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INTRODUCTION |
The Ti plasmids of the plant
pathogen Agrobacterium tumefaciens possess two DNA transfer
systems. The first, called Vir, mediates the transfer of a segment of
Ti plasmid DNA called T-DNA to the cells of a susceptible host plant.
The T-DNA then incorporates into the plant chromosome, and the genes it
encodes, when expressed, cause the unregulated growth of the plant
cells that leads to the formation of a crown gall tumor. While it
mediates trans-kingdom DNA transfer, the Vir system
functions in a manner similar to that of the bacterium-to-bacterium
transfer of conjugal plasmids (9, 34, 41, 67). Consistent
with this, the Vir system also can mediate the transfer of DNA to
bacteria (6). However, the conjugal transfer of the Ti
plasmid between bacteria normally is dependent upon the second DNA
transfer system, called Tra. Expression of the genes of the Tra system
is tightly regulated through a complex circuitry that involves opines
produced by the crown gall tumors, as well as a LuxR-LuxI type
quorum-sensing regulatory mechanism (4, 18, 26, 35, 36, 48).
Genetic and sequence analysis indicates that the Tra system is
physically separated and functionally independent from the Vir system
(15).
Beck von Bodman et al. localized the conjugal transfer system of the
nopaline-type Ti plasmid pTiC58 to three distinct regions, tra1, tra2, and tra3 by Tn5
mutagenesis and trans-complementation (5).
Mutations in any of these regions abolished conjugal transfer. The
tra1 region contains only a single gene, traR,
required for conjugal transfer (49). This gene codes for a
homologue of LuxR and is the transcriptional activator responsible for
the initiation of expression of the tra genes (4,
48). The tra2 region, now called tra,
contains the origin of conjugal transfer (oriT) and two sets
of genes organized as divergently expressed operons (14,
19). Three of the six genes flanking the oriT region are homologous to essential tra genes from IncP and IncQ
plasmids. The products of some of these genes comprise the DNA transfer and replication function (Dtr) of the Ti plasmid Tra system and most
probably are responsible for the formation of the relaxosome complex at
the oriT site. With the possible exception of
traF, which might code for a pilin processing protease
(30), tra does not code for any identifiable
mating-pair formation (Mpf) functions.
The tra3 region of pTiC58, is located at the 2-o'clock
position on the plasmid and is flanked by noc, the locus
conferring catabolism of nopaline and oriV/rep, the locus
for vegetative replication (Fig. 1). In
our previous studies we isolated six Tn5 insertions spanning
approximately 3 kb within this region, all of which abolished conjugal
transfer of the Ti plasmid (5) (see Fig. 2C). However, the
region between noc and oriV/rep occupies more
than 10 kb. Moreover, when compared to other systems, such as the Vir
system of the Ti plasmid and the Tra system of IncP plasmids, a 3-kb
region seems inadequate to encode the remaining Mpf functions that are
essential to a conjugal transfer system. Our study of the
Agrobacterium autoinducer (AAI) synthesis gene, traI, which is located in the region between noc
and oriV/rep, also identified a partial open reading frame
(ORF) just downstream from traI (36). This ORF
showed significant similarity to trbB of RP4, which is one
of the essential genes of the IncP Mpf system. traI and this
partial ORF are located about 5 kb upstream from the Tn5
insertion mutations defining the minimal trb region
(5). This finding led us to propose that the tra3
region of pTiC58 extends beyond 3 kb and encodes the Mpf function of
the Ti plasmid conjugal transfer system. Sequence analysis of a
corresponding region of the octopine-type Ti plasmid
(1) and our recent description of a binary transfer system
we developed to characterize the tra region of the
nopaline-type Ti plasmid (15) further supported this
hypothesis.
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FIG. 1.
