Transsexuality in the rhizosphere: quorum sensing reversibly converts Agrobacterium tumefaciens from phenotypically female to male.

Abstract

Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.

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Published Ahead of Print 20 March 2009.
2009, 191(10):3375. DOI: 10.1128/JB.01608-08.
J. Bacteriol.
Hongbaek Cho, Uelinton M. Pinto and Stephen C. Winans
Phenotypically Female to Male
from
Agrobacterium tumefaciens
Sensing Reversibly Converts
Transsexuality in the Rhizosphere: Quorum
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JOURNAL OF BACTERIOLOGY, May 2009, p. 3375–3383
0021-9193/09/$08.00?0 doi:10.1128/JB.01608-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 10
Transsexuality in the Rhizosphere: Quorum Sensing Reversibly
Converts Agrobacterium tumefaciens from Phenotypically
Female to Male?
Hongbaek Cho,†‡ Uelinton M. Pinto,‡ and Stephen C. Winans*
Department of Microbiology, Cornell University, Ithaca, New York 14853
Received 12 November 2008/Accepted 5 March 2009
Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar
plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti
plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK,
can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb
operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine
lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells
lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block
the entry of Ti plasmids and instead become efficient conjugal donors.
Many conjugative plasmids are able to block the entry of
identical or closely related types of plasmids by creating a
functional barrier at the cell surface. This phenomenon is
known as entry (or surface) exclusion. Two different types of
exclusion determinants are known to cause this phenomenon.
Surface-exposed outer membrane proteins, exemplified by
TraT of the F plasmid, are thought to block the formation of
stable mating aggregates between two donor cells (24). Other
proteins, such as TraS of the F plasmid and TrbK of RP4, are
located in the inner membrane and inhibit conjugative DNA
transfer (8, 24).
Entry exclusion of Agrobacterium Ti plasmids has not been
documented, but it is plausible that they too have such a system
(17). These plasmids are capable of efficient conjugation and
carry a complete suite of conjugative transfer genes, desig-
nated tra and trb genes (1, 5, 17, 25). One of these genes, trbK,
resembles the trbK genes of the IncP plasmids RP4, RK2, and
R18 (all of which are virtually identical), which mediate entry
exclusion of the corresponding plasmids (8, 9, 15, 18). Another
Ti plasmid gene, trbJ, resembles the trbJ gene of RP4, which
may or may not contribute to entry exclusion. Lessl et al. and
Lyras et al. reported that TrbJ proteins from IncP? plasmids
mediate low-level entry exclusion (15, 18). Haase et al. pre-
sented somewhat conflicting data about the role of TrbJ from
RP4 (8, 9). The reasons for these conflicting data are unclear.
The trbJ and trbK genes of RP4 and of Ti plasmids lie within
operons of genes that direct mating-pair formation (Mpf
genes) (1, 17). The structure encoded by Mpf genes is some-
times referred to as a mating bridge and resembles the family
of type IV systems that are able to translocate DNA and/or
protein into foreign cells (3). TrbK of RP4 is not required for
conjugation (9), so its sole function may be in entry exclusion.
Similarly, TrbK of pTiC58 is dispensable for conjugation (17).
In contrast, the TrbJ proteins of pTiC58 and of RP4 are es-
sential for conjugation (9, 17).
TrbK of RP4 is a lipoprotein that has a lipid attachment
motif and is localized mainly to the cytoplasmic membrane (8).
Its signal sequence is removed proteolytically, and one or more
acyl groups are added to a cysteine residue at the newly created
amino terminus. This cysteine is required for wild-type levels of
entry exclusion, although residual levels were detectable when
this cysteine was altered (8). The alteration of the cysteine
residue causes decreased affinity for the cytoplasmic mem-
brane. Significantly, all known Ti plasmid TrbK proteins lack
this cysteine residue. They are therefore unlikely to be acy-
lated. Both TrbK and TrbJ proteins are strongly predicted to
have cleaved signal sequences (see below), though this predic-
tion has not been experimentally confirmed and the localiza-
tion patterns of the proteins have not been determined.
All Ti plasmid tra and trb genes are regulated by the TraR
and TraI quorum-sensing system (6), and a variety of plasmids
of Rhizobium, Mesorhizobium, and Sinorhizobium spp. regulate
conjugation genes in similar fashions (7). TraR resembles the
transcription factor LuxR of Vibrio fischeri, while TraI resem-
bles the V. fischeri LuxI protein and synthesizes the pheromone
3-oxo-octanoylhomoserine lactone (OOHL). This pheromone
binds to and activates TraR. Significantly, both TraR and TraI
are encoded on Ti plasmids, and therefore, this system detects
a quorum of conjugal donors rather than of conjugal recipi-
ents. As this system detects only conjugal donors, it seemed
plausible that conjugation in V. fischeri had evolved to occur
preferentially between conjugal donors. Although conjugation
between donor cells may seem futile, it may have the poten-
tially useful effect of increasing the plasmid copy number, as
transfer requires conjugative DNA replication. Furthermore, it
has been well established that TraR-OOHL complexes in-
crease the plasmid copy number by enhancing vegetative rep-
lication (16, 19). However, the findings of the present study
* Corresponding author. Mailing address: Department of Microbi-
ology, 360A Wing Hall, Cornell University, Ithaca, NY 14853. Phone:
(607) 279-3886. Fax: (607) 255-3904. E-mail: scw2@cornell.edu.
† Present address: Department of Microbiology and Molecular Ge-
netics, Harvard Medical School, 200 Longwood Avenue, Boston, MA
02115.
‡ These authors made equal contributions.
