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Journal of Bacteriology, June 1999, p. 3618-3625, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A High Incidence of Prophage Carriage among Natural
Isolates of Streptococcus pneumoniae
Mario
Ramirez,
Elena
Severina, and
Alexander
Tomasz*
The Rockefeller University, New York, New
York
Received 3 March 1999/Accepted 7 April 1999
 |
ABSTRACT |
The majority (591 of 791, or 76%) of Streptococcus
pneumoniae clinical isolates examined showed the presence of two
or more chromosomal SmaI fragments that hybridized with the
lytA-specific DNA probe. Only one of these fragments,
frequently having an approximate molecular size of 90 kb, was shown to
carry the genetic determinant of the pneumococcal autolysin
(N-acetylmuramic acid-L-alanine amidase).
Strains carrying multiple copies of lytA homologues included both antibiotic-susceptible and -resistant isolates as well as
a number of different serotypes and strains recovered from geographic
sites on three continents. Mitomycin C treatment of strains carrying
several lytA-hybridizing fragments caused the appearance of
extrachromosomal DNA hybridizing to the lytA gene, followed
by lysis of the bacteria. Such lysates contained phage particles
detectable by electron microscopy. The findings suggest that the
lytA-hybridizing fragments in excess of the host lytA represent components of pneumococcal bacteriophages.
The high proportion of clinical isolates carrying multiple copies of
lytA indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.
| |
INTRODUCTION |
As part of a study of the expression
of the autolysin gene in clinical isolates of Streptococcus
pneumoniae, we probed SmaI digests of total DNA
separated by pulsed-field gel electrophoresis (PFGE) with a probe
specific for lytA. The sequence of lytA has no
SmaI recognition site (16). Therefore, it was
surprising that the majority of clinical isolates tested had more than
one lytA-hybridizing band, indicating that there were two or
more highly homologous genes in the chromosome.
In this communication, we present evidence that the supernumerary
lytA-hybridizing fragments detected signal the presence of
prophages and that the incidence of prophage carriage in natural isolates of pneumococci is high.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
S. pneumoniae isolates were obtained from The Rockefeller
University collection. S. pneumoniae strains were grown in
semisynthetic medium (21) at 37°C without aeration or in
tryptic soy agar (Difco, Detroit, Mich.) supplemented with 3% sterile
sheep blood incubated at 37°C.
Escherichia coli HB101 (pGL80) (17) was used as a
source of DNA for the lytA gene probe. E. coli
cells were grown in Luria-Bertani medium (Difco) with aeration or in
Luria-Bertani agar (Difco) solid medium incubated at 37°C and
supplemented with 75 µg of ampicillin/ml.
Probes for the lytA gene.
The probe for the
lytA gene was obtained from plasmid pGL80 (17).
Plasmid DNA was prepared by using the Wizard Midiprep kit (Promega,
Madison, Wis.), digested with HindIII (New England Biolabs, Beverly, Mass.), and separated by agarose gel electrophoresis. The 1.2-kb fragment from pGL80 contained the entire lytA
gene (957 nucleotides [nt]), a fragment of upstream sequence (199 nt), and 51 nt downstream of the gene. The 1.2-kb fragment containing the lytA gene was purified from the gel by using the Wizard
DNA cleanup kit (Promega). A second, PCR-generated DNA probe included an internal, 890-nt lytA fragment lacking the first 57 and
the last 10 nt of the gene. The 890-bp product was generated with primers Lytd-1 and Lytr-1 (Table 1), with
DNA from strain R36A as the template. The PCR product was purified by
using the Wizard DNA cleanup kit (Promega) before being labeled with
either the ECL random prime labeling kit or the ECL direct labeling kit
(Amersham, Little Chalfont, Buckinghamshire, United Kingdom).
Probe for the recA gene.
The recA
probe was an internal fragment of 616 bp obtained by PCR with primers
RecAd and RecAr (Table 1) and with DNA from strain R36A as the
template. The PCR product was purified by using the Wizard DNA cleanup
kit (Promega), and labeling was performed by using the ECL direct
labeling kit (Amersham).
PFGE.
Chromosomal DNA fragments, generated by
SmaI digestion, were separated and analyzed as previously
described (40). SmaI PFGE patterns were assigned
arbitrary numbers. For the visualization of extrachromosomal phage DNA,
cultures were grown and induced with mitomycin C as described below.
