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Journal of Bacteriology, September 1999, p. 5355-5364, Vol. 181, No. 17
Molecular Microbiology Unit, Women's and
Children's Hospital, North Adelaide, South Australia
5006,1 and Department of Microbiology
and Immunology, University of Adelaide, Adelaide, South Australia
5005,2 Australia
Received 7 April 1999/Accepted 16 June 1999
The genetic basis for the structural diversity of capsule
polysaccharide (CPS) in Streptococcus pneumoniae serogroup
19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for
the first time. In this study, the genetic basis for the 19A and 19C
serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared.
Transformation studies show that the structural difference between the
19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI
and cps19fI). The CPS of type 19C differs from that of type
19B by the addition of glucose. We have identified a single gene
difference between the two cps loci (cps19cS),
which is likely to encode a glucosyl transferase. The arrangement of
the genes within the cps19 loci is highly conserved, with
13 genes (cps19A to -H and cps19K
to -O) common to all four serogroup 19 members. These
cps genes encode functions required for the synthesis of
the shared trisaccharide component of the group 19 CPS repeat unit
structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures
of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene
complementation or similarity to other proteins with known functions,
and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.
Streptococcus pneumoniae
(the pneumococcus) is an important cause of invasive disease in human
populations throughout the world, resulting in high morbidity and
mortality. Control of pneumococcal disease is being complicated by the
increasing prevalence of antibiotic-resistant strains and the
suboptimal clinical efficacy of existing vaccines. S. pneumoniae produces a polysaccharide capsule, which is
essential for virulence because it protects the pneumococcus from the
nonspecific immune defenses of the host during an infection
(2). All fresh isolates from patients with pneumococcal
infection are encapsulated, and spontaneous nonencapsulated (rough)
derivatives of such strains are almost completely avirulent.
There are now 90 recognized serotypes of S. pneumoniae
(18), each of which produces a structurally distinct
capsular polysaccharide (CPS). Classical genetic studies carried out by
Austrian et al. (3) demonstrated that the S. pneumoniae genes required for biosynthesis and expression of CPS
are closely linked on the pneumococcal chromosome. This fact enabled us
to clone and sequence the capsule locus from S. pneumoniae
type 19F (designated cps19f) (15, 31). Our
studies were initially concentrated on S. pneumoniae type 19F because it is one of the commonest causes of invasive disease in
children and because the type 19F CPS is one of the poorest immunogens
in this group (11). We have since characterized the type 19B
capsule locus (designated cps19b) and the 5' portion of the
type 19A capsule locus (designated cps19a) (32,
33). The immuno-cross-reactive types 19F, 19A, 19B, and 19C are
all members of group 19. In one study, group 19 pneumococci accounted for 7% of isolates from cases of invasive disease (40). Of
these, 65% were caused by type 19F, 34% were caused by type 19A, and 1% were caused by type 19B; type 19C was a very rare cause of disease
in this study.
The CPS structures of types 19F and 19A are quite similar, as are those
for types 19B and 19C (Fig. 1). However,
the latter two have an extra sugar in the backbone and a disaccharide
side chain. When compared with cps19f, the cps19b
locus has been shown to contain extra genes required for biosynthesis
of the more complicated type 19B CPS repeat unit, as well as a
different polysaccharide repeat unit transporter and polysaccharide
polymerase (32).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparative Genetics of Capsular Polysaccharide
Biosynthesis in Streptococcus pneumoniae Types Belonging to
Serogroup 19
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
View larger version (15K):
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FIG. 1.
Biological repeat units of pneumococcal type 19F, 19A,
19B, and 19C CPSs. The orders of the sugars in the repeat units have
been altered compared to the published chemical structures for 19F
(36), 19A (21), and 19B and 19C (6),
reflecting the fact that glucose is the first sugar in the biological
repeat unit. D-Glc, glucose;
D-ManNAc, N-acetylmannosamine;
L-Rha, rhamnose; D-Rib, ribose.
