INTRODUCTION

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The Ti plasmids of Agrobacterium tumefaciens code for two distinct conjugal transfer systems. One, mediated by the Vir system, transfers T-DNA into the plant cells but also can mobilize transfer of a suitable plasmid to recipient bacteria (for a recent review, see reference 17). The second, which constitutes the major pathway for Ti plasmid transfer, operates through a functionally and physically separated system called Tra. Expression of the Tra system on at least two Ti plasmids is tightly regulated at the transcriptional level through a complex signalling circuitry that involves opines produced by the crown gall tumors plus a LuxR-LuxI-type quorum-sensing mechanism (5, 22, 31, 37, 38, 46).

The Tra system of pTiC58 consists of two physically separated gene sets, tra and trb, which contain all of the genes essential for conjugal transfer (15). The tra region encodes the origin of conjugal transfer (oriT) and two sets of genes organized as divergently expressed operons (14, 24). Three of the six genes flanking the oriT region are related to essential tra genes from IncP and IncQ plasmids. The products of some of these genes comprise the DNA transfer and replication (Dtr) function of the Ti plasmid Tra system and most probably form the relaxosome complex at the oriT site. The second region, trb, 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.

The trb genes are believed to encode the mating pair formation (Mpf) apparatus required for the physical translocation of DNA from donors to recipients. Sequence analysis and genetic studies have shown that this region contains 12 genes, traI and trbB, -C, -D, -E, -J, -K, -L, -F, -G, -H, and -I, organized in a single operon (42). Expression of this operon is controlled by the quorum-sensing activator, TraR, and the acyl-homoserine lactone signal, Agrobacterium autoinducer [AAI; N-(3-oxooctanoyl)-L-homoserine lactone], which is synthesized by the gene product of traI (38, 42, 43). The trb genes of pTiC58 are closely related in sequence and organization to the 11 trb genes from the tra2 core region of the IncP plasmids RP4 and R751. Genes of the trb system also are related to those of several other bacterial conjugation or protein secretion systems (42), including the VirB system of A. tumefaciens (49), the Ptl system of Bordetella pertussis (21, 55), the Tra system of plasmid F (27), the cag system of Helicobacter pylori (10, 36, 50), and the Dot system of Legionella pneumophila (48, 53).

In RP4, all but one of the 11 core trb genes are required for conjugal transfer (34). Similarly, 10 of the 11 virB genes are essential for the transfer of T-DNA to plant cells (8). Although the trb system of the Ti plasmid is related to these two systems, which of the Ti plasmid trb genes are essential for conjugal transfer remains unknown. In this report we describe the construction of a minitransposon carrying the promoter region of the traI-trb operon and the use of this element to generate complementable mutations in the trb genes of pTiC58. Results from matings with these mutants indicate that unlike the case for the RP4 trb system, only 9 of the 11 Ti plasmid trb genes are required for conjugal transfer. Our results also indicate that the large catabolic plasmid pAtC58 harbored by A. tumefaciens C58 not only can mobilize the IncQ plasmid RSF1010 at low frequency (14) but also can complement the mutations in most of the trb genes of the Ti plasmid.

	MATERIALS AND METHODS

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Bacterial strains, growth conditions, and plasmids. The strains of A. tumefaciens and Escherichia coli and the plasmids used in this study are listed in Table 1. A. tumefaciens strains were grown at 28°C in L broth (LB [47]) (Gibco-BRL, Gaithersburg, Md.), 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 at 100 or 200 µg/ml, gentamicin at 30 µg/ml, kanamycin at 100 µg/ml, rifampin at 50 µg/ml, streptomycin at 200 µg/ml, erythromycin at 100 µg/ml, chloramphenicol at 30 µg/ml, and tetracycline at 2 µg/ml; for E. coli, ampicillin at 100 µg/ml, kanamycin at 50 µg/ml, rifampin at 50 µg/ml, and tetracycline at 10 µg/ml. X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; Gibco-BRL) was included in media at 40 µg/ml to assess the production of -galactosidase.

DNA manipulation and plasmid constructions. Ti plasmids were isolated as described by Hayman and Farrand (35). Other plasmids were isolated by an alkaline lysis method (47). Standard recombinant DNA techniques were used as described by Sambrook et al. (47). Digestions with restriction endonucleases were conducted according to the manufacturers' instructions, and digestion products were separated by electrophoresis in agarose gels, using Tris-borate-EDTA buffer.