The optical density of the culture at 620 nm (OD620) was
monitored. Cells were harvested immediately after a decrease in
OD620 or just before lysis. Agarose disks for PFGE were
prepared as previously described (40). The unrestricted DNA
was separated in a 1% agarose gel, with 0.5× Tris-borate-EDTA as the
buffering agent. The gel was run on a Chef DRII apparatus (Bio-Rad,
Hercules, Calif.) under the following conditions: 6 V/cm, ramping of
the pulse between 5 and 35 s, and a total running time of 23 h. The buffer was maintained at 7°C during the run.
Southern blot hybridization.
DNA fragments separated by PFGE
were transferred to nylon membranes (Hybond N+; Amersham)
by using the vacuum gene system (Pharmacia LKB Biotech, Uppsala,
Sweden), according to the manufacturer's instructions. Membranes were
hybridized to specific DNA probes labeled with the ECL system
(Amersham), as described above. Hybridization conditions for the ECL
direct labeling system were as recommended by the manufacturer, using a
sodium chloride concentration of 0.5 M. Hybridization conditions for
the ECL random prime system were as recommended by the manufacturer.
The molecular weights of the hybridization signal(s) (39,
41) and the corresponding SmaI fragments were determined.
Induction of the lytic cycle by mitomycin C in putative lysogenic
strains.
Cultures were grown at 37°C until the OD620
reached approximately 0.2 to 0.3. Mitomycin C was then added to a final
concentration of 0.1 µg/ml to induce the putative lysogenic strains.
Incubation was continued, and growth was monitored by optical density.
Effect of high concentrations of choline on lysis of induced
cultures.
Cultures were grown at 37°C until the
OD620 reached approximately 0.2 to 0.3. Mytomicin C and
choline were then added to final concentrations of 0.1 µg/ml and 2%
(wt/vol), respectively. As a control for the prevention of lysis by
high choline concentrations, penicillin was added at a concentration 10 times the MIC, together with choline. Incubation was continued, and
growth was monitored by OD.
Preparation of samples for electron microscopy.
Cells for
electron microscopy were harvested by centrifugation, washed twice with
0.9% sodium chloride, and suspended in 10 times the volume of the
pellet in a solution composed of 100 mM sodium cacodylate, 100 mM
sodium arsenate, and 2.5% glutaraldehyde (pH 7.4) (CAG). After
embedding and sectioning, the preparations were stained with uranyl
acetate for electron microscopic observation as described previously
(44).
Phage particles were prepared for electron microscopy by allowing
induced cells to lyse until the reduction in OD
620 in
1
h was less than 10%. Cellular debris was removed by
centrifugation
at 3,750 rpm for 15 min. The supernatant was centrifuged
in a
Beckman ultracentrifuge model L5-50 with rotor model SW50.1 at
42,000 rpm for 90 min. The pellet was suspended in 100 µl of 0.9%
NaCl, diluted in CAG, and prepared for negative staining as described
previously (
23).
| |
RESULTS |
Multiple copies of lytA in clinical isolates of
S. pneumoniae.
Each one of the 791 clinical isolates of
S. pneumoniae tested showed at least one chromosomal
SmaI fragment that hybridized to the lytA DNA
probe. Also, each of the 791 clinical isolates showed lysis by
deoxycholate, a property involving the activity of the autolytic
amidase, indicating that at least one of the lytA copies
present in all pneumococcal isolates must be the determinant of the
autolysin. The ubiquitousness of lytA, the genetic
determinant of the major pneumococcal autolysin, an
N-acetylmuramic acid-L-alanine amidase, among
pneumococci, was demonstrated earlier by dot blot hybridization
(31).
The presence of multiple copies of
lytA in the majority of
the pneumococcal isolates examined was an unexpected finding of
these
hybridization studies (Table
2). The
frequency of
S. pneumoniae strains carrying one to four
lytA-hybridizing
SmaI bands is shown
for isolates
collected from seven different countries (Table
2).
Variation in the number and molecular size of SmaI
fragments hybridizing with the lytA DNA probe.
