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Journal of Bacteriology, April 1999, p. 2124-2131, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Stabilization of the Relaxosome and Stimulation of
Conjugal Transfer Are Genetically Distinct Functions of the R1162
Protein MobB
Tariq
Perwez and
Richard J.
Meyer*
Department of Microbiology and Institute for
Cellular and Molecular Biology, University of Texas, Austin, Texas
78712
Received 28 October 1998/Accepted 19 January 1999
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ABSTRACT |
MobB is a small protein encoded by the broad-host-range plasmid
R1162 and required for efficient mobilization of its DNA during conjugation. The protein was shown previously to stabilize the relaxosome, the complex of plasmid DNA and mobilization proteins at the
origin of transfer (oriT). We have generated in-frame
mobB deletions that specifically inactivate the stabilizing
effect of MobB while still allowing a high rate of transfer. Thus, MobB has two genetically distinct functions in transfer. The effect of
another deletion, extending into mobA, indicates that both functions require a specific region of MobA protein that is distinct from the nicking-ligating domain. The mobB mutations that
specifically affected stability also resulted in poor growth of cells,
due to increased transcription from the promoters adjacent to
oriT. The effects of the mutations could be suppressed not
only by full-length MobB provided in trans, as expected,
but also by additional copies of oriT, cloned in pBR322. In
addition, in the presence of MobA both the full-length and truncated
forms of MobB stimulated recombination between
oriT-containing plasmids. We propose a model in which MobB
regulates expression of plasmid genes by altering the stability of the
relaxosome, in a manner that involves the coupling of plasmid molecules.
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INTRODUCTION |
Proteins required to process plasmid
DNA for conjugal transfer assemble at a unique locus, the origin of
transfer (oriT), to form a complex called the relaxosome.
For the broad-host-range plasmid R1162, and the nearly identical
RSF1010, there are three mobilization (mob) genes (Fig.
1) (4, 7), each encoding a
protein important for the activity of the relaxosome. The largest and
best characterized of these proteins, MobA, locally disrupts the
helical structure of the oriT DNA in the relaxosome and then cleaves one of the strands (31). MobC, a second component of the relaxosome, enhances strand separation at the site of cleavage (32). The linear strand, with MobA covalently attached to
the 5' end (1, 28), is probably unwound from its complement
and inserted into a recipient cell in the 5'-to-3' direction
(14). During a late stage in transfer, MobA rejoins the ends
of this strand to regenerate a circular molecule.
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FIG. 1.
Organization of genes for mobilization and of
oriT in plasmid R1162 and locations of different in-frame
deletions. The genes mobA and mobB overlap in
separate reading frames. The carboxy-terminal domain of mobA
is termed repB' and encodes a primase that is also
translated separately (29). Initiation and termination sites
for translation are indicated by open triangles and rectangles,
respectively. The locations of the promoters p1 to p3 (29)
are shown, with the direction of transcription from each indicated by
the arrowheads. The base pair coordinates are distances from the unique
EcoRI site in R1162.
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Reconstitution experiments in vitro have indicated that the third
mobilization protein, MobB, stimulates nicking of oriT DNA in the relaxosome (28). In agreement with this, we have
found that in vivo MobB stabilizes the assembly of MobA and MobC at oriT (23). This stabilization is shown by the
greater proportion of oriT DNA sensitive to oxidation by
permanganate, due to strand separation of this DNA within the
relaxosome (31). In the absence of MobB, the frequency of
mobilization of R1162 decreases 2 to 3 orders of magnitude. The higher
transfer frequency in the presence of MobB could simply reflect
stabilization of the relaxosome by the protein. We isolated mutations
that resulted in unstable relaxosomes but which nevertheless permitted
a high level of transfer (23). Although there were fewer
complexed molecules, each appeared to be more active in nicking, which
could account for the high transfer frequency. However, none of the
mutations eliminated the requirement for MobB in transfer. Since
mutations could be easily found that compensated for unstable
relaxosomes but did not relieve the requirement for MobB, it seemed
unlikely that this requirement could be explained solely by the
stabilizing effect of the protein.
We have isolated and characterized a set of R1162 derivatives
containing in-frame deletions in mobB and overlapping
mobA (Fig. 1). The properties of these mutations indicate
that stabilization of the relaxosome by MobB is genetically separable
from a second role of this protein in transfer and also that a distinct
region of MobA is required for MobB activity. In addition, the
deletions have revealed that stabilization of the relaxosome can be
enhanced by additional copies of oriT in the cell. We
propose a model in which coupling of plasmid molecules at
oriT, brought about by MobA and MobB, stabilizes the relaxosome.