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disproved this hypothesis, as we documented that octopine-
type and nopaline-type Ti plasmids have entry exclusion sys-
tems and that both TrbJ and TrbK can carry out entry exclu-
sion independently and synergistically. In this sense, our
findings tend to support the results of the studies of RP4 by the
Lessl and Lyras groups (15, 18) rather than those of the studies
by Haase et al. (8, 9). However, like all tra and trb genes, trbJ
and trbK are tightly regulated by activated TraR (6, 11, 21), and
in the absence of activated TraR, neither TrbJ nor TrbK is
significantly expressed and host cells exhibit little or no entry
exclusion. These cells, therefore, are efficient recipients, de-
spite the fact that they have Ti plasmids.
MATERIALS AND METHODS
Strains, oligonucleotides, and reagents. Bacterial strains and plasmids used in
this study are described in Table 1, while oligonucleotides used for PCR ampli-
fication and site-directed mutagenesis and for nuclease S1 protection assays are
described in Table 2. Antibiotics and ONPG (o-nitrophenyl-?-D-galactopyrano-
side) were purchased from Sigma-Aldrich. X-Gal (5-bromo-4-chloro-3-indolyl-
?-D-galactopyranoside) was purchased from Gold Biotechnologies. Restriction
endonucleases, T4 DNA ligase, and T4 polynucleotide kinase were purchased
from New England Biolabs. Taq polymerase was purchased from Promega, and
[?-32P]ATP was purchased from Perkin Elmer.
Quantitative conjugation assays. Conjugative donors and recipients were cul-
tured in AT minimal broth (24a) at 27°C for 5 h, concentrated by centrifugation,
combined in a ratio of 50 recipients per donor, spotted onto AT agar medium,
and incubated for 2.5 h for R10-derived donor strains or 18 h for C58-derived
donor strains. Mating was stopped by resuspending the cells from the agar in 1?
AT buffer, and then the cells were serially diluted and plated onto selective AT
defined agar medium containing the appropriate antibiotics.
Site-directed mutagenesis in trbJ and trbK. Site-directed mutagenesis in trbJ
and trbK was performed by using a synthetic overlap extension PCR (23). For the
mutation of trbJ, a 1,100-bp fragment of pUP404 including a unique EcoRI site
located upstream of trbJ and a unique BamHI site located downstream of trbJ
was amplified using Platinum Taq Hi Fi DNA polymerase (Invitrogen). For the
mutation of trbK, a 530-bp fragment of pHC368 including the same restriction
sites listed above was amplified. All oligonucleotides used in this study are listed
in Table 2 and were obtained from Integrated DNA Technologies (Coralville,
IA). For trbJ, the flanking primers TrbJKF-N and pTacR2 were used in separate
reactions with two complementary mutagenic primers, with pUP404 as the tem-
plate. For trbK, the flanking primers were R10-trbKF-N and pTacR1 and the
template was pHC368. In both cases, the two PCR products were combined and
used as the template in a second round of PCR with the same flanking primers
to generate the complete trbJ or trbK gene. The second set of PCR products was
digested with EcoRI and BamHI and ligated into pUP404 or pHC368, digested
with the same enzymes. These mutations caused a one-codon deletion at the 3?
ends of both genes. Mutated sequences were confirmed by automated DNA
sequencing.
Nuclease S1 protection assays. RNA was isolated from cells cultured to late
log phase and harvested in the presence of 2 volumes of RNAprotect bacterial
reagent (Qiagen) per volume of culture. Cell pellets were frozen at ?80°C.
Lysozyme (200 ?l of a 10-mg/ml solution) and 700 ?l of buffer RLT (Qiagen)
were added to the frozen cell pellets, and the tubes were subjected to a vigorous
vortex. Lysates were clarified by centrifugation for 2 min, and RNA was precip-
itated from the supernatant by the addition of 500 ?l of ethanol. Samples were
applied to RNeasy spin columns (Qiagen) and centrifuged for 15 s at 10,000 rpm.
Buffer RW1 (350 ?l) was added to each column, and columns were centrifuged
for 15 s at 10,000 rpm. DNase I was diluted eightfold in RDD buffer (Qiagen),
and 80 ?l per column was added. After 15 min of incubation, columns were
washed successively with 350 ?l of buffer RW1 and 500 ?l of buffer RPE, and
RNA was eluted using 40 ?l of RNase-free water.
Oligonucleotides were radiolabeled with [?-P32]ATP and T4 DNA kinase. A
500-pg sample of radiolabeled oligonucleotides was hybridized with 20 ?g of
total RNA for 10 h at 42°C and then digested with 250 U of nuclease S1 for 1 h
at 37°C. Reaction mixtures were then ethanol precipitated and suspended in 5 ?l
of 0.1 M NaOH, and 5 ?l of formamide loading dye was added. Five microliters
of each sample was size fractionated using 18% denaturing Tris-borate-EDTA
polyacrylamide gels and quantified using a Storm PhosphorImager (model 840;
Molecular Dynamics). A 2.5-pg aliquot of32P-labeled nondigested oligonucleo-
tide was added to one lane of each gel.
RESULTS
Two Ti plasmids encode functional and tightly regulated
entry exclusion systems. The overexpression of TraR in
strains containing the native traI gene causes constitutive
expression of all genes of the quorum-sensing regulon (6).
We reasoned that any entry exclusion gene may also be
regulated by TraR and, if so, would most likely be expressed
constitutively in strains overexpressing TraR. The overex-
pression of TraR during conjugation also relieves the re-
quirement for octopine, which is otherwise needed to induce
the transcription of the native traR gene (6) and therefore
tends to make conjugation data more reproducible. We mea-
sured the efficiency of Ti plasmid transfer from R10 derivative
WCF5(pJZ381), which overexpresses TraR (Table 1), to two
recipient strains: R10(pHC335), which also overexpresses
TraR, and R10(pPZP201), which does not. Both recipient
strains carried the Ti plasmid pTiR10, which is virtually iden-
tical to other so-called octopine-type Ti plasmids (25). The
former strain gave rise to 300-fold fewer transconjugants than
the latter strain (Table 3, first two lines), indicating that either
TraR itself or, more likely, the product of a TraR-regulated
gene mediated a robust level of entry exclusion.