Figure
1A shows PFGE profiles of
SmaI-restricted total DNA isolated from a group of 18 S. pneumoniae clinical isolates selected to illustrate the
variability of the lytA hybridization pattern (Fig. 1B). The
isolates tested were from the United States (Alaska and Ohio) and
Iceland and included penicillin-susceptible and penicillin-resistant
isolates and multidrug-resistant isolates along with three different
serotypes and several clonal types, as defined by PFGE pattern. The
properties of these strains are listed in the legend to Fig. 1. Some
parallels between PFGE patterns and the corresponding lytA
hybridization patterns are apparent in a comparison of Fig. 1A and B.
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FIG. 1.
Multiple patterns of lytA hybridization among
clinical isolates of S. pneumoniae. Total DNA was isolated
from a group of pneumococcal clinical isolates. After digestion with
SmaI and separation by PFGE (A), Southern hybridization was
done to identify fragments hybridizing with lytA (B).
Pneumococcal strains originated in Iceland (Ic), Alaska (Ala50,
intermediate level resistance to penicillin), and Cleveland (Clev2,
serotype 23F, resistance to penicillin, tetracycline, and
chloramphenicol). Except for strain Ic189, which belongs to serogroup
19, and strains Ic165, Ic183, Ic202 (resistant to penicillin,
erythromycin, tetracycline, chloramphenicol, and
sulfamethoxazole-trimethoprim), and Ala50, which belong to serogroup 6, all strains belong to serogroup 23. The Icelandic isolates with
serogroup 23 showed intermediate level resistance to penicillin. Lanes
1 to 18, respectively, Ic165, Ic183, Ala50, Ic202, Ic162, Clev2, Ic107,
Ic118, Ic130, Ic155, Ic226, Ic134, Ic173, Ic189, Ic204, Ic156, Ic191,
and Ic221. Arrows indicate molecular sizes in kilobases.
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Induction of lysis by mitomycin C.
Mitomycin C is known to
induce the lytic cycle of some prophages (29), although not
all prophages respond to this inducing agent (2). A group of
18 pneumococcal strains selected for variation in genetic background
and lytA hybridization pattern was tested for lysis
induction by mitomycin C. The strains, from Hungary and the United
States, included 14 different clonal types (as defined by PFGE
pattern), nine serotypes, and both penicillin-susceptible and
antibiotic-resistant strains. Mitomycin C treatment was shown to induce
lysis in 11 of the 18 isolates (Table 3).
Prevention of lysis by high concentrations of choline.
High
concentrations of choline are known to inhibit lysis of pneumococcal
autolysin, the product of lytA (8, 45). The dependence of the lytic activity of several pneumococcal bacteriophages on the presence of choline residues in the cell wall of the host bacterium was also established (13). Cultures of two
S. pneumoniae strains, SVMC28, responding to mitomycin C
treatment with culture lysis, and SVMC17, which did not lyse upon
treatment with mitomycin C, were tested for the effect of high
concentrations of choline on culture lysis induced by mitomycin C or
penicillin. Antibiotics and choline were added to the cultures at time
0 in Fig. 2. The addition of high
concentrations of choline inhibited both the mitomycin-induced and the
penicillin-induced lysis in strain SVMC28. A high concentration of
choline was found to inhibit each one of six additional cultures from
lysing during mitomycin treatment (data not shown). The culture of
SVMC17 showed no lysis during mitomycin treatment in spite of the fact
that it possessed two SmaI fragments positive for the
lytA gene probe (Table 3).
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FIG. 2.
Lysis of S. pneumoniae induced by mitomycin
C. Cultures of strains SVMC28 and SVMC17 received mitomycin C (0.1 µg/ml) or penicillin (0.1 µg/ml) with or without the addition of
choline (2%) at time zero, and the OD of cultures was monitored at 620 nm, as described in Materials and Methods.
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Electron microscopic detection of phage particles in culture
supernatants and in thin sections of cells induced with mitomycin
C.
Mitomycin-treated cultures harvested before lysis as well as
supernatants of mitomycin-lysed cultures were examined by electron microscopy for the presence of phage particles. Phage particles were
detected in all three strains examined either by negative staining with
phosphotungstic acid or by thin sectioning of the mitomycin-treated
bacteria (Fig. 3). Mature phage particles
were also detected in thin sections of SVMC28 in which the mitomycin C-induced lysis was blocked by the presence of high concentrations of
choline in the medium (Fig. 4).
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FIG. 3.
