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Journal of Bacteriology, December 1999, p. 7552-7557, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Separate Roles of Escherichia coli
Replication Proteins in Synthesis and Partitioning of pSC101
Plasmid DNA
Christine
Miller and
Stanley N.
Cohen*
Department of Genetics, Stanford University
School of Medicine, Stanford, California 94305-5120
Received 2 August 1999/Accepted 27 September 1999
 |
ABSTRACT |
We report here that the Escherichia coli replication
proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured
under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid
replicons from half of the bacterial population during a single cell
division. As was observed previously for DnaA, overexpression of the
wild-type DnaB protein conversely stabilized the inheritance of
partition-defective plasmids while not increasing plasmid copy number.
The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the
separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding
domain) that is physically separate from the DnaA domain that interacts
with other host replication proteins. Our results identify bacterial
replication proteins that participate in partitioning of the pSC101
plasmid and provide evidence that these proteins mediate plasmid
partitioning independently of their role in DNA synthesis.
| |
INTRODUCTION |
Replication of the Escherichia
coli chromosome is initiated when the DnaA protein binds to
specific sequences (DnaA boxes [2]) within the
oriC (replication origin) region of the chromosome and opens
the DNA helix, after which the DnaB helicase and the DnaC protein
(46) are loaded at 13-mer sequences within the origin
(8) through interaction with DnaA (26). The DnaB
protein then proceeds to unwind the helix in advance of the replication fork (23), interacting also with the primase, DnaG
(25), and DNA polymerase III (PolIII) holoenzyme (21,
31), and consequently helping to bring other components of the
replication machinery to the origin.
Replication of plasmid pSC101 additionally requires the plasmid-encoded
RepA protein, which binds to a region (the plasmid origin of
replication) that includes binding sites for DnaA and 13-mers for
DnaB/C entry. Also included in the origin region is a binding site for
integration host factor, which is conditionally required for plasmid
DNA replication (4), and the par locus, which
accomplishes inheritance of plasmids in populations of dividing cells
(28). Replication of pSC101 has been demonstrated to require the host DnaA and DnaG proteins (18, 14), and pSC101
replication is inhibited more rapidly than chromosome replication in
dnaB and dnaC temperature-sensitive strains grown
at an elevated temperature (17). The plasmid-encoded RepA
protein has been shown to interact with the host-encoded DnaA, DnaB,
and DnaG proteins in vitro as well as with the tau subunit of the
PolIII holoenzyme (24, 25, 31, 33).
Earlier work has led to the proposal that partitioning of pSC101 is
mediated by the same origin region multicomponent DNA-protein complex
that accomplishes plasmid DNA replication (i.e., the Rep-Par complex
[4, 20, 29]) but that the partition and replication functions of this complex are distinct (13, 20, 29). Factors affecting overall plasmid DNA superhelicity (30) or altering localized supercoiling in the pSC101 origin region (3, 13) can stabilize the inheritance of par deletion plasmids,
possibly by enhancing the formation of the complex (20).
Additionally, mutant forms of the RepA protein, which is a component of
the origin complex, can promote plasmid stability (4)
independently of its role in plasmid DNA replication (13,
45). Here we show that other components of the pSC101 origin
region complex, the host replication proteins DnaA and DnaB, also
mediate pSC101 partitioning and that their effects on
partitioning are distinct from their roles in DNA replication.
| |
MATERIALS AND METHODS |
Bacterial strains, media, and general methods.
Bacterial
strains and plasmids are listed in Table
1. Unless otherwise stated, bacteria were
grown in Luria broth (LB) medium. Antibiotics (Sigma) were used at
concentrations of 20 µg/ml (ampicillin and chloramphenicol) and 30 µg/ml (kanamycin). Plasmid DNA was isolated either by the method of
Holmes and Quigley (19) or by Triton X-100 (12)
or alkaline lysis (37).
Plasmid construction.