PCR. The traI-trb promoter region was amplified from pCF1 by using AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, Calif.) and oligomers 5'-GGGCGGCCGCCCGATTCTTCAAATGC-3' and 5'-GAGCGGCCGCATCGTAATCTCCGC-3'. Pfu DNA polymerase (Stratagene, La Jolla, Calif.) was used to amplify each of the 11 trb genes, and the products were cloned into pKK38ASH or pKK38. In each case, the 5' primer was designed to generate an NcoI, RcaI, StuI, or AflIII site allowing for in-frame fusion of the second codon of the trb gene to an ATG initiation codon provided by the vector. The sequences of the primers used for these amplifications are available upon request.

Mini-Tn5 mutagenesis and homogenotization. A method based on mutagenesis with a mini-Tn5 transposon as described by de Lorenzo and Timmis (20) was used to transpose mini-Tn5Ptrb from pHM1 into the promoterless trb reporter clone pHM25 and into the full-length trb clone pRKtrb. The transposon delivery strain S17-1-pir(pHM1) was mated with the target strain, DH5(pHM25) or DH5(pRKtrb), on a 0.22-µm-pore-size filter, and the filter was incubated at 37°C for 6 h on the surface of an L agar plate. Following this incubation, the cells on the filter were suspended in 3 ml of LB, serial dilutions were prepared, and 0.1-ml volumes were spread on L agar plates containing kanamycin and tetracycline. The plates were incubated at 37°C, and colonies that appeared were combined. Plasmid DNA was extracted from the pool and used to transform E. coli DH5 with selection for resistance to kanamycin and tetracycline on L agar plates. Independent colonies were isolated and purified, and the locations and orientations of insertions of mini-Tn5Ptrb in the target plasmid were mapped by restriction endonuclease analysis. Insertion mutations of interest in pRKtrb were homogenotized into pTiC58accR by using pPH1JI as the eviction plasmid as previously described (24). Proper marker exchanges in the Ti plasmids were confirmed by restriction endonuclease analysis. pPH1JI was cured from these strains by continuous growth of the strain in LB without gentamicin, the selection marker of pPH1JI. Alternatively, the marker-exchanged Ti plasmid was isolated and introduced into A. tumefaciens NT1 via electroporation. Transformants resistant to kanamycin but remaining susceptible to gentamicin were retained for further study.

-Galactosidase assay. Quantitative assays for -galactosidase activity were conducted as described previously (37). Each sample was analyzed in triplicate, and activity was expressed as units of -galactosidase per 10⁹ CFU.

Conjugation assays. Conjugal transfer of the Ti plasmid and of the oriT-tra plasmids, pFRtra and pPLtra, of the binary transfer system (15) to the A. tumefaciens recipient strains C58C1RS and C58C1EC was assayed by a filter mating method as described previously (14). Samples were plated in triplicate, and the values obtained were used to calculate the average number of transconjugants that arose for each mating. Transfer frequencies were expressed as numbers of transconjugants obtained per input donor cell. Each set of matings was repeated once or twice. Although absolute transfer frequencies usually differed, the patterns of transfer were similar from one experiment to the next. Thus, in each case we present data from a single experiment in which all of the matings shown were conducted in parallel.

Analysis of AAI production. AAI production was assayed by the semiquantitative plate method using A. tumefaciens NT1(pDCI41E33) as the indicator strain as previously described (15). A diffuse blue zone on the assay plate indicates the production of an active acyl-homoserine lactone by the strain being tested.

	RESULTS

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Construction and evaluation of mini-Tn5Ptrb. To construct a mini-Tn5 transposon carrying the traI-trb promoter, a 236-bp fragment containing the promoter region of the traI-trb operon and the first two codons of traI was amplified by PCR using primers containing a NotI site as described in Materials and Methods. The amplified fragment was cloned into the unique NotI site in pUTmini-Tn5Km (20), and the fidelity of the sequence and orientation of the insert were confirmed by nucleotide sequencing. The resulting plasmid is designated pHM1, and the minitransposon is designated mini-Tn5Ptrb (Fig. 1).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Structure of mini-Tn5Ptrb. The 236-bp traI-trb promoter region was amplified from pCF1 by PCR, and the product was cloned into the NotI site of mini-Tn5Km (19, 20). tra box II and tra box III, the 18-bp almost perfect inverted repeat sequences that are conserved in LuxRI-type quorum sensing regulatory systems (29, 38); 10 and 35, the promoter elements of the traI-trb operon identified in pTiR10 (29); rbs, the putative ribosomal binding site of traI; S, translational stop; T, transcriptional terminator; Km, kanamycin resistance gene; I and O, the 19-bp I and O ends of Tn5.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Physico genetic organization of pPLE2-25 and construction of pHM25 and pHM25-70. The restriction map is according to the published sequence (42). pPLE2-25 contains the entire trb region of pTiC58 and a Tn3HoHo1 insertion in trbE. pHM25 is derived from pPLE2-25 by cloning the HindIII fragment 8 including the Tn3HoHo1 insertion into pRK415 (38). The open circle represents the traI-trb promoter region, which is just upstream of the HindIII site. pHM25-70 contains a mini-Tn5Ptrb insertion just upstream of the truncated traI gene. The transposons are not drawn to scale.