Functional map of pTiC58. The outer arcs represent the
Ti plasmid DNA inserted in clones pTHH6 and pPLE2. The inner boxed arcs
indicate two of the four regions of heteroduplex homology between the
nopaline-type Ti plasmid and the octopine-type Ti plasmid as defined by
Engler et al. (17). The shaded arc, labeled B, corresponds
to the replication region, and the region that we have now defined as
trb. The stippled arcs represent region C and code for the
tra-Tra regulatory region (1, 19, 49).
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In this study we delineated the tra3 region of the
nopaline-type Ti plasmid pTiC58 by transposon mutagenesis and
trans-complementation. DNA sequence analysis
identified this as the trb region of the Ti plasmid.
Coupled with our previous findings with the binary transfer system
(15), these results indicate that this locus of pTiC58 is
responsible for the Mpf functions of the Ti plasmid conjugal transfer system.
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MATERIALS AND METHODS |
Bacterial strains, growth conditions, and plasmids.
Strains
of Agrobacterium tumefaciens and Escherichia
coli, bacteriophages, and the plasmids used in this study are
listed in Table 1. A. tumefaciens strains were grown at 28°C in L broth (LB)
(53), in AB minimal medium (11), or on nutrient
agar plates (Difco Laboratories, Detroit, Mich.). Mannitol or glucose, at a final concentration of 0.2%, was used as the sole carbon source
in the minimal medium. 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 A. tumefaciens,
carbenicillin (100 or 200 µg/ml), gentamicin (30 µg/ml),
kanamycin (Km, 100 µg/ml), rifampin (50 µg/ml),
streptomycin (200 µg/ml), and tetracycline (Tc; 2 µg/ml);
and for E. coli, ampicillin (100 µg/ml), Km (50 µg/ml), and Tc (10 µg/ml). X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside; Gibco-BRL, Bethesda, Md.) was included in the media at 40 µg/ml to assess for the production of -galactosidase.
DNA manipulation and plasmid constructions.
Ti plasmids were
isolated as described by Hayman and Farrand (32). Other
plasmids were isolated by an alkaline lysis method (53).
Standard recombinant DNA techniques were used as described by Sambrook
et al. (53). Digestions with restriction endonucleases were
conducted according to the manufacturers' instructions, and digestion
products were separated by electrophoresis in agarose gels by using
Tris-borate-EDTA buffer.
Tn3HoHo1 mutagenesis and homogenotization.
Recombinant clones were mutagenized with Tn3HoHo1,
and the sites of insertions were mapped as previously described
(19). Insertion mutations of interest on clones were
homogenotized into pTiC58
accR by using pPH1JI as the
eviction plasmid as previously described (19). Proper marker
exchanges in the Ti plasmids were confirmed by restriction endonuclease analysis.
DNA sequence analysis.
DNA fragments were sequenced by the
dideoxy method with the Sequenase kit (version 2; United States
Biochemical Co., Cleveland, Ohio) or by automated sequencing using
dye-terminator methodology by the Biotechnology Center at the
University of Illinois at Urbana-Champaign. Nucleotide sequences were
assembled and analyzed, and ORFs were identified and translated by
using the DNA Strider program (43) and the Map program of
the GCG software package (version 8.1; Genetic Computer Group, Madison,
Wis.). Nucleotide and deduced amino acid sequences were compared to
those in the databases by using the BLAST search protocol (2,
3). PileUp, Gap, and BestFit programs of the GCG package were
used to compare sequences and to identify regions conserved among
several protein sequences. The deduced amino acid sequences were
analyzed for hydropathic properties, putative motifs, or domains by
Motif, PepPlot, and PlotStructure programs of the GCG package.
Potential secretion signal sequences were identified by the
method described by von Heijine (62) and by the
SignalP V1.1 World Wide Web Prediction Server (http://www.cbs.dtu.dk/)
(46). Potential transmembrane (TM) domains were identified
by using two TM domain prediction servers, each based on a different
algorithm: PHDhtm (http://www.embl-heidelberg.de/predictprotein/) (52) and TMpred
(http://ulrec3.unil.ch/software/TMPRED_form.html) (33).
Conjugation assays.
