Journal of Bacteriology, October 1999, p. 6556-6559, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Novel Gene Required for Rhamnose-Glucose
Polysaccharide Synthesis in Streptococcus mutans
Yoshihisa
Yamashita,1,*
Yukie
Shibata,1
Yoshio
Nakano,1
Hiromasa
Tsuda,1
Nobuo
Kido,2
Michio
Ohta,3 and
Toshihiko
Koga1
Department of Preventive Dentistry, Kyushu
University Faculty of Dentistry, Fukuoka
812-8582,1 Biosystems, School of
Informatics and Sciences, Nagoya University, Nagoya
464-8601,2 and Department of
Bacteriology, Nagoya University, School of Medicine, Nagoya
466-8550,3 Japan
Received 7 June 1999/Accepted 30 July 1999
 |
ABSTRACT |
Gene rgpG is required for biosynthesis of
rhamnose-glucose polysaccharide (RGP) in Streptococcus
mutans. Its deduced amino acid sequence had similarity to WecA,
which initiates syntheses of enterobacterial common antigen and some O
antigens in Escherichia coli. Gene rgpG
complemented a wecA mutation of E. coli,
suggesting that rgpG may function similarly in RGP synthesis.
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TEXT |
Cell wall antigens of
Streptococcus mutans are rhamnose-glucose polysaccharides
(RGPs), which are composed of 
1,2- and 1,3-linked rhamnan
backbones and glucose side chains (8, 14). The biofunction of RGP is receiving increasing attention because of the facts that the
serotype f-specific RGP induces the release of inflammatory cytokines (19) and also provokes nitric oxide production
(9).
We recently cloned three loci that are related to RGP synthesis. Four
rml genes (rmlA, rmlB,
rmlC, and rmlD) that are directly involved in the
synthesis of dTDP-L-rhamnose (22, 23) and a gene
(gluA) encoding glucose-1-phosphate uridylyltransferase, which synthesizes UDP-D-glucose (28), were
identified in these loci. The two nucleotide sugars were found to
be immediate precursors for RGP synthesis (22, 23, 28).
In addition, we found six rgp genes (rgpA,
rgpB, rgpC, rgpD, rgpE, and
rgpF) required for RGP synthesis in the region downstream
from rmlD (27). Some of these rgp
genes are probably involved in the transport and assembly of RGP.
Here, we describe the identification and characterization of an
additional gene required for RGP synthesis, which is located in a
new locus.
Isolation of an RGP-defective mutant of S. mutans.
A
complete Sau3AI digest of the S. mutans Xc
chromosome was ligated to BamHI- and
BglII-digested pResEmBBN. pResEmBBN was produced by Shiroza
and Kuramitsu in the process of constructing pResEmMCS11
(18) to prepare an S. mutans genomic
library and is equivalent to pResEmMCS11, except that it lacks the
restriction sites from XbaI to NotI in the
multicloning site. S. mutans Xc (22) was randomly
mutated by transformation with the S. mutans genomic
library. Transformants were selected on tryptic soy agar plates
containing 10 µg of erythromycin per ml. Both rml and
rgp mutants of S. mutans, which are completely
unable to synthesize RGP, have a characteristic colony morphology
(22, 23, 27). The fact enables us to visually distinguish a
mutant defective in RGP synthesis from other mutants.
Fifteen transformants that appeared to be RGP-defective mutants were
selected after visually screening 9,000 transformants. Immunodiffusion
analysis of their cell wall polysaccharide extracts with serotype
c-specific rabbit antiserum (23) and a high-pressure liquid
chromatography analysis (23) of sugar components in their cell wall fractions revealed that 7 of the 15 transformants actually lost serotype c-specific polysaccharides.
Southern blot analyses of XbaI- and
BstEII-digested chromosomes of the seven transformants with
defective RGP synthesis were performed using digoxigenin (DIG)-labeled
PCR probes specific for rmlC and rmlD as
previously described (22, 23). The results indicated that
one of seven transformants did not have an insert in the known
rgp or rml gene cluster. We designated this
transformant Xc51.
Characterization of plasmid insertion point in Xc51.
