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Journal of Bacteriology, August 2001, p. 4927-4931, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4927-4931.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The licC Gene of
Streptococcus pneumoniae Encodes a
CTP:Phosphocholine Cytidylyltransferase
Charles O.
Rock,1,2
Richard J.
Heath,1
Hee-Won
Park,2,3 and
Suzanne
Jackowski1,2,*
Protein Science Division, Department of
Infectious Diseases,1 and Department of
Structural Biology,3 St. Jude Children's
Research Hospital, Memphis, Tennessee 38105, and Department of
Molecular Biosciences, University of Tennessee Health Science
Center, Memphis, Tennessee 381632
Received 12 April 2001/Accepted 23 May 2001
 |
ABSTRACT |
The licC gene product of Streptococcus
pneumoniae was expressed and characterized. LicC is a
nucleoside triphosphate transferase family member and possesses
CTP:phosphocholine cytidylyltransferase activity. Phosphoethanolamine
is a poor substrate. The LicC protein plays a role in the biosynthesis
of the phosphocholine-derivatized cell wall constituents that are
critical for cell separation and pathogenesis.
| |
TEXT |
Streptococcus pneumoniae
requires choline for growth, and it decorates the teichoic and
lipoteichoic acids of the cell wall with this essential nutrient
(5). It is established that choline metabolism plays an
indispensable role in cell separation, transformation, autoloysis, and
pathogenicity of S. pneumoniae. For example, the cell
surface phosphocholine (P-Cho) participates in the interaction with the
host surface and induces attachment and invasion (4, 15).
The importance of choline in pathogenesis is not confined to S. pneumoniae but is also found in Haemophilus influenzae
(9, 10, 18, 19), Pseudomonas aeruginosa, and
Neisseria gonorrhoeae (12, 17). In addition,
extracellular P-Cho serves as the scaffold for a group of
choline-binding proteins that are secreted from the cells and are
subsequently attached to the cell surface by their homologous
choline-binding domains (see reference 11 and references therein).
The pathway for choline metabolism in S. pneumoniae and
H. influenzae has been hypothesized to consist of a choline
transport system, choline kinase, CTP:phosphocholine
cytidylyltransferase (CCT), and a choline phosphotransferase that
transfers P-Cho to lipoteichoic acid or lipopolysaccaride
(18). The existence of this pathway is supported by the
detection of choline kinase and CCT activity in crude extracts of
S. pneumoniae (1, 20). Genetic elements
required for choline incorporation into the lipopolysaccharide of
H. influenzae are found in the lic1 locus, which
contains four open reading frames. The hypothesis drawn from the
bioinformatic analysis of the lic1 locus (19)
is that licA corresponds to choline kinase based on a 31%
identity to the choline kinase of Saccharomyces cerevisiae
over the short span of 40 amino acids between residues 222 and 262. The
licB gene has several predicted transmembrane domains and is
thus postulated to be a choline transporter. The hydrophilic
licC gene product is a candidate for the CCT due to the
resemblance of its amino terminus to the amino-terminal 60 residues of
nucleoside triphosphate (NTP) transferase family members, leaving the
licD gene as a candidate for the choline phosphotransferase.
A homologous licC gene exists in S. pneumoniae (21), and the predicted LicC proteins of H. influenzae and S. pneumoniae are 37% identical and
60% similar. There are no examples of bacterial CCTs, and both of the
predicted LicC proteins lack homology to either the prototypical
metazoan CCTs (7, 14) or the CTP:glycerol-3-phosphate
cytidylyltransferase from Bacillus subtilis
(16). Rather, licC is related to members of the
NTP transferase superfamily that primarily activate phosphosugars, such
as galU of Escherichia coli and rmlA
of P. aeruginosa (Fig. 1),
opening the possibility that licC is involved in
carbohydrate metabolism. The goal of this study was to determine if the
licC gene encodes a novel CCT.
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FIG. 1.
Similarity of LicC to NTP transferases. The predicted
amino acid sequence of the licC gene of S.
pneumoniae is aligned with the predicted amino acid sequences
of the E. coli galU and P. aeruginosa
rmlA genes, two nucleotidyltransferases involved in the
activation of phosphosugars for the biosynthesis of cell wall
polysaccharides. Identical amino acids between LicC and either of the
two NTP transferases are highlighted. Members of the NTP transferase
family contain the KAX5GXGTRX8PK
amino-terminal motif.
|
|
Similarity of LicC to NTP transferases.