Conjugal transfer of
pTiC58accR and of the oriT/tra plasmid,
pFRtra, of the binary transfer system to the recipient strain, A. tumefaciens C58C1RS, were conducted either by the spot plate mating method as described by Beck von Bodman et al.
(5) or by the filter mating method as described by Cook et
al. (14). Transfer frequencies were expressed as numbers of
transconjugants obtained per input donor cell.
Phage infection and adsorption assays.
About 107
exponential-phase bacteria were mixed with 3 ml of 0.7% melted agar
(1× nutrient broth), and the suspension was layered over the surface
of nutrient agar plates. Then, 10-µl volumes of serial 10-fold
dilutions of a given phage suspension (stock concentration,
~1010 PFU/ml) were spotted onto the soft agar surface,
and the plates were incubated at the respective growth temperatures of
the test strains until plaques appeared. Plaquing efficiency was
calculated by dividing the relative titer of the phage suspension as
determined on the test strains by the relative titer as determined on
the reference strain, HB101(RP4). Adsorption of phage to various
A. tumefaciens strains was assayed as follows.
Exponential-phase bacteria were mixed with phages of known
concentration at a multiplicity of infection (MOI) of 
0.1. The
mixtures were incubated for 15 min without shaking, followed by 15 min
with gentle shaking. The cells were collected by centrifugation, and
the number of phage remaining in the supernatant was determined by
plaquing on the susceptible host strain HB101(RP4). Adsorption
efficiency (
) was expressed as = (x 
n)/x, in
which x is the titer of the input phage and n is
the titer of the phage remaining in the supernatant.
Nucleotide sequence accession numbers.
The nucleotide
sequence of the trb region of pTiC58 was deposited in the
GenBank database under the accession number AF057718.
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RESULTS |
Tn3HoHo1-induced mutants of pTHH6 and the conjugation
properties of pTiC58accR homogenotized with these
mutants.
Cosmid clone pTHH6 overlaps the region previously
identified as tra3. We mutagenized this clone with
Tn3HoHo1, and 10 independent insertions were isolated and
mapped (Fig. 2A). Each of the mutations was marker exchanged in pTiC58accR as described in
Materials and Methods. Eight of the ten insertions spanning a region of about 6 kb completely abolished conjugal transfer (Fig. 2A and Table
2). Strains harboring these mutant Ti
plasmids all were able to catabolize nopaline. Insertion 11, which is
located at the leftmost end of the region, had no effect on conjugal
transfer, but it did abolish the ability of the strain to utilize
nopaline (Fig. 2A). Among the eight Tra
mutants, one,
with the lacZ gene of the insertion oriented from right to
left, expressed -galactosidase activity (Fig. 2A). The remaining
seven mutant plasmids, all with insertions oriented in the opposite
direction, did not express detectable levels of the enzyme. However,
none of the corresponding insertion derivatives of pTHH6,
regardless of their orientation, expressed -galactosidase activity
when tested in strains with or without a Ti plasmid (data not shown).
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FIG. 2.
Mutational analyses of the trb region of
pTiC58. (A) Phenotypes of 10 Tn3HoHo1 insertional mutants in
pTHH6. The indicated phenotypes are given for each insertion mutation
tested following introduction into the Trac Ti plasmid
pTiC58accR by marker exchange as described in the text.
(B) Phenotypes of 25 Tn3HoHo1 mutants of pPLE2. Assays for
conjugation were performed by using the binary transfer system
UIA143(pFRtra, pPLE2-x) as the donor and C58C1RS as the recipient, as
described by Cook et al. (15) and in Materials and Methods.
(C) The restriction map of the region between 12- and 3-o'clock on
pTiC58. The map, which is based on the nucleotide sequence of the
region, differs somewhat from that of the standard map as reported by
Depicker et al (16). The locations of Tn5
insertion mutations isolated in pTiC58accR and their
phenotypes were described by Beck von Bodman et al. (5).
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Cloning and mutagenesis of pPLE2.
