Department of Microbiology and Institute for
Cellular and Molecular Biology, University of Texas, Austin, Texas
78712
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 MgSO4; 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 108 cpm per µg. About 0.5 µg
of the probe DNA was diluted in 150 µl of H2O, 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).
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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.
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Abstract
Introduction