DNA digestion by restriction
endonucleases was performed according to protocols obtained from the
supplier (New England Biolabs). Derivatives of pACYCdnaA
(43) that contained the DnaA deletion D237-378
(41) and the DnaA mutation T435M (42) were
constructed by digesting each plasmid with EcoNI and
RsrII, exchanging the gel-purified (Qiagen) wild-type
DnaA-containing fragment for the mutant fragment, and ligating the ends
with T4 DNA ligase (Life Technologies, Inc.). The fragment encoding the
membrane binding domain of DnaA was inserted into the same plasmid and
sites; however, the sites were made blunt ended with T4 DNA polymerase
(Life Technologies) and ligated to the similarly made blunt-ended
EcoRI-to-HindIII fragment of pMZ001-31 (which
contains DnaA with mutations R360E, R364E, and K372E
[16]).
Transformation assays, segregation rates, and plasmid copy
number.
Cells containing the pSC101 derivatives were transformed
by using standard procedures (11) with plasmids expressing
the various host replication proteins and plated on LB plates
containing antibiotics selective for either the incoming plasmid or
both plasmids. Plasmid stability (28) and copy number
(5, 45) were determined as described previously; segregation
analysis was performed at the temperatures indicated. The
LD50 (the antibiotic concentration required to kill 50% of
the cells) was used to assess the copy number for Apr
plasmids (45). The rate-of-loss experiments were performed as previously described (45).
Protein concentrations.
Replication proteins were expressed
at intermediate levels from constructs made for overexpression of those
proteins but maintained in a noninduced state. pCM707 is an
Apr derivative of a pACYC plasmid shown previously to
produce five times the normal amount of DnaA protein (1).
Various DnaB-overexpressing plasmids were tested, and the amounts of
DnaB produced under noninduced conditions were monitored by Western
blotting. For detection of DnaB and DnaG proteins, we used antibodies
kindly provided by D. Bramhill and K. Marians, respectively. For
Western blot analysis, routinely 0.7 OD600 (optical density
at 600 nm) units of exponentially growing cells were lysed and
separated on a sodium dodecyl sulfate-10% polyacrylamide gel
(37) run on a Bio-Rad Protean II; Western analysis was
performed as recommended by Promega, using a 1:10,000 dilution of the
anti-DnaB or anti-DnaG antibody in Tris-buffered saline-Tween 20 (20 mM
Tris [pH 7.6], 0.137 M NaCl, 0.17% Tween 20) with 1% bovine serum
albumin (Sigma) and a 1:20,000 dilution of the horseradish
peroxidase-conjugated anti-rabbit antibody (Promega). Protein
recognized by the antibody was visualized by enhanced chemiluminescence
(Renaissance; NEN Dupont) and scanned on a PDI densitometer
(20).
| |
RESULTS |
pSC101 stability in cells having limited DnaB activity.
We
wished to examine the effects of mutations in the essential E. coli replication proteins DnaA, DnaB, DnaC, DnaG, and DnaN
some of which have been shown to interact specifically with the
plasmid-encoded RepA proteinon plasmid partitioning. While
temperature-sensitive mutations in these proteins have been isolated,
it is not possible to carry out extended culture of the mutant bacteria
at temperatures that inactivate the proteins, which are required for
both chromosomal and plasmid DNA replication. However, it has long been
recognized that temperature-sensitive proteins have a gradation of
activity throughout a wide temperature range and that bacteria
containing temperature-sensitive mutations in proteins essential for
growth commonly show detectable phenotypic effects even when cultured at permissive temperatures (14, 47). We therefore
investigated whether temperature-sensitive mutations in host
replication proteins affected plasmid partitioning during continuous
culture at temperatures that permit DNA replication, choosing for each
mutant the highest temperature that allows cell growth to proceed
(17).
As seen in Table
2, the wild-type pSC101
plasmid replicon was entirely stable in
E. coli cells
carrying temperature-sensitive
mutations in the
dnaA,
dnaC,
dnaG, or
dnaN gene when cultured
at 35°C, the highest tested temperature that permits cell growth.
However, the same plasmid was highly unstable in all tested
dnaB mutant strains cultured at 30°C, which was the
highest temperature
that allowed cell growth in these mutants. The
observed instability
in the
dnaB(Ts)
8 strain was
reversed by concomitant overexpression
of the DnaA protein.