Similar experiments were carried out using strains har-
boring the nopaline-type Ti plasmid pTiC58. Here, strain
HC158(pJZ381) was used as a Ti plasmid donor. This strain
contains a nopaline-type Ti plasmid that has a kanamycin re-
sistance gene to facilitate the selection of transconjugants.
This strain also overexpresses TraR from pJZ381. Strains
C58(pHC335) and C58(pPZP201) were used as recipients.
The former recipient yielded approximately 300-fold fewer
transconjugants than the latter (Table 3, third and fourth
lines). We conclude that TraR or a TraR-regulated gene in
pTiC58 can exclude the conjugal entry of the same type of
plasmid.
We also tested the abilities of nopaline-type Ti plasmids to
exclude octopine-type Ti plasmids and vice versa. The octo-
pine-type Ti plasmid present in WCF5(pJZ381) conjugated
approximately 60-fold less efficiently into a strain containing a
nopaline-type Ti plasmid and expressing TraR than into a
congenic strain not expressing TraR [Table 3, lines for heter-
ologous recipients C58(pPZP201) and C58(pHC335)]. Similar
results were obtained with the reciprocal cross (Table 3, last
two lines for heterologous recipients). In the first cross, entry
exclusion appeared to be slightly weaker than that in either
homologous cross [Table 3, compare line for heterologous
recipient C58(pHC335) with lines for homologous recipients
R10(pHC335) and C58(pHC335)], suggesting that entry exclu-
sion determinants of the nopaline-type Ti plasmid may func-
tion more effectively in blocking a homologous donor than in
blocking a heterologous one. For the second cross [Table 3,
line for heterologous recipient R10(pHC335)], no such con-
clusion is possible. No transconjugant colonies were detected,
suggesting very strong entry exclusion. However, relatively few
transconjugants were detected with the negative control [Table
3, line for heterologous recipient R10(pPZP201)], suggesting
either that TraR-independent entry exclusion acted in the re-
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TABLE 1. Strains and plasmids used in this study
Strain or plasmid(s)Descriptiona
Source and/or
reference
Strains
WCF5
R10
C58
C58C1RS
HC158
R10 traR traR-lacZ Kmr
Octopine type strain; pTiR10
Nopaline type strain; pTiC58
Ti plasmid-less derivative of C58; RifrSmr
C58 containing pHC320 inserted into the nopaline-type Ti plasmid pTiC58 by Campbell-type
integration; traR traR-lacZ Kmr
R10 cured of pTiR10 and containing cosmid pYDH902
Strain with polar mutation of trbD by the insertion of pHC327
Strain with polar mutation of trbJ by the insertion of pHC328
Strain with polar mutation of trbK by the insertion of pHC329
Strain with polar mutation of trbF by the insertion of pHC330
6
S. K. Farrand
S. K. Farrand
S. K. Farrand
This study
HC159(pYDH902)
HC161
HC162
HC163
HC164
This study
This study
This study
This study
This study
Plasmids
pCF218
pBBR1MCS5
pVIK111
pKNG101
pPR1068
PtetR-traR Tcrrep-RP4
Broad-host-range vector; rep-pBRR1 Gmr
Carries promoterless lacZ; oriR6K Kmr
sacB?Smrrep-R6K oriT-RP4
pMAL2 derivative with NdeI site at ATG codon of malE; Ptac-MBP-lacZ? lacIqApr
ori-ColE1
Broad-host-range vectors; rep-pVS1 Spr
traR from pTiA6NC cloned into pPZP201
EcoRI fragment containing traR cloned into pBBR1MCS5
Cosmid containing rep and traI-trb operons; rep-RP4 Tcr
pPR1068 digested with NdeI and SacI, with replacement by a linker containing
NdeI-KpnI-SacI sites to delete the malE gene; Ptac is fused to
NdeI-KpnI-SacI-AvaI-XmnI-EcoRI-BamHI-XbaI-SalI-PstI-HindIII; rep-ColE1 Apr
pHC011 digested with EcoRV and KpnI and ligated to pBBRMCS5 after digestion with SphI
and KpnI, with 3?-end fill-in of the SphI site with the Klenow fragment of DNA polymerase
I; Ptac is fused to NdeI-KpnI-ApaI-XhoI-SalI-Bsp106I-ClaI-HindIII-EcoRI-PstI-SmaI-
BamHI-SpeI-XbaI-BstXI-SacI; rep-pBBR1 Gmr
pVIK111 containing an EcoRI-XbaI fragment including the 5? end of traR and upstream
sequences; rep-R6K Kmr
PCR fragment of trbD made using oligonucleotides TDF and TDR and cloned into pKNG101
for Campbell recombination mutagenesis; rep-R6K Smr
PCR fragment of trbJ made using oligonucleotides TJF and TJR and cloned into pKNG101
for Campbell recombination mutagenesis; rep-R6K Smr
PCR fragment of trbK made using oligonucleotides TKF and TKR and cloned into pKNG101
for Campbell recombination mutagenesis; rep-R6K Smr
PCR fragment of trbF made using oligonucleotides TFF and TFR and cloned into pKNG101
for Campbell recombination mutagenesis; rep-R6K Smr
pJZ335 digested with BamHI and ligated to remove a small BamHI fragment between Plac
and traR; carries traR cloned into pPZP201; rep-pVS1 Spr
PCR fragment containing trbJK made using oligonucleotides TrbJKF-N and R10-trbKJK3,
digested with BamHI, and cloned into pHC012; Ptac-trbJK rep-pBBR1 Gmr
trbBCDEJK cloned as a HindIII-BamHI fragment into pHC012; Ptac-trbBCDEJK
rep-pBBR1 Gmr
PCR fragment containing trbK made using oligonucleotides R10-trbKF-N and R10-trbKJK3,
digested with BamHI, and cloned into pHC012; Ptac-trbK rep-pBBR1 Gmr
1,236-nucleotide DNA fragment made by PCR amplification using pHC012 as the template
and oligonucleotides MfeI-For and NsiI-Rev as primers, cloned into the EcoRI-PstI gap of
pPZP200, with the Ptac promoter upstream of the multiple-cloning site of pHC012;
rep-pVS1 Gmr
PCR fragment containing trbJ made using oligonucleotides TrbJKF-N and TrbJR-N and
cloned into pHC012; Ptac-trbJ rep-pVS1 Gmr
PCR fragment containing trbJ made using oligonucleotides TrbJKF-N and TrbJR-N and
cloned into pUP200; Ptac-trbJ rep-pVS1 Gmr
trbK from pHC368 cloned into pUP200; Ptac-trbK rep-pVS1 Gmr
Derivative of pHC368 lacking the 3? codon of trbK
Derivative of pUP404 lacking the 3? codon of trbK
Derivative of pUP402 lacking the 3? codon of trbJ
Derivative of pUP403 lacking the 3? codon of trbJ
6
14
12
13
Paul Riggs (20)
pPZP200 and pPZP201
pJZ335
pJZ381
pYDH902
pHC011
10
26
2
4
This study
pHC012 This study
pHC320This study
pHC327
This study
pHC328 This study
pHC329 This study
pHC330This study
pHC335This study
pHC361This study
pHC364
This study
pHC368This study
pUP200This study
pUP402This study
pUP403This study
pUP404
pUP405
pUP406
pUP407
pUP408
This study
This study
This study
This study
This study
aMBP, maltose-binding protein.