Analysis of purified type 19A CPS has yielded two distinct putative
structures. One is the same as type 19F except for a 1
3 linkage
(rather than 12) between Glc and Rha (Fig. 1) (21). This
difference would necessitate an alteration only in the specificity of
the polysaccharide polymerase (Cps19fI). The alternative
structure involves the same trisaccharide backbone and interunit
linkage as type 19F but with additional
-D-GlcpNAc-(13)--D-Galp-(1-PO4
2)
and
-L-Fucp-(1-PO43)
side chains attached to the Glc and Rha, respectively (24). This would necessitate a number of additional enzyme activities not
found for the cps locus of type 19F strains. Interestingly, individual type 19A strains were subsequently reported to be capable of
producing either structural type, depending on the growth conditions (25). Analysis of the 5' portion of the cps19a
locus revealed that it is similar to cps19f, with the first
seven genes arranged in the same order. However, many of these genes
have only 70 to 80% nucleotide sequence identity with their
cps19f counterpart, suggesting either that the two loci
diverged long ago or that portions of these loci have separate origins
(33).
The last member of serogroup 19 is type 19C. The cps19c locus is predicted to contain both the extra genes present in cps19b (32) and an additional gene to encode an additional transferase required for the glucose side chain present in the type 19C CPS.
In this study, DNA sequence analysis for both the remainder of the type 19A cps locus and the cps19c locus was undertaken, and in conjunction with transformation studies, the identities of the type 19A- and 19C-specific genes were determined. These data complete the characterization of the genetic loci for all members of S. pneumoniae group 19. The data explain the genetic mechanisms used by S. pneumoniae to generate diversity in CPS structure and are of relevance to the evolution of other S. pneumoniae cps loci.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Bacterial strains and plasmids. S. pneumoniae Rx1-19F-I, an unencapsulated insertion-duplication mutant (in which the cps19fI gene has been interrupted) of Rx1-19F (a derivative of Rx1 expressing type 19F capsule), was constructed as described elsewhere (31). A clinical isolate of S. pneumoniae type 19A, strain 1777/39, was obtained from Jorgen Henrichsen, Statens Seruminstitüt, Copenhagen, Denmark, and was designated 19A1. Clinical isolates of S. pneumoniae type 19A (designated 19A2) and 19C were obtained from Chi-Jen Lee, Center for Biologics, Food and Drug Administration, Bethesda, Md. Six Australian clinical isolates of S. pneumoniae type 19A were obtained from Mike Gratten, Acute Respiratory Infections Research and Reference Unit, Centre for Public Health Sciences, Queensland Health, Brisbane, Australia. All other clinical isolates were from the Women's and Children's Hospital, Adelaide, South Australia, Australia. Pneumococci were routinely grown in Todd-Hewitt broth with 0.5% yeast extract or on blood agar. Where appropriate, erythromycin was added to media at a concentration of 0.2 µg/ml.
Escherichia coli K-12 DH5 (Bethesda Research
Laboratories, Gaithersburg, Md.) was grown in Luria-Bertani broth
(27) with or without 1.5% (wt/vol) Bacto-agar (Difco
Laboratories, Detroit, Mich.). Where appropriate, ampicillin was added
to the growth medium at a concentration of 50 µg/ml.
The vector pBluescript KS(+) was obtained from Stratagene, La Jolla, Calif.
Bacterial transformation. Transformation of E. coli with plasmid DNA was carried out as described by Brown et al. (7). The unencapsulated S. pneumoniae strain Rx1-19F-I was transformed as described previously for strain D39 (5).
Assessment of encapsulation. Production of capsule by pneumococci was assessed by the quellung reaction, using factor-specific antisera obtained from Statens Seruminstitüt, Copenhagen, Denmark. This was performed by Mike Gratten.
DNA manipulations. S. pneumoniae chromosomal DNA was extracted and purified by using the Wizard genomic DNA purification kit (Promega Corporation, Madison, Wis.). Chromosomal DNA was purified according to the manufacturer's instructions except that cell lysis was induced by the addition of 0.1% (wt/vol) deoxycholate followed by incubation at 37°C for 10 min. Plasmid DNA was isolated from E. coli by the alkaline lysis method (28). Analysis of recombinant plasmids was carried out by digestion of DNA with one or more restriction enzymes under the conditions recommended by the supplier. Restricted DNA was electrophoresed in 0.8 to 1.5% agarose gels with a Tris-borate-EDTA buffer system as described by Maniatis et al. (27).