During our analysis of the
traI gene, which is responsible for the synthesis of AAI
(36, 44), we also sequenced a portion of a gene,
located immediately downstream of traI, that is homologous to trbB of RP4. Based on an analysis of the mutants
described above, we cloned the 14-kb EcoRI fragment 2 from
cosmid clone pTHE9 into pDSK519 to generate pPLE2 (15).
Analysis with the binary system indicates that this clone encodes the
entire trb region (15). Furthermore, when
placed in trans, pPLE2 restored conjugal transfer to
each of the Tn3HoHo1 trb mutants of
pTiC58accR (Table 2).
We subsequently isolated and mapped 25 independent
Tn3HoHo1 insertions in pPLE2 (Fig. 2B). Attempts to
homogenotize these insertion mutations into pTiC58accR by
marker exchange were not successful. As an alternative, we utilized the
binary conjugal transfer system (15) to determine the effect
of mutations in pPLE2 on the mobilization of the tra/oriT
plasmid, pFRtra. This construct contains the tra/oriT region
of pTiC58 and the traR gene cloned in an IncP1
vector,
pRK415, and is mobilized at high frequency when a functional
trb region is provided in trans
(15). Nineteen independent pPLE2::Tn3HoHo1
insertion mutants were introduced into UIA143(pFRtra), and the
resulting strains were mated with C58C1RS. Fourteen mutants, with
insertions that span about 10 kb, abolished the conjugal mobilization
of pFRtra (Fig. 2B). Of the five mutants that retained the ability to
mobilize pFRtra, four contained inserts that mapped to the far left end
of the clone while one insert mapped to the far right end. This defined an 11-kb interval between these two sets of insertions which contains genes required for conjugal transfer.
The Tn3HoHo1 mutant derivatives of pPLE2 were introduced
into a series of A. tumefaciens strains to examine
expression from any fusions of the lacZ reporter gene to
genes within the region. These included: NT1, which lacks a Ti plasmid;
NT1(pSVB33), which expresses traR from a recombinant plasmid
(48); NT1(pTiC58accR), which contains a
transfer-constitutive derivative of pTiC58 and expresses
traR; and wild-type C58, in which traR expression
is repressed by AccR in the absence of the conjugal opines
(49). Only those clones with inserts oriented from
right to left conferred production of -galactosidase in their
host strains (Fig. 2B). Furthermore, active reporters were expressed
only when these mutant plasmids were tested in strains that
constitutively express traR (Fig. 2B and data not shown).
These results indicate that the entire trb region is
expressed from right to left, or counterclockwise on the Ti
plasmid, and that expression of this region requires TraR.
DNA sequence analysis of the trb region.
We
determined the complete double-stranded DNA sequence of an 11,003-bp
segment between the rep/oriV region (27) and the noc operon (54) by using the sequencing
strategy shown in Fig. 3. Analysis of
this sequence allowed us to make some corrections in the standard map
of pTiC58 (16) with respect to the order of
BamHI and HindIII fragments within this
region of the plasmid (Fig. 2C). The region between repA and
nocR of pTiC58 is 68% identical at the nucleotide sequence
level to the trb region of the octopine-type Ti plasmid
pTi15955 (1). This region of the Ti plasmid also is highly
related to the putative trb region of pNGR234a, the 536-kb
Sym plasmid present in Rhizobium sp. NGR234 (21).
We identified 13 complete ORFs and 1 partial ORF in this 11-kb
region. All, except the partial ORF, are translatable in the
counterclockwise direction. The first ORF corresponds to
traI, which we have described previously (36).
Limited sequence from the region upstream of traI
(42) showed strong homology to the repA
genes from an octopine-type Ti plasmid pTiB6S3 (59) and the
newly reported Ti plasmid pTi-SAKURA (58). No
identifiable ORFs are present between traI and
repA. At the left end of the sequenced region, the partial
ORF, which is oriented from left to right, is identical at the
nucleotide sequence level to the 3' end of the known sequence of
nocR, the transcriptional activator required for expression
of the nopaline catabolism operon of pTiC58 (63). The 3'
ends of nocR and ORF13 are separated by 3 bp.