VOL. 191, 2009TRANSSEXUALITY IN THE RHIZOSPHERE 3377
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cipient to block entry or that the plasmid from the donor
conjugated inefficiently into this recipient.
To confirm that TraR mediates entry exclusion indirectly, we
measured conjugation using recipient strains lacking Ti plas-
mids. Strains C58C1RS(pHC335) and C58C1RS(pPZP201)
lack Ti plasmids, and the former strain expresses TraR while
the latter one does not. Neither strain excluded the entry of
either Ti plasmid (Table 3, lines for recipients lacking a Ti
TABLE 2. Oligonucleotides used in this study
OligonucleotideSequencea
Oligonucleotides used to make polar mutations in trb genes
TDF ...........................................................................................................5?-GCTGGATCCATCGCTGGTGCGATGCTG-3?
TDR...........................................................................................................5?-GCTTCTAGAACCATCTCGACCCCTTCAG-3?
TJF.............................................................................................................5?-GCTGGATCCAATGGGCAATGTCGAAGATG-3?
TJR............................................................................................................5?-GCTTCTAGAGCGCGAGAATGACGATCAG-3?
TKF............................................................................................................5?-GCTTCTAGAGCAGGCACAAAAGGATCTG-3?
TKR...........................................................................................................5?-CGGCGATACCGACCTCGATG-3?
TFF............................................................................................................5?-GCTGGATCCTCATCCCCTACATCGTTGAG-3?
TFR............................................................................................................5?-GCTTCTAGACCATGCCTTTCAAAGCTGTG-3?
Oligonucleotides used to subclone trb genes
TrbJKF-N..................................................................................................5?-GCTGAATTCGCAAAGGGGGATCGCCCATG-3?
TrbJR-N....................................................................................................5?-GTCGGATCCGAGAATGACGATCAGACGCG-3?
R10-trbKF-N.............................................................................................5?-GCTGAATTCGACGATGGAGCCACGCTGGTG-3?
R10-trbKJK3.............................................................................................5?-ATGAACATGATGCGTTTGAC-3?
Oligonucleotides used to clone Ptac-lacZ on pPZP200
MfeI-For....................................................................................................5?-GTCCAATTGTATACGCAAGGCGACAAGGTG-3?
NsiI-Rev ....................................................................................................5?-GTCATGCATACTTATTCAGGCGTAGCACCA-3?
Oligonucleotides used for mutagenesis
pJ_W269Stop-F........................................................................................5?-GAGCCACGCTGATGAGCTCGC-3?
pJ_W269Stop-R........................................................................................5?-GCGAGCTCATCAGCGTGGCTC-3?
pK_W75Stop-F.........................................................................................5?-GAAACCGAGATGATAGTTCACC-3?
pK_W75Stop-R ........................................................................................5?-GGTGAACTATCATCTCGGTTTC-3?
pTacR1......................................................................................................5?-ACGACGTTGTAAAACGACGGC-3?
pTacR2......................................................................................................5?-GCCATTCAGGCTGCGCAACTG-3?
Oligonucleotides used for nuclease S1 protection assay
trbKS1........................................................................................................5?-GGCTGGACAGTAATCCAGGTGCCGATGCCTGCACTACGAC-3?
23SRNAS1................................................................................................5?-AGGCTCGGGCTCCGACTGTTTGTAGGCATCCGGTTTCAG-3?
aItalics indicate restriction endonuclease cleavage sites used in plasmid construction.