Southern
blotting with a DIG-labeled PCR probe specific for the Emr
gene revealed that the probe hybridized with a 4.6-kb EcoRI
fragment from Xc51 but not with any fragments in the wild-type strain
Xc. A PCR probe specific for the Emr gene was prepared by
PCR with a set of primers (5'-CTTAGAAGCAAACTTAAG-3' and
5'-TTATTTCCTCCCGTTAAA-3'), using pResEmBBN as a
template. Strain Xc was transformed with the chromosome DNA prepared
from Xc51. Every Emr transformant showed the same colony
morphology as that of Xc51. Five randomly selected transformants were
further confirmed to be defective in RGP synthesis. Southern blot
analysis with the specific probe for the Emr gene of these
transformants gave identical results to that for Xc51. These findings
indicated that integration of pResEmBBN in a specific locus on the
Xc chromosome, which was different from the rml and
rgp loci involved in RGP synthesis, produced a novel defect in RGP synthesis.
Cloning and sequencing of the region flanking the plasmid insertion
in strain Xc51.
The EcoRI-digested chromosome of
Xc51 was self-ligated. Escherichia coli DH5 was
transformed with this DNA, and transformants were isolated on
Luria-Bertani agar plates containing 200 µg of erythromycin per
ml. All plasmids isolated from transformants were identical (4.6 kb) in
size. One of them was designated pKU51. The plasmid labeled
with DIG-dUTP via random primer labeling hybridized with
the 4.6-kb EcoRI fragment in Xc51 and a 2.6-kb
EcoRI fragment in Xc. The 2.6-kb EcoRI-fragment
of Xc was cloned in pBluescript SKII+ by colony hybridization. The
resulting plasmid, pBluescript SKII+ carrying the 2.6-kb
EcoRI-fragment, was designated pKU52 and the
nucleotide sequence of the insert was determined with a 373 STRETCH
automated sequencer (Applied Biosystems, Inc., Foster City,
Calif.) as described previously (26).
The nucleotide sequence analysis revealed the presence of two open
reading frames (ORFs), as shown in Fig.
1. They were oriented in the same
direction as the plasmid lacZ gene. No ORF on the opposite
strand of this region was more than 250 bp in length. A possible
Shine-Dalgarno sequence was identified just upstream from the potential
initiation codons of orf1. A consensus 
10 and 35
E. coli promoter-like sequence
(TTGAAA-N17-TATAAT; positions 302 to 330) for
orf1 and an inverted repeat structure (positions 2257 to
2274) with a free energy of 16.8 kcal/mol followed by a polyT
sequence, which may act as a transcriptional terminator for
orf2, were recognized. On the other hand, there was no
typical ribosomal binding motif for orf2.
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FIG. 1.
Restriction map of the EcoRI 2.6-kb insert
fragment cloned in pKU52. The large arrows indicate the locations of
the two ORFs. The hatched bar shows the 0.9-kb PpuMI
fragment that was replaced with the Emr gene in Xc52. The
small arrows labelled RgpGF2 and RgpGR1 show the positions of primers
RgpGF2, 5'-GATAAGCTAGATGATACCTT-3' (complementary to
positions 1078 to 1097), and RgpGR1, 5'-AAATTACTTTTTCTTCTTAC-3'
(complementary to positions 2244 to 2225), respectively. The
locations of the inserted fragments are indicated in the lower portion
of the diagram.
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|
The nucleotide database was searched for orf1 and
orf2 using the program FASTA (13) on the DDBJ
server at the National Institute of Genetics, Mishima, Japan. The amino
acid sequence deduced from orf1 showed 25.5% identity with
that from the mecA gene of Bacillus subtilis,
which is involved in the negative regulation of genetic competence
(7). The amino acid sequence deduced from orf2
showed 19.1% identity with that from the wecA
(rfe) gene, involved in initiating the syntheses of
enterobacterial common antigen (ECA) and O8 and O9a antigens in
E. coli (5, 10, 15).
The insert fragment of the integration plasmid should locate within the
target gene to disrupt the gene by Campbell-type recombination. Two
Sau3AI fragments (fragments A and B) located within
orf1 and orf2 (Fig. 1) are candidates as the
insert fragment in the integration plasmid that produced Xc51. However,
pKU51 possessed a unique PstI site. This suggests that the
Sau3AI fragment within orf2 (fragment B) must be
the insert fragment in the integration plasmid that produced Xc51,
because the insert fragment must be duplicated when it is recovered in
pKU51. Based on these findings, we hypothesize that the disruption of
orf2 caused the RGP-defective phenotype of S. mutans.
Insertional inactivation of orf2.
