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Journal of Bacteriology, January 2000, p. 179-188, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Replicator of the Nopaline-Type Ti Plasmid pTiC58 Is a Member
of the repABC Family and Is Influenced by the TraR-Dependent
Quorum-Sensing Regulatory System
Pei-Li
Li1 and
Stephen K.
Farrand1,2,*
Departments of Crop
Sciences1 and
Microbiology,2 University of Illinois
at Urbana-Champaign, Urbana, Illinois 61801
Received 1 July 1999/Accepted 8 October 1999
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ABSTRACT |
The replicator (rep) of the nopaline-type Ti plasmid
pTiC58 is located adjacent to the trb operon of this
conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034-1041,
1985) identified functions involved in partitioning, origin of
replication and incompatibility, and copy number control. In this
study, we determined the nucleotide sequence of a 6,146-bp segment that
encompasses the rep locus of pTiC58. The region contained
four full open reading frames (ORFs) and one partial ORF. The first
three ORFs, oriented divergently from the traI-trb operon,
are closely related to the repA, repB, and
repC genes of the octopine-type Ti plasmid pTiB6S3 as well
as to other repA, -B, and -C genes
from the Ri plasmid pRiA4b and three large plasmids from
Rhizobium spp. The fourth ORF and the partial ORF are
similar to y4CG and y4CF, respectively, of the
Sym plasmid pNGR234a. The 363-bp intergenic region between traI and repA contained two copies of the
tra box which is the cis promoter recognition
site for TraR, the quorum-sensing activator of Ti plasmid conjugal
transfer. Expression of the traI-trb operon from the
tra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an
intact tra box III. On the other hand, the region
containing the two tra boxes was required for maximal
expression of repA, and this expression was enhanced
slightly by TraR and AAI. Copy number of a minimal rep
plasmid increased five- to sevenfold in strains expressing
traR but only when AAI also was provided. Consistent with
this effect, constitutive expression of the quorum-sensing system
resulted in an apparent increase in Ti plasmid copy number. We conclude
that Ti plasmid copy number is influenced by the quorum-sensing system,
suggesting a connection between conjugal transfer and vegetative
replication of these virulence elements.
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INTRODUCTION |
The Ti and Ri plasmids of
Agrobacterium spp. are primary pathogenicity determinants
and are responsible for crown gall or hairy root diseases caused by
these bacteria. These plasmids are large (
200 kb), unit copy, and
stably maintained in the bacterial cells. Molecular and genetic studies
of these plasmids have led to an understanding of functions associated
with tumorigenicity, opine catabolism, and conjugal transfer (reviewed
in references 12, 14, and 40).
However, other functions, most notably the vegetative replication of
these plasmids, have been studied in considerably less detail. The
rep region of pTiB6S3, a classical octopine-mannityl
opine-type Ti plasmid, has been mapped (25, 26), and the DNA
sequence has been determined (47). Similarly, the
rep region of pRiA4b, the Ri plasmid from
Agrobacterium rhizogenes A4, and the rep region
of an otherwise undescribed Ti plasmid, pTi-SAKURA, have been sequenced
(34, 35, 46).
Recent studies of several plasmids from Rhizobium spp. have
spurred interest in the replication regions of these large plasmids. These plasmids include pNGR234a, the Sym plasmid from
Rhizobium sp. strain NGR234 (15), pRL8JI, a
cryptic plasmid from Rhizobium leguminosarum 3841 (50), and p42d, the Sym plasmid from Rhizobium etli CFN42 (39). Comparative sequence analysis
indicates that the rep genes and their products, and the
organization of the genes on these plasmids, are similar and are
related to those of pTiB6S3, pRiA4b, and pTi-SAKURA. These replication
systems all contain three genes, repA, repB, and
repC, organized in tandem. Surveys based on the presence of
a repC homologue have indicated that this replicator may
exist in many other large rhizobial plasmids (6, 41, 49).
Furthermore, in addition to these agrobacterial and rhizobial plasmids,
the second rep region from pTAV1, a large cryptic plasmid
from the unrelated soil bacterium Paracoccus versutus UW1,
also belongs to this group (3).
The rep region of pTiC58 is located near the 2 o'clock
position on the plasmid and is adjacent to the trb operon,
the locus responsible for the mating-pair formation (Mpf) functions of
the Ti plasmid conjugal transfer system (17, 27, 28). In our sequence analysis of the Mpf region of pTiC58 we identified the 5' end
of an open reading frame (ORF) that is oriented divergently from
traI, the first gene of the trb operon
(27). This ORF, which is separated from traI by a
363-bp intergenic region, is almost identical to the repA
genes of pTi-SAKURA and the octopine-type Ti plasmid pTiB6S3.