TABLE 3. Entry exclusion of octopine-type and nopaline-type Ti plasmids by homologous and heterologous recipients
RecipientDonor Relevant protein(s) expressed in recipient
No. of transconjugantsa
per donor (SD)
Exclusion
coefficientb
Homologous recipients
R10(pPZP201)
R10(pHC335)
C58(pPZP201)
C58(pHC335)
WCF5(pJZ381)c
WCF5(pJZ381)
HC158(pJZ381)d
HC158(pJZ381)
None
TraR, TraI, TraA to TraH, TrbB to TrbL
None
TraR, TraI, TraA to TraH, TrbB to TrbL
0.94 (0.2)
0.003 (0.001)
0.22 (0.03)
0.0008 (0.0004)
1
313
1
275
Heterologous recipients
C58(pPZP201)
C58(pHC335)
R10(pPZP201)
R10(pHC335)
WCF5(pJZ381)
WCF5(pJZ381)
HC158(pJZ381)
HC158(pJZ381)
None
TraR, TraI, TraA to TraH, TrbB to TrbL
None
TraR, TraI, TraA to TraH, TrbB to TrbL
0.032 (0.01)
0.005 (0.002)
0.001 (0.001)
?0.00001e
1
64
1
?100
Recipients lacking a Ti plasmid
C58C1RS(pPZP201)
C58C1RS(pHC335)
C58C1RS(pPZP201)
C58C1RS(pHC335)
WCF5(pJZ381)
WCF5(pJZ381)
HC158(pJZ381)
HC158(pJZ381)
None
TraR
None
TraR
0.47 (0.22)
0.55 (0.08)
0.35 (0.035)
0.42 (0.13)
1
0.85
1
0.8
aTransconjugants were selected using the Kmrgene of the Ti plasmid and the Sprgene of pPZP201 or pHC335. In mock conjugations, we did not detect spontaneous
resistance to either kanamycin or spectinomycin. The data are the averages of results from three independent experiments, with the standard deviations shown in parentheses.
bThe exclusion coefficient is the number of transconjugants per donor for the no-exclusion control (lines with exclusion coefficients of 1) divided by the number of
transconjugants of the tested recipient strain per donor.
cR10-derived strain containing a Kmrgene on the octopine-type Ti plasmid.
dC58-derived strain containing a Kmrgene on the nopaline-type Ti plasmid.
eNo transconjugants were detected in an assay mixture containing 100,000 donor bacteria.
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plasmid), indicating that TraR is not sufficient for entry exclu-
sion and that it functions by activating one or more entry
exclusion genes.
These data also allow us to compare a strain containing a Ti
plasmid but lacking TraR with a strain lacking a Ti plasmid.
Strains C58(pPZP201) and C58C1RS(pPZP201) are identical
except for the presence or absence of a Ti plasmid. Neither
strain overexpresses TraR. These two strains showed little if
any difference in their inability to exclude either Ti plasmid
(Table 3, compare the first line with the fourth-to-last line and
the third line with the second-to-last line). This finding indi-
cates that entry exclusion determinants are not significantly
expressed in the absence of active TraR.
Identification of the entry exclusion determinants encoded
in the Ti plasmid. As described above, plasmid RP4 has a trb
operon that resembles those of Ti plasmids (Fig. 1). Within the
RP4 operon, the trbK gene encodes a product required for
entry exclusion (8, 9, 15, 18), while the trbJ product may (15,
18) or may not (8, 9) play an accessory role. TrbK of RP4 is
23.5 and 18.2% identical to the TrbK proteins of octopine- and
nopaline-type Ti plasmids, respectively, while TrbJ of RP4 is
20.7% identical to both Ti plasmid TrbJ proteins. TrbK pro-
teins of Ti plasmids lack the acylation site of TrbK of RP4,
suggesting that they may be nonfunctional or weakly func-
tional. Both TrbJ and TrbK were strongly predicted by the
program SignalP-HMM to have cleaved signal sequences
(probability, 1.0).
The cleavage of TrbJ was predicted to remove 33 residues,
while the cleavage of TrbK was predicted to remove 21 resi-
dues.
We sought to determine whether TrbK and/or TrbJ of an
octopine-type Ti plasmid plays a role in entry exclusion. To
address this question, we compared strain R10(pHC335), which
contains a native octopine-type Ti plasmid and overexpresses
TraR, with strain HC159(pYDH902)(pHC335), which lacks
the Ti plasmid, overexpresses TraR, and contains a cosmid
(pYDH902) that carries the trb and rep operons (4). The donor
strain in this experiment was WCF5(pJZ381). Both recipient
strains exhibited entry exclusion, and in both cases, TraR overex-
pression was required (Table 4, first four lines). A similar strain,
HC159(pYDH902)(pPZP201), which does not express TraR,
showed a low but detectable level of exclusion [Table 4, lines for
R10(pPZP201) and HC159(pYDH902)(pPZP201)], due possibly
to elevated basal expression of entry exclusion determinants from
the multicopy plasmid pYDH902. These data indicate that all
FIG. 1. Genetic organization of the trb operon of an octopine-type
Ti plasmid. Short thick lines above the genetic map represent DNA
fragments that were used in suicide plasmids to create transcriptionally
polar mutations upon Campbell-type integration. Fragments of the trb
region overexpressed by fusion to the Ptac promoter are shown be-
neath the genetic map.
TABLE 4. Mapping of the entry exclusion locus of the Ti plasmid by using polar insertion mutations within the trb operon
Recipienta
Relevant protein(s) expressed in recipient
Conjugation
efficiencyb(SD)
Exclusion
coefficientc
Recipients without trb mutations
R10(pPZP201)
R10(pHC335)
HC159(pYDH902)(pPZP201)
HC159(pYDH902)(pHC335)
None (vector control)
TraR, TraI, TraA to TraH, TrbB to TrbL
None
TraR, TraI, Trb
0.90 (0.1)
0.006 (0.0007)
0.11 (0.015)
0.0005 (0.00008)
1
150
8.2
1800
Recipients with trb genes mutated using
transcriptionally polar insertion mutations
HC161(pHC335)
HC162(pHC335)
HC163(pHC335)
HC164(pHC335)
TraR, TrbBCD
TraR, TrbBCDEJ
TraR, TrbBCDEJK
TraR, TrbBCDEJKLF
0.84 (0.1)
0.19 (0.02)
0.007 (0.001)
0.007 (0.001)
1.07
4.7
129
129
Recipients with Trb proteins expressed from a
multicopy plasmid via a tac promoter
R10(pHC012)
R10(pJZ381)
R10(pHC364)
R10(pHC361)
R10(pHC368)
None (vector control)
TraR, TraI, TraA to TraH, TrbB to TrbL
TrbBCDEJK
TrbJK
TrbK
0.18 (0.02)
0.0004 (0.0002)
0.008 (0.004)
0.0012 (0.0005)
0.025 (0.006)
1
450
22.5
150
7.2
aThe donor strain in each experiment was WCF5(pJZ358), which overexpresses TraR and has an octopine-type Ti plasmid marked with a Kmrdeterminant (6).