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MATERIALS AND METHODS |
Strains and plasmids.
The Escherichia coli K-12
strains used were MV10 (C600 
trpE5) (11) and
JW151 (thi endA polA1 T3s), obtained from I. Molineux. The recipient strain in mating experiments was DF1019, a C600
derivative resistant to nalidixic acid (8). M13 derivatives
were propagated in RV lacX42 (F'lac)
(24), and isolated plaques formed on lawns of JM103
(19).
To construct deletion derivatives of R1162, a CG-to-GC mutation was
first introduced in
mobB at bp 4468 (Fig.
1) by
oligonucleotide-directed
mutagenesis (
15). The mutation
created an
Acc65I site and caused
a substitution of proline
for alanine in MobB. A set of plasmids
(Fig.
1) containing deletions
that extend from the site of this
mutation were constructed by first
filling in the
Acc65 site with
Klenow fragment to create a
unique
SnaBI site. The
XhoI-
StuI-
XhoI
linker
CTCGAGGCCTCGAG was introduced at the
SnaBI site,
and deletion
derivatives were generated by digestion with
StuI and partial
digestion with
HaeIII. Only
deletions rightward, as shown in Fig.
1, were obtained by this
procedure, because the first
HaeIII site
leftward was in a
region encoding a primase essential for plasmid
replication
(
26). The correct reading frame in the resulting
plasmids,
pUT1529, pUT1530, pUT1531, and pUT1532, was restored
by filling in the
remaining
XhoI site, which also creates a
PvuI
site. The plasmid pUT1562 was constructed by deletion of DNA from
this
site to a second
PvuI site at bp 4767, created by
oligonucleotide
mutagenesis. Plasmid pUT1533 is identical to pUT1530
except that
it contains a 48-bp deletion that includes all of the
oriT DNA
but not the adjacent promoters. The plasmid was
constructed by
exchanging a
Bst1107I/
EcoO109
fragment from another R1162 derivative
containing the deletion
(
23).
The derivation of pUT530 (
5), pUT1371, pUT1376, and pUT221
(
23) has been described elsewhere. The plasmid pUT1585 was
constructed by joining pUT221 and pUT1530 at their unique
AflIII
sites within R1162 DNA and then screening for
spontaneous, second-site
recombinants to regenerate a plasmid identical
to pUT221 but with
the
mobB deletion of pUT1530. The plasmid
pUT1440 consists of
an 802-bp
HpaII-
EcoO109
fragment of R1162 DNA (coordinates, 5135
to 5936 [Fig.
1]) cloned by
replacement of the small
ClaI-
EcoRV
fragment of
pBR322 (
3).
The plasmid used for measurement of intracellular amounts of specific
mRNAs was constructed by first cloning a 2,667-bp
ScaI-
EcoO109
fragment, containing the entire
mob region from R1162, into the
EcoRV site of
pBR322. The fragment and adjacent DNA were then
excised by digestion
with
SalI and
EcoRI and cloned into pLG339
(
30) by replacement of the small
SalI-
EcoRI fragment. The deletion
from pUT1530
was introduced by fragment exchange following digestion
with
BlpI and
Bst1107I.
Assaying recombination between plasmids.
MV10 cells
containing R1162 or a derivative (see Fig. 6) were transformed with
pUT1440, and colonies of cells resistant to ampicillin and streptomycin
were obtained by plating. Three unrelated colonies were separately
inoculated in 5 ml of broth medium containing antibiotics, and the
culture was grown overnight to stationary phase. Plasmid DNA, isolated
by the Qiagen miniprep procedure, was used to transform JW151.
Transformants were selected by plating them on medium containing
streptomycin and, separately, on medium containing ampicillin. The
number of colonies in each case was determined after incubation at
37°C for approximately 48 h.
Determining relative amounts of specific mRNAs in cells.
Relative amounts of specific mRNAs were determined by hybridization of
total RNA, immobilized on a nitrocellulose membrane, with radiolabeled
DNA probes. Total cellular RNA from 10 ml of log-phase cells was
extracted by a commercially available procedure (Rneasy; Qiagen), and 3 to 5 µg of this preparation was dissolved in 25 mM MgCl2
and digested at 37°C for 1 h with 10 U of DNase I (Sigma). Half
of this sample was stored frozen at 
70°C; the other half was
further digested with 1 µg of DNase-free RNase A under the same
conditions. Serial dilutions of the samples were then applied to a
nitrocellulose membrane (BA85; Schleicher and Schuell) prepared
according to the method of Sambrook et al. (25) in a slot
blot apparatus.