Notwithstanding the destabilizing effects
of
dnaB mutations
on plasmid inheritance during growth at 30°C,
plasmid copy number, as
measured by LD
50 (
45), was unchanged
compared to
dnaB+ strains cultured at the same temperature
and was higher than
the copy number seen at the same temperature in
dnaA and
dnaC temperature-sensitive strains
{LD
50 for pPM30, 200 µg/ml in wild-type
cells, 250 µg/ml in PC8 [
dnaB(Ts)
8], 100 µg/ml in
WM448 [
dnaA(Ts)
46],
and 125 µg/ml in PC2
[
dnaC(Ts)]}.
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TABLE 2.
Effects of temperature-sensitive mutations in
dnaA or dnaB on stability of the wild-type pSC101
plasmid, pDLC8
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|
Partition without replication.
Cessation of pSC101 DNA
replication consequent to a temperature-sensitive mutation in RepA
yields plasmid-free cells only after the intracellular pool of
nonreplicating plasmids has been diluted during several cell divisions,
i.e., equipartition (45). However, when the pSC101 replicon
lacks a par locus, shutoff of replication by inactivation of
RepA causes loss of plasmids from half of the population during the
first cell division (45) (Fig. 1A). This phenotype (i.e., the
segregation of a nonreplicating cellular pool of pSC101 plasmids as a
unit), which reflects loss of plasmid partition functions
(20), was used to investigate the effects of host
replication mutations on plasmid partitioning.
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FIG. 1.
Rate of loss of pSC101 plasmids when replication ceases.
Cells containing the plasmids were grown at the permissive temperature
(30°C) with selection for the plasmid and then diluted into warm
medium without antibiotic and grown at 42°C for the indicated times.
The percentage of plasmid-containing cells was monitored by plating the
cells at 30°C without selection and then picking onto plates with
selection for the pSC101 plasmid; the number of total colonies was
monitored to determine the number of cell divisions after the shift in
temperature. Note that the y axis is a log scale. (A) pSC101
repA(Ts) plasmids. Squares, percentage of cells containing
par+ pCM301; diamonds, percentage of cells
containing par deletion plasmid pCM302 (45). (B)
Host replication mutant temperature-sensitive strains with wild-type
pSC101. The percentages of cells containing pDLC8 in dnaA
(WM448, dnaA46 [44]), dnaB (PC8,
dnaB8 [9], and HfrH165, dnaB70
[7]), dnaG (PC3, dnaG3
[9]), or dnaN (HC194, dnaN159
[35]) strains were identical and are represented by
diamonds; the percentages of cells containing pDLC8 in dnaC
(PC2, dnaC2 [9]) are represented by
squares; triangles represent pDLC8 in the
dnaB(Ts)252 strain (RS162
[38]).
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In bacteria containing temperature-sensitive mutations in host
replication genes, DNA synthesis stops either immediately upon
shift to
a nonpermissive temperature (fast-stop mutants) or at
initiation of the
next replication round (slow-stop mutants) (
22).
As the
nonreplicating mutant cells continue to divide for several
generations
in both cases (
48), the effects of host replication
gene
mutations on plasmid partitioning can be assessed by investigating
plasmid segregation during this period. As seen in Fig.
1B, cessation
of replication following a shift to the nonpermissive temperature
of
bacteria mutated in
dnaA,
dnaC,
dnaG,
or
dnaN, whether slow-stop
or fast-stop mutations, allowed
equipartition of the plasmid pool.
Two
dnaB mutants that we
tested, PC8 (dnaB8 [
9]) and HfrH165
(dnaB70
[
7]), both of which showed the fast-stop phenotype,
also showed equipartition of pSC101 plasmids (Fig.
1B). However,
loss
of plasmids from half of the population occurred immediately
upon the
temperature shift of RS162, the
dnaB252 (
38)
mutant
strain, indicating a unique effect of this
dnaB
mutation on plasmid
partitioning. Interestingly, the slow-stop mutation
producing
this partitioning defect has less immediate effects on DNA
replication
than the two
dnaB fast-stop mutations that we
studied (
38) (Fig.
1B).
Stabilization of plasmids by DnaB overexpression.