Transconjugants were selected using the Kmrgene of the Ti plasmid and the Sprgene of pPZP201 or pHC335 (first eight lines) or using the Gmrgene of pHC012 and
its derivatives (last four lines).
bNumber of transconjugants per donor.
cThe exclusion coefficient is the number of transconjugants per donor for the no-exclusion control (lines with exclusion coefficients of 1) divided by the number of
transconjugants of the tested recipient strain per donor.
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genes essential for entry exclusion lie within pYDH902 and prob-
ably within the trb operon.
To more closely localize the genes responsible for entry
exclusion, we constructed four insertion mutations in the trb
operon that are predicted to exert strong transcriptional po-
larity effects on downstream genes. We used derivatives of the
suicide plasmid pKNG101 containing various trb fragments.
The insertion in HC161 expresses TrbB, TrbC, and TrbD but
not TrbE, TrbJ, TrbK, TrbL, TrbF, TrbG, TrbH, or TrbI (Fig.
1). This mutation blocked virtually all entry exclusion [Table 4,
line for HC161(pHC335)]. Strain HC162(pHC335) expresses
TrbB, TrbC, TrbD, TrbE, and TrbJ and showed approximately
fourfold fewer transconjugants than the negative control (Ta-
ble 4), suggesting a role for TrbJ and/or TrbE in entry exclu-
sion. Strain HC163(pHC335), which expresses TrbB, TrbC,
TrbD, TrbE, TrbJ, and TrbK (Fig. 1), strongly expressed entry
exclusion (Table 4), indicating that TrbK plays a major role.
This strain expressed entry exclusion levels similar to those
expressed by HC164(pHC335), which expresses two additional
Trb proteins, and by R10(pHC335), which expresses all Trb
proteins (Table 4), suggesting that the genes downstream of
trbK do not have any role in entry exclusion.
We also tested the expression of Trb proteins from a Ptac
promoter of a multicopy plasmid. Plasmid pHC364 expresses
TrbB, TrbC, TrbD, TrbE, TrbJ, and TrbK (Fig. 1) and ex-
pressed entry exclusion, albeit at a reduced level [Table 4, lines
for R10(pJZ381) and R10(pHC364)] compared to that caused
by overexpression of TraR on pJZ381. Plasmid pHC361, which
expresses only TrbJ and TrbK (Fig. 1), expressed high levels of
entry exclusion, while plasmid pHC368, which expresses only
TrbK, expressed a low level of entry exclusion (Table 4, last
two lines).
To further measure the effects of TrbJ and TrbK on entry
exclusion, we expressed these proteins using separate, compat-
ible plasmids in recipient strains. We made a series of fusions
using plasmids pHC012 and pUP200, both of which have Ptac
promoters and lacZ? genes. Ptac-trbJ fusions were constructed
in such a way that the lacZ? gene was translationally fused to
the stop codon of trbE (which lies immediately upstream of trbJ
in the native Ti plasmid). This was done to mimic any possible
translational coupling between trbE and trbJ. Similarly, Ptac-
trbK fusions were made in such a way that the lacZ? gene was
translationally fused to the stop codon of trbJ.
Expressing TrbJ alone from a derivative of pBBRMCS5
(pUP402) decreased conjugation approximately ninefold, while
expressing it from a derivative of pPZP200 (pUP403) caused a
fourfold decrease (Table 5, third and fourth lines). This dif-
ference is most likely attributable to a difference in copy num-
ber, as the Ptac-trbJ fusions of the two plasmids are identical in
sequence. The expression of TrbK alone in these two vectors
caused similar decreases in conjugation (Table 5, fifth and sixth
lines). Most importantly, coexpressing these two proteins from
compatible plasmids caused a strong additional decrease in
conjugation (Table 5, seventh and eighth lines). We conclude
that TrbJ and TrbK make independent contributions to entry
exclusion and that the presence of both proteins has a syner-
gistic effect.
Interestingly, a strain expressing TrbJ and TrbK from sepa-
rate plasmids showed less entry exclusion than a strain express-
ing these proteins from a single plasmid (Table 5, last three
lines). To ensure that TrbK was expressed at similar levels in
these strains, we assayed for the accumulation of TrbK mRNA.
Plasmid pHC368, which has a Ptac-trbK fusion, expressed con-
siderably more TrbK mRNA than pHC361, which has a Ptac-
FIG. 2. Results of nuclease S1 protection assays showing trbK tran-
script levels in recipients containing fusions between Ptac and the
indicated trb genes (top) and rpoD transcript levels for each strain
(bottom). All strains are derivatives of A. tumefaciens strain R10, which
contains pTiR10. Plasmid pJZ381 carries the trbK gene in the Ti
plasmid background.
TABLE 5. Expression of TrbJ and TrbK in recipients of multicopy
plasmidsa
pBBRMCS5 derivative
(description or
genotype)
pPZP200 derivative
(description or
genotype)
Conjugation
efficiencyb(SD)
Exclusion
coefficientc
pHC012 (vector)
pJZ381 (traR)d
pUP200 (vector)
pPZP200 (vector)
0.69 (0.2)
0.002 (0.001)
1
345
pUP402 (Ptac-trbJ)
pHC012 (vector)
pUP200 (vector)
pUP403 (Ptac-trbJ)
0.08 (0.009)
0.18 (0.03)
8.6
3.8
pHC368 (Ptac-trbK)
pHC012 (vector)
pUP200 (vector)
pUP404 (Ptac-trbK) 0.21 (0.04)
0.10 (0.04) 6.9
3.3
pUP402 (Ptac-trbJ)
pHC368 (Ptac-trbK)
pUP404 (Ptac-trbK) 0.007 (0.002)
pUP403 (Ptac-trbJ)
98.6
31.40.022 (0.004)
pHC361 (Ptac-trbJK) pPZP200 (vector)0.002 (0.0008)345
aThe donor strain in each experiment was WCF5(pCF218), which overex-
presses TraR and has an octopine-type Ti plasmid marked with a Kmrdetermi-
nant. Transconjugants were selected using the Kmrgene of the Ti plasmid, the
Sprgene of pUP200 or its derivatives, and the Gmrgene of pHC012 or its
derivatives.
bNumber of transconjugants per donor.
cThe exclusion coefficient is the number of transconjugants per donor for the
no-exclusion control (top line) divided by the number of transconjugants of the
tested recipient strain per donor.
dThe overexpression of TraR by pJZ381 induces the expression of all tra and
trb genes (6).