Double-stranded, radiolabeled DNA probes were prepared by PCR in a
reaction mixture (100 µl) that contained 100 to 200 ng
of plasmid
DNA; 2 mM MgSO
4; a 0.2 µM concentration of each primer;
dGTP, dCTP, and dTTP (50 µM each); 45 µM dATP; 10 µl of
[
-
32P]dATP (3,000 Ci/mmol; 10 µCi/µl); and 1 U of
Vent DNA polymerase
(New England Biolabs). Unincorporated nucleotides
were removed
with a spin column (Qiagen). The specific activity of the
product
was approximately 10
8 cpm per µg. About 0.5 µg
of the probe DNA was diluted in 150
µl of H
2O, heated to
100°C for 15 min, and then chilled on ice
and added to 15 to 20 ml of
hybridization buffer (
25) containing
the immersed
nitrocellulose membrane. After incubation at 42°C
for 16 to 20 h, the membrane was washed once at room temperature
with 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium
dodecyl sulfate (SDS)
and then three times with 0.2× SSC-0.1%
SDS at 68°C. The membrane
was dried at room temperature. The amounts
of hybridized probe were
quantified with a phosphorimager; the
hybridized DNA was also
visualized by
autoradiography.
Other procedures.
Bacteria were mated on semisolid medium by
a standard procedure (4). The recombination frequency of
M13mp9 derivatives containing two directly repeated copies of
oriT was determined as previously described (21).
DNA in whole cells and cleared lysates (12) was treated with
permanganate as described by Zhang and Meyer (31) and Perwez
and Meyer (23). DNA prepared from cleared lysates was also
used to assay site-specific nicking at oriT by primer
extension (23). In this assay, half of the DNA sample was
digested with BsmAI, which cleaves the plasmid DNA between
oriT and the priming site. The other half was digested with
BstZ17, which cleaves distal to oriT in the
direction of priming. The samples were then mixed, and the DNA was
denatured and annealed to 32P-, end-labeled primer. Strand
extension was carried out by thermal cycling with Taq DNA
polymerase, as previously described (23), and the sample was
applied to a 0.35-mm, 8% polyacrylamide gel. The relative amounts of
DNA in the bands after electrophoresis were determined with a
phosphorimager. The fraction of nicked molecules was taken as the
amount of DNA due to termination at the oriT nick site
divided by the amount of DNA resulting from termination at the
BsmAI cleavage site.
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RESULTS |
Two functions of MobB in conjugal mobilization can be distinguished
by mutation.
The R1162 derivative pUT1371 (Fig. 1) contains a
162-bp, in-frame deletion in mobB, a mutation that
inactivates the gene and causes at least a 100-fold decrease in the
mobilization frequency of the plasmid (23) (Table
1). The mutation is complemented when
MobB, encoded by the plasmid pUT221 (23), is provided in trans (Table 1). This indicates that the mutation only
affects mobB and not the overlapping gene, mobA
(Fig. 1).
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TABLE 1.
Effect of internal in-frame deletions in mobA
and mobB on plasmid mobilization frequency and on generation
time of the host
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We generated additional in-frame
mobB deletions, starting at
bp 4475, near the middle of the gene and extending toward the
N-terminal coding end (Fig.
1). Three of the resulting plasmids,
pUT1529, pUT1530, and pUT1531, contain deletions of 12, 120, and
141 bp, respectively, that are entirely within
mobB. These
deletions,
as well as the one in pUT1371, were all complemented to the
same
level by pUT221 (Table
1). However, in the absence of
complementation,
pUT1529, pUT1530, and pUT1531 were each mobilized at a
substantially
higher frequency than pUT1371. This higher rate of
transfer was
observed even for pUT1531, the plasmid that contained a
deletion
removing about one-third of
mobB.
The mutation in pUT1371 results not only in a low rate of transfer but
also in relaxosomes that are unstable. There is a smaller
proportion of
nicked molecules in the cell and, as a consequence,
an increased yield
of supercoiled DNA compared to those with R1162
following alkaline
extraction (
23) (Fig.
2). Like
the low frequency
of transfer, this instability was also reversed by
providing MobB
in
trans (Fig.
