Molecular Microbiology (2007)
doi:10.1111/j.1365-2958.2006.05558.x
Transformation in Streptococcus pneumoniae:
formation of eclipse complex in a coiA mutant
implicates CoiA in genetic recombination
Bhushan V. Desai† and Donald A. Morrison*
Laboratory for Molecular Biology, Department of
Biological Sciences, University of Illinois at Chicago,
Chicago, IL 60607, USA.
Summary
CoiA is a transient protein expressed specifically
during competence and required for genetic transformation in Streptococcus pneumoniae, but not for
DNA uptake. It is widely conserved among Grampositive bacteria but its function is unknown. Here we
report that although the rate of DNA uptake was not
affected in a coiA mutant, the internalized donor DNA
did not recombine into the host chromosome to form
a physical and genetic heteroduplex. Instead, DNA
taken up by a coiA mutant accumulated in the form of
a single-stranded (ss) DNA–protein complex indistinguishable from the eclipse complex formed as a
recombination intermediate in wild-type competent
cells. Internalized donor DNA in a dprA mutant did not
accumulate either as ss DNA or as an eclipse
complex. Together, these results establish that a coiA
mutant exhibits a phenotype different from that of
dprA or recA mutants, and that CoiA functions at a
later step in promoting recombination during genetic
transformation in Streptococcus pneumoniae.
Introduction
Competence for genetic transformation in Streptococcus
pneumoniae is a transient physiological state associated
with a brief global shift to the synthesis of new sets of
proteins (Campbell et al., 1998; Peterson et al., 2000;
2004; Bartilson et al., 2001; Dagkessamanskaia et al.,
2004). During this transition at least two sets of genes are
transcriptionally activated. While one set includes genes
involved in triggering an intercellular signalling circuit
(early genes), the other set includes genes involved in
Accepted 5 December, 2006. *For correspondence. E-mail
DAMorris@uic.edu; Tel. (+1) 312 996 6839; Fax (+1) 312 413 2691.
†
Present address: Department of Pathology, Northwestern University
Feinberg School of Medicine, 303 E. Chicago Ave., Chicago, IL
60607, USA.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
DNA processing and genetic recombination (late genes)
(Claverys and Martin, 2003; Lacks, 2004). Expression of
some, but not all, of the genes required for DNA processing and recombination is under the control of an alternative sigma factor, ComX (Lee and Morrison, 1999; Luo
and Morrison, 2003). Based on sequence similarities with
counterparts in the similar Bacillus subtilis transformation
mechanism or on direct experimental results, roles for
some of these genes have been assigned (Dubnau, 1999;
Bergè et al., 2002; Claverys and Martin, 2003; Chen and
Dubnau, 2004), including the products of the cfl (comF),
cel (comE), cgl (comG), endA loci, which may form the
membrane pore for entry of donor DNA. Upon binding of
DNA to the surface of a competent pneumococcal cell, a
double-strand (ds) break is created, probably by the
membrane-bound endonuclease, EndA (Morrison and
Guild, 1973; Lacks et al., 1974; 1975; Puyet et al., 1990).
DNA degradation and DNA uptake then take place concomitantly and at approximately equal rates, with half of
the donor DNA degraded by EndA in a 5′ to 3′ direction,
while a single strand is transported in the 3′ to 5′ orientation across the cell envelope (Méjean and Claverys, 1988;
1993). As the transforming activity of single-stranded (ss)
DNA is very low, donor markers in this state are described
as being in eclipse (Ephrussi-Taylor, 1960). Markers that
enter the resident chromosome by homologous recombination form a heteroduplex by strand displacement (Fox
and Allen, 1964; Gurney and Fox, 1968) and thereby
recover full transforming activity (Fox, 1960). Donor DNA
label is also incorporated via a pathway of degradation
and DNA synthesis, with loss of its genetic information.
After uptake, the ss DNA is found associated with protein(s) in a structure termed the ‘eclipse complex’ (Morrison, 1977). Based on the size of the single pulse-labelled
protein found in this complex (Morrison et al., 1979), it is
thought that the eclipse complex may contain SsbB.
