologous bacteria as revealed by Tn916 mutagenesis (36, 37). Furthermore, a gene very similar to the
pneumococcal galU gene described here has been found in
the partial nucleotide sequence of the S. pyogenes genome
that is currently available. It should be mentioned that,
both in S. pneumoniae and S. pyogenes, the galU is immediately preceded by a gene (gpsA) that is presumably involved
in the synthesis of membrane lipids (28). The significance of
this finding is not currently understood but it is interesting to
point out that gpsA and galU genes are completely unlinked in other bacteria such as B. subtilis or E. coli.
A relevant role of UDPG:PP for virulence has been recognized in various gram-negative bacteria (38). However, to the best of our knowledge, there are no data available in gram-positive organisms concerning the importance
of this protein in pathogenicity. In this work, we have constructed galU mutants of two strains of S. pneumoniae of different serotypes, namely types 1 and 3. Both mutants were
unencapsulated according to a series of criteria, i.e., colony
morphology, growth in liquid medium, agglutinability with anti-R serum, and lack of recognition by type-specific
antiserum. On the other hand, galU+ revertants of strains
M23g and M25g synthesized type 1 and type 3 capsules,
respectively. The unencapsulated phenotype of the galU
mutants was expected in the case of the type 1 strain that apparently does not harbor any other gene encoding a
UDPG:PP in addition to galU (20). However, in type 3 pneumococci the unencapsulated phenotype of the galU
mutants was somehow surprising since these bacteria contain an active copy of cap3C that also codes for the same
enzymatic activity (16). Since Cap3C UDPG:PP activity is not required for type 3 capsule formation and it cannot replace the activity lost in the galU mutants we conclude that,
at least under laboratory conditions, either Cap3C is poorly
translated or its enzymatic activity is very low. As the HasC
protein of S. pyogenes is also not needed for hyaluronate
biosynthesis (see above), it can be predicted that galU mutants of GAS will be also unencapsulated. This observation
might be of remarkable clinical relevance since loss of capsule is associated with a 100-fold reduction in virulence of
S. pyogenes (41).
The continuous dissemination of multiply resistant S. pneumoniae clones throughout the world is the cause of
great concern, and much effort is currently dedicated to the
search for new antibacterial drugs (42, 43). The polysaccharide capsule of pneumococcus is the main virulence factor
of this bacterium (3) and drugs inhibiting its biosynthesis
should potentially render S. pneumoniae virtually avirulent.
Unfortunately, the noticeable genetic variability found in
the genes responsible for the capsular polysaccharide biosynthesis has precluded until now the search for such drugs. Remarkably, the galU gene has been found in all the pneumococcal types tested so far (Fig. 3). The UDPG:PP,
which is directly involved in the synthesis of the capsular
polysaccharide in S. pneumoniae and other bacterial pathogens, might represent a suitable target for the search of inhibitors of such an important virulence factor. In this sense,
it should be emphasized that eukaryotic UDPG:PPs appear
to be completely unrelated to their prokaryotic counterparts (for review see reference 44). As depicted in Fig. 8,
the structural arrangement of the domains found in prokaryotic UDPG:PP is remarkably similar. In contrast, the
eukaryotic enzymes exhibit a completely different arrangement as well as a different amino acid sequence. This interesting feature suggests the possibility that putative inhibitors
of the bacterial enzymes would not be harmful for the host.

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Fig. 8.
Domain arrangement of several UDPG:PP from prokaryotic
and eukaryotic organisms. Bars represent domains and identical shading
indicates high amino acid sequence similarity. The proteins were aligned
according to programs available at the ProDom database (reference 50).
The number assigned to each domain (#) as well as its length in amino
acid residues (aa) are indicated at the top right. The UDPG:PP aligned
correspond to the following species: E. coli (EcoGalU); B. subtilis (BsuGtaB);
S. pyogenes (SpyHasC and SpyGalU); S. pneumoniae (SpnCap3C and
SpnGalU); Bos taurus (UDPGBovin); Homo sapiens (UDPGHuman); S. cerevisiae (UDPGYeast and UDPHYeast); Solanum tuberosum (UDPGSolTu);
and Dictyostelium discoideum (UDPGDicDi).
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Address correspondence to Rubens López, Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Velázquez 144, 28006 Madrid, Spain. Phone: 34-91-561-1800; Fax: 34-91-562-7518;
E-mail: cibl220{at}fresno.csic.es
We thank J.L. García, and R. Muñoz for critical reading of the manuscript. The technical assistance of E. Cano and M. Carrasco, as well as the artwork by V. Muñoz and A. Hurtado are greatly acknowledged.
This work was supported by grant PB96-0809 from the Dirección General de Investigación Científica y
Técnica and the Programa de Cooperación Científica con Iberoamérica from the Subdirección General de
Cooperación Internacional.
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