Full text of DOI:10.1089/10766290152652756

MICROBIAL DRUG RESISTANCE
Volume 7, Number 3, 2001
Mary Ann Liebert, Inc.
Molecular Characterization of the Pneumococcal Teichoic
Acid Phosphorylcholine Esterase
BLANCA DE LAS RIVAS, JOSÉ L. GARCÍA, RUBENS LÓPEZ, and PEDRO GARCÍA
ABSTRACT
A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-bind-
ing proteins) has permitted the localization, cloning, sequencing, and overexpression of a gene (pce), coding
for a protein (Pce) that liberates phosphorylcholine from purified cell walls of Streptococcus pneumoniae. The
pce gene of the pneumococcal strain R6 encodes a protein of 627 amino acids with a predicted Mr of 72,104.
Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid. In silico analy-
sis of Pce shows a modular organization of the enzyme where the choline-binding domain, involved in cell
wall substrate recognition, and the catalytic domain are located at the carboxy- and amino-terminal moieties
of the protein, respectively. Remarkably, a long tail of 85 amino acids follows the carboxy-terminal domain,
a structural feature that had not been described for the published choline-binding proteins. The carboxy-ter-
minal moiety of Pce is assembled by 10 repeated motifs, and the protein has also a cleavable signal peptide
of 25 amino acids that renders after its cleavage a mature protein of 69,426 Da (602 amino acids). The pce
gene has been expressed in Escherichia coli, and Pce was active when assayed on pneumococcal walls. We
have also found that the signal peptide of Pce was functional in E. coli. Biochemical analysis suggested that
Pce is the teichoic acid phosphorylcholine esterase of S. pneumoniae that had been biochemically character-
ized previously. Construction of two pce pneumococcal mutants (R6D and M31D) by insertion-duplication
mutagenesis revealed that these mutants grew at a doubling-time similar to those of the parental strains of
the wild-type R6 and the lytA-mutant M31, respectively. R6D and M31D were morphologically indistin-
guishable from the parental strains when whole-mounted cells were observed under the electron microscope
and exhibited levels of competence for genetic transformation slightly lower than those reported for R6 and
M31.
choline residues and 22% with one.6 The choline residues of
the TA and LTA, among many other functions, serve as ligand
INTRODUCTION
TREPTOCOCCUS PNEUMONIAE (PNEUMOCOCCUS) is an impor- for the attachment of a number of proteins to the cell surface,
tant human pathogenas a common etiologicalagent in com- named choline-binding proteins (CBPs).9 It has been pointed
S
munity-acquired pneumoniae and meningitis in adults and in out that pneumococcus has evolved this unique mechanism to
acute otitis media in children. Pneumococcus is unique among associate proteins to the cell surface.4 In this sense, we had
the Gram-positive bacteria because of the nutritional require- shown that two CBPs, the major pneumococcalautolysin(LytA
ment of this bacterium for choline31 and because the teichoic
amidase) and the Cpl1 lysozyme encoded by the pneumococ-
acid (TA), and lipoteichoic acid (LTA) polymers of the cell cal Cp-1 phage, contain a homologous carboxy-terminal do-
wall membrane complex have the same chain structure. Inter- main (choline-binding domain, ChBD) of six 20-amino acid
estingly, the aminoalcohol choline is a structural component of tandem repeats (motifs) that allows the binding of these en-
the TA and LTA and, for a given strain, an identical number zymes to the cell wall substrate through the choline residues of
of phosphorylcholine residues have been found per repeat. This TA.7 Up to now all the described CBPs contain this typical
S. pneumoniae
is, in
R6 strain, on the average, 78% of the re-
ChBD with a variable number of repeats either in its carboxy-
peats in LTA and TA are substituted with two phosphoryl- terminal or its amino-terminal position.11 Moreover, the criti-
Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain.
213

214
DE LAS RIVAS ET AL.
