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Many, if not all Ti plasmids of
Agrobacterium tumefaciens are self-conjugal (8,
20) and can transfer in situ on crown gall tumors (19,
20). In addition to disseminating the Ti plasmids, transfer
certainly is involved in the evolution of these elements (reviewed in
reference 9). Conjugation is strongly regulated at
the transcriptional level by a hierarchical system involving induction
by opines (8), followed by activation by TraR in concert
with a self-produced signal called Agrobacterium autoinducer
(AAI) (12, 23). This latter level of regulation, called
autoinduction or quorum-sensing (15), requires that AAI accumulate to some critical extracellular level before TraR can induce
expression of the tra genes (28). Induction by
opines is required for expression of traR and also for
high-level production of AAI by the donor cells (13, 24,
28). The tra system itself is composed of three
operons organized into two clusters. Two of these operons,
traAFB and traCDG, are divergently expressed from
a central intergenic promoter region that also contains the oriT site (1, 7, 10). The third operon,
trb, contains 12 genes and is located between 75 and 100 kb
from the tra locus, depending upon the Ti plasmid (1,
21). This operon codes for production of AAI (12, 17)
as well as for the mating pair formation system including the conjugal
pilus (1, 21).
Some conjugal plasmids transfer with similar efficiency when matings
are conducted in liquid or on solid surfaces (11, 18, 26).
Donors harboring other types of plasmids mate best on solid surfaces
(3, 26). To our knowledge, the effect of stratum on the
efficiency of Ti plasmid transfer has not been determined. This is of
particular interest because stratum preferences give clues as to the
nature of the habitat in which conjugation of these plasmids occurs.
Moreover, because transfer is controlled by the availability of opines
and the accumulation of AAI in the environment, it is conceivable that
the regulatory system is influenced by whether the bacteria are
colonizing solid surfaces or interacting in a liquid environment. Thus,
we designed a series of experiments to determine the influence of a
liquid or a solid environment on the efficiency of Ti plasmid
conjugation as well as on the induction of expression of the
tra regulon.
Optimum conditions for Ti plasmid transfer.
To maximize
conjugal transfer efficiency, we first determined the influence of
bacterial density on transfer frequency. Late-exponential-phase cultures of NT1 harboring the transfer-constitutive
nopaline-agrocinopine A+B-type Ti plasmid pTiC58
accR
(2) were mated with C58C1RS (2) on sterile
nitrocellulose filters (7) at various cell densities, with
the input ratios of donor to recipient kept uniform at approximately
1:1. Filters were incubated on the surfaces of AB-mannitol (ABM)
minimal agar medium (6) for 2 h, and the cells were
resuspended by vortexing the filters in 1-ml volumes of 0.9% NaCl in
water. The mating mixtures were diluted, and transconjugants were
selected for by plating 0.1-ml volumes on AB minimal medium containing
9 mM arginine and 1 mM nopaline as the sole carbon source
(2). Rifampin (50 µg per ml) and streptomycin (200 µg per ml) were included to select against the donors. Under these conditions, frequencies of transfer were maximal at total cell densities above 2 × 107 CFU per cm2 (Fig.
1). Transfer frequency dropped by 3 orders of magnitude at a density of 2 × 106 CFU per
cm2 and was undetectable at densities of 2 × 105 CFU per cm2 or lower.
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FIG. 1.
Minimum cell densities are required for mating. Donor
and recipient bacteria at ratios of 1:1 were mated on filters at
increasing total cell densities as described in the text. Transfer
frequencies are expressed as transconjugants recovered per input donor,
and culture densities are expressed as CFU per square centimeter. Each
mating was quantified in triplicate, with less-than-10% differences in
colony counts. The experiment was repeated two times. Although the
actual frequencies of transfer differed between the three experiments,
the patterns were identical. The results from a representative
experiment are presented.
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We also determined the optimum ratio of donors to recipients by mating
late-exponential-phase cultures of NT1(pTiC58accR) at
input densities ranging from 2 × 102 to 2 × 108 CFU per cm2 with cultures of C58C1RS at
densities ranging from 2 × 108 to 2 × 102 per cm2. The resulting donor-to-recipient
ratios on the filters ranged from 10
6 to 106,
while total cell density was maintained above 2 × 107
CFU per cm2. Transfer frequencies, measured as
transconjugants per input donor, were maximum and remained constant
when the recipient was present in 10-fold or greater excess over the
donor (Fig. 2). However, frequencies
dropped by 10-fold increments as the donor-to-recipient ratio was
increased by 10-fold increments. Thus, we performed subsequent matings
by using donor-to-recipient ratios of 1:10 and total cell densities
greater than 107 unless otherwise noted.
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FIG. 2.