Although it is known that donor DNA is cut endonucleolytically on binding to competent cells, and is reduced to
single strands during transport into the cell, less is understood about the proteins that interact with the donor DNA
once it is inside the cell in a ss state. Although RecA is well
known for strand exchange activities during homologous
recombination (Dubnau et al., 1973; Kowalczykowski
et al., 1994), its specific role in transformation has not
2
B. V. Desai and D. A. Morrison
Fig. 1. DNA uptake in coiA and dprA mutants. Stages of DNA processing were assayed during competence induction at 30°C in CP1250
(wild-type), CP1793 (coiA–) and CP1389 (dprA–) strains. One-millilitre samples were harvested every 5 min after addition of CSP and 3H-DNA
for determination of cumulative DNA degradation (䉬), DNA uptake (䉱) and transformation (䊐) as described in Experimental procedures. Note
that competence induction is slower at 30°C than at 37°C (Peterson et al., 2004; Desai and Morrison, 2006).
been established. Transformation is indeed abolished in a
recA mutant (Martin et al., 1995; Mortier-Barriere et al.,
1998), but recent studies demonstrated that recA and
dprA/dalA (Karudapuram and Barcak, 1997; Lee and Morrison, 1999) are essential for DNA protection during
uptake (Bergè et al., 2003). In recA and dprA (another late
competence gene) mutants, donor DNA is degraded
immediately upon uptake. A non-induced protein,
MmsA (RecG), is also known to be important for DNA
processing (Martin et al., 1996), but its specific role is also
unknown.
coiA is a late competence gene required for efficient
transformation and expressed under the control of ComX
(Pestova and Morrison, 1998; Peterson et al., 2004;
Desai and Morrison, 2006). A coiA mutant exhibits a deficiency of approximately 95% in transformability at 37°C
(Desai and Morrison, 2006). Although disruption of coiA
reduces the yield of transformants, it does not interfere
with DNA uptake or general regulation of competence
genes (Desai and Morrison, 2006). Here we show that
DNA taken up by a coiA mutant is protected from degradation and is recoverable as a ssDNA–protein complex,
suggesting a role for CoiA in DNA processing after eclipse
complex formation.
Results
Because of the apparently similar phenotypes of coiA and
dprA mutants, the kinetics of DNA processing and the
properties of intracellular donor DNA were compared
directly between them. Using single gene-deletion mutations of both coiA and dprA, described previously (Desai
and Morrison, 2006), and a purified 11 kb donor gyrA DNA
fragment, the maximal yield of transformants among
viable cells were 25%, 1.3% and 0.00007% for wild-type,
coiA and dprA strains respectively (data not shown).
Kinetics of DNA uptake is not affected in a coiA mutant
strain
Although total DNA uptake in coiA and dprA mutants is
normal (Bergè et al., 2002; 2003; Desai and Morrison,
2006), its kinetics have not yet been analysed. To
compare the kinetics of uptake and degradation in a coiA
mutant with that in wild-type and a dprA mutant, the fate of
labelled donor DNA was measured at 5 min intervals
during parallel exposure of the three strains to DNA and
competence-stimulating peptide (CSP). As expected,
DNA degradation and uptake proceeded in parallel in the
wild-type cells (Fig. 1). Although CP1793 (coiA–) and
CP1389 (dprA–) yielded few or no transformants respectively, DNA degradation and uptake proceeded in both
mutants at rates similar to those in the wild type.
A coiA mutant is unable to form heteroduplex
As the coiA mutant took up DNA but yielded few transformants, we asked whether the donor marker might recover
from eclipse but then fail to replicate or to be transmitted
to the progeny. To look directly for such recovery of transforming activity, chromosomal DNA was extracted immediately after transformation of CP1250, CP1793 and
CP1389 with NovR DNA and used to transform a second
NovS recipient. In the first round of transformation, the
NovR point marker was integrated in high yield in the
wild-type strain (CP1250) (Table 1 – NovR establishment).
Both the coiA and dprA mutants incorporated normal
amounts of donor DNA label, but exhibited their characteristic defects in inheritance of the nov marker. In the
second round of transformation when a NovS recipient
was transformed with chromosomal DNA from the firstround recipients, transformation was reduced in the cases
of coiA or dprA mutants (Table 1 – Recovery from
eclipse). Heteroduplex formation was affected to the
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
DNA uptake and recombination 3
Table 1. Determination of heteroduplex formation in coiA and dprA mutant strains.