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FIG. 3.
Organization of the trb region of pTiC58.
Each arrow in the upper part of the figure represents the length of a
single sequencing reaction. Relevant restriction sites are shown as
vertical lines. Shaded arrows at the bottom represent the ORFs which
are named after their RP4 homologues. The coordinates of the start and
stop codons of each ORF are with respect to the BglII
cleavage site in the region just upstream of traI as
position 1. The arrow labeled P just before traI represents
the location of the traI/trb promoter. The level of each ORF
arrow indicates its reading frame.
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Of the remaining ORFs, the translational product of the last, ORF13,
does not show significant similarity to those of any known genes in the
databank. The deduced amino acid sequences of the remaining 11 ORFs
between traI and ORF13 are related at the amino acid
sequence level to the gene products of trbB, -C, -D, -E, -J, -K,
-L, -F, -G, -H, and
-I of the trb region of pTi15955 and of the
tra2 core region of RP4 (1, 39, 47) (Table 3). Like that of pTi15955, the gene organization of the trb
region of pTiC58 is very similar to that of RP4, except that the
trb region of pTiC58 has trbJKL placed between
trbE and trbF (Fig. 4). The translational products of the
trb genes of the Ti plasmid also are similar at the deduced
amino acid sequence level to those of genes associated with other
mating systems and with protein secretion systems (Table
3). These include virB of the
Ti plasmid (56), tra of plasmid F
(23), and ptl of Bordetella pertussis (37, 66) (Table 3).
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FIG. 4.
Comparison of the gene organizations among the
trb regions of pTiC58, pTi15955, pNGR234a, and the
tra2 core region of RP4. Regions shaded in the same pattern
represent homologous segments.
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TABLE 3.
Relatedness among the predicted gene products of the Ti
plasmid trb region and those of the other mating bridge
or secretion systemsa
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That the trb genes all are transcribed in the
counterclockwise orientation on the Ti plasmid is consistent with the
transposon mutagenesis studies described above. We could not locate any
identifiable promoter elements between any of these genes suggesting
that they form a single transcriptional unit. In certain of the
consecutive ORFs, the stop codon of the preceeding ORF overlaps
the start codon of the succeeding ORF, with no identifiable
ribosomal binding site present before the start codon. This
characteristic suggests that these ORFs, which include
trbB-C, trbC-D, trbK-L, and
trbF-G, are translationally coupled.
Properties of the putative trb gene products.
We
analyzed the amino acid sequence of each of the putative trb
gene products for their properties and searched for any
significant domains or motifs by using several computer programs (Table
4). Two proteins, TrbB and TrbE, contain
a domain related to the sequence of the canonical nucleoside
triphosphate (NTP)-binding motif A (Fig.
5A). TrbH contains a putative
membrane lipoprotein lipid attachment site.
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FIG. 5.
Significant motifs and domains of Trb proteins. (A)
Filled areas represent the locations of the NTP-binding motif A in TrbB
and TrbE. (B) Shaded areas represent the putative TM domains in six Trb
proteins as predicted by PHDhtm and TMpred (see Materials and Methods).
Only the regions that are predicted by both programs are considered
significant. The protein export signal peptide (Table 5) often is
predicted as a TM domain by these programs. Therefore, in these
representations we have excluded those that also could be signal
peptides. N, amino terminus; C, carboxy terminus.
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We also examined the deduced amino acid sequences of each of the
trb gene products for Sec-dependent export signal
peptides and cleavage sites by using the "3,1" rule
described by von Heijne (62) and the signal peptide
prediction server, SignalP V1.1 (46). Due to the inherent
error in both methods (46, 62), we considered as significant
only those sequences that gave strong matches using both algorithms. Of
the 11 trb gene products of pTiC58, 5 contain a
potential signal peptide (Table 5). We
also analyzed the Trb proteins for possible TM domains with two
programs, PHDhtm (52) and TMpred (33), which
utilize different databases and algorithms. Of the 11 proteins, 6 contain at least one potential TM domain (Fig. 5B).