Ratio between donor and recipient cell densities
influences conjugation. Donors harboring pTiC58accR were
mated with recipients on nitrocellulose filters at various ratios as
described in the text. The total cell density in each mating was
greater than 107 CFU/cm2. Frequencies of
transfer are expressed as transconjugants recovered per input donor.
Each mating was quantified in triplicate, with less-than-10%
differences between colony counts at each dilution. The experiment was
repeated three times. As in Fig. 1, results from a representative
experiment are presented.
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Conjugation requires a solid surface.
We determined whether Ti
plasmid transfer is surface dependent by comparing the transfer
frequency of pTiC58accR in matings conducted in liquid
and on solid media. Donor and recipient cultures were grown to late
exponential phase in ABM liquid medium. Samples of a series of 10-fold
dilutions of the donor culture, prepared in ABM liquid medium, were
incubated with an equal volume of the recipient culture for 2 h.
Concurrently, samples of the donor and recipient cultures were mated
for 2 h on filters placed on ABM agar medium to determine the
transfer frequency on a solid surface. Following the matings, samples
of each mixture were collected and diluted, and 0.1-ml volumes from
these dilutions were spread onto AB plates supplemented as described
above to select for transconjugants. In addition, samples of donor and
recipient cultures incubated separately in liquid medium were spread
together onto selection plates to assess for matings that occurred
after plating on the surface of the selection medium.
While transconjugants appeared on plates from both matings, cells mated
on solid medium yielded progeny at a frequency 3 to 4 orders of
magnitude higher than cells mated in liquid medium (Table
1). Moreover, the efficiency of transfer
on solid medium remained constant, even with decreasing numbers of
donors. Transconjugant colonies also arose on the control plates,
indicating that at least some of the progeny appearing on plates spread
with the liquid mixtures arose from matings that occurred on the
selection plates. However, transfer frequencies from matings in liquid
consistently were nearly double the transfer frequencies from matings
that occurred on the selection plate, suggesting that donors harboring Ti plasmids can form productive mating pairs in liquid, albeit at a
very low frequency.
Preference for solid versus liquid strata correlates with the type of
sex pilus encoded by the plasmid. Those that code for short, rigid pili
transfer best on solid surfaces, while plasmids encoding flexible pili
transfer equally well in liquid and on solid surfaces (3,
4). By analogy, our results suggest that the sex pilus encoded by
pTiC58 is the rigid type. Consistent with this hypothesis, the
trb genes of RP4, which are closely related to those of
pTiC58 (21), direct the synthesis of a rigid-type pilus
(16). More importantly, the results suggest that Ti plasmid transfer occurs on the surfaces of soil particles or crown gall tumor
cells rather than free in the interstitial aqueous environment formed
by films on the surface of these strata or bridging these strata. This
preference for solid surfaces also is consistent with the fact that
opines produced by the tumors and exuded to the cell surfaces and
rhizosphere serve as specific inducers of the conjugal transfer
apparatus for many Ti and opine-catabolic plasmids (8, 20).
Presumably, opine concentrations are at their highest close to the
surface of the crown gall tumor cells.
Conjugation is inducible in liquid medium.
While productive
matings occurred at highest frequencies on solid surfaces, previous
results with lacZ fusions indicated that the tra
genes of pTiC58accR are expressed at high levels in
liquid media (10, 17, 23, 24), prompting us to determine
whether the tra regulon is inducible in a liquid
environment. Strain C58(pJM749), which harbors a wild-type Ti
plasmid as well as a recombinant plasmid containing a
traA::lacZ reporter fusion (10,
23), was incubated with a preparation of agrocinopines A+B
(25) on solid and in liquid media. For the former, the donor
was grown overnight on sterile filters (13-mm diameter) placed on small towers of ABM agar medium (1 cm ht by 0.5 cm in diameter) impregnated with agrocinopines A and B (25) at a concentration of 2 mM. After induction, the cells were resuspended in 500 µl of 0.9% NaCl.
For the latter, cells were grown overnight in 800 µl (a volume
corresponding to that of the agar towers) of ABM medium containing the
agrocinopines. The cells were harvested by centrifugation and
resuspended in 500 µl of 0.9% NaCl. In each case the donors were
spot mated (2) at various dilutions directly on selection plates previously spread with a culture of C58C1RS. In addition, samples of the donor cultures were assayed for 
-galactosidase activity (17). Both induction conditions yielded
transconjugants, with the cells induced in liquid medium transferring
at a slightly higher frequency than those induced by growth on solid
medium (Table 2). Moreover, the
traA::lacZ reporter fusion was induced to similar levels in the two donor cultures (Table 2). Thus, the
hierarchical regulatory system can function with equal efficiency in
liquid and on solid strata.