Furthermore, the position of this ORF coincides with the par
locus of pTiC58 genetically defined by Gallie et al. (17)
(Fig. 1). These findings, coupled with
the facts that the nopaline- and octopine-type Ti plasmids are
incompatible (19) and that the rep loci of these
two Ti plasmids belong to the same heteroduplex region (13),
led us to predict that the rep region of pTiC58 belongs to
the repABC-type replicator family.
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FIG. 1.
Physicogenetic map of the rep region of
pTiC58 and structure of pH13Km. The phenotype designations
par, ori/inc, and cop are according to
Gallie et al. (17). The restriction map and gene
designations are based on nucleotide sequence analysis as described in
the text.
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In this study, we determined the nucleotide sequence of the region
responsible for vegetative replication of pTiC58. Comparison of the
sequence with the genetic map described by Gallie et al. (17) allowed us to assign functions to some of the genes and also to potential cis-acting regions. We also show that the
conserved cis-acting tra box elements residing in
the intergenic region between traI and repA can
affect expression of the traI-trb operon as well as
repA. Furthermore, we provide evidence for the coregulation of conjugal transfer and the copy number of the Ti plasmid by the
TraR-dependent quorum-sensing regulatory system.
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MATERIALS AND METHODS |
Bacterial strains, growth conditions, and plasmids.
The
Agrobacterium tumefaciens and Escherichia coli
strains and the plasmids used in this study are listed in Table
1. A. tumefaciens strains were
grown at 28°C in L broth (LB) (42), in ABM minimal medium
(9), or on nutrient agar plates (Difco Laboratories,
Detroit, Mich.). E. coli strains were grown at 37°C in LB
or on L agar plates. Antibiotics were added at the following concentrations when required: for Agrobacterium,
carbenicillin, 100 or 200 µg/ml; kanamycin, 100 µg/ml; and
tetracycline, 2 µg/ml; for E. coli, ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; and tetracycline, 10 µg/ml. X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside; Gibco-BRL, Gaithersburg, Md.) was included in media at 40 µg/ml to
assay for the production of -galactosidase.
DNA manipulation and strain constructions.
Plasmids in
E. coli and A. tumefaciens were isolated by an
alkaline lysis method (42, 45). Preparations for plasmid
copy number assessment were conducted as follows. A. tumefaciens strains containing the plasmid being tested were
inoculated into 2 ml of LB without antibiotics and incubated overnight
at 28°C with aeration. The cultures were used to reinoculate fresh
2-ml volumes of LB, and the optical density at 600 nm
(OD600) of each culture was adjusted to 0.1. The cultures
were incubated to late exponential phase (OD600 ~ 1), and cells from a 1-ml volume were harvested by centrifugation for
plasmid preparation. The viable titer of each culture was determined at
the time of harvest to ensure that all samples contained roughly the
same number of cells (ca. 109 cells). The cells were washed
with a 1-ml volume of Agrowash (18), and plasmids were
extracted by a modified alkaline lysis method as described previously
(18). Following lysis, all extractions, mixings, and other
manipulations were performed as gently as possible to minimize shearing.
Standard recombinant DNA techniques were used as described by Sambrook
et al. (
42). Digestions with restriction endonucleases
were
conducted according to the manufacturers' instructions. Plasmids
were
introduced into
E. coli by using standard techniques as
described
previously (
42). IncP-based plasmids were
introduced into
A. tumefaciens strains via
E. coli S17-1-mediated biparental mating
(
10). Other
plasmids, including pH13Km, were introduced into
A. tumefaciens strains by electroporation (
7).
Agarose gel analysis.
Immediately after preparation, closed
circular Ti plasmid DNA and HindIII-digested pH13Km were
electrophoresed in 0.8% (wt/vol) agarose gels at 4 V/cm for 3 to
6 h, using Tris-borate-EDTA buffer. To compare copy numbers among
the Ti plasmids or among samples containing pH13Km, relative
intensities of the appropriate bands on the agarose gel were analyzed
by densitometry as follows. After electrophoresis, the agarose gel was
stained in a solution containing ethidium bromide (5 µg/ml) for 30 min. The stained gel was rinsed with distilled water and exposed to UV
light, and the gel image was captured and digitized with a
charge-coupled device camera (Fotodyne, Heartland, Wis.). The bands on
the digitized gel image were analyzed using the Gel Plot macro of the
public domain NIH Image program (version 1.62; National Institutes of
Health, Bethesda, Md. [http://rsb.info.nih.gov/nih-image/]) on a
Macintosh computer.