2). In the same assay, plasmid
DNA yields for pUT1529,
pUT1530, and pUT1531 were also greater than the
yield of R1162,
indicating that the relaxosomes of these plasmids were
likewise
unstable (Fig.
2), and again, the yield was reduced when
full-length
MobB was present in the cell.
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FIG. 2.
DNA yields following alkaline extraction (17)
for R1162 and derivatives containing deletions in mobB. The
cells also contained pACYC184 (6) or pUT221, a pACYC184
derivative containing mobB (23). Plasmid DNAs
were linearized by digestion with EcoRI and displayed by
0.8% agarose gel electrophoresis.
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If the effect of MobB on transfer is solely through stabilizing the
relaxosome, then the different
mobB deletions should have
approximately commensurate effects on the proportion of molecules
nicked at
oriT and on the frequency of mobilization. We
measured
the fraction of molecules nicked at
oriT for
pUT1371, pUT1530,
and R1162. Plasmid DNA in cleared lysates
(
12) was treated with
SDS and phenol to disrupt the
relaxosome, and the proportion of
molecules specifically nicked within
oriT was determined by primer
extension and measurement of
radioactivity in DNA bands after
gel electrophoresis (Fig.
3A). The proportion of nicked molecules
was 0.23 for R1162; this decreased to 0.14 for pUT1530 and 0.12
for
pUT1371 (Fig.
3B). Thus, although the deletions in pUT1530
and pUT1371
affected the frequency of plasmid mobilization to
different extents
(Table
1 and Fig.
3B), they had similar effects
on nicking. Moreover,
the decrease in transfer frequency and the
proportion of nicked
molecules were similar for pUT1530, but transfer
of pUT1371 was lower
than could be accounted for by the level
of nicked molecules. We
conclude that mutations in
mobB can differentially
affect
the frequency of transfer and the stability of the relaxosome.
Mutations within the N-terminal half of MobB destabilize the
relaxosome,
and this probably accounts in large part for the small but
detectable
decrease in the transfer frequency of these plasmids (Table
1).
The C-terminal region of MobB, conserved in these deletions and
inactivated in pUT1371, is important for transfer in a second
way,
unconnected with relaxosome stability.
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FIG. 3.
(A) Proportion of plasmid DNA molecules nicked at
oriT for R1162, pUT1371, and pUT1530. The bands on the
polyacrylamide gel reflect primer extension with termination at the
nick site of oriT (nic) and on an equal amount of template
with termination at a BsmAI site proximal to the primer. (B)
Relative transfer frequency (obtained from Table 1) and relative
fraction of nicked molecules (by quantitative analysis of the bands in
the gel), with values set at 1.0 for R1162.
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A region of mobA is required for activity of MobB.
We also isolated and characterized two plasmids that contained in-frame
deletions extending further from bp 4475, across the beginning of
mobB and into mobA (pUT1532 and pUT1562 [Fig.
1]). Because DNA required for initiation of translation of
mobB was deleted from these plasmids, we did not expect them
to be mobilized at high frequency by R751. The mobilization frequency
of pUT1562 was low and similar to that of pUT1371, and conjugal
transfer of pUT1532 was not detected (Table 1). However, unlike the
plasmids having deletions entirely within mobB, pUT1532 and
pUT1562 could not be complemented for transfer by MobB in
trans (Table 1). In addition, the high yield of pUT1562 and
pUT1532 DNA following alkaline extraction was unaffected when MobB was
in the cell (Fig. 2).
The MobA proteins encoded by pUT1532 and pUT1562 could simply fail to
bind to
oriT DNA. To test this possibility, we took
advantage of the facts that this binding locally disrupts the
DNA
duplex and the resulting unpaired pyrimidines can be detected
by their
increased sensitivity to permanganate (
31). Strand
separation still occurs in the absence of MobB, although because
MobB
stabilizes the relaxosomes, sensitivity to permanganate is
enhanced
when this protein is present in the cell (
23). We compared
the permanganate sensitivity of the
oriT DNA in the
relaxosomes
of pUT1530, pUT1562, and pUT1532. In each case, a cleared
lysate
of plasmid-containing cells was prepared and exposed to
permanganate,
and the oxidized bases on the negative strand, the one
not transferred,
were mapped by primer extension. As reported elsewhere
for R1162
(
31), three adjacent thymine residues in the
oriT DNA of pUT1530
were sensitive to permanganate, and this
sensitivity was enhanced
by MobB (Fig.