NovR establishmenta
Recovery from eclipseb
Strain
DNA uptakec
(cpm ml-1)
Transformantsd
(NovR ml-1)
DNA uptakec
(cpm ml-1)
Transformantsd
(NovR ml-1)
CP1250 (wild-type)
CP1793 (coiA–)
CP1389 (dprA–)
26400
38600
14900
100%
6%
< 0.001%
383 ⫾ 31
511 ⫾ 12
228 ⫾ 7
100%
7%
0.0001%
a. Initial transformation.
b. Assay of the recovery of novr marker from eclipse.
c. DNA uptake was measured as total DNase I resistant 3H.
d. NovR transformants were measured as novobiocin resistant colonies after overnight incubation at 37°C. (100% = 3 ¥ 106 for a, and 1.4 ¥ 104
for b).
⫾ Standard deviation between three independent experiments.
same extent as transformation both in the coiA mutant,
and, as reported previously (Bergè et al., 2003), in the
dprA mutant. We conclude that the CoiA requiring step is
before, not after, heteroduplex formation.
Internalized donor DNA undergoes eclipse complex
formation in wild-type cells
DNA processing during genetic transformation involves
three distinguishable stages: (i) DNA uptake, (ii) eclipse
complex formation and (iii) heteroduplex formation
(recombination). To determine the physical properties of
donor DNA immediately after uptake, a protocol described
previously (Morrison, 1977) can be used to characterize
the state of donor DNA shortly after a brief period of uptake
at 25°C, before much integration occurs (Collins and
Guild, 1972; Shoemaker and Guild, 1972). DNA in eclipse
can be distinguished from nucleotides and from ss DNA or
ds DNA by hydroxylapatite chromatography. Donor DNA in
eclipse is in the form of single strands (Lacks et al., 1967)
which can be recovered in a complex that has a lower
affinity for hydroxylapatite than ss DNA and that contains a
protein of MW 15–20 kDa (Morrison and Mannarelli, 1979;
Morrison et al., 1979). Short chains elute at 0.07 M PO4
buffer, eclipse complex, at 0.1–0.13 M PO4, ss DNA, at
0.17–0.18 M PO4, and ds DNA at 0.25–0.28 M PO4 (Morrison, 1977). This property was exploited to compare the
effects of dprA and coiA mutations on the form of internalized donor DNA in more detail. As displayed in Fig. 2A,
after 5 min of uptake at 25°C, the majority of donor DNA
label in wild-type cells was in the form of eclipse complex
and eluted from a hydroxylapatite column at 0.12 M PO4,
earlier than 14C-labelled ss DNA or ds DNA internal standards. As the eclipse complex protein is non-covalently
bound to ss DNA (Morrison, 1977; 1978; Morrison and
Mannarelli, 1979), treatment of the complex with protein
denaturants or proteases releases the donor DNA as
single strands that elute at 0.17–0.18 M PO4 buffer (Morrison, 1977). Treatment of a parallel sample to that of
Fig. 2A with proteinase K and SDS caused such a shift of
donor label to the ssDNA position (Fig. 2B). The same shift
was caused by treatment of the eclipse complex with
GuHCl (data not shown). Together, these results characterize the bulk of donor DNA immediately after uptake in
the wild type as being in the form of eclipse complex.
Eclipse complex fails to form in a dprA mutant
Single-stranded DNA is not formed (or is rapidly
degraded) in dprA and recA mutants, as 32P-labelled
donor DNA is reported to be recovered only as small
fragments, nucleotides or products of DNA synthesis after
exposure to DNA for as little as 1 min (Bergè et al., 2003).
This failure to detect any macromolecular DNA in eclipse
suggests an important role of these gene products in DNA
protection. To determine the state of donor label in the
dprA mutant, the early fate of donor DNA was determined
as above. Immediately after uptake during 5 min at 25°C,
approximately 30% of the 3H-DNA donor label was in a
form that eluted at 0.12 M PO4, suggesting the possible
presence of eclipse complex (Fig. 3A). However, upon
treatment with proteinase K or GuHCl (data not shown),
the material in this peak did not shift to a position corresponding to ss DNA (0.18 M PO4) (Fig. 3B). This implies
that the material eluting at 0.12 M PO4 buffer (Fig. 3A)
was not eclipse complex, but a different product whose
affinity for hydroxylapatite is not determined by a protein
component, perhaps very short DNA fragments. This
result is consistent with the one described by Bergè et al.