DNA sequence analysis and bioinformatics.
DNA fragments were
sequenced on both strands by the dideoxy method as described previously
(27). Nucleotide sequences were assembled and analyzed, and
ORFs were identified and translated by using the DNA Strider program
(30) and the Map program of the Genetics Computer Group
(GCG) software package (version 10; GCG, Madison, Wis.). Nucleotide and
deduced amino acid sequences were compared to those in the databases by
using the BLAST2 search protocol (2). PileUp, Gap, and
BestFit programs of the GCG package were used to compare sequences and
to identify regions conserved among several nucleotide or protein
sequences. Unrooted phylogenetic trees of RepC and the repBC
intergenic sequence (igs) were constructed by using the
maximum parsimony algorithm with bootstrap method by the PAUPSearch
program of the GCG package.
-Galactosidase assay.
-Galactosidase activity was
determined qualitatively on ABM agar medium containing 40 µg of X-Gal
per ml. Quantitative assays were conducted as described previously
(20). Each sample was assayed in duplicate, and each
experiment was repeated at least twice. Levels of activity were
expressed as units of -galactosidase per 108 or
109 CFU.
Nucleotide sequence accession numbers.
The nucleotide
sequence of the entire rep region of pTiC58 was deposited in
the GenBank database under accession no. AF060155. The accession
numbers of rep genes from other plasmids are listed in Table
2.
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RESULTS |
DNA sequence analysis of the rep region of pTiC58.
The nucleotide sequences of both strands of the entire rep
region of pTiC58 as defined by Gallie et al. (17), including BamHI fragment 13 and EcoRI fragments 27 and 25 (Fig. 1), were determined. This yielded 6,146 bp of new sequence,
continuing from the BglII site located 155 bp upstream of
traI (27) to the EcoRI site at the
right end of EcoRI fragment 25 (Fig. 1). The nucleotide
sequence is 99.7% identical to that of a corresponding region of
pTi-SAKURA (46) and 78% identical to that of the
octopine-type Ti plasmid pTiB6S3 (47). The sequenced region
contains four significant ORFs and one partial ORF. The first three
ORFs, which are oriented opposite to those of the traI-trb
operon or clockwise on the Ti plasmid, are very similar at both
nucleotide and deduced amino acid sequence levels to the replication
genes repA, repB, and repC,
respectively, from pTi-SAKURA and pTiB6S3 (Table
2). On this basis, we designated these
three ORFs repA, repB, and repC. These
three genes also are similar to replicator genes from several other
plasmids from the family Rhizobiaceae, including pRiA4b from
A. rhizogenes A4 (35), pNGR234a, the Sym plasmid from Rhizobium sp. NGR234 (15), pRL8JI, a cryptic
plasmid from R. leguminosarum 3841 (50), and
another Sym plasmid, p42d from R. etli CFN42
(39). The three genes also are related to three genes found
in the second replicator region from pTAV1, a large cryptic plasmid
from P. versutus UW1 (3). The fourth ORF, which is oriented opposite to repABC (Fig. 1), showed similarities
to the y4CG gene from pNGR234a and to genes coding for DNA
invertases or resolvases from other bacteria, phages, or transposons
(Table 2). The partial ORF (Fig. 1) showed similarity to
y4CF (Table 2), a gene of unknown function also from
pNGR234a, but to no other sequences in the databanks. Whereas
repC is separated from y4CG by 8 bp in pTiC58,
these two genes are separated by about 3 kb in the corresponding region
of pNGR234a (15).
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TABLE 2.
Relatedness among the predicted gene products of the Ti
plasmid rep region and those of the other
repABC-type replicatorsa
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Influence of the traI-repA intergenic region on the
expression of traI.