Detection of phage particles in the supernatant of
strain SVMC28 induced to lyse by mitomycin C. The preparation of the
sample and negative staining for electron microscopy were performed as
described in Materials and Methods. The larger electron micrograph
shows a pneumococcal cell with multiple phage tails attached and a
cluster of phages and phage tails in the top left corner. This cluster
is probably held together by a cellular fragment containing phage
receptors. The inset shows a complete phage with a morphology that is
typical of the Siphoviridae family and similar to that of
the pneumococcal phage HB-3 (3). Black bar, 0.3 µm; white
bar (inset), 0.1 µm.
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FIG. 4.
Intracellular phage particles in pneumococci induced
with mitomycin C but protected from lysis by a high concentration of
choline in the medium. Samples for electron microscopy were prepared as
described in Materials and Methods. The strain used in this experiment
was SVMC28. Collapse of the membrane is evident from the white
background in the cytoplasmic space. Escape of phage particles is
prevented by the integrity of the cell wall. Arrowheads indicate fully
assembled phage particles. Bar, 0.3 µm.
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Production of extrachromosomal phage DNA in mitomycin C-treated
bacteria.
Four S. pneumoniae clinical isolates showing
induction of lysis during mitomycin C treatment were tested for the
presence of extrachromosomal phage DNA after exposure to mitomycin C. The chromosome of the bacterium is about 2.2 Mb (19) and
migrates in the compression zone of the gel under the running
conditions of PFGE. In most but not all strains, unrestricted total DNA
from control cultures (not treated with mitomycin) showed no DNA
fragments smaller than the chromosome (Fig. 5, lanes a). In contrast,
DNA fragments generated by mitomycin treatment were smaller than the chromosome and separable by PFGE. Particularly striking was the appearance of the ladder-like pattern of DNA fragments in
mitomycin-treated cultures of SVMC28. Like the bacterial chromosome,
all mitomycin-induced DNA fragments hybridized with the lytA
gene probe, suggesting that they correspond to extrachromosomal phage DNA.
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DISCUSSION |
Incidence of multiple bands hybridizing to the lytA
probe and localization of the lytA gene.
A
surprisingly large proportion (76%) of the 791 pneumococcal clinical
isolates examined showed more than one SmaI DNA fragment hybridizing with the DNA probe specific for the pneumococcal autolysin gene. The number and molecular sizes of the lytA homologues
were independent of the geographic origin, serotype, or antibiotic resistance of the isolates (Table 2). Since lytA does not
have an SmaI cutting site (16), the observation
raised questions concerning the nature of the multiple
lytA-hybridizing bands.
All pneumococcal isolates had at least one
lytA-hybridizing
fragment, as expected from the ubiquitousness of the autolytic
enzyme
in pneumococci (
31). The laboratory strain R6 had a single
lytA-hybridizing band, and strain M31 (
38), a
lytA deletion
mutant derivative of R6 selected for defective
autolytic activity,
gave no hybridization signal with the
lytA DNA probe. Moreover,
it was shown that the pneumococcal
recA gene (accession no.
Z34303)
is located approximately 2 kb upstream of
lytA and that, at least
in some
circumstances,
recA is transcribed together with
lytA (
24). Each of a randomly selected group of
79 pneumococcal isolates
probed with
recA showed a single
band that always colocalized
with a
lytA-hybridizing band
(results not shown). In most cases,
the size of this band was about 90 kb (Table
3), although fragments
in the range of 80 to 130 kb were also
observed. These findings
indicate that the
lytA-hybridizing
band representing the genetic
determinant of the host autolytic
activity was associated in most,
if not all, of the isolates with a
SmaI fragment of approximately
90
kb.
Two possibilities could explain the multiplicity of
lytA
homologues in most of the clinical isolates. The
lytA gene
may have
one or more
SmaI recognition sites in some clinical
isolates.
Alternatively, the chromosome of pneumococcal isolates may
carry
multiple genes with a high degree of identity to
lytA.
The sequence
of the
lytA gene of the isolates presenting
multiple positive
fragments was not determined, and thus the first
possibility cannot
be formally excluded. However, several observations
described
in this communication strongly suggest an alternative
explanation,
namely, that the supernumerary
lytA-hybridizing
bands represent
pneumococcal prophages. Consistent with this hypothesis
is the
observed variation in the molecular size and number of the
lytA-hybridizing
bands in different isolates, suggesting
independent acquisition
of the
lytA-hybridizing
genes.
Lysis induction by mitomycin C and detection of phage
particles.