(2003), and supports the conclusion that donor DNA is
immediately broken down upon uptake by a dprA mutant.
Eclipse complex is formed in a coiA mutant
Although the competence defect of the coiA mutant was
not as severe as that of the dprA mutant, both mutants
exhibited normal kinetics of DNA uptake but formed little
heteroduplex. Due to this phenotypic resemblance, it was
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
4
B. V. Desai and D. A. Morrison
Fig. 2. Formation of eclipse complex (EC) by donor DNA in the wild type. CP1250 was grown, induced to competence, and exposed to
3
H-DNA (䊏). After 5 min at 25°C, cells were collected, lysed and fractionated on hydroxylapatite columns. Both an untreated lysate (A) and an
equal portion treated with proteinase K and SDS and boiling at 95°C for 5 min (B) were eluted in parallel with a linear gradient of PO4 buffer
(------). Elution of donor 3H (䊏) was compared with internal standards of 14C-labelled ds DNA (䊐) (A) or ss and ds DNA (B).
of interest to know the state of donor DNA immediately
after entry in a coiA mutant, and to distinguish degradation, as in recA and dprA mutants, eclipse complex formation, as in wild-type, or some other state. To ask if the
effect of the coiA mutation on the fate of donor DNA was
similar to that of a dprA mutation, the fate of donor label
was similarly followed in a coiA mutant strain. As displayed in Fig. 4A, shortly after uptake, the majority of
donor label eluted at 0.12 M PO4. To determine if this
material possessed other properties characteristic of
eclipse complex, a parallel lysate was treated with proteinase K and SDS. The majority of donor label then
shifted to the position of free ss DNA (0.18 M PO4)
(Fig. 4B). Thus the majority of donor label in the coiA
mutant immediately after uptake appears to be in the form
of eclipse complex, a pool not seen in dprA or recA
mutants.
The ss donor DNA in the eclipse complex does not
recombine in a coiA mutant
To determine directly the fate of DNA in the eclipse
complex that accumulates in a coiA mutant, we used
HpUra [6-(p-hydroxyphenylazo)uracil], which blocks DNA
synthesis but not recombination (Tiraby and Fox, 1974;
Vijayakumar and Morrison, 1983; Méjean and Claverys,
1984), so that any label recovered in ds DNA after incubation in HpUra represents incorporation strictly due to
homologous recombination. To distinguish the incorporation of donor label into ds DNA via DNA synthesis from
incorporation via recombination in a coiA mutant, a
portion of the culture used for determining the presence of
eclipse complex in Fig. 4A was incubated with HpUra at
37°C for 15 min after DNase I addition. A second portion
was incubated for 15 min without HpUra, and both portions were lysed and loaded onto hydroxylapatite
columns. Although the untreated control incorporated all
donor DNA label in ds DNA (Fig. 5A), in the culture with
HpUra no additional label was incorporated into ds DNA
during post-uptake incubation (Fig. 5B). As shown for replicate experiments in Table 2, although donor DNA was
incorporated into the eclipse complex, only approximately
2% of it was incorporated into a recombination product,
consistent with the low yield of transformation in this
mutant. This contrasts with the parallel wild-type cultures,
in which 20–30% of the label initially present in eclipse
complex was eventually incorporated in ds DNA by
recombination.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
DNA uptake and recombination 5
Fig. 3. Lack of formation of eclipse complex in a dprA mutant. CP1389 was grown, induced to competence, and exposed to 3H-DNA (䊏).
After 5 min at 25°C, cells were collected, lysed and fractionated on hydroxylapatite columns. Both an untreated lysate (A) and an equal portion
treated with proteinase K and SDS and boiling at 95°C for 5 min (B) were eluted in parallel with a linear gradient of PO4 buffer (------). Elution
of donor 3H (䊏) was compared with internal standards of 14C-labelled ss DNA (䊐) (A) or ds DNA (B).