Transcription of the traI-trb
operon is activated by TraR in a quorum-dependent manner
(21). The 363-bp intergenic sequence between traI
and repA, which presumably constitutes a divergent promoter
system, contains two copies of an 18-bp inverted repeat sequence called
the tra box (Fig. 2). This
element constitutes the cis-acting site recognized by TraR
and its coinducer, Agrobacterium autoinducer [AAI;
N-(3-oxooctanoyl)-L-homoserine lactone
(HSL)] (16, 29, 54). Previously we showed that pKP19, which
contains the upstream promoter region of traI and a
lacZ gene fused to traI (Fig. 2), expresses
-galactosidase activity but only in the presence of TraR and AAI
(21). In this construct, the BglII site, which is
located upstream of the promoter region, is situated in the middle of
the distal tra box (tra box III [Fig. 2]). As a
result, pKP19 contains the intact tra box II but only half
of tra box III. Using PCR, we modified the 5' end of this
region to reconstitute the intact tra box III. The resulting
plasmid, pTB12, is identical to pKP19 except that it contains both
copies of the tra box (Fig. 2). Strains harboring each
plasmid were tested for expression of the reporter. Similar to the case
for pKP19, expression of the traI::lacZ
fusion in pTB12 requires both TraR and AAI (Fig.
3 and data not shown). However, the
traI::lacZ fusion in pKP19 was induced
with significantly faster kinetics than the same reporter in pTB12
(Fig. 3). Thus, the presence of an intact tra box III
down-modulates the rate of induction of traI from the
tra box II-associated promoter mediated by TraR and AAI.
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FIG. 2.
Structure of the traI-repA intergenic region
and of the traI and repA expression clones.
Shaded boxes indicate the extents and locations of the conserved
tra box elements. Hatched arrows represent the
lacZY genes from pLKC482. The vector for pKP19 and pTB12 is
pLAFR6; the vector for pPLrep4, -5, and -6 is pJB3. The gene sizes of
trbB, traI, repA, and lacZY
are not drawn to scale.
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FIG. 3.
Influence of tra boxes II and III on
expression of traI. Overnight cultures of the test strains
were diluted into and incubated in fresh ABM medium supplemented with
synthetic AAI (40 nM). Samples were removed at the indicated times and
assayed for -galactosidase activity as described in Materials and
Methods. The experiment was repeated twice. Bars show the standard
deviation for each data point.  , NT1(pKP19, pSVB33);  , NT1(pTB12,
pSVB33);  , NT1(pKP19);  , NT1(pTB12).
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Influence of TraR-AAI on the expression of rep.
In our
studies, we consistently have observed increased amounts of Ti plasmid
DNA isolatable from strains harboring the transfer-constitutive (Trac) mutant pTiC58
accR compared to those
containing its wild-type transfer-inducible parent, pTiC58.
pTiC58accR contains a deletion in accR which
codes for a repressor responsible for the regulation of expression of
traR (4, 37, 38). Strains harboring this mutant
plasmid express traR constitutively, produce large amounts of AAI, and transfer the Ti plasmid in the absence of induction by the
conjugal opine. We examined this anecdotal observation more closely by
comparing the amount of Ti plasmid DNA recoverable from strains
harboring pTiC58accR and pTiC58. Following
electrophoresis of plasmid DNA prepared from similar numbers of cells
as described in Materials and Methods, the band intensity of
pTiC58accR was three- to fivefold greater than that of
the wild-type parent plasmid (Fig. 4).
While the increase in plasmid yield could be due to other reasons, we
suspected that constitutive production of TraR and AAI in some way
affects the copy number of this Ti plasmid. We also examined another
Trac mutant of pTiC58, pCMA1, which is wild type for
accR but contains a deletion in the antiactivator,
traM (20). This Ti plasmid expresses
traR at its repressed level, but the activator is not inhibited by TraM (20). Like that of
pTiC58accR, the band corresponding to pCMA1 was six- to
sevenfold more intense than that of the wild-type parent plasmid (Fig.
4). These observations, along with the negative effect of an intact
tra box III on the expression of the divergently expressed
traI-trb operon, prompted us to explore the possibility that
TraR, together with its signal ligand, influences vegetative replication of the Ti plasmid.
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FIG. 4.
Electrophoretic analysis of wild-type pTiC58 and its
Trac mutants. Cells were grown and plasmids were isolated
and subjected to electrophoresis in agarose gels as described in
Materials and Methods. Lanes contain equal loading volumes of plasmid
DNA isolated from 9.0 × 108 CFU of NT1(pTiC58),
8.5 × 108 CFU of NT1(pCMA1), and 1.4 × 109 CFU of NT1(pTiC58aacR). The arrowhead
indicates the position of the Ti plasmid. The bands below the Ti
plasmid in NT1(pCMA1) correspond to the open circular and closed
circular forms of pPH1JI present in this strain (20). The
experiment was repeated four times with indistinguishable results; in
all repetitions, the intensities of the bands corresponding to
pTiC58accR and pCMA1 always were brighter than those of
pTiC58.