LicC is classified as
a member of the NTP transferase (nucleotidyltransferase) superfamily of
proteins based on the presence of the signature sequence
(KAX5GXGTRX8PK)
for this group of enzymes at its amino terminus. Figure 1 shows the
relationship of the primary sequence of LicC to two representative
members of this family, a phosphosugar uridylyltransferase (GalU) and
thymidylyltransferase (RmlA) from gram-negative bacteria. LicC
possessed a high degree of similarity with these two
nucleotidyltransferases in the amino-terminal domain (Fig. 1). The
crystal structures of members of the NTP transferase family verify that
this amino-terminal sequence motif is involved in nucleotide binding
(2, 3).
Expression, purification, and activity of LicC.
The
licC gene was amplified using primers designed to introduce
an NdeI site at the initiator codon and a BamHI
site after the stop codon. The fragment was sequenced and cloned into
pET-15b for expression as a His-tagged protein. The construct began at Ile1 and ended at Asn234 (Fig. 1). The construct was sequenced to
verify the absence of PCR artifacts.
The protein was purified on Ni-nitrilotriacetic acid agarose followed
by gel filtration on Superdex-200 26/60 (Fig.
2). The enzyme was homogeneous as judged
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The
apparent molecular mass calculated from the gel filtration column was
30 kDa, which agreed with the predicted molecular mass of LicC plus the
His tag based on the amino acid sequence (Fig. 1). This result
indicates that LicC exists as a monomer (Fig. 2). The protein was
stored in 50% glycerol-20 mM Tris-HCl, pH 7.5, at 
20°C and
retained full activity for several months.
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FIG. 2.
Purification of LicC. The His-tagged LicC protein was
purified by Ni-nitrilotriacetic acid affinity chromatography as
described previously (6). The protein was immediately
applied to a Superdex-200 26/60 column (Pharmacia Biotech) equilibrated
with 50 mM Tris-HCl (pH 7.0), 1 mM dithiothreitol, and 1 mM EDTA and
was eluted at a flow rate of 2.5 ml/min. LicC was monitored at 280 nm
and eluted at 226 ml. The molecular mass was estimated to be 30 kDa by
graphic analysis of a standard curve based on the elution volumes
(arrows) of protein molecular mass standards (Sigma).  A, -amylase
(200 kDa); ADH, alcohol dehydrogenase (150 kDa); BSA, bovine serum
albumin (66 kDa); CA, carbonic anhydrase (29 kDa); CytC, cytochrome
c (12.4 kDa). LicC was pure, based on analysis with a
sodium dodecyl sulfate-12% polyacrylamide gel followed by Coomassie
blue staining (inset).
|
|
LicC catalyzed the formation of CDP-Cho as identified by thin-layer
chromatography (Fig. 3). LicC
cytidylyltransferase activity was linear with time and protein and
exhibited a specific activity of 2.5 µmol/min/mg of protein in the
standard assay conditions (Fig. 3, inset).
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FIG. 3.
LicC is a CCT. The LicC assay contained 2 mM CTP, 10 mM
MgCl2, 1 mM phospho[methyl-14C]choline
(specific activity of 3.68 mCi/mmol), 150 mM bis-Tris-HCl (pH 7.0), and
62.5 ng of LicC in a final volume of 50 µl. LicC was added last to
initiate the reaction, and after 10 min at 37°C, the reaction was
stopped by the addition of 5 µl of 0.5 M EDTA. A 40-µl aliquot of
the reaction mixture was applied to a preabsorbent silica gel G plate
that was developed with 95% ethanol-2% ammonium hydroxide (1:1,
vol/vol) (8). The single product was visualized with a
Bioscan Imaging detector and migrated with authentic CDP-Cho. The
formation of CDP-Cho per assay was linear with LicC (inset).
|
|
Biochemical properties.
We compared the activity of LicC using
either ATP, TTP, UTP, or GTP in place of CTP, all at 2 mM
concentration. No activity was detected with TTP, UTP, or GTP. The
activity with ATP was 0.25% of that with CTP (data not shown). The
high degree of nucleotide selectivity was consistent with the
identification of LicC as a cytidylyltransferase rather than another
nucleotidyltransferase. The Km for
CTP was 133 µM (Fig. 4A) and for
P-Cho was 83 µM (Fig. 4B). In both cases, the
Vmax was 7.7 ± 0.2 µmol/min/mg. There was no indication of cooperativity in the binding
of either substrate consistent with LicC existing as a monomer.