Phenotype of pRKtrb::mini-Tn5Ptrb mutants. pRKtrb, which contains the entire trb region, was mutagenized with mini-Tn5Ptrb as described in Materials and Methods. Using restriction endonuclease analysis, we identified 14 independent insertions, all oriented in the correct direction and representing at least one insertion in 9 of the 11 trb genes (Fig. 3B). To generate nonpolar mutations in the two remaining genes, trbC and trbK, the following cloning strategies were used (Fig. 3A). For trbC, a cassette containing Ptrb and the nptII gene was constructed from mini-Tn5Ptrb. This cassette, called Km-Ptrb, retains the kanamycin resistance gene and the traI promoter region but lacks some restriction sites and the insertion sequence elements of the transposon. The cassette was cloned between the internal BamHI and NruI sites in trbC, thus replacing 112 bp of the gene with the Km-Ptrb cassette. Subsequent cloning resulted in the replacement of wild-type trbC by the trbC deletion-insertion allele within the full-length trb clone, pRKtrb, to generate pRKtrbC. For trbK, an nptI cassette coding for resistance to kanamycin was excised from pSB315 and cloned between the internal NruI sites within the gene. This cassette lacks a transcriptional terminator, and the promoter of the nptI gene is known to express genes downstream of the insertion (32). This resulted in an allele of trbK deleted for 133 internal residues and containing the nptI cassette oriented such that the trb genes downstream from trbK will be expressed from the promoter of nptI.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Mutational analysis of the trb genes of pTiC58. (A) Cloning strategies for constructing nonpolar insertions in trbC and trbK. (B) Locations of mini-Tn5Ptrb insertions in pRKtrb. Each vertical bar represents an independent insertion, and the horizontal arrow indicates the orientation of the traI-trb promoter of the transposon. (C) Restriction map of the trb region and locations of the Tn3HoHo1 insertions in pTiC58accR::Tn3HoHo1 which were used in complementation assays to test polarity of the mini-Tn5Ptrb insertion mutants of pRKtrb.

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View this table: [in this window] [in a new window]   TABLE 4.   Conjugal transfer frequency of pTiC58accR::mini-Tn5Ptrb mutants and complementation of these mutants with trb gene clones

Complementation analysis. Each of the trb genes was amplified by PCR using primers containing NcoI, RcaI, StuI, or AflIII sites, depending on the 5'-end sequence of the gene, and either HindIII, PstI, or BamHI sites immediately following the stop codon of the gene. These PCR products were cloned into pKK38ASH, which is a derivative of pKK38 into which we inserted extra cloning sites for HindIII, PstI, and BamHI (Fig. 4). Each of the trb open reading frame (ORF) clones was introduced in trans into UIA5 harboring a derivative of pTiC58accR with a mini-Tn5Ptrb insertion in the corresponding trb gene. When mated with C58C1EC, all complemented donor strains transferred the mutant Ti plasmids, although the efficiency of complementation varied for different mutations (Table 4). Complemented donors with mutations in trbC, trbD, trbE, trbL, and trbF transferred the test plasmid at near-wild-type levels. On the other hand, the trbB and trbJ mutants were only poorly complemented, while mutants with insertions in trbG, trbH, and trbI transferred their Ti plasmids at intermediate frequencies (Table 4).

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Construction of individual trb gene ORF clones. PCR products corresponding to each of the trb genes were cloned into the expression vector as described in Materials and Methods. Due to the lack of suitable restriction sites, the trbB clone was obtained by first amplifying the 5' and 3' halves of the gene separately to generate trbB1 and trbB2 and then cloning trbB2 into trbB1. All PCR products were cloned in pKK38ASH except for trbK, which was cloned in pKK38.