Long-range PCR. The Expand Long Template PCR System (Boehringer, Mannheim, Germany) was used for long-range PCR according to the manufacturer's instructions.
Southern hybridization analysis. Chromosomal DNA (2.5 µg) was digested with appropriate restriction enzymes, and the digests were electrophoresed on agarose gels in Tris-borate-EDTA buffer. DNA was then transferred to a positively charged nylon membrane (Hybond N+; Amersham, Amersham, England) as described by Southern (39), hybridized to digoxigenin (DIG)-labelled probe DNA, washed, and then developed with anti-DIG-alkaline phosphatase conjugate (Boehringer) and 4-nitroblue tetrazolium-X-phosphate substrate according to the manufacturer's instructions. DIG-labelled lambda DNA, restricted with HindIII, was used as a DNA molecular size marker.
DNA sequencing and analysis.
DNA sequencing of various PCR
products was carried out by using dye terminator chemistry with
specifically designed primers on an Applied Biosystems model 373A
automated DNA sequencer. Nested deletions of pJCP484, which contains
the type 19C cps locus, were constructed by the method of
Henikoff (17) with an Erase-a-Base kit (Promega). This DNA
was transformed into E. coli DH5, and the resulting
plasmid DNA was characterized by restriction analysis. Double-stranded
template DNA for sequencing was prepared as recommended in the Applied
Biosystems sequencing manual. The sequences of both strands were then
determined by using dye-labelled primers on an Applied Biosystems
model 373A automated DNA sequencer. The sequence was analyzed by using
DNASIS and PROSIS version 7.0 software (Hitachi Software Engineering,
South San Francisco, Calif.). The program BLASTX 2.0 (1) was
used to translate DNA sequences and conduct homology searches of the
protein databases available at the National Center for Biotechnology
Information, Bethesda, Md. The program PROFILEGRAPH (19) was
used to align hydropathy plots generated by the method of Kyte and
Doolittle (23).
Nucleotide sequence accession numbers. The nucleotide sequences for the cps19a locus and the 5' intergenic region in S. pneumoniae 19A1 have been deposited with GenBank under accession no. AF094575. The nucleotide sequences for the cps19a2 and cps19c loci have been deposited with GenBank under accession no. AF105113 and AF105116, respectively. The nucleotide sequences for the 5' intergenic regions from strains 19A2, 19B, and 19C have been deposited with GenBank under accession no. AF105112, AF105114, and AF105115, respectively.
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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PCR amplification of the 3' region of the type 19A cps locus. The number and arrangement of the genes in the 5' portion of the cps19a locus were found to be identical to those in the cps19f locus (33). It was assumed that the arrangement of the genes in the remainder of the two loci would also be similar. Thus, a series of overlapping DNA fragments containing type 19A-specific genes flanked by conserved sequences was generated by long-range PCR with primers based on the cps19f sequence. A map of the PCR products spanning the entire cps19a locus is shown in Fig. 2A. DNAs from two different type 19A clinical isolates (19A1 and 19A2) were used as templates. Interestingly, the PCR products amplified from regions between cps19A and cps19J were identical in size for both type 19A isolates and type 19F, but the 19A2 isolate differed in the 3' region of the cps19a locus; the PCR products obtained from the cps19J-to-cps19O region were either smaller or absent from 19A2. This suggests that part of this region of the cps19a locus from this strain may have been deleted.
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Sequence analysis of the cps19a locus.
The
sequences of the PCR products from 19A1 were determined by using
specifically designed primers. Analysis of the compiled sequence
revealed that the entire cps19f and cps19a loci
are indeed very closely related. The cps19a locus has the
same number of open reading frames (ORFs) organized in an order
identical to those in cps19f, with homologies to the
cps19f genes ranging from 70.1 to 99.4% identity (Fig. 2B).