Phage plaquing and adsorption on A. tumefaciens
strains expressing Tra functions.
Propagation of
donor-specific phages is one of the criteria that serve to define
the Mpf system of RP4 (31). We tested three such phages,
PRD1, PRR1, and Pf3 (Table 1), for their ability to plaque on
four Agrobacterium strains: NT1, C58,
NT1(pTiC58accR), and NT1(pFRtra, pPLE2). As
controls, we also tested NT1(RP4) and E. coli
HB101(RP4). All three bacteriophages plaqued on NT1(RP4) with an
efficiency of about 10% compared to HB101(RP4). None of these
phages produced visible plaques on any of the other A. tumefaciens strains, even at very high MOIs (data not shown). We
then determined if PRD1, which recognizes the RP4 mating bridge complex
(28), adsorbs to A. tumefaciens strains
expressing the trb components of pTiC58 by using strains
NT1(pTiC58accR) and NT1(pFRtra, pPLE2). Less than 1%
of the input bacteriophage were adsorbed to these strains, whereas
adsorption levels for HB101(RP4) and NT1(RP4) were about 60 and 42%,
respectively (data not shown).
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DISCUSSION |
Identification and sequence analysis of the trb
region.
Our genetic and sequence analyses clearly show that the
11-kb region between noc and oriV/rep of
pTiC58 codes for trb, the major portion of the Mpf component
of the conjugal mating apparatus of this Ti plasmid. As predicted by
the heteroduplex analysis of Engler et al. (17), the
trb region of pTiC58 is strongly related at the
nucleotide sequence level to the trb region of pTi15955, an octopine-type Ti plasmid (1). Moreover, this
region from pTiC58 is 99% identical to a recently reported segment of a new Ti plasmid, pTi-SAKURA (reference 58 and data
not shown). Although no information is available concerning the
phenotypic characteristics of this plasmid, given the high degree of
homology we predict that this region encodes the Mpf for the
conjugal transfer system of pTi-SAKURA.
The trb region of pTiC58 consists of 13 significant ORFs.
The first, corresponding to traI, is responsible for the
synthesis of AAI
[N-(3-oxooctanoyl)-L-homoserine lactone] and
is essential for the quorum-sensing mediated regulation of the
conjugal transfer of the Ti plasmid (26, 36, 48, 68).
Of the remaining 12 ORFs, 11 could encode proteins that are
similar to those from genes of several DNA-transfer and
protein-secretion systems (Table 3). Among these systems, the
tra2 core region of RP4 showed significant relatedness with
the trb region from pTiC58 in several aspects. First, the
trb region of pTiC58 contains homologues of all of the
essential trb genes of RP4, suggesting that the
trb system of pTiC58 codes for an Mpf system very similar to
that of RP4. Second, the Trb proteins from the two systems show degrees
of relatedness ranging from identities of 30 to 50% and similarities of 50 to 70% (Table 3), indicating that these two systems are closely
related. Third, the homology between trb of pTiC58 and tra2 of RP4 exists not only at the individual gene level but
extends to the gene organization (Fig. 4). Both of these systems
are organized with a leading regulatory gene followed by 11 trb genes, trbB through trbI.
Only the order of three of the genes, trbJ, -K, and -L, differs between these two plasmids (Fig. 4).
In RP4, all of the 11 shared trb genes except
trbK are required for conjugal transfer of the plasmid
(31). While our insertion mutations in the trb
region of pTiC58 abolish transfer, the possibility of polarity
precludes speculation on the requirements for most of these genes in
the conjugal process. However, transposon insertions in the distal ORF,
ORF13 do not affect conjugal transfer of the Ti plasmid (Table 2) or
the mobilization of pFRtra in the binary transfer system (Fig. 2B).