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FIG. 4.
Kinetic constants for LicC. The kinetic constants for
the two LicC substrates are shown. Double reciprocal plots were used to
calculate the apparent Km for CTP (A),
P-Cho (B), and P-Etn (C) by employing the radiochemical assay described
in the legend to Fig. 3. (A) The CTP
Km was 133 µM measured in the
presence of 1 mM P-Cho. (B) The P-Cho
Km was 83 µM measured in the
presence of 2 mM CTP. (C) The Km for
P-Etn of 1.43 mM was determined in the presence of 2 mM CTP. Assays for
CDP-Etn formation were the same as the standard assay, except that
P-Etn (specific activity of 3.68 mCi/mol) was substituted for P-Cho and
that the LicC concentration was 10 µg/assay. The error bars represent
the range of data.
|
|
We assessed the ability of LicC to use phosphoethanolamine (P-Etn) as a
substrate by substituting 1 mM P-[14C]Etn
(specific activity of 3.68 mCi/mmol) for
P-[14C]Cho in the standard assay described for
Fig. 3 and raising the LicC concentration to 10 µg/assay. LicC was
capable of utilizing P-Etn as the substrate but was much less
efficient. The protein exhibited a Km of
1.43 mM for P-Etn and a Vmax with this
substrate of 0.06 µmol/min/mg. Thus, LicC was able to convert P-Etn
to CDP-Etn, but it used P-Etn much less efficiently as a substrate.
Conclusions. Our study establishes that the third
gene in the lic1 cluster, licC, encodes a
functional CCT responsible for the formation of CDP-Cho. LicC does not
have similarity in primary sequence to the known metazoan CCTs and thus
represents a unique member of this functional enzyme class. However,
LicC is a member of the NTP transferase (nucleotidyltransferase)
superfamily of enzymes that transfer the nucleoside
monophosphate moiety to a phosphorylated acceptor
substrate concomitant with the release of pyrophosphate (National
Center for Biotechnology Information Conserved Domain Database, protein
family no. 00483). The inclusion of LicC in this family is illustrated
by its similarity to the amino termini of GalU and RmlA, two
nucleotidyltransferases that are involved in the formation of
nucleoside diphosphate-hexose derivatives that are used in the
biosynthesis of cell wall components (Fig. 1). The less conserved
regions in the middle of these proteins represent the substrate-binding
regions. Many family members are dimers or tetramers; however, LicC
appears to be a monomer, based on the results of gel filtration
chromatography (Fig. 2). The structure of RmlA (2) shows
that the subunit interaction surface is encoded by the carboxy-terminal
domain. Interestingly, LicC is smaller than typical NTP transferases
due to the lack of a carboxy-terminal tail (Fig. 1), consistent with
its monomeric behavior in solution.
The biochemical properties of LicC are consistent with its proposed
role in the pathway for the decoration of teichoic acids with P-Cho.
Etn does replace Cho as a nutritional requirement and is incorporated
to approximately the same extent into the cell wall (13).
However, the replacement of Cho with Etn results in several severe
phenotypic changes, including the lack of daughter cell dissociation
and autolysis, resistance to deoxycholate-induced lysis, and the
inability to undergo genetic transformation. Significantly, the
presence of Cho in the medium completely blocks the incorporation of
Etn into cell walls, illustrating that the pathway is highly selective
for Cho. This physiological observation suggests that the Cho
incorporation pathway is highly selective for Cho but is capable of
using Etn. This conclusion is reflected in the substrate specificity of
LicC, and we suggest that the other enzymes in the pathway will have
the same dual specificity and Cho selectivity. The importance of
choline metabolism to viability and virulence suggests that LicC would
be a excellent target for the development of antibacterial therapies.
| |
ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health Grants
GM34496 and GM45737, Cancer Center (CORE) Support Grant CA21765, and
the American Lebanese Syrian Associated Charities.
We thank Pam Jackson and Matthew Frank for their expert technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Danny Thomas
Research Tower, Rm. 4007B, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3494. Fax:
(901) 525-8025. E-mail: suzanne.jackowski{at}stjude.org.
| |
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Journal of Bacteriology, August 2001, p. 4927-4931, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4927-4931.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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