HpUra prevents incorporation of donor label into the
host chromosome via DNA synthesis
Some 3H of donor DNA origin (~20% of macromolecular
label) was observed in the ds state (in the host chromosome) after 5 min of uptake at 25°C in all strains examined here (Figs 2A, 3A and 4A). In contrast, Shoemaker
and Guild (1972) reported that after 5 min of uptake at
25°C, only approximately 1.5% of donor DNA has been
integrated in ds form. To resolve the discrepancy, we
blocked DNA synthesis during exposure of competent
cells to DNA using HpUra, as it inhibits DNA synthesis
without affecting cell viability, growth or competence
(Brown et al., 1972; Vijayakumar and Morrison, 1983). As
shown in Fig. 6, treatment of CP1250, CP1793 and
CP1389 with HpUra before and during competence induction and DNA exposure prevented the initial accumulation
of donor label in the ds state during DNA uptake (compare
Fig. 6 with Figs 2A, 3A and 4A). These results explain the
presence of 3H-labelled ds DNA of donor origin in Figs 2,
3 and 4, as due to rapid degradation and re-incorporation
via DNA synthesis rather than recombination.
Table 2. Fate of internalized donor label in a coiA mutant.
After 15 min of further incubation at 37°C inb
After 5 min of uptake at 25°C
0 mM HpUra
40 mM HpUra
Incorporation via recombinationc
Strain
Experiment #
Eclipse complexa
ds DNAb
ds DNA
ds DNA
(cpm)
(% of eclipse complex)
CP1250
1
2
1
2
20 800
11 500
23 400
34 900
4800
3200
5900
5900
23 700
12 700
26 200
38 200
11 200
5 500
6 200
6 500
6400
2300
300
600
30
20
1
2
CP1793
a. cpm in peak at 0.12 M PO4 (EC) per ml of culture.
b. cpm in peak at 0.21 M PO4 (ds DNA) per ml of culture.
c. Increase in ds DNA label during 15 min at 37°C in HpUra (column 6 minus column 4).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
6
B. V. Desai and D. A. Morrison
Fig. 4. Formation of eclipse complex (EC) in a coiA mutant. CP1793 was grown, induced to competence, and exposed to 3H-DNA (䊏). After
5 min at 25°C, cells were collected, lysed and fractionated on hydroxylapatite columns. Both an untreated lysate (A), and an equal portion
treated with proteinase K and SDS and boiling at 95°C for 5 min (B) were eluted in parallel with a linear gradient of PO4 buffer (------). Elution
of donor 3H (䊏) was compared with internal standards of 14C-labelled ss DNA (䊐) (A) or ss and ds DNA (B).
Taken together, these results establish the presence of
ss donor DNA inside a coiA mutant, in amounts characteristic of the wild type and in the form of a nucleoprotein
complex, although its integration into the resident chromosome is reduced by 90%.
Discussion
Many of the late competence gene products that are
required for transformation are thought to have functions
at the entry pore during DNA uptake, including the products of the cel, cgl, cfl and ccl loci. Some of the basis for
this understanding is in work with B. subtilis homologues
of these genes (Dubnau, 1999), but Bergè et al. (2002)
demonstrated directly in S. pneumoniae that the product
of comGA (cglA) is involved in DNA binding, that those of
comEC (celC) and comFA (cflA) are required for DNA
uptake, and that that of comEA (celA) is required for both
degradation and uptake. As CoiA appears not to act in
regulating early or late competence genes (Desai and
Morrison, 2006), we analysed its role in the DNA processing pathway. Although the transformability of a coiA
mutant was reduced, it took up normal amounts of donor
DNA and produced TCA-soluble extracellular products
with kinetics comparable to its wild-type parent, suggest-
ing that the donor DNA in a coiA mutant was processed
normally by EndA, the membrane-bound endonuclease
required for DNA degradation (Lacks et al., 1974; 1975;
Puyet et al., 1990), just as in recA and dprA mutants
(Bergè et al., 2002).
