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Journal of Bacteriology, May 1999, p. 2973-2978, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The MobA-Linked Primase Is the Only Replication
Protein of R1162 Required for Conjugal Mobilization
Dorian
Henderson and
Richard
Meyer*
Department of Microbiology and Institute for
Cellular and Molecular Biology, University of Texas, Austin, Texas
78712.
Received 21 December 1998/Accepted 1 March 1999
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ABSTRACT |
Cells newly transformed with plasmid R1162 DNA were used as donors
in conjugal matings to determine if the plasmid replication genes are
necessary for transfer. An intact system for vegetative replication is
not required for transfer at normal frequency, but the plasmid primase,
in the form linked to the nickase, must be present in donor cells.
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TEXT |
The basic features of
intercellular DNA transport during bacterial conjugation are
similar for a wide variety of different plasmids in gram-negative
bacteria (35). In the donor cell, one DNA strand is cleaved
at a unique site within a locus called the origin of transfer
(oriT) and is then unwound and passed in a 5'-to-3'
direction into a recipient cell. The cleaving protein remains
covalently linked at the 5' end of the strand and recircularizes the molecule after a round of transfer. Because only one strand is
transferred, synthesis of the complementary strand is required for
survival of the plasmid in the recipient. Early studies demonstrated such synthesis for the F factor and also revealed that the transferred strand was replaced by synthesis in the donor (30, 31).
However, it should be noted that replacement synthesis in donor cells
is not obviously required, particularly for multicopy plasmids where the overall transfer frequency is low, since the occasional loss of a
molecule through conjugation would have little impact on plasmid maintenance.
Apart from strand replacement, replication could play another
role in conjugation. The DNA strands are unwound ahead of the replication fork, and the machinery of DNA replication could be conscripted to separate strands during conjugation. Such an idea was
generally discarded, once it was shown that the F factor and several
other large plasmids were transferred at the nonpermissive temperature
from donor cells containing a temperature-sensitive dna
mutation (9, 13, 16, 31, 34). However, these early genetic
experiments suffered from the shortcoming that the ts mutation might be particularly leaky for conjugation, when limited amounts of replication would be required. An additional problem is that
the mutation might be suppressed by other, overlapping functions
encoded by the chromosome or plasmid. The latter problem is
particularly relevant for a large plasmid such as F, which contains
multiple replicons and which probably encodes more than one mechanism
for initiation of DNA synthesis (17, 20). Moreover, a
general requirement for replication cannot be determined by cloning,
since the vector replicon could provide a substitute system for replication.
We decided to reinvestigate the possible role of the plasmid
replication genes in conjugation, either for strand replacement or for
initiation of a round of transfer. We selected the plasmid R1162, which
is simpler than plasmid F, as a model system and used conditions where
initiation of replication was stringently inhibited.
R1162 encodes three proteins required for its conjugal
mobilization: MobA, which cleaves and ligates the transferred
strand (6), and two accessory proteins, MobB and MobC.
MobC assists in localized separation of the DNA strands at
oriT (38); MobB stabilizes the complex of Mob
proteins at oriT and also has an additional function in
transfer (23). In addition, R1162 encodes three
replication proteins (25), a helicase and an
iteron-binding protein, the products of the repA
and repC genes, respectively, and a primase (see Fig.
2). The primase is encoded in the repB' region and is
translated both as the C-terminal domain of MobA and separately
(28). Both forms of the primase are active and sufficient
for plasmid replication both in vitro (26) and in vivo
(14a). Each DNA strand has an initiation site for the
primase within the origin of replication; there are no known secondary sites in R1162 for this primase or for other priming systems
(3).
Replication of plasmid DNA in the donor is not required for
transfer.
To investigate the role of replication in
conjugal transfer of R1162, we carried out the
experiment outlined in Fig. 1. We first
constructed by electroporation a population of potential donor cells
lacking some or all of the R1162 replication proteins. These were then
immediately mated, and transconjugants were selected by plating on
medium containing antibiotics. The plasmid introduced by
electroporation and then transferred was pUT1557, a derivative of R1162
lacking all the replication genes (Fig.