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We tested the influence of TraR and AAI, as well as the intergenic
region between
traI and the replication region, on the
expression of
repA by constructing three clones containing
the
lacZ gene translationally fused to the 5' end of
repA (Fig.
2).
pPLrep4, which contains the entire
traI gene and an intact
traI-repA intergenic
region, confers production of AAI but only in cells
expressing TraR.
pPLrep5 contains the entire
traI-repA intergenic
region and
the first 17 codons of
traI and does not confer production
of the acyl-HSL. The shortest clone, pPLrep6, contains the upstream
region of
repA but extends only to the
BglII site
at the center
of
tra box III. Expression of the
repA::
lacZ fusion in each of
these
plasmids was assessed in the presence and absence of TraR
and AAI.
pPLrep6, which contains the shortest intergenic region,
consistently
expressed the reporter fusion at two- to threefold
lower levels
compared to the clones with the full intergenic region
(Table
3). Expression of the
repA::
lacZ fusion in each of the
three
reporter plasmids was not affected by TraR in the absence
of AAI.
However, when signal molecule was added, expression of
the reporter in
pPLrep5 was slightly but consistently enhanced
(Table
3), and this
enhancement was dependent on TraR. Similarly,
expression from the
reporter in pPLrep4, which itself codes for
the production of AAI, was
enhanced slightly when TraR was coexpressed
in the cells (Table
3). On
the other hand, expression of the
reporter from pPLrep6, which lacks
half of
tra box III, as well
as upstream untranslated DNA
was slightly but consistently inhibited
when TraR and AAI were provided
(Table
3).
Construction of a minimal rep plasmid and the effect of
TraR and AAI on copy number.
The 4,906-bp HindIII
fragment 13, containing the first 17 codons of traI, the
traI-repA intergenic region, and all three rep genes as well as 496 bp from the 3' end of y4CG (Fig. 1),
was cloned into pBluescript SK(+) to generate pPLH13. This plasmid replicates in A. tumefaciens as well as in E. coli (data not shown). Thus, all functions essential for
replication of the Ti plasmid are contained in this
HindIII fragment. To eliminate any influence from the
rep region of the vector plasmid, we ligated
HindIII fragment 13 with the 3,424-bp
HindIII internal fragment from Tn5 to
generate a minimal rep plasmid pH13Km (Fig. 1). This
construct is identical with respect to pTiC58 DNA to pUCD510
constructed by Gallie et al. (17). pUCD510 replicates in
A. tumefaciens with the same copy number and stability as
wild-type pTiC58. Although not carefully examined, pH13Km, like
pUCD510, replicates at the anticipated low copy number and is very
stable in our A. tumefaciens test strain (data not shown).
We then assessed the influence of TraR and AAI on the copy number of
pH13Km. pRKE33, which codes for
traR, or its vector,
pRK415,
was introduced into NTL4(pH13Km). We then compared the
copy numbers of
pH13Km in these three strains grown in the presence
or absence of AAI
by measuring the relative intensities of bands
on an agarose gel
corresponding to
HindIII fragment 13 (Fig.
5).
In these comparisons, the intensities
of the bands corresponding
to pRK415 and the vector portion of pRKE33
served as internal
controls. The addition of AAI had virtually no
effect on the copy
number of pH13Km or pRK415 in strains lacking TraR.
However, in
strain NTL4(pH13Km, pRKE33), which contains the
TraR-producing
plasmid, the copy number of the
rep plasmid
was increased five-
to sixfold but only upon addition of AAI (Fig.
5
and Table
4).
This increase is similar to
the apparent increase in copy number
that we observed for the two
Tra
c Ti plasmids (Fig.
4). This was not a generalized
effect; the
quorum-sensing regulators had no effect on the copy number
of
pRKE33 or its parent vector, pRK415 (Fig.
5 and data not shown),
nor
did these factors affect the copy number of pAtC58, the 450-kb
catabolic plasmid present in C58 and its derivatives. The
HindIII
digestion products of this plasmid are visible
as faint bands
in each lane of Fig.
5.
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FIG. 5.