The sequences and properties of the cps19aA to -G
genes have been reported previously (33). The sizes
and percent identities of the cps19aH to -O and
cps19fH to -O protein products are shown in Table
1.
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Comparison of Cps19aI and Cps19fI.
The putative polysaccharide
polymerases, Cps19aI and Cps19fI, are predicted to form different
glycosidic linkages in type 19A (13) and type 19F (12) CPS,
respectively. As these two proteins are 80.7% identical, their amino
acid sequences were examined to identify any potentially significant
differences between them. A cluster of nonconservative amino acid
substitutions is located in the region between amino acids 290 and 320. No such clustering of nonconservative amino acid substitutions was
observed when either Cps19fH and Cps19aH or Cps19fJ and Cps19aJ were
compared. The region where these clustered substitutions occur is
predicted to be in a loop located on the outer surface of the
cytoplasmic membrane, based on the topology of the O-antigen polymerase
(Rfc/Wzy) from Shigella flexneri (10). The CPS
repeat units are predicted to be transported across the cytoplasmic
membrane prior to polymerization (41). Thus, the external
location of the nonconserved regions in Cps19aI and Cps19fI is
consistent with that of the putative catalytic site in these proteins.
Capsule transformation from type 19F to 19A. To confirm that the cps19a locus was sufficient for type 19A CPS biosynthesis, a 16.5-kb PCR product from the 5' end of cps19aA to the 5' end of aliA was amplified by using the primers CPS5' and J36 (Fig. 1). This DNA product was used to transform Rx1-19F-I, an unencapsulated, erythromycin-resistant derivative of Rx1-19F in which the cps19fI gene had been disrupted by insertion-duplication mutagenesis with pVA891, as described previously (31). Several smooth transformants were checked for erythromycin sensitivity, indicating loss of the pVA891 sequence. Southern hybridization analysis confirmed the absence of both pVA891 and the cps19fI gene and the presence of the cps19aI gene. The presence or absence of both the cps19aC (located in the 5' region of the cps19a locus) and cps19aK (located in the 3' region of the cps19a locus) genes in the three individual transformants was also investigated to determine the sites of recombinational exchange between the cps19f locus and the type 19A PCR product (data not shown). The production of a type 19A capsule by these three smooth transformants, designated Rx1-19A.1 to -3, was then confirmed by the quelling reaction.
Based on the Southern hybridization data, the crossover points between the cps19f locus and the type 19A PCR product were then identified by sequencing the regions where recombination was predicted to have occurred. A diagrammatic representation indicating the positions of the recombination points is shown in Fig. 3. Two transformants (Rx1-19A.1 and -3) were similar, resulting in the exchange of a large region of the cps19f locus, from cps19fG to cps19fN (including cps19fO in Rx1-19A.3) for the homologous region from cps19a. On the other hand, Rx1-19A.2 was derived from exchange of a much smaller region of the cps19f locus, involving only cps19H and cps19I (Fig. 3). The cps19aH gene has 90.8% nucleotide identity to cps19fH, and the encoded highly conserved putative rhamnosyl transferases (95.2% amino acid identity) are predicted to be functionally identical in both type 19F and 19A CPS biosynthesis. The cps19aI and cps19fI genes are less conserved, with only 78.5% nucleotide identity, and the encoded putative polysaccharide polymerases (80.7% amino acid identity) are predicted to form different glycosidic linkages (as described above). Thus, these data show that it is possible to alter capsule production from type 19F to type 19A by replacing no more than two genes in the cps19f locus and that the presence of the cps19aI gene probably determines the 19A serotype.
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Characterization of a gene rearrangement in the cps locus from strain 19A2. The 19A2 PCR product obtained by using primers J9 and J36 (Fig. 1), which amplified the 3' region of the cps19a locus containing the dTDP-Rha biosynthesis genes (cps19L to -O), was smaller than those from both Rx1-19F and 19A1. To identify the deletion present in 19A2, this PCR product was sequenced, and this region of the locus was designated cps19a2.