TABLE 1. BACTERIAL STRAINS, PLASMIDS, AND PRIMERS
a
Material
Description
Reference or source
Bacterial strains
S. pneumoniae
R6
Wild type
lytA
Rockefeller University
M31
R6D
M31D
M222
D
R6 mutant
(22)
This study
This study
(5)
pce erm
R6 ( ;; C)
pce erm
M31 ( ;; C)
hex-4, end-1, exo-2,
1
2
R
Lyt , S2 , Str
E. coli
supE44 hsdR17 recA1 endA1 lacU169 ( 80 lacZ M15)
DH5a
D
D
D
(21)
(27)
hsdR17 recA1 endA1 gyrA96 thi-1 relA1
BL21 (DE3)F2
with DE3
ompT gal [cdm] [lon] hsdSB
Plasmids
pUC18
pT7-7
ApR
BioLabs
(28)
(8)
(11)
Unpublished
(1)
This study
This study
This study
This study
ApR
lytA
lytC
lytB
R
R
pGL100
pLCC14
pRGR5
, Ap
, Ap
, Ap
R
pUCE191 LnR/ErR, ApR
R
pce
pRGR11
pRGR12
pRGR18
, Ap
R
pce
2.5-kb
with deleted signal peptide encoding sequence, Ap
R
Eco
Hin
Bam pce
HI fragment containing , Ap
cII internal fragment of
RI–
R
R
R
pce
, Ln /Er , Ap
pRGR191 1.1-kb
Primers
LytDEco
LytDBam 59-TTGGATCCATCAGCAATCTTAGTCGGACGC-3 9
LytDN1
LytDN2
LytDC
59-CCGAATTCGGCTGTAACTGCTAAAGGGAAG-3 9
This study
This study
This study
This study
This study
59-CCGAATTCTGGAGATTGAGAAAGTAAGAATG -39
59-CCGAATTCAAGGAGATTAACATATGCAAGAAAGTTCAGGAAATAAAATCC-3 9
59-TTGGATCCCTACTACTGTTCTGATTCCGATTTG-3 9
aStr, streptomycin; Ap, ampicillin, Ln, lincomycin; Ery, erythromycin. The nucleotides underlined denote the restriction sites
and start codons.
cal role of several CBPs in the physiology and pathogenicity
of pneumococcus has been recently documented in the litera-
ture.13
MATERIALS AND METHODS
Bacterial strains, plasmids, growth conditions,
and transformation
The functions of some of the CBPs remain to be determined.
In this sense, a teichoic acid phosphorylcholine esterase had
The bacterial strains, plasmids and oligonucleotide primers
S. pneumoniae14
Streptococcusoralis20
been describedin
and in
E. coli
used in this study are listed in Table 1.
was grown in
as an enzyme that liberated phosphorylcholine residues from
the choline-containing cell walls, but in spite of the fact that
this enzyme might be a clear CBP candidate, it remained to be
characterized at a molecular level. It should be pointed out that
pneumococcus undergoes spontaneous and reversible phase
variationmarked by switching from opaque to transparentmor-
photypes, and vice versa, that implies changes in the amount
Luria-Bertani medium.21
was grown in C
S. pneumoniae
medium16 supplemented with yeast extract (0.8 mg/ml; Difco
Laboratories) (C1Y medium). The procedures used for the
preparation of the competent pneumococci and the transform-
ing DNA (Strr) were essentially those described for analysis of
S. pneumoniae.
genetic transformationin
32 Transforming DNA
was prepared from strain M222, and transformation frequency
was calculated as the number of streptomycin-resistant trans-
formants per milliliter times 100 divided by the total number
i.e.,
of choline in the cell wall,
high amount in transparent and
low amount in opaque variants33 and it is tempting to specu-
late on a role of the esterase in this conversion.However, a fine
characterizationof the turnover of choline has not yet been de-
termined although this aminoalcohol is essential for the biol-
ogy of pneumococcus.
E. coli
of viable cells per milliliter.
was transformed following
21
a procedure previously described.
In this study, we have isolated, cloned, and overexpressedin
Preparation of choline-binding proteins from
autolytic walls
Escherichia coli
pce
that en-
a new pneumococcalgene named
codes an enzyme capable of liberating phosphorylcholinefrom
choline-containing cell walls and that, according to its molec-
ular structure, represents a new member of the CBP family. In
a parallel and independent effort, Vollmer and Tomasz have
To purify the choline-binding proteins bound to the cell en-
velope, we followed the procedure described by García
In short, an exponentialculture of the M31 strain was disrupted
et al.11
pce
also characterized the product of
its biological properties.
gene and studied some of in a French Pressure Cell Press (American Inst. Comp.) and
34
g
centrifuged (10,000 3 , 5 min), and the pellet washed twice

215
PNEUMOCOCCAL PHOSPHORYLCHOLINE ESTERASE
in 50 mM potassium phosphate buffer pH 6.5 containing 10 Munich Information Center for Probe Sequences server
mM MgCl2 and 0.1% Brij-58. After centrifugation, the pellet (http://pedant.mips.biochem.mpg.de).