Electrophoretic analysis of pH13Km in cells with and
without traR. Cells were grown, plasmids were isolated and
digested with HindIII, and equal loadings of DNA were
subjected to electrophoresis in agarose gels as described in Materials
and Methods. The band labeled Sm/Bleo/Km represents the 3.4-kb
HindIII fragment from Tn5 cloned in pH13Km.
The weakly staining bands in the background come from pAtC58, a 450-kb
catabolic plasmid present in C58 and its derivatives (14).
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DISCUSSION |
Sequence and function of the rep region of pTiC58.
The repABC-type replicators are common among the large,
low-copy-number plasmids present in members of the family
Rhizobiaceae. These replicators exhibit similarities in
individual genes and also in overall organization. In addition to the
three rep genes, the igs between repB
and repC is believed to serve as a cis-acting site, perhaps as the origin of replication (oriV) or for
incompatibility (inc) (47, 50). Alignment of the
igs regions indicates that several domains are strongly
conserved among the different repABC-type replicators for
which complete sequence is available (Fig.
6). On the other hand, a similar
alignment of the intergenic sequence between repA and
repB does not show such a strong conservation in primary
sequence (data not shown). That the rep region of pTiC58 possesses all of these characteristics clearly indicates that this
replicator is a member of the repABC family. Furthermore, the rep region of pTiC58 is closely related to the
rep regions of other Ti plasmids but more distantly related
to those of the Ri plasmid pRiA4b, the two Sym plasmids, and a cryptic
plasmid from R. leguminosarum (Table 2). Phylogenetic
analysis of the RepC protein, which has been used as an indicator of
plasmid diversity (6, 41, 49) (Fig.
7A), and the igs (Fig. 7B)
indicate that the rep complexes of the three Ti plasmids
form a tight cluster, whereas those of the Ri plasmid and the three
Rhizobium plasmids form a second cluster less closely
related to that of the Ti plasmid replicators. The rep
regions of pRmeGR4a, which contains a RepC homologue, and of pTAV1,
present in a host bacterium outside of the family
Rhizobiaceae, are more distantly related to the members of
this replicator family (Fig. 7). The close relatedness between the
replicators of pTiC58 and pTiB6S3 is consistent with the observation that these two plasmids belong to the same incompatibility group, IncRh1 (19, 33). The incompatibility properties of
pTi-SAKURA have not been reported, but we anticipate that this plasmid
also is a member of the IncRh1 group. In this regard, the divergence between the rep regions of pRiA4b and the Ti plasmids also
is consistent with the observation that the Ri plasmid is compatible with the IncRh1 Ti plasmids (52). Turner et al.
(49) and Rigottier-Gois et al. (41) reported that
the repC sequence is widely distributed among strains of
Rhizobium spp. and that these repC sequences cluster into different groups. They suggested that the different repC families correspond to incompatibility groups and that
divergence in RepC may be responsible for the compatibility properties
exhibited by many members of this family. Our analysis of RepC from the Ti and Ri plasmids supports this hypothesis. However, even though RepC
of pTAV1 is distantly related to that of the Ti plasmids, a recombinant
clone containing only this replicator is reportedly incompatible with
pTiB6S3 (3). On the other hand, Rigottier-Gois et al.
(41) also reported that two plasmids classified to the same
repC group coexist in a field isolate of R. leguminosarum bv. viciae. Therefore, incompatibility properties of
the repABC-type family may involve functions in addition to
RepC. Consistent with this conclusion, Tabata et al. reported that a
clone containing only the igs region exerted incompatibility
against an IncRh1 plasmid (47).
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FIG. 6.
Alignment of the igs region from seven
repABC-type replication systems. Identical bases are shown
as white letters on a black background. Boxed sequences indicate the
last codon and the translational stop codon of repB and the
first two codons of repC. The consensus sequence is based on
identities in at least five of the eight aligned igs
regions.
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FIG. 7.
Phylogenetic relationships among cis and
trans elements of repABC-type replicators. The
unrooted phylogenetic trees for RepC amino acid sequences (A) and the
igs regions (B) are presented. Note that pRmeGR4a codes for
a RepC homologue but does not contain genes related to repA
or repB (31).
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By aligning the functional map of the pTiC58 replicator described by
Gallie et al. (
17) with our sequence, the
par
region,
which is involved in partitioning of the plasmid, corresponds
to
repA (Fig.
1). Consistent with this conclusion,
mutational
analysis indicates that RepA and RepB are required for
stable
maintenance of pTiB6S3 and pRiA4b but are not essential for
replication
per se (
35,
47).
repC, which is
believed to code for the major
replicator protein, corresponds to
ori/inc, the origin of replication
and the incompatibility
region (Fig.