Upon entry into competent wild-type cells, the internalized DNA enters a state of eclipse, losing transforming
activity as it is reduced to a ss form (Fox, 1960). DNA in
eclipse is recoverable as a complex which may contain
SsbB (MW 14795), as it contains a single protein of
15–20 kDa mass (Morrison, 1977; 1978; Morrison and
Mannarelli, 1979; Morrison et al., 1979; Vijayakumar and
Morrison, 1983). Indeed, recent results indicate that
eclipse complex formation is decreased in a DssbB
mutant (B.V. Desai, B. Leach and D.A Morrison, unpubl.
results). But it is unclear whether SsbB has a direct role in
eclipse complex formation or has a role similar to DprA
and RecA in DNA protection; either proposed function for
SsbB could be consistent with a phenotype of reduced
eclipse complex formation and reduce yield of transformants in ssbB mutants. Upon further incubation, the DNA
in eclipse complex is integrated, to form a heteroduplex in
the recipient chromosome. Although RecA is well known
as a protein intimately involved in genetic recombination,
recent studies reveal that in addition, RecA also plays a
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
DNA uptake and recombination 7
Fig. 5. Lack of recombination in a coiA mutant. CP1793 was grown, induced to competence, and exposed to 3H-DNA (䊏). After 5 min at
25°C, cells were incubated further at 37C for 15 min either in the absence (A) or the presence (B) of 40 mM HpUra, and collected, lysed and
fractionated using a linear gradient of PO4 buffer (------) on parallel hydroxylapatite columns. Elution of donor 3H (䊏) was compared with
internal standards of 14C-labelled ss and ds DNA (䊐).
role in donor DNA protection inside the competent cell
(Bergè et al., 2003). Immediately upon entry into a recA
mutant strain, donor DNA is degraded. Interestingly, it was
recently found that during transformation in B. subtilis,
RecA is localized near the site of donor DNA entry (Kidane
and Graumann, 2005). As DNA uptake was not impaired
in a coiA mutant, we analysed properties of the internalized DNA in the mutant strain. Although the phenotype of
a coiA mutant included reduced transformation but normal
DNA uptake, similar to that of dprA and recA mutants, the
fate of internalized DNA in the coiA mutant was distinguishable from that in dprA or recA mutants. Specifically,
in a coiA mutant internalized donor DNA was recoverable
as single strands complexed with protein in a form indistinguishable from wild-type eclipse complex, as illustrated
in Fig. 7. This distinction establishes a role for CoiA in a
different step from DNA protection, i.e. in some step after
eclipse formation but before heteroduplex formation,
implying a role for CoiA in genetic recombination.
CoiA contains two adjacent N-terminal motifs implicated in interactions with DNA: a C2H2 type zinc (Zn)
finger motif (aa, 26–47) and an endonuclease-like fold
(aa, 64–136) (B.V. Desai and D.A. Morrison, unpubl.
results; Kinch et al., 2005). These two DNA interaction
domains of CoiA might participate in interaction with
either the ss DNA (in eclipse) or the recipient ds DNA
(during heteroduplex formation). Although rare in
prokaryotes, the Zn finger motif is very common in
eukaryotes and often plays a role in protein–DNA interaction. The endonuclease-like fold domain is shared with
a wide variety of sequence- or structure-specific endonucleases, suggesting that CoiA could act on the heteroduplex DNA during strand cleavage and separation.
Together, the present results and the presence of the
putative DNA interaction domains are consistent with a
role for CoiA in recombination.
Figure 7 summarizes functions for three ComXdependent proteins that have been linked to the DNA
processing pathway. DprA and RecA are proposed to be
involved in the protection of incoming donor DNA (Bergè
et al., 2003), while the properties of internalized donor
DNA in a coiA mutant, described here, imply that CoiA
acts at a later step in transformation, i.e. in promoting
recombination during genetic transformation in this
species. While participation of RecA in the later step has
not been directly demonstrated, it must be considered
likely, as the pneumococcal RecA protein does form heteroduplex joints in vitro (Steffen and Bryant, 2000) and
does participate in gene conversion in non-competent
cells (Sung et al., 2001).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
8
B. V. Desai and D. A. Morrison
Fig. 6. Inhibition of replicative re-incorporation by use of HpUra. CP1250 (A), CP1793 (B) and CP1389 (C) were induced to competence in
the presence of 40 mM HpUra at 37°C. After 13 min of treatment, cells were equilibrated to 25°C and exposed to the 3H-DNA (䊏) for 5 min.
DNA uptake was terminated and cells were collected, lysed and fractionated on hydroxylapatite columns. All three columns were developed in
parallel with a linear gradient of PO4 buffer (------). Elution of donor 3H (䊏) was compared with internal standards of 14C-labelled (䊐) ss and ds
DNA (A) or ss DNA (B and C).