was resuspended in the same buffer containing 0.1% choline
and allowed to autolyse by overnight incubation at 30°C (“au-
tolytic walls”). Afterwards, the concentration of choline was
raised to 2%, and, after incubation at 4°C for 15 min, the mix-
Purification of Pce
E. coli
E. coli
BL21(DE3)
BL21(DE3) (pRGR11) or
(pRGR12) strains were incubatedup to an OD600 of 0.3. At this
time, isopropyl-b-D-thio-galactopyranoside(40 nM) was added,
and incubationproceededfor 6 hr at 22°C to minimize the pres-
ence of inclusionbodies. The culturewas centrifuged(10,000 3
g
ture was ultracentrifuged (120,000 3 , 1 hr). The supernatant
was dialyzed against 50 mM potassium phosphate buffer pH
6.9 applied to a DEAE-cellulose column and washed with 1.5
M NaCl. CBPs were eluted from the column upon addition of
2% choline.11 Sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) was carried out with the buffer sys-
tem describedby Laemmli17 in 12.5% polyacrylamidegels, and
protein bands were detected by staining with Coomassie bril-
liant blue R250.
g
, 5 min) and the pelleted bacteria were resuspendedin 20 mM
sodium phosphatebuffer pH 6.9 and disruptedin a French Pres-
sure Cell Press. The insoluble fraction was separated by cen-
g
trifugation(15,000 3 , 15 min) and the supernatantwas loaded
onto a DEAE-cellulose column to purify the Pce, in a single
23
step, following a procedure previously described.
Amino-terminal amino acid sequence
Assay of Pce activity
Amino-terminal amino acid sequence analyses were per-
formed through Edman degradation by using an Applied
Biosystem (Foster City, CA) model 470A gas-phase sequencer radioactively labeled with
Pce activity was determined using pneumococcal cell walls
3
methyl
[
- H]choline, as de-
fitted with an on-line PTH analytical system, after transferring scribed.14,19 One unit (U) of activity was defined as the amount
proteins to a membrane (Immobilon-P; Millipore), according to of enzyme needed to release 700 cpm of labeled material per
a published procedure.26 Proteins were identifiedby brief stain- 10 min.14 The Pce activity was also tested using as substrate
ing with amido black, and paper strips were excised for se- [14C]ethanolamine-labeled pneumococcal cell walls.30 The hy-
3
methyl
quencing.
drolytic products released from the [
- H]choline-labeled
pneumococcal cell walls were analyzed by gel filtration in a
Sephadex G-75 column.14 In addition, to identify the labeled
low-molecular-weight material released by Pce, a thin-layer
chromatography (TLC) was carried out on silica gel using 0.2
M Tris-HCl buffer pH 8.0 in 50% ethanol as solvent.14 Sam-
ples of 15 ml (about 20,000 cpm) of the soluble fraction re-
leased from the pneumococcal cell walls after digestion with
Pce were spotted on the chromatograms together with samples
of authentic phosphorylcholine and choline and developed for
about 4 hr at room temperature. The chromatogramswere dried
in an oven at 100°C for 5 min and the single strips were cut
into segments of 1 cm length. The radioactivity of each seg-
ment was determined by using a 1219 Rackbeta scintillation
counter (LKB Wallack). Pce activity was also determined us-
DNA manipulations
Routine DNA manipulations were performed essentially as
21
described. The oligonucleotide primers LytDEco and LytD-
Bam (Table 1) were used to amplify and sequence the 2.5-kb
fragment encoding the
designedaccordingto the sequenceof contig number 4,139 that
is included in The Institute for Genome Research (TIGR)
pce
gene. These oligonucleotides were
S.
pneumoniae
genome database (http://www.ncbi.nlm.nih.gov/
BLAST/tigrbl.html). The oligonucleotide primers LytDN1,
pce
LytDN2, and LytDC (Table 1) were used to clone the
gene,
with or without the region encoding the signal peptide, by us-
ing the pT7-7 vector. All primers for PCR amplification and
nucleotide sequencing were synthesized on a Beckman model
Oligo 1000M synthesizer. DNA fragments were purified by us-
ing the Geneclean II kit (Bio 101). DNA probes were labeled
with the DIG Luminescent Detection Kit (Boehringer
Mannheim). Southern blots and hybridizationswere carried out
p
ing -nitrophenylphosphorylcholine(Sigma) as substrate as de-
scribed for phospholipase C.15 The time course of the reaction
was followed at 410 nm (e 5 1.5 3 104 M21) in a UV-260 Shi-
madzu spectrophotometer equipped with a thermostatized
holder. The assay was performed in a final volume of 0.5 ml
S. pneumoniae
according to the manufacturer’s instructions.