4A, lanes a and b). The sensitive bases
are located
at bp 25, 26, and 27 in the
oriT base sequence
shown at the top
of the figure. These bases were unreactive in the
oriT of pUT1532
(Fig.
4A, lanes e and f), indicating that
the MobA encoded by
this plasmid binds poorly to
oriT DNA or
is unstable, thus accounting
for the low frequency of mobilization of
the plasmid. In contrast,
although pUT1562 was mobilized poorly, the
bases in the
oriT DNA
of pUT1562 were still sensitive to
oxidation (Fig.
4A, lanes c
and d). The sensitivity was about half that
detected in the
oriT DNA of pUT1530. However, unlike the
relaxosome of pUT1530 (or
pUT1371 [
23]), permanganate
sensitivity was not increased when
MobB was present in the lysate (Fig.
4A, lane d). Thus, for both
stabilization of the relaxosome and
mobilization of the plasmid,
the region of MobA affected by the
deletion in pUT1562 is required
for MobB to be active.
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FIG. 4.
(A) Permanganate-sensitive bases on the unnicked
oriT DNA strand for pUT1530 (lanes a and b), pUT1562 (lanes
c and d), and pUT1532 (lanes e and f). The locations of the bases were
determined from a sequencing ladder (not shown) generated with the same
template and primer. Plasmid DNAs in cleared lysates were treated with
permanganate prior to primer extension by PCR (31, 32). The
cells also contained either pACYC184 (lanes a, c, and e) or pUT221
(lanes b, d, and f). (B) Permanganate sensitivity of the unnicked
oriT DNA strand for pUT1530 isolated from cells also
containing pBR322 (lane c) or pUT530 (lane d). Whole cells were exposed
to permanganate prior to extraction of the DNA (31). For
comparison, the permanganate sensitivity of pUT1530 DNA isolated from
cells treated under identical conditions but containing pACYC184 (lane
a) or pUT221 (lane b) is also shown. The base sequence of
oriT is given at the top of the figure; the
permanganate-sensitive bases are identified by asterisks. The inverted
repeat (opposing arrows), the site cleaved by MobA in the top
(transferred) strand, and the direction of transfer are also
indicated.
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MobA not only interacts with duplex DNA in the relaxosome but also
binds the single
oriT DNA strand normally transferred during
conjugation and can both cleave this DNA at the site nicked in
the
relaxosome and ligate the ends (
1,
27). This property
is
reflected in vivo by site-specific recombination between
oriTs
cloned in M13 single-stranded DNA, when MobA is
provided in the
cell (
21). The reaction can be monitored by
infecting plasmid-containing
cells with M13mp9 phages (
20)
having two directly repeated copies
of
oriT cloned in the
lacZ(
) cloning region. These phages form
white
plaques on medium containing
isopropyl-
-
D-thiogalactopyranoside
(IPTG) and
5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside (X-Gal).
After recombination between the two
oriTs, the phages form
blue
plaques, due to translation through the remaining
oriT
and production
of an altered but active
-complementing fragment. We
tested pUT1562,
as well as pUT1531, for the ability to support
oriT recombination
in M13. In both cases, the recombination
frequency of the M13
derivative with two directly repeated
oriTs was 4% in cells of
RV and above the background level
of 0.3% in Rec
+ cells. These frequencies were unchanged
when MobB was present
in the cell. Thus, the activity of MobA on
single-stranded DNA
was unaffected by either MobB or the region of MobA
needed for
the activity of this
protein.
Unstable relaxosomes cause long generation times.
The
generation time of MV10 (R1162) cells in broth was approximately 35 min
(Table 1). However, cells containing R1162 derivatives with deletions
in mobB had substantially longer generation times (Table 1)
and formed long filaments (not shown). This was true for pUT1371, in
which mobB is completely inactivated, and also for pUT1529,
pUT1530, and pUT1531, which are still mobilized at high frequency. For
each of these plasmids, MobB in trans restored the
characteristics of normal growth (Table 1). The plasmids pUT1562 and
pUT1532 also caused long generation times, but in these cases the
effect was not complemented by MobB (Table 1). These observations
suggested that it was the unstable relaxosomes, resulting from the
deletion of either the N-terminal region of MobB or the region of MobA
required for MobB activity, that caused the poor growth.
MobA and MobC proteins act together as repressors at
oriT
(
9) and regulate the activity of the adjacent promoters p1,
p2,
and p3 (
29) (Fig.