2). This plasmid also contains a 952-bp
DNA fragment (3) encoding resistance to chloramphenicol. The
Cmr gene was introduced because of the very low background
following selection. The additional DNA contains no pas
sites and is inactive for initiation of R1162 DNA replication when the
normal system of replication is disabled (3).
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FIG. 1.
Experimental strategy to examine conjugal transfer in
the absence of plasmid vegetative replication.
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FIG. 2.
Map of R1162 (top) and fragments of R1162 DNA in the
different plasmids used in this study. The horizontal bar, interrupted
at the site of a deletion, indicates the R1162 DNA present in each
plasmid. The filled regions of the bar designate the locations of the
origin of replication (oriV) and the origin of transfer
(oriT). The locations and direction of transcription of the
genes for replication (rep), mobilization (mob), resistance to
streptomycin (Sm1 and Sm2), and resistance to sulfonamides (Su) are
shown by the arrows on the map of R1162. Those genes retained in the
other plasmids are indicated in each case. The inverted triangles
indicate the locations of cloned DNA containing either attP,
a gene for chloramphenicol resistance (Cm), or a 14-bp oligonucleotide
insertion. Construction of these plasmids is outlined in Table 1.
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Plasmid pUT1557 was introduced into cells that contained additional
plasmids, which are needed to create potential donors
for mating. These
cells contained R751, an IncP1 plasmid that
conjugally mobilizes R1162
at a high frequency (
36), and pUT1584
(Fig.
2), which
provided a source of MobB, one of the R1162 mobilization
proteins. In
addition, the cells contained one of the helper plasmids
shown in Fig.
2. These all consist of different fragments of R1162
DNA, containing
the remaining R1162
mob genes (
mobA and
mobC)
and different subgroups of the R1162 replication
genes, cloned
into pBR322. Donor strains were all derivatives of the
Escherichia coli K-12 strain MV10 (C600
trpE5)
(
15). The recipient strain
was the nalidixic
acid-resistant derivative of MV12 (C600
trpE5 recA56) (MV12 Nal
r) (
5) containing pUT459,
which provided the Rep proteins for
replication of
pUT1557.
For electroporation, approximately 10
10 cells, grown to
mid-log phase in broth, were collected by centrifugation, washed
sequentially
with 1.0, 0.5, and 0.25 culture volumes of 10% glycerol,
and resuspended
in 40 µl of this solution. The cells were then mixed
with 0.1
µg of pUT1557 DNA, which had been extracted by the alkaline
lysis
method (
19) from cells also containing pMS40
(
21), a helper
plasmid supporting replication of pUT1557. To
eliminate cotransformation
with pMS40 DNA, the preparation was digested
with the restriction
enzyme
SmaI before being added to the
cells. The mixture was placed
in a 0.1-ml cuvette, and the cells were
electroporated with a
pulse of 1.8 kV. The cells were then resuspended
in 1 ml of 2%
Bacto Tryptone-0.5% Bacto Yeast Extract-10 mM
NaCl-2.5 mM KCl-10
mM MgCl
2-20 mM glucose and mixed with
5 × 10
8 recipient cells in the same medium. The
donors and recipients
were concentrated by centrifugation, resuspended
in 0.1 ml of
this medium, and deposited as a spot on a broth plate.
Cells were
allowed to mate for 90 min at 37°C and were then
resuspended in
1 ml of broth, and dilutions were plated on medium
containing
chloramphenicol and nalidixic acid (25 µg/ml each). The
number
of potential donors was determined by plating, on medium
containing
chloramphenicol, the mating mixture in which the donor
strain
contained the Rep
+ helper plasmid pUT1543. This
number was also used to estimate
the frequency of mobilization for the
matings involving the other
donor strains. Electroporation with
pUT1385, the replication-proficient
parent of pUT1557, was carried out
in parallel to detect any significant
differences in the
transformability of the donor strains. These
strains were all
transformed at essentially the same frequency
by this DNA (data not
shown).
When cells containing pUT1543 (Fig.