Fuqua and Winans (12) reported that the closely related
tra genes of the octopine-mannityl opine-type Ti plasmid
pTiR10 are not strongly expressed in liquid medium when induced by
octopine alone but are expressed in cultures to which AAI also is
added. They proposed that the low expression levels result from the
lack of production of AAI by cells induced for transfer in liquid and concluded that tra gene activation requires factors specific
for growth on solid surfaces. In contrast, we recently showed that another virtually identical Ti plasmid, pTi15955, is inducible for
transfer and produces high levels of AAI in liquid medium containing
octopine (22). These latter results are consistent with our
observation that the tra genes of pTiC58 are expressed and
AAI is produced at high levels during growth under inducing conditions
in liquid medium (17, 24). Furthermore, strains harboring
Trac Ti plasmids produce large amounts of AAI when grown in
liquid medium, indicating that production of the signal is not
dependent on solid surfaces (5, 17, 23, 27, 29, 30). It
should be noted that while we measured conjugal transfer frequencies as
well as gene induction, Fuqua and Winans (12) measured only the expression levels of the tra genes. Thus, while the
tra regulon may not be strongly induced in liquid medium,
the levels of expression are sufficient for conjugation to occur at
normal frequency. In support of this hypothesis, the data of Fuqua and
Winans (14) demonstrate that modest increases in
tra gene expression result in maximal plasmid transfer and
that induction of the tra regulon to higher levels does not
necessarily result in elevated transfer rates. These results are
consistent with our observations with pTiC58 and suggest that the low
expression levels which may be seen in liquid environments do not
necessarily limit conjugal transfer.
We conclude that the tra genes of both types of Ti plasmids
are inducible and that the conjugation apparatus can be expressed in
liquid environments. However, the formation of stable mating pairs
requires a solid surface. We think it likely that given a regulatory
system dependent upon the accumulation of AAI and a conjugation
apparatus reliant on short rigid pili, that the majority of the Ti
plasmid conjugal transfer events occur on the surface of the crown gall
tumor or on root surfaces and soil particles sufficiently close to be
influenced by opines produced by these plant neoplasias.
We thank Ingyu Hwang, Pei-Li Li, David M. Cook, Susanne Beck von
Bodman, Clay Fuqua, and Stephen Winans for helpful discussions.
This work was supported by grant no. GM52465 from the NIH and grant no.
AG93-337301-8943 from the USDA to S.K.F.
| 1.
|
Alt-Mörbe, J.,
J. L. Stryker,
C. Fuqua,
P.-L. Li,
S. K. Farrand, and S. C. Winans.
1996.
The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.
J. Bacteriol.
178:4248-4257[Abstract/Free Full Text].
|
| 2.
|
Beck von Bodman, S.,
G. T. Hayman, and S. K. Farrand.
1992.
Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.
Proc. Natl. Acad. Sci. USA
89:643-647[Abstract/Free Full Text].
|
| 3.
|
Bradley, D. E.
1981.
Conjugative pili of plasmids in Escherichia coli K12 and Pseudomonas species, p. 217-226.
In
S. B. Levy, R. C. Clowes, and E. L. Koenig (ed.), Molecular biology, pathogenicity and ecology of bacterial plasmids. Plenum Press, New York, N.Y.
|
| 4.
|
Bradley, D. E.,
D. E. Taylor, and D. R. Cohen.
1980.
Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12.
J. Bacteriol.
143:1466-1470[Abstract/Free Full Text].
|
| 5.
|
Cha, C.,
P. Gao,
Y.-C. Chen,
P. D. Shaw, and S. K. Farrand.
1998.
Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria.
Mol. Plant-Microbe Interact.
11:1119-1129[Medline].
|
| 6.
|
Chilton, M.-D.,
T. C. Currier,
S. K. Farrand,
A. J. Bendich,
M. P. Gordon, and E. W. Nester.
1974.
Agrobacterium tumefaciens and PS8 bacteriophage DNA not detected in crown gall tumor DNA.
Proc. Natl. Acad. Sci. USA
71:3672-3676[Abstract/Free Full Text].
|
| 7.
|
Cook, D. M., and S. K. Farrand.
1992.
The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders.
J. Bacteriol.
174:6238-6246[Abstract/Free Full Text].
|
| 8.
|
Ellis, J. G.,
A. Kerr,
A. Petit, and J. Tempé.
1982.
Conjugal transfer of nopaline and agropine Ti-plasmids the role of agrocinopines.
Mol. Gen. Genet.
186:269-273.
|
| 9.
|
Farrand, S. K.
1998.
Conjugation in the Rhizobiaceae, p. 199-233.
In
H. P. Spaink, A. Kondorosi, and P. J. J. Hooykaas (ed.), The Rhizobiaceae: molecular biology of model plant-associated bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
|
| 10.
|
Farrand, S. K.,
I. Hwang, and D. M. Cook.
1996.