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trbJK fusion (Fig. 2). Despite this result, the former plasmid
expressed entry exclusion more weakly than the latter plasmid.
This finding underscores the importance of TrbJ in this process
and supports the conclusion that these proteins act preferen-
tially in cis.
In the course of searching for proteins homologous to TrbK,
we fortuitously noticed sequence similarity between TrbK and
TrbJ. The C-terminal 12 amino acid residues of these proteins
are identical or similar (Fig. 3). This similarity is found among
a variety of plasmids of Agrobacterium, Rhizobium, and Sino-
rhizobium and in two plasmids found in Nitrobacter hambur-
gensis and Oligotropha carboxidovorans (Fig. 3). The latter two
bacteria express TrbJ and TrbK proteins that are strongly
similar to those of A. tumefaciens and its close relatives. The
last amino acid residues of the proteins show remarkable con-
servation, even in more distantly related proteins from the
IncP-type plasmids. A small number of other cognate TrbJ and
TrbK proteins also show sequence similarities at their C ter-
mini, but the majority do not (Fig. 3 and data not shown).
Since the last amino acid residues of TrbJ and TrbK, both
tryptophans, are completely conserved not only in all Ti plas-
mid proteins but also in more distantly related proteins from
other plasmids, it seemed plausible that these residues may
play a crucial role in protein function. To test the functional
importance of the similar C termini of TrbK and TrbJ, we
deleted the last amino acid residue by using site-directed mu-
tagenesis and tested the mutated proteins for their roles in
entry exclusion. A truncated TrbK protein had a virtually null
phenotype when expressed alone and had little if any synergis-
tic effect when coexpressed with TrbJ (Table 6). The corre-
sponding mutation on TrbJ also had a strong impact on the
ability of the protein to mediate entry exclusion, though the
mutant TrbJ still mediated a low level of exclusion when ex-
pressed together with wild-type TrbK (Table 6, 2nd, 3rd, 6th,
7th, 11th, and 12th lines). When both mutant proteins were
expressed together in the cell, entry exclusion was negligible
compared to that mediated by wild-type proteins (Table 6, last
four lines). Overall, these results confirm the prediction that
the C termini of the two proteins play a crucial role in entry
exclusion.
DISCUSSION
It is well established that strains lacking active TraR do not
conjugate or conjugate at extremely low levels (6, 11, 21). We
now show that such strains also do not express entry exclusion
functions and therefore readily act as conjugative recipients
of Ti plasmids. The fact that C58(pPZP201) and C58C1
(pPZP201) were virtually identical in their abilities to receive a
Ti plasmid, even though the former has a Ti plasmid while the
latter lacks it, indicates that entry exclusion genes are tightly
regulated. Strains containing conjugal plasmids but not ex-
pressing conjugation or entry exclusion functions are some-
times referred to as “female phenocopies” (22). Female phe-
nocopies are generally detected after long-term culturing of a
strain at stationary phase. In the case of A. tumefaciens, cul-
tures that do not express active TraR are female phenocopies,
even when actively growing, a consequence of the extremely
tight regulation of the tra-trb regulon.
The finding that TrbJ and TrbK mediate entry exclusion was
initially surprising. On the one hand, TrbJ and TrbK of RP4
have been described previously as mediating this property.
However, as described above, there is considerable controversy
about the role of TrbJ (8, 9, 15, 18). Furthermore, A. tumefa-
ciens TrbK lacks a cysteine residue that is critical for the
normal function of the RP4 protein, suggesting that trbK of A.
tumefaciens may be a pseudogene. It seemed plausible that A.
tumefaciens might not exhibit entry exclusion, as described
above. Finally, it seemed counterintuitive for entry exclusion
functions to be encoded within a tightly regulated operon. One
may imagine a priori that exclusion genes may be needed even
when the Tra-Trb regulon is not expressed, and it would seem
a simple evolutionary step for these genes to be expressed
constitutively.
As described above, pHC368, which expresses just TrbK,
makes considerably more TrbK mRNA than pHC361, which
expresses TrbJ and TrbK (Fig. 2). Despite this fact, the former
plasmid expresses the entry exclusion phenotype more weakly
than the latter. This finding highlights the importance of TrbJ
in entry exclusion. However, pHC368 expresses entry exclusion
more weakly than pHC361 even in the presence of a second
plasmid expressing TrbJ (Table 5, lines for pHC368 and
pHC361). The Ptac-trbJ fusions in pHC361, pUP402, and
pUP403 are identical, making it unlikely that TrbJ is expressed
at greatly different levels by these three plasmids. The most
likely interpretation is that TrbJ and TrbK function more ef-
fectively when expressed in cis than in trans. An alternative
interpretation is that TrbJ and TrbK interact and do so more
effectively if expressed at the same location.