Moreover, a smaller trb clone, pPLtrb, which lacks ORF13,
remains fully functional in the binary transfer system (data not
shown). Thus, this ORF, even if translated into a protein product, is
not essential for conjugal transfer. In a similar fashion, several
genes located downstream of trbL in the trb
operon of RP4 are not required for conjugal transfer of this IncP1
plasmid (40). Homologs of these genes, including trbM, trbN, trbO, and trbP
are not present in the Ti plasmid trb region. Finally, ORF13
is not related to any of these genes, and we could not detect a similar
ORF in the sequences of the trb regions from pTi15955
and pNGR234a. We conclude that ORF13, while perhaps part of the
trb operon, is not required for conjugal transfer. Transposon insertions in trbI, the gene that precedes ORF13,
abolished conjugal transfer when tested in both the whole Ti plasmid
system and in the binary transfer system (Fig. 2; Table 2). Since there can be no polar effect resulting from these insertions except on ORF13,
trbI may be essential for conjugal transfer of pTiC58. Similarly, trbI is required for the transfer of RP4
(31). However, pTiA6NC, which contains a deletion of most of
trbI, can still undergo conjugal transfer, albeit at a
severely reduced frequency (1). Hence, the essential nature
of trbI in conjugal transfer of the Ti plasmids remains to
be elucidated.
Our analyses suggest that all of the 12 genes in the trb
region of pTiC58 are expressed from the promoter located immediately upstream of traI. Correctly inserted Tn3HoHo1
insertions in pTHH6, the insert of which lacks the 5' end of
traI, do not express -galactosidase activity under any
conditions. However, properly oriented fusions formed by these
insertions express enzyme activity when marker exchanged into
pTiC58-accR (Fig. 2A). Similar insertions in pPLE2, which contains the promoter region upstream of traI, do
express -galactosidase activity, but this expression requires TraR
(Fig. 2). Therefore, the traI/trb operon is regulated as
part of the TraR-AAI quorum-sensing regulon. Fuqua and Winans reported
that traI of the octopine-type Ti plasmid also requires TraR
for expression, but there is no information available concerning
expression of the rest of the trb region from this
octopine-type Ti plasmid (25). Given the similar gene
organization, we predict that expression of the entire trb
region of the pTiR10/pTi15955 also requires TraR and AAI.
As is the case for their RP4 homologues, the putative products of
trbC, -D, -K, -L,
-F, and -I of pTiC58 contain potential transmembrane domain(s) (Fig. 5). This finding is consistent with this
locus encoding the mating bridge. Several of the putative gene
products also contain amino-terminal Sec-dependent signal peptides (Table 5). In addition, TrbB and TrbE contain the
NTP-binding motif A, "GXXXXGKT/S" (64) (Fig. 5A).
This domain is conserved in other homologues, including TrbB
and TrbE of RP4 (41), and in VirB11 and VirB4 of the Ti
plasmid Vir transfer system. VirB11 and VirB4 exhibit
ATPase activities (13, 55), and the
NTP-binding motifs are essential to their functions (7, 24,
51, 57). Unlike all of the other TrbE homologues so far
described, the trbE gene of pNGR234a is composed of
two ORFs: trbEa and trbEb (21). These
two proteins of pNGR234a may function cooperatively in the same way as
the single full-length protein by a LacZ-like complementation
mechanism. It also is possible that only one of the two trbE
gene products is required for the Mpf. Alternatively, trbE
of pNGR234a may be defective, with its function being provided by some
other conjugal system present in Rhizobium sp. NGR234. All
transport systems to which trb of pTiC58 is related contain a TrbE homologue, and the function encoded by this gene is essential for conjugal transfer of RP4 (31).
Differences between the trb systems from pTiC58 and
RP4.