Sma Sac Apa
p
of 0.1 M Tris-HCl buffer pH 8.0 in the presence of 3 mM
nitrophenylphosphorylcholine.
-
DNA digested with either
I, II, or
I was analyzed
by pulsed-field gel electrophoresis (PFGE) using a contour-
clamped homogeneous electric field DRII apparatus (Bio-Rad
Labs) as previously described.2 DNA sequencewas determined
by the dideoxy chain-termination method24 with an automated
Abi Prism 377™ DNA sequencer (Applied Biosystems). The
amino acid sequence was analyzed with the Protein Analysis
Tool at the World Wide Web molecular biology server of the
Geneva University Hospital and the University of Geneva. Pro-
tein sequence similarity searches were done with the BLASTP
program via the National Center for Biotechnology Informa-
tion server (http://www4.ncbi.nlm.nih.gov). Pairwise and mul-
tiple protein sequence alignments were done with the ALIGN
and CLUSTAL W programs, respectively, at the Baylor Col-
Electron microscopy
Samples of bacteria were taken, washed three times with 0.1
M ammonium acetate pH 7.0 and mixed (1:1) with 2% potas-
sium phosphotungstate, and a drop was placed on carbon-coated
copper grids and kept there 1 min. The excess was then blot-
ted off, and the grids were placed in a desiccator.Samples were
examined in a Philips 300 electron microscope at 80 kV.
Nucleotide sequence accession number
pce
-containing frag-
The nucleotide sequence data for the
lege of Medicine-HumanGenome Center server (http://kiwi.im- ment have been deposited in GenBank under accession num-
gen.bcm.edu:8088). Analyses of blocks were performed at the ber AJ272039.

216
DE LAS RIVAS ET AL.
the regulators of the LysR family, strongly suggesting that the
Orf1 protein could play a regulatory function. In this sense, we
have detected a repeated sequence (AAAGATAAAG) two nu-
cleotides downstream from the putative 210 promoter box that
could act as a recognition sequence for the Orf1 regulator.
Whether Orf1 plays a regulatory function controlling the ex-
pression of its own operon or other genes should be investi-
gated.
RESULTS
gene
Identification of the
pce
In an effort to identify CBPs of pneumococcus using a pro-
cedure previously publishedto extract proteins tightly bound to
the cell envelope,10 we have recentlyidentified,by SDS-PAGE,
four protein bands with strong choline affinity.11 One of these
bands, of about 70 kDa, was excised from the acrylamide gel,
and the amino-terminal sequence was determined. Comparison
of this sequence with the translated version of the nucleotide
orf2
The
acids) that showed a 41.8% identity with the signal peptidase
Lactobacillus lactis
gene encodes a protein of 17,150 Da (153 amino
type II from
The ATG start codon and the putative ribosome binding site of
orf2 orf1.
(accession number U63724).
S. pneumoniae
sequence of the
leased by TIGR (http://www.tigr.org) allowed the localization
pce
type 4 genome that has been re-
the
gene overlap the 39 coding region of
The high
of a gene designed as
for phosphorylcholine esterase. A
pce
similarity of the Orf2 sequence with the enzymes of the type II
signal peptidase family29 supports the hypothesis that Orf2
2.5-kb DNA fragment embracing the
gene was PCR am-
plified using the primers LytDBam and LytDEco and pneumo-
coccal R6 DNA as template. The resultingfragment was cloned
into the pUC18 vector to produce the plasmid pRGR18. The
sequenceof the PCR-amplified fragment revealed the existence
of 10-nucleotide changes compared to the equivalent type 4
pneumococcal sequence, which originates seven changes in
amino acid composition (S99G, T162A, T164K, D407A,
S. pneumoniae.
could play a similar role in
A detailed analysis
of the pneumococcal genome did not reveal the existence of
other homologous signal peptidase II proteins in this microor-
ganism.
gene encodes a protein of 32,850 Da (295 amino
acids) that showed a 59.7% identity with the YlyB protein of
orf3
The
Bacillus subtilis
of unknown function (accession number
U48870) that is apparently co-transcribedwith the
pce
V424I, D488G, G494E). The
gene encodes a putative pro-
lps
gene en-
tein of 72,104 Da (627 amino acids). According to the amino-
terminal amino acid analysis, Pce contains a typical signal pep-
tide of 25 amino acids that renders, after its cleavage, a mature
protein of 69,426 Da (602 amino acids) (Fig. 1A). Further
analysis of the nucleotide sequence revealed that Pce displays
a modular organization typical of CBPs. This is, after the sig-
nal peptide, Pce contains an amino-terminal region that most
probably represents the catalytic domain, followed by a car-
boxy-terminal moiety built up of 10 repeated motifs that is
highly similar to the ChBDs of other characterized CBPs (data
not shown) and a carboxy-terminaltail of 85 amino acids (Fig.