Analysis of the sequence identified a gene rearrangement in the 3' region of the cps19a2 locus, as well as a deletion of 1.4 kb of DNA between the end of cps19aO and the start of aliA (Fig. 4). The first 3,347 nucleotides of the cps19a2 sequence have 99.8% identity to cps19a, followed by 1,185 nucleotides with 80% identity to cps19a and 84% identity to cps19f. The remainder of the sequence then diverges until the final 94 nucleotides, which are 90% identical to the same region in cps19a. The conserved regions contain the genes cps19a2JKLMN, with a recombination point approximately 120 nucleotides from the end of cps19a2M. The next 1.1 kb of DNA contains an inverted copy of cps19a2O (Fig. 4) with 76.4% identity to cps19aO and cps19fO. A potential promoter was identified upstream of cps19a2O in the same region (but on the opposite DNA strand) as that for aliA. There are 61 nucleotides between the stop codons of cps19a2N and cps19a2O, and a stem-loop structure which could be a transcription terminator (
G = 
30.5 kcal/mol) was identified in this region (Fig. 4).
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Isolation of the type 19C-specific cps region. The exact location of the extra gene predicted to be present in the type 19C cps locus was investigated by using long-range PCR with a variety of primer pairs (Fig. 5). PCR products obtained with the type 19C template appeared to be approximately 2 kb larger than respective type 19B PCR products with any primer combination that spanned the cps19K-L region. Accordingly, the PCR product amplified from type 19C DNA by using primers J49 and J27 was purified and cloned into pBluescript KS(+), generating pJCP484 (Fig. 5). A map of the 5.3-kb PCR product was also constructed by using the restriction enzymes BamHI, ClaI, HindIII, NsiI, NdeI, and EcoRI (Fig. 5).
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Characterization of Cps19cS.
The type 19C-specific ORF
cps19cS is located between cps19cK and
cps19cL in the cps19c locus (nucleotides 3276 to
4385) and encodes a putative 43.2-kDa protein containing 343 amino
acids. This hydrophilic protein has a hydrophobicity index (according to Kyte and Doolittle [23]) of 0.23 and a predicted
pI of 5.18. The region from the 3' end of cps19cK to the 5'
end of cps19cL has a G+C content of 30.4%, increasing
slightly to 31.4% for the cps19cS coding region. This is
lower than the G+C contents of the two flanking genes
cps19cK (35.3%) and cps19cL (42.6%).
(13)
glucosyl transferase involved in lipopolysaccharide core biosynthesis
in E. coli and Salmonella enterica serovar
Typhimurium (16). Thus, Cps19cS could function as the
glucosyl transferase required for the addition of the (16)-linked
Glc side chain to the backbone in type 19C CPS biosynthesis.
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Serotype distribution of cps19cS.
To examine the
relationship between cps19cS and encapsulation loci of other
S. pneumoniae serotypes, a
SacI-HindIII DNA fragment from a nested
deletion derivative of pJCP484 corresponding to nucleotides 3160 to
4269 of the cps19c sequence was labelled with DIG and used
to probe (at high stringency) Southern blots of restricted chromosomal
DNAs from representative pneumococci belonging to serotypes and
serogroups 2, 3, 4, 6, 7F, 7B, 8, 9N, 9V, 12, 14, 16, 17, 18, 19F, 19A,
19B, 20, 22, 23F, and 24. None of these serotypes had a high-stringency
homologue to cps19cS (result not shown). However, this is
not surprising when the structures for their CPSs are examined, because
none contain a Glc side chain with a (16) linkage
(40).
Transformation of S. pneumoniae type 19F to type 19C. We have previously demonstrated that capsule production was altered from type 19F to type 19B by replacing cps19fIJ with the central region of cps19b, which contains the cps19bPIQRJ genes and determines the 19B serotype (32). A similar approach was taken to determine whether cps19cS is indeed the gene responsible for the additional Glc side chain which distinguishes type 19C CPS. A large PCR product of the cps19c region between cps19cF and aliA was amplified by using primers J5 and J36 (Fig. 5) and transformed into Rx1-19F-I (as described above). The resultant transformant, expressing type 19C CPS, would be predicted to contain the cps19cPIQRJ genes required for both type 19B and 19C CPS biosynthesis as well as cps19cS. The cps19cK gene, which is located between cps19cJ and cps19cL, would also replace the almost identical cps19fK gene (94.9% identity). However, the encoded UDP-GlcNAc-2-epimerase, while essential for CPS biosynthesis in all group 19 members, is not serotype determining (31).
