Abstract
A search to identify proteins with high affinity for choline-containing pneumococcal cell walls (choline-binding 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
M
r
of 72,104. Pce can remove a maximum of 20% phosphorylcholine residues from the cell wall teichoic acid.
In silico
analysis 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-terminal
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 characterized 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 indistinguishable 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.