1). Destabilization of the relaxosome
probably
decreases the level of repression, resulting in greater
synthesis
of plasmid proteins, which in turn stresses the cell and
causes
slower growth. Several other observations are consistent with
this interpretation. The plasmid pUT1533 is identical to pUT1530
but
contains a deletion that removes
oriT but not the adjacent
promoters. In contrast to pUT1530, this plasmid resulted in poor
growth, whether or not pUT221 was also present (Table
1). Thus,
MobB
cannot be acting simply as an antidote by binding to MobA
and reducing
the toxicity of this or some other protein. In addition,
oriT must be present in its normal location. The plasmid
pUT1376
contains the
mobB deletion present in pUT1371, but
oriT is at
a new position distant from the promoters p1 to
p3. Although the
oriT in this plasmid was active in
mobilization, the cells had
a long generation time, whether or not
pUT221 was present (Table
1). Finally, point mutations restoring good
growth were isolated
by serially culturing cells containing pUT1530 in
broth. Four
independent point mutations were identified, and in each
case
these mapped in the promoters adjacent to
oriT.
Derepression of transcription by the
mobB mutation in
pUT1530 was confirmed by hybridization of radiolabeled probes to total
cellular RNA. In order to avoid changes in copy number due to
changes
in transcription from the promoters adjacent to
oriT,
the
mob DNA from pUT1530 was first cloned into the vector pLG339
(
30), which has a replicon derived from pSC101.
Transcription
was measured in cells containing the resulting plasmid
(pUT1596
[Fig.
5]) and also either
pUT221 (MobB
+) or pACYC184 (no MobB present). Two
radiolabeled probes were
used, one hybridizing to part of the
mobA transcript initiated
from p1 and p3, and thus regulated
at
oriT, and another, as an
internal control, hybridizing to
transcripts of the kanamycin
resistance (
kan) gene of the
vector (Fig.
5). The results (Fig.
5A and B) show that MobB in the cell
did not affect transcription
of the
kan gene but reduced the
amount of
mobA transcript in the
cell. We conclude that when
MobB stabilizes the relaxosome, it
also increases repression of
transcription at
oriT.
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FIG. 5.
Hybridization of radiolabeled DNA probes to RNA
immobilized on a nitrocellulose membrane. An undiluted, DNase I-treated
sample and 1:10, 1:20, and 1:40 dilutions were applied in each column
of slots. In the bottom slot of each column, three times the amount of
undiluted sample was digested with RNase A prior to binding to the
membrane. At the top is the reporter plasmid with the approximate
locations of the probes for the kan and mobA
transcripts.
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Multiple copies of oriT in trans suppress
poor cell growth and stabilize relaxosomes in strains containing
certain defective MobB proteins.
The plasmid pUT530 (5)
consists of a copy of the R1162 oriT cloned into the vector
pBR322. When this plasmid was introduced into cells containing pUT1529
or pUT530, the resulting strains became healthy and grew with short
generation times (Table 2). This effect
depended on the cloned oriT in pUT530, because suppression by pBR322 was not observed (data not shown). Thus, copies of
oriT in trans suppressed the poor growth
phenotype caused by the mobB deletion in pUT1530. A copy of
oriT in pUT1530 was also required, since the poor growth of
cells containing pUT1533, a derivative of pUT1530 lacking
oriT, was unaffected by pUT530 (Table 2).
Plasmid pUT530 did not suppress the poor growth of cells containing
pUT1371, in contrast to those containing pUT1529 and pUT1530
(Table
2).
This could mean that suppression required MobB and
that the necessary
activity was conserved on the MobB fragments
encoded by pUT1529 and
pUT1530 but not pUT1371. To test this,
we constructed the plasmid
pUT1585, analogous to pUT221 but with
the
mobB deletion of
pUT1530. When cells contained this plasmid
as well as pUT1371, the
introduction of pUT530 resulted in healthy
cells (Table
2). There was
no suppression when the cells contained
the parental vector pACYC184
instead of pUT1585 (Table
2). In
contrast, pUT530 was nonsuppressing
when introduced into cells
that contained pUT1585, but with pUT1562
instead of pUT1371 (Table
2). Thus, suppression required the region of
MobB conserved in
pUT1530 (and pUT1529) and also the domain of MobA
required for
MobB
activity.