A smooth transformant was found to be erythromycin sensitive, indicating loss of the pVA891 sequence. Southern hybridization confirmed the absence of both pVA891 and the cps19fI gene and the presence of the cps19cP, -J, and -S genes (data not shown). The production of a type 19C capsule by the transformant, designated Rx1-19C, was then confirmed by the quellung reaction. This result showed that it is possible to alter capsule production from type 19F to type 19C by replacing cps19fIJ with the cps19cPIQRJ genes (required for both type 19B and 19C CPS biosynthesis) and the cps19cS gene. Hence, cps19cS determines the 19C serotype.Sequence variation in the 5' intergenic region of serogroup 19. The PCR products from the 5' intergenic regions of 19A1, 19A2, Rx1-19F, 19B, and 19C, amplified by using the DEXB and CPSA2 primers (Fig. 2), were highly variable in size, ranging from approximately 1.2 kb for 19A2 to 2 kb for 19A1 and 19B and 4 kb for Rx1-19F and 19C. The PCR products from 19A1, 19A2, 19B, and 19C were sequenced by using specific primers, and the 5' intergenic regions were compared to that from Rx1-19F.
Interestingly, the 5' intergenic regions of 19A1, 19B, and 19C are all almost identical, with three conserved deletions compared to 19F, of 58, 742, and 321 bp, respectively. These three deletions remove all but 150 nucleotides of the 1-kb intergenic region between dexB and IS1202, as well as the 3' end of IS1202 (up to the stop codon of the putative transposase). Another mutation at nucleotide position 2151 introduces a stop codon which interrupts the putative transposase in 19A1, 19B, and 19C. The 5' intergenic region of the S. pneumoniae type 23F Mexican drug-resistant strain Him18 (37) is almost identical to that of 19A1, 19B, and 19C, suggesting that these strains may have a shared clonal origin. The larger size of the PCR product obtained from 19C is due to the presence of an additional IS element, designated IS19C. This 1.2-kb IS element is inserted into the inverted repeat of IS1202, adjacent to the cps19c locus, and is flanked at both ends by a 13-bp direct repeat, followed by 14 bp of unique DNA and then a 14-bp inverted repeat. The ORF which encodes the putative transposase in IS19C lies in the same orientation as that for IS1202 and opposite to that of the cps19c genes. This putative transposase has 67.5% amino acid identity to the transposase encoded by IS1239 from Streptococcus pyogenes, but at the DNA sequence level these IS elements exhibit negligible similarity. The transposases from these two IS elements also have 28 to 36% amino acid similarity to other transposases found in several different bacterial species, including IS30 from E. coli. Analysis of the sequences indicated that the 5' intergenic region of 19A2 is almost identical to that of 19F, except that it does not contain a copy of IS1202 in the 5' intergenic region, although Southern hybridization data have previously shown that this type 19A strain does contain a copy of IS1202 in its chromosome (29). When PCR products from the 5' intergenic regions from the six Australian type 19A isolates were examined by electrophoresis, they were all the same size as that from 19A2 (data not shown). A type 19F strain which lacks IS1202 has also been previously reported (29). The 5' intergenic regions of four different S. pneumoniae strains belonging to serotypes 2, 3, 14, and 23F were also found to be almost identical to that from 19A2 (13, 20, 22, 30).Conclusions. S. pneumoniae group 19 is the first group for which the cps loci from all of the members (19F, 19A, 19B, and 19C) have been completely characterized (Fig. 6). Functions have been assigned to the majority of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions (15, 31, 32). The ability of PCR products containing either complete or partial cps loci to transform pneumococci from one serotype to another demonstrated that the cps19 loci contain sufficient genetic information for expression of type-specific CPSs.