The majority (11 of 17) of the pneumococcal isolates
tested carrying multiple copies of lytA lysed within a 5-h
period following the addition of mitomycin C to the medium. Conditions
that inhibit the activity of the pneumococcal amidase (the
lytA gene product) are known to prevent phage-mediated lysis
of pneumococcal cells (37). The inhibition of
mitomycin-induced lysis by choline suggests that the phage lytic enzyme
has biochemical properties similar to those of the host autolytic
amidase. Electron microscopy of thin sections of mitomycin C-induced
cells just before lysis or of cells in which mitomycin-induced lysis
was blocked by high concentrations of choline revealed the presence of
phage particles inside the cells. Phage particles were also detected in
supernatants of mitomycin-lysed cultures. The morphology of the phage
observed in the culture supernatant of strain SVMC28 was characteristic of the Podoviridae family (1), similar to the
previously described pneumococcal temperate phage HB-3 (3)
(Fig. 3).
Detection of extrachromosomal phage DNA.
Total DNA prepared
from cells induced with mitomycin C was separated by PFGE, in order to
confirm prophage induction. In strains that lysed upon mitomycin C
induction, PFGE analysis allowed detection of discrete DNA fragments
with molecular sizes smaller than that of the chromosome, and all
extrachromosomal bands hybridized with the lytA probe. These
results are compatible with the excision of the phage genome from the
bacterial chromosome induced by mitomycin C.
In the mitomycin-treated strain SVMC28, PFGE identified a ladder-like
pattern of DNA fragments, all of which hybridized to
the
lytA probe. These fragments appeared to be multiples of the
smallest fragment (30 kb), suggesting that, like
E. coli 
phage,
the prophage infecting SVMC28 has a genome with cohesive ends
(
42). The estimated 30-kb genome size of the hypothetical
prophage
in SVMC28 is similar to the genome sizes of other pneumococcal
phages (
18). The multiple
lytA-hybridizing
fragments seen upon
induction with mitomycin C in strain MA62 could
correspond to
multiple prophages. However, the possibility that the
larger bands
correspond to closed circular forms of one of the smaller
bands
cannot be clarified, since circular molecules are known to have
unusual migration properties in PFGE (
7).
The diversity of molecular sizes of DNA fragments obtained by mitomycin
C induction and the fact that more than one fragment
was obtained from
several strains suggest that clinical isolates
of pneumococci are
lysogenic for more than one phage. This conclusion
is supported by the
fact that a number of isolates have more than
two
SmaI
fragments positive for the
lytA gene probe. In some cases
(for instance, MA49 in Fig.
5), signals
were also detected in
uninduced controls with the same molecular size
as that observed
in mitomycin C-treated cells. This finding is
compatible with
a low-level spontaneous induction of prophage, similar
to what
was described previously in other systems (
12,
14,
29).
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FIG. 5.
Extrachromosomal phage DNA in S. pneumoniae
induced with mitomycin C. Total DNA was isolated from cultures of
strains SVMC12, SVMC28, MA49, and MA62 treated with mitomycin C or left
untreated. Preparations were separated by PFGE without prior treatment
with restriction enzymes. Lanes a and b show PFGE profiles in the UV,
and lanes c and d show hybridization with the lytA probe.
Lanes a and c are from control (untreated) cultures, and lanes b and d
are from mitomycin C-treated cultures. Open triangles indicate the
position of the bacterial chromosome that migrates in the compression
zone of the gel. Arrowheads on the right indicate molecular sizes in
kilobases. Arrows on the left indicate DNA fragments smaller than the
bacterial chromosome.
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Pneumococcal isolates that did not lyse upon mitomycin C treatment in
spite of having multiple
lytA-hybridizing fragments
may
carry defective prophages. Large fragments related to phage
DNA have
already been identified in the
E. coli chromosome
(
34)
as well as in
S. pneumoniae (
35).
The phage-related fragment
described in
S. pneumoniae 8R1
encompassed the phage lytic genes
precisely and provided the insertion
site for temperate phage
HB-746 (
35). Pneumococci carrying
defective prophages may represent
bacterial hosts that survived cycles
of infection and lysogenization
or have lost part of the phage genome,
as proposed for
E. coli K12 (
34).
Frequency of prophage carriage.