Cells containing pUT1530 grow poorly because the R1162 promoters
adjacent to
oriT are partially derepressed; the cells become
healthy when full repression is restored by providing MobB in
trans to stabilize the relaxosomes. Do copies of
oriT in
trans suppress poor growth by a similar
mechanism? We asked first whether
pUT530, like MobB, increased the
permanganate sensitivity of the
oriT DNA in pUT1530. In
order to minimize disruption of any macromolecular
complexes in the
cytoplasm, we treated whole cells rather than
cleared lysates with
permanganate (
31). Sensitive bases on the
nonnicked strand
were again identified by primer extension. As
before, full-length MobB,
encoded by pUT221, enhanced strand separation
within
oriT
(Fig.
4B, lanes a and b). In addition, we found that
copies of
oriT cloned in pUT530 likewise increased the permanganate
sensitivity of the
oriT DNA of pUT1530 (Fig.
4B, lanes c and
d).
Extra copies of
oriT also repressed transcription initiated
from the promoters adjacent to
oriT. As before with MobB, we
measured
the relative amounts of the
kan and
mobA
transcripts for plasmid
pUT1596, but this time in cells containing
either extra copies
of
oriT (pUT530) or only the vector
pBR322. The results (Fig.
5C and D) indicate that although pUT530 had
no effect on the amount
of
kan transcript, it repressed
transcription of
mobA. Moreover,
the levels of repression,
determined by comparing the relative
amounts of bound radioactive probe
at the same dilution of RNA,
were similar for MobB and
oriT.
We estimate that the amount of
mobA transcript decreased
about 12-fold when MobB was present
and 5- to 6-fold in the presence of
the additional copies of
oriT.
The relaxosome is recombinogenic at oriT by a mechanism
that requires MobB.
Additional copies of oriT in the
cell can act in trans, in a way that depends on MobA and
MobB, to repress transcription from neighboring promoters. If
repression came about by a direct interaction between plasmid
molecules, then it might increase the frequency of their recombination.
To test this, we used the plasmid pUT1440, which is similar to pUT530
but contains a larger, 802-bp fragment of cloned R1162 mob
DNA. This DNA includes oriT and adjacent parts of
mobA and mobC. The plasmid was introduced by
transformation into MV10 cells containing R1162 or a derivative, and
the DNA was then extracted and used to transform JW151
(polA1) for resistance to ampicillin and, separately, for
resistance to streptomycin. Because the replicon of pUT1440 is inactive
in JW151, ampicillin-resistant colonies were primarily the result of
recombination that had taken place between pUT1440 and R1162 prior to
extraction of the DNA. The ratio of ampicillin-resistant transformants
to streptomycin-resistant transformants was thus a measure of the
recombination frequency in MV10.
The recombination frequency between pUT1440 and R1162 was 0.004, and
this frequency was little changed when pUT1530 instead
of R1162 was in
the cell (Fig.
6). In contrast,
recombination
between pUT1440 and either pUT1371 or pUT1562, plasmids
which
do not encode an active MobB, was undetectable under our assay
conditions. In addition, no recombination was observed when
oriT was deleted from pUT1530 (pUT1533 [Fig.
6]). These
results suggested
that MobB promotes an interaction between plasmid DNA
molecules
at
oriT and that this interaction influences the
rate of recombination.
However, it was also possible that a greater
overall level of
nicking in the relaxosomes of R1162 and pUT1530 was
resulting
in a substrate more favorable for recombination. The plasmid
pUT1579
is identical to R1162 but contains a mutation causing a
substitution
of phenylalanine for tyrosine-24, the active nucleophile
in strand
cleavage (
27). The protein forms a normal complex
as assayed
by sensitivity of the DNA to permanganate, but nicking is
undetectable
in vivo (
33). Nevertheless, R1162 and pUT1579
recombined with
pUT1440 at essentially the same frequency (Fig.
6). We
conclude
that the relaxosome enhances plasmid recombination by a
mechanism
that does not depend on nicking at
oriT.
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|
FIG. 6.
Recombination frequency between pUT1440, a derivative of
pBR322 containing a cloned fragment of R1162 mob DNA that
includes oriT, and R1162 or a derivative. In each case, the
result is the average and standard deviation of three experiments.
|
|
| |
DISCUSSION |
We show here that two effects of MobB, stabilization of the
relaxosome and enhancement of the frequency of transfer, can be genetically distinguished by mutations within the gene. The
mobB deletions in pUT1529, pUT1530, and pUT1531 destabilize
the relaxosome but have only a small effect on the frequency of
mobilization (Table 1). These deletions map in the region of the gene
encoding the amino-terminal half of the protein.