1B). Neither the amino-terminal region nor the terminal tail
showed significant similarities with any other proteins of the
data banks and, consequently, we could not assign through this
analysis a precise catalytic role to this protein.
B. subtilis.
coding the signal peptidase II of
orf3
Similar genes to
of unknown function have also been found contiguous to
the signal peptidase II-coding genes in other microorganisms,
e.g., Thermotoga maritima
(accession number AE001724). In
addition, Orf3 protein showed a 40% identity with the RluD
Pseudomonas aeruginosa
pseudouridine synthase from
(accession number AE004868) and 38.5% identity with the
E. coli
PAO1
SfhB (RluD) pseudouridine synthase from
(accession
number ECU50134) suggesting that Orf3 could play a similar
role in pneumococcus. The ATG start codon and the putative
orf3
ribosome binding site of the
gene overlap the 39-coding
strongly suggesting that these genes could be co-
orf2
region of
transcribed.
pce
The start codon of the
gene was located two nucleotides
orf3
downstream from the stop codon of the
the putative ribosome binding site overlaps the 39 coding re-
orf3. pce
gene, and, thus,
analysis of the
-containing region
pce
In silico
gion of
This finding suggests that the
gene most prob-
orf1orf2orf3
pce
Comparison of the
nucleotide sequence with the type 4
ably would be also transcribed together with the
pneumococcal genome sequence provided by TIGR revealed genes, forming a single transcription unit. Supporting this hy-
that this gene mapped in contig 4,139. A detailed analysis of pothesis, we have not found any palindromic sequence within
pce
-surrounding regions showed that this gene is located at the
orf1orf2orf3
-coding region that could form a hairpin loop
the
the 39 end of a putative transcriptional unit that contains three with a significant free energy value that might account for a
additional open reading frames (orfs) that we have named as rho
putative
-independent terminator. However, as pointed out
orf1, orf2, orf3
and
TTGGAA-16 bp-TATAAT was found 28 bp upstream from the the
orf1. orf1orf2orf3pce
(Fig. 1C,D). A putative promoter sequence
above, a palindromic sequence was found just downstream of
pce
gene that appears to constitute the terminator of the
operon.
ATG start codon of
In addition, a palindromic sequence
G
that forms a hairpin loop of D 5 27.7 kcal/mol that might
act as a putative rho-independent terminator was located just
Localization of the
S. pneumoniae
gene in the chromosome of
pce
pce.
downstream of the two tandem stop codons found in
In-
proABC
we have found the
pce
terestingly, downstream of
S. pneumoniae
pro-
vides a useful framework for gene localization. PFGE and
operonthat encodesthe enzymesresponsiblefor prolinebiosyn-
thesis.
The developmentof the physical map of
orf1
The
gene encodes a putative protein of 34,976 Da (302 Southern blot hybridization using pRGR18 as probe showed
pce
Sma
amino acids) that showed a 32.5% identity with CpsY, a LysR- that the
gene was localized in the 380-kb
I, the 330-kb
II fragments (Fig. 2). The accuracy of
pce
Streptococcus agalactiae
like regulatory protein of
number AF163833). The analysisof the Orf1 sequencerevealed
that the protein contains all of the typical sequence motifs of some is best illustrated if we take into account the
(accession Apa
Sac
I, and the 160-kb
this physical localization of
in the pneumococcal chromo-
in silico

217
PNEUMOCOCCAL PHOSPHORYLCHOLINE ESTERASE
S. pneu-
FIG. 1. Amino acid sequence and structural organization of Pce protein and genetic organization of contig 4,139 of
moniae
pce
gene. (A) The signal peptide and the motifs that build up the ChBD (P1–P10) are over-
chromosome containing the
lined and marked with doubleheaded arrows. (B) Domain organization of Pce. s.p., signal peptide. (C) Genetic organization of
pce
the region of contig 4,139 containing the
the diamond represents a putative transcription terminator. (D) Nucleotide sequence of the junction regions between the differ-
pce proABC
gene. Arrows represent putative genes, rectangles indicate putative promoters, and
ent orfs of the operon containing the
(235 and 210) are underlined and in boldface, and the start codons of
and marked. Stop codons are underlined and marked as “end.” Potential ribosome binding sites are indicated by RBS. The pu-
pce
gene and the upstream region of the
operon. The consensus promoter sequences
orf1, orf2, orf3, pce,
proA
and
genes are also in boldface
tative transcription terminator located downstream of
gene is in boldface.