Deletion of
the par locus (28) (Table
3) or overproduction of the RepA protein
(20) (Table 4) can result in
unstable inheritance of pSC101 plasmids. Overexpression of DnaB at 4 to 10 times the normal level (as determined by Western blot analysis) was
found to reverse both instances of plasmid instability (Tables 3 and
4), as was observed previously for DnaA (20). In contrast, overexpression of other host proteins that also interact directly with
RepA (i.e., DnaG and the tau subunit of PolIII holoenzyme) had no
stabilizing effect on inheritance of unstable pSC101 replicons (Tables
3 and 4). Importantly, the enhanced stabilization by excess DnaA or
DnaB was not accompanied by enhanced plasmid DNA replication as
measured by the copy number (Fig. 2),
providing further evidence of the separateness of the partition and
replication functions of these host proteins.
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FIG. 2.
Lack of effect of excess DnaA or DnaB on amount of
par deletion plasmid DNA. Cultures of PM191 with the
par deletion pSC101 plasmid, pPM24, and another plasmid were
grown with selection for both plasmids. Equal amounts of cells were
harvested at an OD of 0.5, lysed rapidly, and run on an agarose gel
(5). Lanes 1 and 2, pPM24 and pBR322; lanes 3 and 4, pPM24
and pCM707 (DnaA); lanes 5 and 6, pPM24 and pMJRDnaB. The arrow
indicates the pPM24 plasmid DNA band; the pBR-based plasmid is the
larger plasmid (higher band) in each lane. Densitometry tracing of the
image indicated less pPM24 plasmid DNA in cells with excess DnaA (66%)
or excess DnaB (44%) than in the cells with the pBR322 control.
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|
Domain specificity of host proteins in the stabilization of
pSC101.
Overexpression of any of the dnaB(Ts) alleles
described above, when cloned on pUC-based vectors, failed to stabilize
par deletion pSC101 plasmids in wild-type hosts cultured at
either 30°C (data not shown) or 37°C (Table
5), consistent with our evidence (Table 2) that dnaB mutant proteins fail to support normal plasmid
partitioning. In contrast, some of the mutant DnaA proteins stabilized
plasmid inheritance when overexpressed, as does the wild-type DnaA
protein (20). DnaA protein mutated in the aggregation domain
(A31T [43]), RepA binding domain (G287S
[43]), DNA binding domain (L447W [43]), or DnaB binding domain (plasmid pACHA200
[42]) all stabilized partition-defective pSC101
plasmids when overexpressed in wild-type bacteria (Fig.
3A and Table 5). However, overexpression of a DnaA mutant protein containing an in-frame deletion (D237-378 [43]) that removes both the RepA binding domain and
cell membrane binding domain failed to stabilize even though cells
carrying this mutant allele can support replication of pSC101
(42) (Fig. 3A and Table 5).
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TABLE 5.
Effects of overproduction of host mutant replication
proteins on the stability of pSC101 par deletion plasmids
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FIG. 3.
The host replication genes DnaA and DnaB (marked to
indicate functional domains) and the mutations used in this study. (A)
Domains of DnaA (41). The mutant amino acid are indicated
above the line; deletion D237-378 does not support oriC
replication, while mutants 31, 287, and 477 do not support pSC101
replication. (B) Domains of DnaB and locations of the
temperature-sensitive alleles (36).
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|
As the above observations suggested a possible role for the membrane
binding domain of DnaA (included in the deletion D237-378)
in plasmid
partitioning, we tested the effects on pSC101 stability
of a DnaA
protein containing mutations in amino acids specifically
implicated in
membrane binding (R360E, R364E, and K372E [
16]).
Hase
et al. have shown that a DnaA protein containing these three
mutations
cannot interact with cardiolipin despite its normal
ATPase activity
(
16). As seen in Table
5, overexpression of
the mutant
protein failed to stabilize partition-defective pSC101
replicons, thus
localizing the DnaA region required for stabilization
of pSC101
specifically to the membrane binding domain. Data showing
the locations
and properties of the DnaA mutants used in these
experiments are
summarized in Fig.
3A.
| |
DISCUSSION |
Plasmid pSC101 normally is stably maintained without selection in
cultures of dividing cells. This stability is mediated by the plasmid
par locus (28). In the presence of
par, pSC101-based plasmids can be equipartitioned, even when
their replication is shut off by loss of plasmid-encoded RepA function
(45), implying that the partition and replication functions
of RepA are distinct. The investigations reported here show that the
E. coli replication proteins DnaA and DnaB also have effects
on pSC101 plasmid partitioning and that these effects are separate from
their functions in DNA replication.