Despite the sequence and organizational similarities between
trb from RP4 and the Ti plasmid, these two systems differ in several ways. Bacteria expressing trb of IncP1 plasmids
plaque bacteriophages PRD1, PRR1, and Pf3 (for a review, see reference 22). However, these bacteriophages do not propagate
on cells expressing trb of pTiC58. This failure is not due
to some defect in A. tumefaciens; strain NT1(RP4) plaques
all three bacteriophages at reasonable efficiencies. Furthermore, PRD1
fails even to adsorb to strains of A. tumefaciens expressing
trb. These results suggest that the epitopes of the Trb
proteins of RP4 responsible for phage sensitivity are not conserved in
the Trb proteins of pTiC58. Mutational analysis implicates TrbC,
TrbE, and especially TrbL in sensitivity to PRD1 conferred by RP4
(28). TrbL is one of the two least-conserved gene
products encoded by the trb operons of pTiC58 and RP4 (Table 3). Whether any one of the differences in the three proteins of the two
systems is responsible for the inability of PRD1 to adsorb to strains
harboring the Ti plasmid remains to be determined.
As a further measure of the disparity between the two trb
systems, the IncP Mpf system cannot substitute for the Ti plasmid Mpf system. Ti plasmids with a mutation in trb do not
transfer from donors also harboring pPH1JI, a self-conjugal
derivative of R751 containing the intact IncP1 tra1 and
tra2 regions (data not shown). Moreover, RP4 does not
mobilize a clone containing the Ti plasmid tra/oriT region,
nor can the tra system of pTiC58 mobilize vectors based on
IncP1 plasmids (14). Thus, the two Mpf components are
specific to their respective transfer systems. However, these results
demonstrate only that the Mpf systems of the IncP and Ti plasmids are
not cross-functional as a whole. We have not ruled out the possibility
that one or more of the individual trb genes are
interchangeable between the two systems.
Although most of the trb genes are closely related,
some of the genes show significant divergence. The most notable
of these is trbK. In RP4, trbK is necessary
and sufficient for the entry exclusion function of the plasmid
(29, 31). Although TrbK of pTiC58 contains
neither a cysteine residue nor a detectable secretion signal
peptide, both of which are important for the functionality of TrbK of
RP4 (29), our preliminary results indicate that cells
containing pTiC58 exhibit a Ti plasmid-specific entry exclusion
phenotype. Moreover, a clone expressing the trbK gene from
pTiC58 confers entry exclusion (45).
trb of the Ti plasmid and the type IV secretion
superfamily.
We previously proposed (1, 15, 19) that
the extant Ti plasmid, which contains both a conjugal transfer system
and the Vir system, arose from the fusion of two conjugal
plasmids. Our results indicate that the conjugal transfer system
itself is chimeric, being composed of both IncP-like elements,
which include TraG, TraF, and the Trb proteins, and also IncQ-like
elements, which include TraA and oriT (1, 19).
In the larger context, the Trb proteins of pTiC58 are related to
proteins from several secretion systems (Table 3). These type-IV
secretion systems (12) transport DNA or protein substrates, or perhaps both (8, 61). They include the VirB system of the
Ti plasmids (56, 65), the Trb system of the IncP plasmids (39), the Trw system of the IncW plasmids (12),
the Tra system of the IncN plasmids (50), the Ptl system of
B. pertussis (66), the Dot system of
Legionella pneumophila (61), and perhaps the Cag
system of Helicobacter pylori (10, 60). These
systems can be further grouped into subfamilies based on their degrees of similarity. Given the extensive sequence and organizational similarities among Trb from the Ti plasmids, Trb from pNGR234a, and Trb
from IncP plasmids RP4 and R751, we propose that these Mpf systems
belong to a common DNA transporter family with the Trb system from RP4
as the representative. Similarly, the VirB operon of the Ti plasmids,
along with Tra of the IncN plasmids and Ptl of B. pertussis, form a second family within this type IV superfamily,
with VirB as the representative.
We are grateful to Audra Smyth for helpful discussion and
critical reading of the manuscript. We also thank Susanne Beck von Bodman, Ingyu Hwang, and David Cook for helpful discussion.
This work was supported by grants R01GM52465 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.
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Abstract
Introduction
Present address: Family Practice Residency Program, Quad City
Genesis, Davenport, IA 52807.