218
DE LAS RIVAS ET AL.
To determine whether Pce could be a novel cell wall lytic
3
A
B
methyl
enzyme, we tested its activity using [
- H]choline-labeled
pneumococcal cell walls as substrate. Interestingly, crude ex-
E. coli E. coli
tracts prepared from
(pRGR11) and
(pRGR12)
cells liberated a significant fraction of the cell wall radioactiv-
ity, indicating that Pce produced in this heterologous system
was active on pneumococcalcell walls. To characterizethe type
of cell wall hydrolytic activity of Pce, the soluble radioactive
cell wall products released by this cell wall hydrolase were an-
alyzed by chromatography on Sephadex G-75, revealing the
presence of a low-molecular-weightmaterial (data not shown).
This material co-eluted with standard phosphorylcholinewhen
pce
S.
FIG. 2. Localization of
pneumoniae
gene in the physical map of
R6. PFGE of the DNA prepared from strain R6 of
S. pneumoniae Apa Sac
digested with
I (lane 1),
II (lane 2), or
pce
Sma
I (lane 3) (A), was blotted and hybridized with a
-con-
taining plasmid pRGR18 as a probe (B). The sizes (in kb) of
Sma
the
I fragments are indicated.
pce
analysis discussed above that revealed that
is located up-
operon, that has been recently postulated
Sma
proABC
stream of the
to map in the 380-kb
chromosome.3
I fragment of the pneumococcal R6
Cloning and overexpression of the
gene and
pce
biochemical characterization of the Pce enzyme
To determine the biological role of Pce, we have cloned and
pce
E. coli.
Plasmids pRGR11 and
overexpressed the
pRGR12 were constructed to express either the unprocessed or
pce
gene in
the mature forms of
of f10 promoter (Fig. 3A).
gene, respectively, under the control
E. coli
cells harboring pRGR11 or
pRGR12 overexpressed a soluble 70-kDa protein, correspond-
ing to the expected size of Pce (Fig. 3B). Determination of the
amino-terminal amino acid sequence revealed that the protein
pce
FIG. 3. Schematic representation of the cloning of the
gene and analysis of the purified Pce enzyme. (A) Construction
of plasmids pRGR11 and pRGR12. The relevant elements and
restrictionsites used to constructthe recombinantplasmidscod-
ing for the unprocessed and the mature form of Pce, respec-
tively, are indicated. (B) SDS-PAGE (12.5%) analysis of the
purified Pce enzyme. All of the CBPs shown have been puri-
E. coli
expressed in
BL21(DE3) (pRGR11) yielded a Pce with
E. coli
QESSG as amino-terminal sequence whereas
BL21(DE3) (pRGR12) expressed a protein with MQESSG as
amino-terminal sequence that corresponds to the mature form
where the methionine codon was included during the construc-
tion of plasmid pRGR12. These results demonstrated that the
E. coli
fied from crude extracts produced in
matography on DEAE-cellulose. Lane 1, Purified Pce esterase
E. coli
by affinity chro-
E. coli.
signal peptide of Pce was functional in
The mature en-
was purified in a single-step by affin-
prepared from
lar weight markers; lane 3, purified Pce esterase prepared from
E. coli
BL21(DE3) (pRGR11); lane 2, molecu-
E. coli
zyme produced in
ity chromatography on DEAE-cellulose (Fig. 3B). The protein
was specifically eluted from the chromatographicsupport with
choline, demonstrating that Pce has a high affinity for choline
as the other members of the CBP family.9
BL21(DE3) (pRGR12); lanes 4, 5, and 6, purified LytA
amidase, LytB glucosaminidase, and LytC lysozyme, respec-
tively. The molecular mass of the markers, in kDa, is indicated
on the left.

219
PNEUMOCOCCAL PHOSPHORYLCHOLINE ESTERASE
p
analyzed by TLC, indicating that Pce is a pneumococcal phos- enzyme on -nitrophenylphosphorylcholine, a typical chro-
phorylcholineesterase. The same characteristics had been pre- mogenic substrate commonly used to assay phospholipaseC.15
S.
viously reported for a minor hydrolytic activity detected in
Pure Pce enzyme prepared as mentioned above hydrolyzed this
pneumoniae14
S. oralis,20
K
and in
which led to the biochemical compound with a
of 0.048 min21 and a
of 0.5 mM.