2), a helper plasmid providing all
the replication proteins of R1162, were transformed
with pUT1557 and
then mated, many transconjugants were obtained
(see Table
2). The
mating frequency, the number of transconjugants
per donor cell, was
6 × 10
3 (Table
1).
This frequency is similar to that observed for mobilization
of pUT1557
in standard matings from donor cells also containing
R751 and pUT1543.
Thus, sufficient time was provided in the period
between transformation
and plating to allow establishment and
processing of plasmid molecules
for DNA transfer.
Significant numbers of transconjugants were also obtained in a second
mating, after electroporation of cells containing the
helper plasmid
pUT1559 (Table
2). This plasmid is
similar to
pUT1543 but lacks
repA and
repC (Fig.
2), so that pUT1557 DNA
was not replicated in the potential donor
cells. Using the number
of pUT1543 potential donors as an estimate of
the number also
available after transformation with pUT1559, we
calculated a mating
frequency of approximately 2.3 × 10
4 (Table
2). This value probably underestimates the
actual transfer
frequency, since the number of pUT1543 donors was
determined after
the 90-min mating period, which is sufficient time for
their number
to increase due to growth on the medium. However, the
number of
potential donor cells containing pUT1559 cannot increase
during
this period.
The appearance of transconjugant colonies in matings involving
pUT1559 indicated that the complete system of vegetative replication
of
R1162 was not required for conjugal transfer. However, we did
additional tests to verify that these colonies did in fact
arise
from bacterial mating. When R751 was absent from the cells
transformed
with pUT1543, no colonies of cells resistant to
chloramphenicol
and nalidixic acid appeared after mating (Table
2).
Thus, these
colonies were not due to chromosomal mutation or to
transformation
of recipient cells by the pUT1557 DNA remaining in the
medium
after transformation. The possibility that transfer of R751
potentiates
a recipient cell for transformation by pUT1557 was also
ruled
out, since no colonies were obtained in other matings, described
below, in which R751 was
present.
The plasmid DNA content of transconjugant cells from five separate
matings involving donor cells containing pUT1559 was analyzed
by gel
electrophoresis (Fig.
3). In each case,
the sizes of the
restriction fragments were the same (Fig.
3, lanes d
to h) and
identical to those obtained after matings involving the
Rep
+ helper plasmid pUT1543 (lane c) or after
transformation of the
recipient strain with pUT1557 (lane b). These
results indicated
that only unaltered pUT1557 and pUT459 were present
(the transferring
vector R751 was also in most of the transconjugants,
but the bands
were very faint because of its low copy number). Thus,
the transconjugant
cells did not arise by recombination between pUT1557
and the helper
plasmid and then transfer of the joint molecule. In
addition,
because all the helper plasmids lack
oriT,
transient chimeric
molecules, due to site-specific recombination at
this locus (
33),
could not have occurred.
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FIG. 3.
Plasmid DNA in MV12 Nalr(pUT459) (lane a)
and in derivatives containing pUT1557, constructed by transformation
(lane b) or by conjugal mating with the donor strain containing helper
plasmid pUT1543 (lane c) or helper plasmid pUT1559 (lanes d to
h). Each transconjugant for the plasmid DNA in lanes d to h was
the result of an independent mating. The DNA was digested with
EcoRI before being applied to an 0.8% agarose gel. In lanes
c to h, faint, slowly migrating bands were observed. These are derived
from the mobilizing vector R751. Lane i contains
HindIII-digested  DNA as marker.
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Our results indicate that mobilization of R1162 can occur at a normal
frequency in the absence of RepA and RepC, and thus
vegetative
replication of R1162 is not required. However, these
proteins, encoded
by pUT459, were present in the recipient cells
in our experiments, in
order to maintain pUT1557 after transfer.
It was possible that RepA and
RepC leaked through the conjugal
pore and into the donor cell of a
mating pair. Although such leakage
would not be expected to allow
general replication of plasmid
DNA in the donor, a transferring
plasmid copy, positioned at the
conjugal pore, might have access to
these proteins. To rule out
this possibility, we modified the
mating experiment by cloning
a 493-bp
DNA fragment containing the
attP gene into pUT1557,
to create pUT1613. We also replaced
pUT459 in the recipient strain
with pUT1612, a pBR322 derivative
encoding the
integrase protein.