The 0.9-kb
PpuMI fragment within orf2 on pKU52 was replaced
with the 1.0-kb Emr gene (18). The resultant
plasmid was linearized by EcoRI digestion and used to
transform strain Xc. Four randomly selected Emr
transformants were examined by immunodiffusion analysis, and all were
ascertained to be defective in RGP synthesis. An appropriate disruption
of orf2 in these transformants was confirmed by PCR amplification of the 1.2-kb region spanning orf2 with
primers RgpGF2 and RgpGR1 (Fig. 1) and the subsequent HincII
digestion of this fragment, because there is a HincII site
within the Emr gene but none in orf2. One of the
transformants was designated Xc52.
When the total amount of hexosamine in the purified cell wall
preparation of Xc52 was determined by using the colorimetric method of
Strominger et al. (21), the hexosamine content in the cell
wall of Xc52 (163 ± 13 µg per mg [dry weight] of the purified
cell wall preparation) was not very different from those in the cell
walls of Xc and the purified cell wall preparations of its
rml mutants (23). It seems unlikely that
rgpG is involved in peptidoglycan synthesis.
Complementation analysis of rgpG.
We constructed a new
E. coli-streptococcus shuttle plasmid, pKU55, containing a
tetracycline resistance marker, the tetM916 gene derived
from pLN2 (2), which functions both in E. coli and in streptococci, and the pUC and pC194 replicons for maintenance in
E. coli and in streptococci, respectively, which are
derived from pTH10 (4). Expression of rgpG
was ensured by the promoter sequence in the 0.35-kb upstream region of
the Emr gene from pAM77 (3). The 0.35-kb region
was amplified by PCR with primers EmF2,
5'-AGAGAGTCTAGAGAAGCAAACTTAAGAGTGTG-3'
(XbaI site underlined), and EmR2,
5'-GTGTGTCTGCAGTTTCGTCGTTAAATGCC-3' (PstI site underlined), using pResEmBBN as a
template. The PCR fragment was digested with XbaI and
PstI and ligated to the XbaI and
PstI sites of pKU52, producing pKU54, which contains
the streptococcal promoter sequence at the 5' terminus of
rgpG. The fragment containing both promoter sequence and
rgpG in the same direction was excised from pKU54 by
digestion with XbaI and HindIII and ligated
to the XbaI and HindIII sites of pKU55; the
resulting plasmid was designated pKU56.
Direct transformation of the rgpG mutant with pKU56 was
abandoned because we tried unsuccessfully to transform Xc52 with
shuttle plasmid pKU55 and also with a tetracycline resistance marker on the chromosomal DNA from strain GS5DD (2). The surface
structure of cells with normal RGP synthesis might be critical to the
genetic competence of S. mutans. As an alternative,
wild-type strain Xc was initially transformed with pKU56 and
subsequently transformed with EcoRI-digested pKU53. Three
transformants which were randomly selected retained their serotype c
antigenicity and had normal rhamnose and glucose contents in cell wall
preparations, suggesting that the rgpG gene located on the
shuttle plasmid complemented the inactivated chromosomal
rgpG. The result ruled out a possible polar effect of the
pResBBN insertion and proved a direct effect of the disruption of
rgpG on the phenotype of Xc52.
Functional analysis of rgpG.
We examined whether
rgpG complements wecA. A plasmid, pKU58, which
has only rgpG on the insert fragment, was constructed by PstI digestion of pKU52 and self-ligation for removal of
orf1 (Fig. 1). The plasmid was introduced into E. coli 21548, which is a wecA-defective mutant of strain
K-12 (10). Strain 21548 was kindly provided by P. D. Rick, Department of Microbiology, Uniformed Services University of
Health Sciences, Bethesda, Md. ECA and O9a antigen production in
E. coli strains were detected by immunoblotting analysis
with anti-ECA serum and anti-O9a monoclonal antibody (MAb F719),
respectively, as previously described (5, 12), because the
wecA gene is known to be involved in the syntheses of ECA
(10) and O9a antigen (5). Strain 21548 transformed with pKU58 and its parental strain AB1133 produced ECA,
whereas strain 21548 transformed with pBluescript SKII+ did not (Fig. 2A). Furthermore, O9a antigen production
was observed in strain 21548 transformed with both pKU58 and pNKB26 and
in strain AB1133 transformed with only pNKB26 but not in 21548 transformed with pNKB26 (Fig. 2B). These findings indicate that
rgpG complemented the wecA-deficient phenotype of
E. coli, suggesting a functional similarity between the gene
products of rgpG and wecA.
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FIG. 2.