Deletion of the region of mobA adjacent to mobB
results in a relaxosome that is no longer responsive to MobB, and as a
result mobilization decreases to the level observed when MobB is
effectively absent (pUT1562 [Table 1]). However, this deletion has a
smaller effect on the DNA-processing reactions carried out by MobA:
cleavage and ligation of single-stranded DNA, as monitored by phage
recombination, or the separation of DNA strands within the relaxosome.
We interpret these results to mean that the region of mobA
adjacent to mobB encodes a domain of the protein required
for recognition of MobB. When this region is absent, MobA becomes blind
to the presence of MobB, and the frequency of mobilization becomes
similar to that of pUT1371. R1162 and pSC101, a plasmid which is
unrelated to R1162 overall, have oriTs with very similar
base sequences (16). In addition, pSC101 encodes a
mobilization protein with tracts of amino acids identical to those in
MobA. The similarities between the two proteins map at the
amino-terminal end of MobA, throughout the DNA-binding region, but do
not extend into the domain required for recognition of MobB.
Interestingly, there is no protein encoded by pSC101 that can be
identified as MobB-like on the basis of sequence similarities. Thus,
MobB and a cognate site within MobA might represent a particular
adaptation within the IncQ plasmid group.
Relaxosomes stabilized by MobB cause maximal repression of
transcription from the promoters adjacent to oriT. When the
relaxosomes are unstable, partial derepression presumably leads to
cells that form filaments and grow with long generation times. Since
derepression affects the expression of not only the mob
genes but also the replication genes downstream from mobA
(9), it is not clear whether overexpression of a particular
gene or overall plasmid gene expression is the basis for the poor
growth. The nicking domain of MobA is not solely responsible, since a
deletion that eliminates this region does not restore normal growth
(pUT1532 [Table 1]). In any case, the effect of derepression is
probably amplified by an increase in plasmid copy number, due to the
increase in expression of the replication genes (10, 13).
The destabilizing effect of the mobB mutations in pUT1529
and pUT1530 could be overcome by additional copies of oriT
in trans. This suppression required the remaining fragment
of MobB as well as MobA protein with its recognition domain for MobB.
An increased frequency of recombination between
oriT-containing plasmid molecules showed identical
requirements. From these observations, we propose that MobA and MobB
link the oriTs on different plasmid molecules by means of a
protein bridge and that this structure is required for stable
relaxosomes and, consequently, for optimal repression of plasmid
promoters and healthy growth of cells (Fig.
7A). As a result, inactivation of MobB
(in the plasmid pUT1371) results in both a low level of transfer and
high levels of transcription, with the cells growing poorly (Fig. 7B).
The same effects are observed when the region of mobA
adjacent to mobB is deleted (Fig. 7C), because MobB is then
no longer able to recognize MobA. The mutations in pUT1529, pUT1530,
and pUT1531 partially impair the ability of MobB to stabilize the
relaxosomes, so that transcription is high and cells again grow poorly
(Fig. 7D). However, an increased dosage of oriT, provided by
pUT530, partially offsets this instability by driving the
oriT interaction toward the coupled complex, thereby decreasing gene repression (Fig. 7E).
View larger version (15K):
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|
FIG. 7.
Model describing the interactions of MobA and MobB with
oriT DNA and the effects of these interactions on transfer,
transcription and cell growth.
|
|
These molecular interactions proposed in Fig. 7 are reminiscent of the
coupling or "handcuffing" which is thought to be part of the
mechanism of replication control for plasmids RK2 and R6K and phage P1
(2, 18, 22). In these cases, excess iterons bind the
plasmid-specific initiation protein, freezing it at the origin of
replication and preventing a new round of replication. For R1162,
control of replication would be more indirect. Copy number is
determined by the level in the cell of the plasmid-specific replication
protein RepC (10, 13), and enhanced repression at
oriT reduces the overall level of transcription through the gene for this protein (9).
Although lacking the amino-terminal region, the MobB proteins encoded
by pUT1529, pUT1530, and pUT1531 are still very active for transfer.
MobB might also be required for the R1162 relaxosome to recognize the
conjugal apparatus of the mobilizing, self-transmissible plasmid. MobB
could act as a bridge, or it could modify the conformation of MobA to
better fit the conjugal machinery. Thus, MobB could mediate
interactions between relaxosomes and also between the relaxosome and a
docking site for mobilization. Experiments to obtain physical evidence
for these interactions are now under way.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the National Institutes
of Health (GM37462).
We thank S. R. Kushner for providing a strain containing pLG339.
| |
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, April 1999, p. 2124-2131, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