Experimental procedures
Streptococcus pneumoniae strains, culture growth and
transformation
Bacterial strains used in this study are listed in Table 3.
Unless otherwise specified, S. pneumoniae was cultured,
induced to competence, and transformed as described previously (Desai and Morrison, 2006).
Preparation of 3H-labelled DNA
CP1016 was grown in 10 ml CAT (Morrison et al., 1983)
containing 0.5 mCi of 3H-thymidine (MP Biomedicals; Sp. Act.
30 Ci mmol-1) for 6.5 generations until OD550 0.2. The culture
was chilled, harvested, lysed in a buffer containing 0.5%
Triton X-100, and processed for DNA purification as recommended by the Qiagen Genomic DNA handbook (Qiagen,
Valencia, CA). The yield of DNA from such preparations was
10–15 mg, with a specific activity of 1.0 ¥ 106 cpm mg-1. 14Clabelled CP1016 DNA was prepared in a similar way, using
10 mCi of 14C-thymidine (Sp. Act. 55 mCi mmol-1).
Assay of the stages of DNA uptake
CP1250, CP1793 and CP1389 were grown in 15 ml CAT at
37°C to OD550 0.08–0.1, chilled if required, and returned to
37°C before adding CSP (250 ng ml-1) and DNA (60 ng ml-1).
To measure competence, 0.15 ml of cells was mixed with
1.5 ml of CAT containing MgCl2 (10 mM) and DNase I
(40 mg ml-1), incubated at 30°C for 60 min, and then plated in
Nov agar plates (2.5 mg ml-1). To measure DNA degradation,
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
DNA uptake and recombination 9
Fig. 7. Stages in DNA processing by competent S. pneumoniae.
DNA uptake (binding, degradation and internalization), eclipse
complex (EC), and heteroduplex (HD) formation are depicted.
Incoming donor DNA and host chromosome are represented in
black and grey lines respectively. Steps where individual proteins
are known or proposed to have a role are indicated. MP,
membrane pore for the entry of incoming donor DNA.
a 1 ml sample was chilled and centrifuged at 10 000 rpm at
4°C for 2 min. The supernatant was mixed with 1/4 vol. of
50% TCA, incubated on ice for at least 10 min, and centrifuged at 10 000 rpm for 10 min. 0.1 ml of the resulting supernatant was mixed with 0.4 ml of water and 10 ml of EcoLite
scintillation fluid. To measure DNA uptake, the pellet from the
first spin was washed twice with 1 ml of cold CAT without
resuspending it (Lacks et al., 1974). The resulting pellet was
resuspended in 0.1 ml SEDS buffer (Shoemaker and Guild,
1972), lysed for 10 min at 37°C, and mixed with 0.4 ml water
and 10 ml EcoLite scintillation fluid.
Assay of heteroduplex formation
To determine the establishment of the donor marker,
CP1793, CP1389 and wild-type CP1250 were grown in CAT
as described before. Cells were incubated at 37°C for 5 min
for competence induction, after which 2 ml of competent cells
was exposed to 1 mg of 3H-labelled DNA. After 15 min at
37°C, uptake was terminated by adding DNase I (40 mg ml-1)
and MgCl2 (final 10 mM). Transformed cells were allowed to
complete recombination and DNA integration by incubation at
37°C for an additional hour. A 0.15 ml aliquot from each
culture was diluted for measuring the transformation
efficiency. The rest of the culture was lysed and used as the
source of donor DNA (see below) for the second round of
transformation. A sample of the cell lysate was also mixed
with 10 ml EcoLite(+) scintillation fluid (MP Biomedicals) and
radioactivity was determined as a measure of DNA uptake.
Lysates were prepared as described by Shoemaker and
Guild (1972). In short, cells were diluted with an equal volume
(2 ml) of cold saline citrate (0.15 M NaCl, 15 mM sodium
citrate) and centrifuged at 10 000 rpm for 3 min at 4°C. The
supernatant was discarded and the cell pellet was washed
twice with 2 ml cold saline citrate. The pellet was resuspended in 0.2 ml of SEDS (0.15 M sodium citrate, 0.15 M
NaCl, 0.1% sodium deoxycholate, 0.01% SDS), and lysed at
37°C for 5 min. The lysate was diluted 10-fold with 0.15 M
NaCl, stored at 4°C and used for transformation within
2 days.