The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F.
J. Bacteriol.
178:4233-4247[Abstract/Free Full Text].
|
| 11.
|
Frost, L. S.,
K. Ippen-Ihler, and R. A. Skurray.
1994.
Analysis of the sequence and gene products of the transfer region of the F sex factor.
Microbiol. Rev.
58:162-210[Abstract/Free Full Text].
|
| 12.
|
Fuqua, W. C., and S. C. Winans.
1994.
A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite.
J. Bacteriol.
176:2796-2806[Abstract/Free Full Text].
|
| 13.
|
Fuqua, C., and S. C. Winans.
1996.
Localization of OccR-activated and TraR-activated promoters that express two ABC-type permeases and the traR gene of Ti plasmid pTiR10.
Mol. Microbiol.
20:1199-1210[Medline].
|
| 14.
|
Fuqua, C., and S. C. Winans.
1996.
Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes.
J. Bacteriol.
178:435-440[Abstract/Free Full Text].
|
| 15.
|
Fuqua, W. C.,
S. C. Winans, and E. P. Greenberg.
1994.
Quorum-sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators.
J. Bacteriol.
176:269-275[Free Full Text].
|
| 16.
|
Haase, J.,
R. Lurz,
A. M. Grahn,
D. H. Bamford, and E. Lanka.
1995.
Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.
J. Bacteriol.
177:4779-4791[Abstract/Free Full Text].
|
| 17.
|
Hwang, I.,
P.-L. Li,
L. Zhang,
K. R. Piper,
D. M. Cook,
M. E. Tate, and S. K. Farrand.
1994.
TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer.
Proc. Natl. Acad. Sci. USA
91:4639-4643[Abstract/Free Full Text].
|
| 18.
|
Ippen-Ihler, K. A., and J. E. G. Minkley.
1986.
The conjugation system of F, the fertility factor of Escherichia coli.
Annu. Rev. Genet.
20:593-624[Medline].
|
| 19.
|
Kerr, A.
1969.
Transfer of virulence between isolates of Agrobacterium.
Nature (London)
223:1175-1176.
|
| 20.
|
Kerr, A.,
P. Manigault, and J. Tempé.
1977.
Transfer of virulence in vivo and in vitro in Agrobacterium.
Nature (London)
265:560-561[Medline].
|
| 21.
|
Li, P.-L.,
D. M. Everhart, and S. K. Farrand.
1998.
Genetic and sequence analysis of the pTiC58 trb locus encoding a mating-pair formation system related to members of the type IV secretion family.
J. Bacteriol.
180:6164-6172[Abstract/Free Full Text].
|
| 22.
|
Oger, P.,
K.-S. Kim,
R. L. Sackett,
K. R. Piper, and S. K. Farrand.
1998.
Octopine-type Ti plasmids code for a mannopine-inducible dominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugal transfer.
Mol. Microbiol.
27:277-288[Medline].
|
| 23.
|
Piper, K. R.,
S. Beck von Bodman, and S. K. Farrand.
1993.
Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction.
Nature (London)
362:448-450[Medline].
|
| 24.
| Piper, K. R., S. Beck von Bodman, I. Hwang, and
S. K. Farrand. Hierarchical gene regulatory systems arising
from fortuitous gene associations: controlling quorum-sensing by the
opine regulon in Agrobacterium. Mol. Microbiol., in press.
|
| 25.
|
Ryder, M. H.,
M. E. Tate, and G. P. Jones.
1984.
Agrocinopine A, a tumor-inducing plasmid-coded enzyme product, is a phosphodiester of sucrose and L-arabinose.
J. Biol. Chem.
259:9704-9710[Abstract/Free Full Text].
|
| 26.
|
Willets, N.
1981.
Sites and systems for conjugal DNA transfer in bacteria, p. 207-215.
In
S. B. Levy, R. C. Clowes, and E. L. Koenig (ed.), Molecular biology, pathogenicity, and ecology of bacterial plasmids. Plenum Press, New York, N.Y.
|
| 27.
|
Zhang, L.
1993.
Molecular biology and biochemistry of a novel conjugation factor in Agrobacterium. Ph.D. dissertation.
University of Adelaide, Australia.
|
| 28.
|
Zhang, L., and A. Kerr.
1991.
A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.
J. Bacteriol.
173:1867-1872[Abstract/Free Full Text].
|
| 29.
|
Zhang, L.,
P. J. Murphy,
A. Kerr, and M. E. Tate.
1993.
Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones.
Nature (London)
362:446-448[Medline].
|
| 30.
|
Zhu, J.,
J. W. Beaber,
M. I. Moré,
C. Fuqua,
A. Eberhard, and S. C. Winans.
1998.
Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens.
J. Bacteriol.
180:5398-5405[Abstract/Free Full Text].
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