K
m
cat
identification of the phosphorylcholine esterases in both The specific activity of Pce on this substrate is about ten-fold
K
higher, and the m almost 1,000-fold lower, than that described
species.
15
Clostridium perfringens.
The enzyme was active between pH values of 7 and 8 and for phospholipase C of
Therefore,
at temperatures from 30 to 37°C, but the maximum activity this substrate was used to determine several biochemical par-
(2,400 U/mg protein) was obtained at pH 7.0 and 30°C using ameters of the enzyme. As expected, the activity was inhibited
choline-containing pneumococcal cell walls as substrate. up to 40% by 5 mM choline chloride, but addition of 20 mM
Ethanolamine-containing cell walls were not degraded by Pce. ethanolamine did not produce any effect. The enzyme did not
The addition of 5 mM choline chloride to the assay inhibited appear to have any metal requirement because 10 mM EDTA
50% of the initial activity whereas the addition of 20 mM only produced a very small reduction of the enzyme activity.
ethanolamine did not.
Moreover, the addition of divalent cations (1 mM) as Mg21
,
In all the conditions assayed, including the presence of high Zn21, Cu21, and Ca21 did not cause a significant alteration of
salt concentration (300 mM NaCl), the enzyme was able to re- the enzyme activity. Only Mn21 and Fe21 showed a significant
lease only about 20% of the total radioactivity present in the inhibition of 20% and 40% of activity, respectively. The en-
cell wall substrate,suggestingthat not all the phosphorylcholine zyme activity remained also unaffected by 10 mM b-mercap-
residues were accessible to the enzyme or that the enzyme was toethanol and 1 M NaCl. The enzyme conserved 50% of its
inactivated during the assay. In this sense, we observed that original activity after incubation for 30 min at 45°C, although
whereas the addition of higher amounts of enzyme did not pro- it was completely inactivated after heating for 30 min at 65°C.
duce a furtherreleaseof phosphorylcholinefrom the Pce-treated
Phenotype of the
mutants
cell walls, the addition to the assay of fresh, untreatedcell walls
produced further release of phosphorylcholine, indicating that
the enzyme was not inactivated during the assay. This unusual
behaviorwas identical to that previouslyobservedfor the phos-
pce
To get insight into the biological role of the Pce enzyme, we
pce
constructed a
mutant strain (R6D) by insertion-duplication
S. pneumo-
mutagenesis using plasmid pRGR191 to transform
S. pneumoniae14
S. oralis20
phorylcholine esterase of
supports the hypothesis that, at least
and
in vitro,
and
only a fraction of
niae
strain R6 (Fig. 4A). Plasmid pRGR191 was obtained by
Hin
pce
cII internal fragment of the
subcloning the 1,081-bp
the phosphorylcholine residues of the cell wall are accessible
to the Pce enzyme.
gene into the pUCE191 vector. A lincomycin/erythromycin-re-
sistant transformant of R6 was isolated and used for further
studies. The accuracy of the construction was tested by South-
ern blot analysis, and Fig. 4B shows the results for R6 and R6D.
Analysis of the Pce activity using a model substrate
To determine if the Pce was able to hydrolyze less complex A similar mutant (M31D) was prepared using M31 as recipi-
substrate than the whole cell wall, we tested the activity of this ent strain for transformation with plasmid pRGR191 (data not
pce
pce
FIG. 4. Construction of a
in the insertion-duplication strain R6D and its parent strain R6. The white arrow represents the
pce Hin
pneumococcalmutant. (A) Schematic representationof the DNA region surrounding the
gene
gene. The hatched rectangle
cII DNA insert of pRGR191 used for insertion-duplication mutagenesis. The thick line corre-
pce
in
indicates the 1,081-bp
Dra erm
sponds to the vector plasmid pUCE191 used to construct pRGR191. Abbreviations: D,
I;
C gene, encodes ery-
Hin
thromycin/lincomycin resistance. (B) Southern blot hybridization analysis using the 1,081-bp
cII DNA fragment as probe.
Dra
Dra
Lane 1,
I-digest of R6 DNA; lane 2, I digest of R6D DNA. The size of the DNA fragments is shown on the right.