1 and reference
17). The
close
relatedness between the three
rep genes of the two Ti
plasmids,
coupled with prior genetic analyses (
17,
47),
prompts us to
conclude that the
rep gene products of pTiC58
play roles identical
to those of pTiB6S3. Gallie et al. (
17)
proposed a third function,
copy number control or
cop,
associated with this region. The
cop locus, which maps
distal to
repC, overlaps
y4CG and perhaps also
y4CF (Fig.
1 and reference
17).
y4CG, which shows similarities
to DNA invertases and
resolvases from several bacteria and phage,
is not part of the minimum
stable, unit-copy replicator as defined
by pH13Km. Furthermore, except
for pNGR234a, most of the
repABC-type
replicators lack this
gene, although our analysis of the limited
available sequence suggests
that a similar ORF may be located
immediately downstream from
repC of pTi-SAKURA and pTiB6S3 (data
not shown). However, in
pNGR234a the
y4CG ORF, while present,
is located nearly 3 kb
downstream of
repC. Thus, it is unlikely
that
y4CG itself plays a role in replication or copy number
control.
Furthermore, studies on other
repABC-type
replicators provide
little support for the involvement of accessory
genes outside
of
repABC. However, Cevallos et al.
(
8) recently reported that
a 152-bp region located immediate
downstream of
repC of p42d exerts
incompatibility and is
essential for replication of this Sym plasmid.
It is possible that a
sequence with similar
cis-acting functions
resides in the
503-bp region downstream of
repC from pTiC58 that
is
contained in
pH13Km.
Relationship between tra boxes and the rep
region.
Two tra boxes are located in the intergenic
region between traI and repA from both octopine-
and nopaline-type Ti plasmids (Fig. 8A)
(1, 27). Fuqua and Winans (16) reported that tra box II is required for the expression of the
traI-trb operon, whereas tra box III exerts no
detectable effect on expression in either direction in the
octopine-type Ti plasmid. Our results suggest that an intact
tra box III somewhat impairs the tra box II-dependent TraR-mediated expression of traI in pTiC58
(Fig. 3). Moreover, the upstream region which contains tra
box III is required for full expression of repA of the
nopaline-type Ti plasmid (Table 3). Intriguingly, expression of
repA, as assessed by our lacZ reporter fusions,
is enhanced slightly by TraR and AAI, but only when the entire upstream
region is present (Table 3).
View larger version (33K):
[in this window]
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|
FIG. 8.
Comparison of structures of the traI-repA
intergenic regions of six plasmids from the family
Rhizobiaceae. (A) Extents of the intergenic regions and
distribution of tra box-like sequences. As noted by the (?),
the designation of traI in p42d and trbB in
pRL8JI is based on similarities from limited available sequence. Roman
numerals II and III in the Ti plasmid sequences indicate the two
tra boxes located in the intergenic regions between
traI and repA. (B) Alignment of the
tra boxes, the lux box of V. fischeri,
and the las box of Pseudomonas aeruginosa.
Arrowheads indicate the symmetry of the sequences; the dashed line
separates sequences that are more related to the tra box
consensus from those more related to the lux or
las box sequences. Symbols in the consensus sequence: R,
purine; Y, pyrimidine; S, G or C; N, any nucleotide.
|
|
tra boxes are present in the region upstream of
repA in all three Ti plasmids examined to date and also in
the corresponding
regions of other plasmids with a
repABC-type replicator (Fig.
8A). The
tra
box-like sequence in pRL8JI is more closely related
to those of the Ti
plasmids, whereas the
cis element in pNGR234a
is more
similar to the
lux box of
Vibrio fischeri (Fig.
8B). Furthermore,
two Sym plasmids encode
traI-like genes
divergently oriented from
repA (Fig.
8A). As with the Ti
plasmids,
traI of pNGR234a is the
first gene of the
trb operon (
15). Apparently, the association
of
the quorum-sensing regulatory system with Tra occurred on the
replicon ancestral to pNGR234a and the Ti plasmids. On the other
hand, pRL8JI, which is self-conjugal (
23), lacks a
traI homologue,
and the
repABC cluster is linked
directly to a gene with relatedness
to
trbB, the second gene
of the Ti plasmid-type
trb operon. This
finding suggests
that quorum sensing is not a de rigueur component
of the conjugal
transfer systems of these types of plasmids. However,
a
tra
box-like sequence is located in the intergenic region between
the
trbB homologue and
repA of pRL8JI (Fig.