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ACKNOWLEDGMENT |
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This work was supported by a grant from the National Health and Medical Research Council of Australia.
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Microbiology Unit, Women's and Children's Hospital, North Adelaide, S.A., 5006, Australia. Phone: 61-8-82046302. Fax: 61-8-82046051. E-mail: patonj{at}wch.sa.gov.au.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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| 1. |
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402 |
| 2. | Austrian, R. 1981. Some observations on the pneumococcus and on the current status of pneumococcal disease and its prevention. Rev. Infect. Dis. 3(Suppl.):S1-S17. |
| 3. | Austrian, R., H. P. Bernheimer, E. E. B. Smith, and G. T. Mills. 1959. Simultaneous production of two capsular polysaccharides by pneumococcus. II. The genetic and biochemical bases of binary capsulation. J. Exp. Med. 110:585-602[Abstract]. |
| 4. | Barnes, D. M., S. Whittier, P. H. Gilligan, S. Soares, A. Tomasz, and F. W. Henderson. 1995. Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo. J. Infect. Dis. 171:890-896[Medline]. |
| 5. |
Berry, A. M.,
J. Yother,
D. E. Briles,
D. Hansman, and J. C. Paton.
1989.
Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae.
Infect. Immun.
57:2324-2330 |
| 6. | Beynon, L. M., J. C. Richards, M. B. Perry, and P. J. Kniskern. 1991. Antigenic and structural relationships within group 19 Streptococcus pneumoniae: chemical characterization of the specific capsular polysaccharides of type 19B and 19C. Can. J. Chem. 70:131-137. |
| 7. | Brown, M. C. M., A. Weston, J. R. Saunders, and G. O. Humphreys. 1979. Transformation of E. coli C600 by plasmid DNA at different phases of growth. FEMS Microbiol. Lett. 5:219-222. |
| 8. | Bugert, P., and K. Geider. 1995. Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol. Microbiol. 15:917-933[Medline]. |
| 9. | Coffey, T. J., M. C. Enright, M. Daniels, J. K. Morona, R. Morona, W. Hryniewicz, J. C. Paton, and B. G. Spratt. 1998. Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Mol. Microbiol. 27:73-83[Medline]. |
| 10. | Daniels, C., C. J. Vindurampulle, and R. Morona. 1998. Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy). Mol. Microbiol. 28:1211-1222[Medline]. |
| 11. | Douglas, R. M., J. C. Paton, S. J. Duncan, and D. Hansman. 1983. Antibody response to pneumococcal vaccination in children younger than five years of age. J. Infect. Dis. 148:131-137[Medline]. |
| 12. |
Fallarino, A.,
C. Mavrangelos,
U. H. Stroeher, and P. A. Manning.
1997.
Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1.
J. Bacteriol.
179:2147-2153 |
| 13. | García, E., and R. López. 1997. Molecular biology of the capsular genes of Streptococcus pneumoniae. FEMS Microbiol. Lett. 149:1-10[Medline]. |
| 14. |
Grebe, T.,
J. Paik, and R. Hakenbeck.
1997.
A novel resistance mechanism against beta-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyl transferase.
J. Bacteriol.
179:3342-3349 |
| 15. |
Guidolin, A.,
J. K. Morona,
R. Morona,
D. Hansman, and J. C. Paton.
1994.
Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F.
Infect. Immun.
62:5384-5396 |
| 16. | Heinrichs, D. E., M. A. Monteiro, M. B. Perry, and C. Whitfield. 1998. The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologues. J. Biol. Chem. 23:8849-8859. |
| 17. | Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351-359[Medline]. |
| 18. | Henrichsen, J. 1995. Six newly recognized types of Streptococcus pneumoniae. J. Clin. Microbiol. 33:2759-2762[Abstract]. |
| 19. |
Hofmann, K., and W. Stöffel.
1989.
PROFILEGRAPH: an interactive graphical tool for protein sequence analysis.
Comput. Appl. Biosci.
5:151-153 |
| 20. |
Iannelli, F.,
B. J. Pearce, and G. Pozzi.
1999.