In the second round of transformation, to assay the
recovery of donor marker from eclipse, 0.3 ml of each cell
lysate was used to transform 2 ml of competent wild-type
cells. The recipient was grown in complete CAT until OD550
0.08, induced with CSP for 10 min at 37°C, and exposed to
the lysate as described above. After 5 min at 37°C, DNA
uptake was terminated by addition of DNase I (40 mg ml-1).
After 60 additional minutes at 37°C, transformants were
assayed by plating 0.15 ml samples in Nov agar plates. The
remaining cells were pelleted in the cold, washed once, and
lysed in 0.1 ml SEDS at 37°C for 5 min. The lysates were
mixed with 10 ml EcoLite scintillation fluid to determine DNA
uptake.
Assay of the state of donor DNA in eclipse
Competent strains were prepared by treating respective cultures (CP1250, CP1793 or CP1389) grown at 37°C in CAT
containing 10 mM HCl to OD550 0.08–0.10 with CSP-1 at
37°C for 10 min and at 25°C for 5 min. After each culture was
exposed to 3H-DNA (~75–100 ng ml-1) for 5 min at 25°C,
uptake was terminated with DNase I (40 mg ml-1) at 25°C for
1 min. Two 1 ml samples were harvested by centrifugation at
7000 g for 5 min at 4°C. The cell pellet was washed once with
1 ml of cold CAT containing 5 mM EDTA without resuspending the cells. After washing, sample (A) was resuspended and
lysed in 0.5 ml of lysis buffer [0.05 M Tris (pH 8.0), 0.03 M
Table 3. Strains used in this study.
Strain
CP1016
CP1250
CP1389
CP1500
CP1793
Genotype and description
–
nov-1 str-1 ery-2 vlt; Thy
hex malM511 str-1 bgl-1; Rx derivative, low b-galactosidase background
CP1250, but DdprA::KmR
hex nov-r1 bry-r str-1 ery-r1 ery-r2; NovR, SmR, EmR, BrR
CP1250, but DcoiA::KmR
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology
Source
Morrison et al. (1984)
Pestova et al. (1996)
Desai and Morrison (2006)
Cato and Guild (1968)
Desai and Morrison (2006)
10 B. V. Desai and D. A. Morrison
EDTA, 0.4% sarkosyl, 0.1% Triton X-100], while sample (B)
was lysed in the same buffer containing 20 mg ml-1 proteinase
K and 0.1% SDS. Lysis for (A) took place at 37°C for 10 min,
while for (B) incubation continued for 30 min. The lysates
were stored at 0°C for 1–2 h and loaded onto hydroxylapatite
columns (below) along with internal ss and/or ds 14C DNA
standards.
In 6 ml disposable polypropylene syringe barrels, 1.5 ml of
hydroxylapatite (Bio-Rad) suspended in 0.01 M sodium phosphate buffer containing 0.05% sarkosyl was supported on a
25 mM pore-size polyethylene disk. Another such disk was
placed on the top of the bed and the column was washed with
20 vols of 0.01 M phosphate buffer containing 0.05%
sarkosyl. Differentially prepared lysates were loaded on
separate hydroxylapatite columns, washed with the same
buffer (5 ¥ 1.5 ml fractions), and developed in parallel with a
linear gradient of 0.01 M to 0.3 M phosphate buffer from a
single mixing chamber. Consecutive 1.5 ml samples were
collected directly into scintillation vials, at a flow rate of
0.3 ml min-1. Fractions were mixed with 15 ml of EcoLite (MP
Biomedicals). 3H channel counts were corrected for overflow
from the 14C channel. A 14C-labelled ds DNA standard was
prepared by mixing a small aliquot of 14C-labelled DNA in
0.01 M PO4 buffer, and standard was made by boiling a
similar preparation for 5 min immediately before loading
directly onto the column.
Acknowledgements
This work was supported in part by the US National Science
Foundation (MCB-0110311). The dedication and persistence
of Benjamin Leach in assisting with hydroxylapatite chromatography is gratefully acknowledged.
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