Immunoblots with anti-ECA serum (A) and monoclonal
antibody against O9 antigen (B). Shown is complementation analysis of
the E. coli wecA mutant (21548) with rgpG. ECA or
lipopolysaccharide was separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and detected by
immunoblotting with the respective antiserum. (A) Lane 1, strain
AB1133; lane 2, strain 21548 transformed with pKU58; lane 3, strain
21548 transformed with pBluescript SKII+. (B) Lane 1, strain AB1133;
lane 2, strain 21548 transformed with pKU58 and pNKB26; lane 3, strain
21548 transformed with pNKB26.
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|
There was no typical preceding ribosomal binding motif for
rgpG. Several potential start codons in the 5'-terminal part
of rgpG in addition to the TTG codon at position 1075 were
expected. Therefore, truncated PCR fragments were amplified and ligated into pBluescript SKII+. Transformants of strain 21548 with the ligated
DNA were obtained, and their ECA production was checked by Western blot
analysis with anti-ECA serum as described above. The transformant with
the plasmid containing the largest rgpG fragment that
covers the TTG codon at position 1075 produced ECA, whereas
all the other transformants with plasmids containing smaller PCR
fragments did not. The fidelity of the PCR amplification was confirmed by sequencing the insert of the plasmids. This
complementation study suggested that translation of the largest
coding frame is essential for the enzyme activity of the
rgpG gene product. This study clearly showed that the TTG at
position 1075 is the initiation codon for rgpG translation.
To date, three distinct pathways have been proposed for the
initiation of cell surface polysaccharide synthesis in some bacteria. The enzyme encoded by wecA is an
N-acetylglucosamine-1-phosphate transferase that forms
lipid I (undecaprenyl pyrophosphoryl
N-acetylglucosamine). In gram-negative bacteria, this
enzyme is involved in the first step of the biosyntheses of ECA and
several lipopolysaccharide O antigens, even when the mature
polysaccharides do not contain N-acetylglucosamine residues
(24). The second pathway, that for the biosynthesis of the
heteropolysaccharide O antigen in Salmonella enterica, is
initiated by the enzyme encoded by wbaP, which catalyzes the
transfer of galactose-1-phosphate to a lipid carrier. A similar type of
initiation of polysaccharide synthesis is speculated to occur in the
capsular polysaccharide production systems of group B streptococci
(17) and Streptococcus pneumoniae (6,
11). Finally, group A streptococci are not believed to utilize a
lipid intermediate in the production of their hyaluronic acid capsule
(20). In addition, it was recently reported that no
lipid-linked intermediates are detected in the synthesis of polysaccharide intercellular adhesin in Staphylococcus
epidermidis (1).
Functional similarity between RgpG and WecA hinted that transferring
N-acetylglucosamine-1-phosphate to a lipid carrier may be
the first step in RGP synthesis, even though repeating units of RGP do
not contain N-acetylglucosamine. It is interesting that a
gene highly homologous (47.3% identity) to rgpG was found
in a locus involved in polysaccharide biosynthesis in
Enterococcus faecalis (25). However, the gene
(orfde3) is not required for antigenic polysaccharide
biosynthesis, and its biological function remains to be determined. The
present study is the first report of functional characterization of a
gene homologous to wecA in gram-positive bacteria, although
intermediates of teichuronic acid biosynthesis in Micrococcus
luteus have earlier been shown to contain undecaprenyl
pyrophosphoryl N-acetylglucosamine structure (3a).
Mechanisms of cell surface polysaccharide synthesis are more
poorly characterized in gram-positive bacteria than in
gram-negative bacteria (16). The lipid carrier
involved in the synthesis of the cell surface polysaccharides of
gram-positive bacteria, including S. mutans RGP, and the way
these polysaccharides are linked to the cell wall still remain to be
clarified. Further characterization of this enzyme is important to
resolve the polysaccharide synthetic pathway in gram-positive bacteria.
Nucleotide sequence accession number.
The 2,614-bp nucleotide
sequence described in this paper has been submitted to the
EMBL/GenBank/DDBJ data bank under accession no. AB022909.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a Grant-in-Aid for Developmental
Scientific Research [(B)09470474] (Y.Y.) from the Ministry of
Education, Science, Sports and Culture of Japan and the Kyushu University Interdisciplinary Programs in Education and Projects in
Research Development.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Preventive Dentistry, Kyushu University Faculty of Dentistry, Fukuoka 812-8582, Japan. Phone: 81-92-642-6353. Fax: 81-92-642-6354. E-mail: yoshidha{at}mbox.nc.kyushu-u.ac.jp.
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Journal of Bacteriology, October 1999, p. 6556-6559, Vol. 181, No. 20
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