In a preliminary experiment, we
found that after transformation
with
SmaI-digested pUT1613
and pMS40 DNA, chloramphenicol-resistant
transformants that did not
contain helper or other replicating
plasmids were formed (data
not shown). Thus, the replication-defective
plasmid could
integrate into the chromosome upon entry into the
cell
(
1). Donor cells were electroporated with pUT1613 and
mated
with MV12 Nal
r(pUT1612). Again, transconjugants were
obtained not only from
donor cells containing the complete set of
R1162 replication proteins
but also from those lacking RepA and RepC
(Table
2). For each
donor, the transfer frequencies were similar,
whether the mobilized
plasmids were rescued by Int-mediated
recombination or by providing
replication proteins in the
recipient.
Although we can only estimate the number of potential donors that
are formed after electroporation of Rep
strains, it is
clear that the efficiency of transfer is similar,
whether or not all
the Rep proteins are present in the donor cells.
It is possible that
each competent cell takes up many molecules
of DNA, so that the plasmid
copy number in Rep
donors is transiently similar to that
in donors in which the
plasmid can replicate (at least 10 copies per
cell [
2]). We
transformed the Rep
+ donor
strain (containing helper plasmid pUT1543) with a mixture
of two
plasmid DNAs, pUT1557 DNA and an equimolar amount of DNA
from pUT1601,
a pACYC184 derivative encoding resistance to tetracycline
but not
chloramphenicol and compatible with all the plasmids in
the donor.
Cells were plated on medium containing chloramphenicol
or tetracycline,
and colonies were then tested for resistance
to the other antibiotic.
In two separate experiments, on average
only 14% of the cells
receiving one plasmid also received the
other. This indicated that
during electroporation, competent cells
do not generally receive a
number of plasmid molecules similar
to the copy number of R1162. We
conclude that after transformation,
plasmid DNA is efficiently targeted
to the conjugal apparatus,
and this accounts for the high frequency of
transfer after
electroporation.
MobA-linked primase in the donor is essential for recovery of
plasmid DNA in recipient cells.
We have shown elsewhere that the
MobA-linked form of the primase increases the efficiency of the
mobilization system encoded by R1162 (14). This
stimulation requires the cognate primase recognition site,
properly oriented for synthesis of the complement to the transferred
strand, suggesting that R1162 can use this priming system for DNA
synthesis during conjugal transfer. We used electroporation and
mating to examine the role of the R1162 primase in transfer. We
constructed two additional helper plasmids, pUT1582 and pUT1548,
similar to pUT1543 (Rep+) and pUT1559
(repAC), respectively, but containing an inactivating, 14-bp insertion in the primase-coding region of the plasmid
(14). After transformation of these cells with pUT1557
and subsequent mating, very few colonies were obtained in each
case (Table 2), indicating that the R1162 primase is involved in transfer.
The results with several other helper plasmids indicate that, as
suggested by earlier results (
14), it is the linked primase
that is principally active. The plasmid pUT1593 is identical to
pUT1559
but contains a frameshifting 10-bp deletion in
mobB,
upstream
from
repB'. Because of the frameshift, only the
short form of
the primase is made. The mutation does not affect the
N-terminal
third of MobA, the region required for transfer
(
14). However,
pUT1593 no longer supported detectable
mobilization of pUT1557
(Table
2). In contrast, a nonshifting, 9-bp
deletion in the same
location still permitted transfer of pUT1557
(pUT1592) (Table
2). Finally, plasmid pUT1581 is similar to pUT1559 but
contains
an in-frame deletion removing the ribosome binding site and
initiation
codon of the short form of the primase, so that this form is
no
longer made (
14a). Like pUT1559, pUT1581 allowed
mobilization
of pUT1557 (Table
2). Thus, of the two forms of primase,
the
long form is sufficient to ensure mobilization at a high
frequency.