The incidence of prophage
carriage in bacteria varies from 4% to almost 100%, depending on the
species, origin of the isolates, and method used to evaluate carriage
rates (9, 12, 20, 28). Early studies on S. pneumoniae that used mitomycin C for induction and relied on
plaque formation for phage detection found that 8 of 12 (4)
or 58 of 139 (3) pneumococcal isolates were lysogenic. The
incidence of prophage based on lytA hybridization of
PFGE-separated SmaI fragments determined in this study
(76%) is higher than earlier estimates (combined average of 42.4%)
(3, 4). Either one of these two values may be realistic,
since the incidence of prophage in bacteria depends on many factors. Our method may overestimate the incidence of functional prophages, since it can also detect defective prophages. Conversely, the method of
Bernheimer (3, 4), which relies on the sensitivity of an
indicator strain, probably underestimates the incidence of prophage.
Lysogeny and competence.
Functional as well as defective
prophages may promote genetic variation and may contribute to the
structure of pneumococcal populations in their natural environment. The
high incidence of lysogeny observed among clinical strains raises the
possibility that some of the exchange of genetic information occurring
in S. pneumoniae in vivo proceeds through transduction or is
assisted by phage function(s). Some temperate phages are known to carry virulence-related genes (6, 15, 27, 46); an analogous process could be of selective advantage for pneumococci.
It has been suggested that lysogeny may inhibit DNA-mediated genetic
transformation (
25), based on a study with the temperate
bacteriophage 304 and a single streptococcal isolate (R6X). However,
tests of this proposition with a large number of clinical strains
and
their infecting prophages showed no correlation between
transformability
and prophage carriage. The results described in Table
4 demonstrate
that strains that carried
an inducible prophage transformed at
a high level in response to
competence-stimulating peptide (CSP
)
(e.g., SVMC54), whereas others
did not transform at all (e.g.,
SVMC52). Isolates presenting two
lytA-hybridizing bands but not
responding to mitomycin C
induction also showed a high-level response
to CSP
(e.g., SVMC23) or
did not respond at all (e.g., SVMC35).
A process similar to transduction, but requiring competence
development, was described previously in pneumococci (
30).
In
this system, the host DNA seems to be packed into the phage heads
but enters the cells through the same route as transforming DNA.
Moreover, Porter and coworkers (
30) concluded that the
efficiency
of pseudotransduction of a deletion of more than 15 kb is
10-fold
more efficient than transformation of the same deletion. This
result was obtained in spite of the median 16-kb fragment size
in the
DNA preparation. Porter et al. argue that protection of
the DNA from
excessive cuts by the DNA binding sites in the surface
of the bacteria
could account for this phenomenon. The possibility
of in vivo DNA
exchange through this mechanism is attractive,
because the higher
efficiency of pseudotransduction of large fragments
of DNA compared to
transformation could favor this route over
transformation for the
observed in vivo capsular switch events
(
10,
11,
26,
33).
The unexpectedly high incidence of phage infection in natural isolates
of
S. pneumoniae could have an impact on the structure
of
natural populations of pneumococci in their ecological niche.
Phages
capable of lysing nonencapsulated (nonlysogenic) indicator
strains have
been readily isolated from carriers or patients (
23,
36,
43). On the other hand, it was shown that the presence
of
capsular polysaccharide protects pneumococci against infection
by
phages of the
group (
5). The overwhelming majority of
pneumococcal clinical isolates express antiphagocytic capsules,
and it
is conceivable that defense against phage infection may
be another
selective pressure that affects the structure and maintenance
of
capsular polysaccharides in
pneumococci.
The population structure of
S. pneumoniae colonizing the
nasopharynx may be modulated by the phage immunity pattern of the
resident flora, which would provide a strong selective pressure
against
incoming strains. Superinfection immunity (
22), restricted
to closely related phages or associated with phages of a broader
scope
(
2), is a frequent property of
prophages.
| |
ACKNOWLEDGMENTS |
Partial support for these studies was received from grant no. RO1
AI37275 from the National Institutes of Health and from the Irene
Diamond Fund. M.R. received partial support from the Gulbenkian
Foundation (PGDBM) and the Fundação Luso Americana para o
Desenvolvimento (FLAD).
We thank Sigurdur Vilhelmsson, Cristina Brandileone, Gabriela Aviles,
and Lena Setchanova for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The Rockefeller
University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8277. Fax: (212) 327-8688. E-mail:
tomasz{at}rockvax.rockefeller.edu.
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Abstract
Introduction