Wild-type pSC101 plasmids were unstable in dnaB(Ts) mutant
strains grown at a temperature permissive for DNA replication (30°C), whereas temperature-sensitive mutations in other essential DNA replication proteins (dnaA, dnaC,
dnaG, and dnaN) failed to affect plasmid
stability at any temperature where the proteins can function for
bacterial DNA replication. At a temperature nonpermissive for plasmid
or chromosomal DNA replication, the entire intracellular pool of pSC101
replicons segregated as a unit in the dnaB slow-stop mutant
strain RS162 (dnaB252), while the plasmids were
equipartitioned in the two other dnaB mutants (the fast-stop
alleles in PC8 [dnaB8] and HfrH165 [dnaB70])
and in bacteria mutated in all other host replication genes tested.
These results suggest a special role in plasmid partitioning for the
region of the DnaB protein mutated in the dnaB252 strain.
This region of the protein is conserved in DnaB homologues, but its
function remains unknown. Interestingly, the two other dnaB
alleles whose inactivation did not prevent equipartitioning show
temperature-sensitive biochemical activities (in helicase, nucleotide
binding, and primase activities) in in vitro assays, whereas the
dnaB252 strain has wild-type DnaB activity in vitro at
elevated temperatures (36).
The DnaB protein, when overproduced, can stabilize unstable pSC101
replicons, as was shown previously for DnaA (20);
stabilization occurred without observable effects on plasmid copy
number, consistent with earlier evidence that changes in pSC101 copy
number are independent of stability (13, 45) and further
supporting the notion that the effects of these host proteins on
replication and partitioning are distinct. Both DnaA and DnaB have been
shown to interact with each other as well as with the pSC101 RepA
protein in vitro (24, 33). However, overproduction of other
host replication proteins that also have been reported to interact with
RepA, i.e., DnaG (25) and the tau subunit of PolIII
holoenzyme (31), had no effect on pSC101 stability, implying
that interaction with RepA per se is not the basis for the observed
effects of DnaA and DnaB on plasmid partitioning.
It previously has been shown that chromosomal DNA replication and
plasmid replication require different domains of the E. coli
DnaA protein (24, 43). Overexpression of DnaA proteins in
wild-type cells indicated that the DnaA region involved in pSC101
partitioning is different from the ones mediating its replication: DNA
mutants that did not support replication of pSC101-stabilized par deletion pSC101 derivatives. Conversely, a DnaA allele
(D237-378 [43]) that is mutated in the region of the
DnaA protein that has been shown to interact with the bacterial
membrane (15) supported pSC101 replication but failed to
stabilize par deletion plasmids when overexpressed. Still
other results that we obtained indicate that a DnaA variant containing
multiple membrane binding domain mutations that eliminate the
interaction of DnaA with cardiolipin (16) lost the ability
to stabilize par deletion pSC101 plasmids when
overexpressed, thus suggesting that the membrane binding domain of DnaA
is the specific region of the protein required for partitioning of
pSC101. Together, these findings establish that the replication and
partition activities of DnaA on pSC101 are physically as well as
functionally separated. Membrane binding by pSC101 plasmid molecules
(39) has been observed to correlate with the stability of
pSC101 plasmids (our unpublished data), raising the possibility that
DnaA can serve as the bridge between the plasmid and host membrane
during partition.
| |
ACKNOWLEDGMENTS |
We thank Hanne Ingmer for stimulating discussions; J. Kaguni, D. Bastia, D. Bramhill, N. Grindley, N. Dixon, R. Diaz, T. Mizushima, and
M. Berlyn at CGSC for supplying plasmids and strains; D. Bramhill for
the DnaB antibody; and K. Marians for the DnaG antibody.
This study was supported by NIH grants GM26355 and AI 08619 to S.N.C.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Genetics, Stanford University School of Medicine, Stanford, CA
94305-5120. Phone: (650) 723-5315. Fax: (650) 725-1536. E-mail:
sncohen{at}stanford.edu.
| |
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Journal of Bacteriology, December 1999, p. 7552-7557, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