8A),
suggesting that
the Tra system and perhaps the
rep genes of
this plasmid were
regulated by quorum sensing at one time. It also is
possible that
conjugal transfer of pRL8JI is controlled by quorum
sensing but
that the
traI homolog is located in some other
region of the genome.
Interestingly, although there is a
traI-like gene present in the
region upstream from
repA of p42d, there is no identifiable
tra box in
this
region.
A plasmid replicator influenced by quorum sensing.
Our results
indicate that copy number of pTiC58 is influenced by TraR and AAI. That
both pTiC58accR and pCMA1 exhibit such an increase
indicates that the effect is not due to the influence of some other
gene in the arc operon (37). Furthermore, the effect can be seen on the minimal rep plasmid, pH13Km,
indicating that TraR influences copy number through
repABC and not by some indirect interaction with another
component of the Ti plasmid. This is the first report of plasmid
replication being influenced by a quorum-sensing system. How TraR and
AAI influence Ti plasmid copy number remains to be determined. While
transcription of the rep genes does not require the
quorum-sensing activator, our analysis suggests that TraR, coupled with
AAI, enhances expression of repA (Table 3). This enhancement
might be due to weak activation by TraR. Alternatively, TraR bound to
tra box III may alter the structure of the DNA around the
origin, leading to an increase in the rate of replication initiation.
Furthermore, the repA promoter most probably contains
cis-acting signals located in the vicinity of tra
box III since removing this region resulted in lowered levels of
expression and the loss of the enhancing effect associated with TraR
and AAI (Table 3).
The connection between DNA replication and quorum sensing is not
without precedence. Withers and Nordström (
53)
reported
that an extracellular factor negatively influences chromosomal
DNA replication of
E. coli in a quorum-dependent manner.
Apparently
replicon copy number needs in some way to respond to changes
in
the environment and the population size itself. Studies with RP4
point to a relationship between plasmid replication and conjugal
transfer. In this IncP1
plasmid, the replicator gene,
trfA, and
the conjugal transfer genes in the Tra2 region are
coregulated
by the product of the
trbA gene (
22,
32). Sia et al. (
43)
suggested that conjugal transfer
contributes to the maintenance
of RK2 by reducing the proportion of
plasmidless segregants in
a growing population when growth conditions
are favorable for
conjugation.
Regulating copy number in a quorum-dependent manner could serve several
purposes. First, since opine availability regulates
quorum sensing
(
37), increasing the copy number of the Ti plasmid
in
response to population density results in an increase in components
of
the opine catabolism systems. Such an increase in opine transporters,
for example, may be advantageous to a bacterium under conditions
in
which availability of these nutritional resources becomes limiting
due
to increased numbers of opine utilizers. Second, elevated
plasmid copy
number could augment conjugal transfer by increasing
the number of
mating pores as well as relaxosome assemblies. This,
in turn, could
compensate for the occasional loss of the Ti plasmid
from some fraction
of the agrobacterial population, especially
at high population
densities. Upregulating plasmid copy number
also may explain the
observation by Veluthambi et al. (
51) that
octopine, the
conjugal opine of pTiA6, enhances the level of
vir gene
induction by acetosyringone some 2- to 10-fold. It is conceivable
that
this stimulation in expression of the
vir regulon results
from an increase in copy number mediated by the octopine-inducible
TraR-AAI quorum-sensing system of this Ti plasmid. These observations
suggest that upregulating plasmid copy number in response to conditions
that signal an environment favorable for transfer is an important
component of the biology of the Ti
plasmid.
| |
ACKNOWLEDGMENTS |
We thank Ingyu Hwang and Philippe Oger for helpful discussions.
This work was supported by grants R01 GM52465 from the NIH and
AG92-3312-8231 from the USDA to S.K.F. P.-L.L. was supported in
part by HATCH project 15-0326 to S.K.F.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Crop Sciences, University of Illinois at Urbana-Champaign, 240 Edward R. Madigan Laboratory, 1201 West Gregory Dr., Urbana, IL 61801. Phone:
(217) 333-1524. Fax: (217) 244-7830. E-mail:
stephenf{at}uiuc.edu.
| |
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Journal of Bacteriology, January 2000, p. 179-188, Vol. 182, No. 1
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Abstract
Introduction