The MobA-linked primase is also important for transfer when the
incoming plasmid is inserted into the chromosome by the
integrase.
Transconjugants of MV12 Nal
r(pUT1612) were formed
efficiently when the donor strain contained
the helper plasmid
pUT1581, which encodes the primase long form.
Only a few apparent
transconjugants were found when the donor
strain contained pUT1582,
which does not encode an active primase
(Table
2). Presumably,
integration of the plasmid by site-specific
recombination required
restoration of the incoming plasmid DNA
to the duplex
form.
In several matings, a small number of transconjugant colonies were
obtained when primase was absent from the donor cells (Table
2).
Colonies were also obtained at a similar frequency when recipient
cells
lacked either of the rescue plasmids, pUT459 or pUT1612.
The cells in
these colonies contained pMS40. This background level
of
transconjugants is presumably the result of matings involving
rare
donor cells that received both pUT1557 and intact pMS40 during
electroporation.
Our results indicate that mobilization of R1162 does not require an
intact system of vegetative replication. However, because
only a
single strand of DNA is transferred, a priming system for
its
complement is required. In the case of R1162, the plasmid
primase,
linked to one of the Mob proteins, is used. Utilizing
for transfer the
plasmid-encoded primase and its cognate recognition
site is an obvious
adaptation for broad-host-range plasmids. By
contrast,
narrow-host-range plasmids, such as F and ColE1, probably
use cellular
mechanisms of priming (
37).
Recipient cells containing pUT459 encode primase in amounts
sufficient to support replication of transferred molecules.
Nevertheless,
primase in the recipient could not substitute for an
absence of
MobA-linked primase in donor cells. Increasing the distance
between
oriT and
oriV reduces the frequency
of transfer (
14). Thus,
we believe that the MobA-linked form
of the primase, attached
to the 5' end of the transferring
molecule, is also uniquely positioned
to prime the complementary strand
efficiently. Possibly, priming
is initiated at the conjugal pore, with
MobA, immobilized at this
site, scanning the DNA for the cognate
priming site during movement
of the DNA strand into the recipient. It
is also possible that
priming might be required for proper termination.
MobA not only
ligates single-stranded
oriT DNA but also
readily cleaves this
DNA (
6,
27). Synthesis of the
complementary strand through
oriT, initiated at the
neighboring
oriV by the primase domain
of MobA,
would result in a substrate that is poorly cleaved by
MobA. This would
ensure that the transferred molecule would remain
circular.
Several observations suggest that fusion of the primase to the nicking
protein evolved after an ancestral plasmid acquired
the mobilization
genes. The plasmid pTF-FC2 is an IncQ-like replicon
and encodes a
primase related to RepB' (
11,
12). However,
there
is no fused form of the primase, and the mobilization genes
of the
plasmid are unrelated to those of R1162 (
24). Another
IncQ
plasmid, pIE1107 (
29), has an arrangement of
mob
and
rep genes similar to that in R1162, and from inspection
of the sequence,
both a short and fused form of the primase would
appear to be
synthesized. However, the amino acid sequence at the
fusion is
different, suggesting that this fusion evolved
independently from
the one in R1162. Finally, pSC101 encodes a MobA
protein similar
to that encoded by R1162, but it is not fused to an
R1162-like
primase (
4). It is therefore likely that the
fusion followed
acquisition of the
mob genes and was
selected because it improved
the frequency of transfer. It is
noteworthy that once a priming
system is captured, a
mob
system can become completely independent
of its plasmid host, allowing
new modes of maintenance. This might
have happened in the case of
mobilizable transposons, at least
some of which might specify their own
priming system for transfer
(
18).
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ACKNOWLEDGMENTS |
This work was supported by a grant from the National Institutes of
Health (GM37462).
We thank Tove Atlung for providing a plasmid as a source of the int gene.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Institute for Cellular and Molecular Biology,
University of Texas, Austin, TX 78712. Phone: (512) 471-3817. Fax:
(512) 471-7088. E-mail: rmeyer{at}mail.utexas.edu.
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Journal of Bacteriology, May 1999, p. 2973-2978, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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