220
DE LAS RIVAS ET AL.
pce
FIG. 5. Comparative analysis of growth, lysis, and genetic transformation of the wild-type strains and
mutants. The strains
were cultivated in C1Y medium at 37°C and growth and lysis for R6 (s), R6D ( ), M31( ), and M31D ( ) were followed
n
d
m
by nephelometry(N). Genetic transformation was tested at a cell concentration of about 2.1 3 108 bacteria/ml (arrow). Open bar,
R6; striped bar, R6D; solid bar, M31; and, stippled bar, M31D.
shown). Both R6D and M31D mutants (the latter lacking the
Biochemical analyses demonstratedthat this protein behaves
LytA amidase and Pce esterase) exhibited normal growth rates as the pneumococcal phosphorylcholineesterase that had been
in C1Y medium (Fig. 5) as well as in other media tested (data biochemicallycharacterized previously.14 The structural analy-
not shown), and formed typical diplo cells (R6D) or small sis revealed that Pce shows a building plan similar to that ob-
i.e.,
chains of about 10 cells (M31D) as reported for the parental served for other CBPs,
the amino-terminal domain em-
strain. Observations at the electron microscope revealed that braces the catalytic region of the enzyme whereas the carboxy-
there were not noticeable changes when whole-mounted cells terminal domain is a typical ChBD responsible for substrate-
of the esterase-mutants were compared to the parental strains binding specificity to choline. The structure of Pce represents
(data not documented). Moreover, R6D mutant normally au- a new example which reinforces our previous observation that
tolysed at the end of the stationary phase of growth and R6D the recruitment and genetic adaptation of the ChBD to provide
and M31D were competent for transformation. Transformation a higher catalytic efficiency to the enzyme recognizing the cell
frequencies were: 1.1% and 1.5% for R6D and M31D, respec- wall as a substrate is extremely versatile in the pneumococcal
tively, as compared with the 3.5% and 2.5% found for the system.18 Moreover, the feasibility of the ChBD to become
parental strains R6 and M31, respectively (Fig. 5). These re- functional when it is located either at the amino or carboxyl
sults indicate that there is only a limited influence of Pce in ends of a protein was anticipated through the preparationof ar-
competence development.
tificial fusion proteins containing the ChBD.23 Recently, it has
been confirmed by the characterization of two novel members
of the CBP family, the LytB and LytC cell wall lytic enzymes,
which contain the ChBD in the amino-terminal position in con-
DISCUSSION
trast with the carboxy-terminalarrangementof Pce and the other
11,12
pce
We have now identified and characterizedthe
ing for a novel member of the CBP family of pneumococcus,
and we have been able to express the gene product as a mature is generated by the action of the signal peptidase at the cleav-
gene, cod- CBPs already described.
pce
The
gene codes for a pre-protein, and the mature form
E. coli.
proABC
pce Q
gene has been mapped in the vicin- age site VQA QES that is in agreement with typical cleavage
operon and most probably is transcribed to- sites previously described. Preparation of recombinant plas-
protein in
ity of the
The
35
orf1orf2orf3
a single transcription unit (Figs. 1 and 2).
pce,
gether with the
genes, located upstream of
as mids coding for the unprocessed and the mature form of Pce
(Fig. 3), together with amino-terminal analysis of the purified

221
PNEUMOCOCCAL PHOSPHORYLCHOLINE ESTERASE
E. coli,
enzymes expressed in
revealed that the signal peptide and V. Muñoz and M. Fontenla for the artwork. This work was
E. coli.
of Pce was also functional in
Similar signal peptides supported by grant PB96-0809 from the Dirección General de
have been also observed in other pneumococcal CBPs such as Investigación Científica y Técnica. B.R. was the recipient of a
the murein hydrolases LytB and LytC.11,12 The fact that we fellowship from the Comunidad de Madrid.
have localized the enzyme bound to the cell wall also indicates
that Pce should play its physiological role in pneumococcus for Genomic Research (TIGR) through the website at
S. pneumoniae
Preliminary sequence data were obtained from the Institute
only after its secretion to the cell envelope. Actually, it is dif- http://www.tigr.org. Sequencing of the
genome
ficult to speculate on the biological role of the pneumococcal was accomplished with support from TIGR, the National Insti-
esterase. It has been suggested that the about 20% of residues tute of Allergy and InfectiousDiseases (NIAID), and the Merck
removable by the enzyme might exist either in an anatomically Genome Research Institute (MGRI).
unique position in the cell wall or might represent terminal
residues in the teichoic acid chains.14 More recently, the prepa-
ration of choline-independent pneumococcalstrains that lead to
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Address reprint requests to:
Dr. Rubens López
Departamento de Microbiología Molecular
Centro de Investigaciones Biológicas, CSIC
Velázquez 144
28006 Madrid, Spain
E-mail:
ruben@cib.csic.es

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