As noted earlier, we found a curious sequence similarity
between the C termini of TrbJ and TrbK. Mature TrbK pro-
teins are predicted to be quite small, approximately 50 amino
acid residues in length, and the C-terminal 15 residues there-
fore constitute a rather large fraction of the entire protein. The
C termini of TrbK proteins are also far more conserved than
other parts of these proteins (data not shown), suggesting that
the C-terminal residues may be crucial for protein function. In
some cases, a TrbK protein from one plasmid may resemble
TrbJ from the same plasmid more strongly than it resembles
TrbK proteins from other plasmids (Fig. 3). This pattern sug-
gests that the TrbJ and TrbK proteins encoded by a particular
plasmid may coevolve by a process resembling gene conver-
sion. In light of the overlapping functions of TrbJ and TrbK, it
seemed tempting to speculate that the C termini of both pro-
teins may play a crucial role in entry exclusion. In fact, the
results of deleting the last amino acid residues of both proteins
confirmed this hypothesis (Table 6). It may be noteworthy that
the C-terminal five amino acid residues of TrbK of RP4 are
essential for activity (8). Interestingly, our results show that
TrbK protein cannot tolerate a truncation eliminating its last
amino acid residue. However, TrbJ can still function in the
presence of wild-type TrbK, albeit rather poorly. These results
also suggest that these two proteins may interact for proper
function, but this hypothesis remains to be tested.
The finding that a bacterium having a Ti plasmid but not
expressing the Tra-Trb regulon is a female phenocopy may
imply interesting ecological consequences. For example, one
could imagine a situation in which two strains of A. tumefa-
ciens, one containing an octopine-type Ti plasmid similar to
pTiA6 and the other having a nopaline-type Ti plasmid similar
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FIG. 3. Alignment of the C termini of TrbJ and TrbK proteins of selected conjugation systems. Sequence similarities between TrbJ and TrbK pairs
were obtained using the MegAlign program (DNASTAR). Colons indicate conservative substitutions. R. leguminosarum, Rhizobium leguminosarum; A.
rhizogenes, Agrobacterium rhizogenes; R. etli, Rhizobium etli; E. coli, Escherichia coli.
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to pTiC58, colonize the same crown gall tumor. One could
imagine furthermore that there is an abundance of octopine
but very little or no agrocinopines A and B (the conjugal
opines for pTiC58). Conjugal opines are required for conjuga-
tion, as they are required for the transcription of both traR
genes (6, 21). In such a scenario, the octopine-type Ti plasmid
would both conjugate and block the entry of a nopaline-type Ti
plasmid, while the nopaline-type Ti plasmid would do neither.
If an octopine-type Ti plasmid conjugated into a strain already
containing a nopaline-type Ti plasmid, the transconjugants
would contain both Ti plasmids. These plasmids are incompat-
ible at the level of DNA replication and would segregate into
different daughter cells upon cell division. As a result, new
combinations of host strains and Ti plasmids may appear.
Thus, an active entry exclusion system would prevent the futile
transfer of Ti plasmids between identical strains but would
allow the reassortment of Ti plasmids and heterologous host
strains even if those strains already contained heterologous Ti
plasmids.
ACKNOWLEDGMENTS
We thank the members of our laboratory for helpful discussions and
for critical reviewing of the text.
This work was supported by a grant from the National Institute of
General Medical Sciences (GM42893). U.M.P. acknowledges the fi-
nancial support of the Brazilian government through a fellowship grant
from the Coordenac ¸a ˜o de Aperfeic ¸oamento de Pessoal de Nível Su-
perior (Capes).
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TABLE 6. Effects of deleting the C-terminal Trp residues of TrbJ
and TrbKa
pBBRMCS5 derivative
(description or
genotype)
pPZP200 derivative
(description or
genotype)
Conjugation
efficiencyb
(SD)
Exclusion
coefficient
pHC012 (vector)
pHC012 (vector)
pHC012 (vector)
pHC012 (vector)
pHC012 (vector)
pUP402 (Ptac-trbJ)
pUP407 (Ptac-trbJ*)
pHC368 (Ptac-trbK)
pUP405 (Ptac-trbK*) pUP200 (vector)
pUP402 (Ptac-trbJ)
pUP407 (Ptac-trbJ*)
pHC368 (Ptac-trbK)
pUP405 (Ptac-trbK*) pUP403 (Ptac-trbJ)
pUP407 (Ptac-trbJ*)
pUP405 (Ptac-trbK*) pUP408 (Ptac-trbJ*)
pUP402 (Ptac-trbJ)
pHC368 (Ptac-trbK)
pUP200 (vector)
pUP403 (Ptac-trbJ)
pUP408 (Ptac-trbJ*)
pUP404 (Ptac-trbK)
pUP406 (Ptac-trbK*) 1.55 (0.69)
pUP200 (vector)
pUP200 (vector)
pUP200 (vector)
2.62 (0.88)
0.81 (0.68)
1.66 (0.51)
0.26 (0.06)
1
3.2
1.6
10.0
1.7
7.4
4.2
8.5
1.7
15.2
28.4
12.7
5.9
5.1
1.9
143.9
47.6
0.35 (0.23)
0.63 (0.22)
0.31 (0.14)
1.58 (0.68)
pUP406 (Ptac-trbK*) 0.17 (0.07)
pUP404 (Ptac-trbK)
pUP408 (Ptac-trbJ*)
0.09 (0.04)
0.21 (0.08)
0.44 (0.12)
pUP406 (Ptac-trbK*) 0.52 (0.26)
1.36 (0.07)
0.018 (0.008)
0.055 (0.018)
pUP404 (Ptac-trbK)
pUP403 (Ptac-trbJ)
aThe donor strain in each experiment was WCF5(pCF218), which overex-
presses TraR and has an octopine-type Ti plasmid marked with a Kmrdetermi-
nant. Transconjugants were selected using the Kmrgene of the Ti plasmid, the
Sprgene of pUP200 or its derivatives, and the Gmrgene of pHC012 or its
derivatives. The symbol * denotes a deletion of the last residue of the corre-
sponding protein.
bNumber of transconjugants per donor.
VOL. 191, 2009TRANSSEXUALITY IN THE RHIZOSPHERE3383
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