The type 2 capsule locus of Streptococcus pneumoniae.
J. Bacteriol.
181:2652-2654 |
| 21. | Katzenellenbogen, E., and H. J. Jennings. 1983. Structural determination of the capsular polysaccharide of Streptococcus pneumoniae type 19A 57. Carbohydr. Res. 124:235-245[Medline]. |
| 22. | Kolkman, M. A. B., W. Wakarchuk, P. J. M. Nuijten, and B. A. M. van der Zeijst. 1997. Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyl transferases required for the biosynthesis of the tetrasaccharide subunit. Mol. Microbiol. 26:197-208[Medline]. |
| 23. | Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydrophobic character of a protein. J. Mol. Biol. 157:105-132[Medline]. |
| 24. |
Lee, C.-J., and B. A. Fraser.
1980.
The structures of the cross-reactive types 19 (19F) and 57 (19A) pneumococcal polysaccharides.
J. Biol. Chem.
255:6847-6853 |
| 25. |
Lee, C.-J.,
B. A. Fraser,
R. A. Boykins, and J. P. Li.
1987.
Effect of culture conditions on the structure of Streptococcus pneumoniae type 19A 57 capsular polysaccharide.
Infect. Immun.
55:1819-1823 |
| 26. |
Lin, W. S.,
T. Cunneen, and C. Y. Lee.
1994.
Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus.
J. Bacteriol.
176:7005-7016 |
| 27. | Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 28. | Morelle, G. 1989. A plasmid extraction procedure on a miniprep scale. Focus 11.1:7-8. |
| 29. |
Morona, J. K.,
A. Guidolin,
R. Morona,
D. Hansman, and J. C. Paton.
1994.
Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.
J. Bacteriol.
176:4437-4443 |
| 30. |
Morona, J. K.,
D. C. Miller,
T. J. Coffey,
C. J. Vindurampulle,
B. G. Spratt,
R. Morona, and J. C. Paton.
1999.
Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 23F.
Microbiology
145:781-789 |
| 31. | Morona, J. K., R. Morona, and J. C. Paton. 1997. Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway. Mol. Microbiol. 23:751-763[Medline]. |
| 32. |
Morona, J. K.,
R. Morona, and J. C. Paton.
1997.
Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 19B.
J. Bacteriol.
179:4953-4958 |
| 33. |
Morona, J. K.,
R. Morona, and J. C. Paton.
1999.
Analysis of the 5' portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae.
J. Bacteriol.
181:3599-3605 |
| 34. | Muñoz, R., M. Mollerach, R. López, and E. García. 1997. Molecular organisation of the genes required for the synthesis of type 1 capsular polysaccharide of Streptococcus pneumoniae: formation of binary encapsulated pneumococci and identification of cryptic dTDP-rhamnose biosynthesis genes. Mol. Microbiol. 25:79-92[Medline]. |
| 35. | Nesin, M., M. Ramirez, and A. Tomasz. 1998. Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo. J. Infect. Dis. 177:707-713[Medline]. |
| 36. | Ohno, N., T. Yadomae, and T. Miyazaki. 1980. The structure of the type specific polysaccharide of pneumococcus type XIX. Carbohydr. Res. 80:297-304. |
| 37. |
Ramirez, M., and A. Tomasz.
1998.
Molecular characterization of the complete 23F capsular polysaccharide locus of Streptococcus pneumoniae.
J. Bacteriol.
180:5273-5278 |
| 38. | Skurnik, M., R. Venho, P. Toivanen, and A. al-Hendy. 1995. A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis. Mol. Microbiol. 17:575-594[Medline]. |
| 39. | Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517[Medline]. |
| 40. | van Dam, J. E. G., A. Fleer, and H. Snippe. 1990. Immunogenicity and immunochemistry of Streptococcus pneumoniae capsular polysaccharide. Antonie van Leeuwenhoek 58:1-47[Medline]. |
| 41. | Whitfield, C. 1995. Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol. 3:178-185[Medline]. |
Journal of Bacteriology, September 1999, p. 5355-5364, Vol. 181, No. 17
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