Conjugal transfer of a nonpolar traI mutant can be restored by adding AAI. We also identified a mini-Tn5Ptrb insertion in traI, the first gene of the trb operon (Fig. 3B). An Agrobacterium strain harboring this mutant plasmid, pRKtrb-14, does not produce AAI at detectable levels even in the presence of TraR (Table 5). Complementation assays using pTiC58accR::Tn3HoHo1 indicated that this insertion mutation is not strongly polar (Table 3). Attempts to marker exchange this mutation in pTiC58accR were not successful. Thus, we assessed the effect of the disruption of traI on conjugal transfer by testing the ability of pRKtrb-14 to mobilize pPLtra, which is a pDSK519 derivative containing the tra operons, the Ti plasmid oriT, and traR of the Ti plasmid, in a binary transfer system. NT1 harboring both pPLtra and pRKtrb-14 mobilizes the tra plasmid from the Ti plasmid oriT only at very low frequency (Table 5). This transfer rate is similar to that observed from strain NT1(pPLtra), which lacks the trb component, and is about 3 orders of magnitude lower than that observed from NT1(pPLtra, pRKtrb), which contains the wild-type Trb system of the Ti plasmid (Table 5). This basal level of mobilization of pPLtra is commonly observed when an RSF1010 derivative is harbored in strain NT1 (14). However, the mobilization frequency of the tra plasmid in NT1(pPLtra, pRKtrb-14) was restored to that of NT1(pPLtra, pRKtrb) by adding exogenous AAI (Table 5). Mobilization of the oriT plasmid from NT1(pPLtra) was not stimulated by addition of AAI.

	DISCUSSION

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traI and 9 of the 11 trb genes of pTiC58 are essential for conjugation. Mutations in all but two trb genes resulted in complete loss of conjugal activity of the Ti plasmid (Table 4). The first gene of the trb operon is traI, the only known function of which is the synthesis of AAI, the essential signal for the quorum-sensing regulation of Ti plasmid conjugal transfer (30, 38, 43). Consistent with this, a mini-Tn5Ptrb mutation in traI abolished normal conjugation in a binary transfer assay but the wild-type phenotype could be restored by supplying exogenous AAI (Table 5). Thus, we conclude that the first gene in the trb operon, traI, is essential for Ti plasmid conjugal transfer but only because it is required for synthesis of the quorum-sensing signal. A nonpolar mutation in trbK has virtually no effect on conjugal transfer, suggesting that the product of the gene is not essential for the trb-encoded Mpf apparatus. However, trbK does play a role in conjugation; when present in a recipient, this gene confers entry exclusion against closely related Ti plasmids (44). This is consistent with studies of RP4 in which trbK is not required for conjugal transfer but is responsible for entry exclusion (33, 34). Hence the conservation between these two systems extends, at least in one case, to the function of the individual genes, even though the TrbK proteins from the two plasmids show considerable sequence divergence (42).

Mini-Tn5Ptrb as a tool to generate complementable mutations in the trb operon. Using transposable elements as mobile promoters to study polycistronic transcriptional units has proved to be useful (for a review, see reference 7). Transposons such as Tn5virB (16) and mini-Tn5-lacI^q/Ptrc (18) have been successfully applied in the genetic analyses of complex operons in A. tumefaciens and Pseudomonas spp. Our analyses indicate that when inserted in the proper location and correct orientation, mini-Tn5Ptrb can provide a promoter capable of expressing downstream genes, and that expression from this promoter is regulated by TraR and AAI. However, the transcriptional activity from the traI-trb promoter of mini-Tn5Ptrb in pHM25-70 was only about 60% of that observed when the trbE::lacZ fusion was expressed from the native traI-trb promoter in the original clone pPLE2-25 (Table 2). This difference in expression probably is due to the location of the insertion and the distance between the insertion and the immediate downstream gene. It also is possible that early termination of transcription occurs due to translational stops in the other reading frames. Therefore, for any given insertion some degree of polarity may be expected, and examination of more than one insertion in each gene may be necessary to obtain a suitable nonpolar mutation. Such factors may account for the difference in the ability of the two trbE mutants to trans complement a downstream mutation in the trb operon. Polar effects also may arise from the disruption of the preceding gene in a translationally coupled gene cluster as observed in other studies (8, 34). However, the promoter in mini-Tn5Ptrb contains a ribosomal binding site which may allow translational reinitiation of downstream, translationally coupled genes. With respect to our analysis, each of the Ti plasmid trb genes is preceded by a sequence that could serve as a ribosomal binding site (reference 42 and data not shown). Even so, that some of our trb mutants could not be complemented to wild-type levels of transfer suggests that mini-Tn5Ptrb insertions can induce some degree of polarity on the expression of downstream genes. Such effects may account for the relatively weak complementation of the trbB and trbJ mutations by the complementing cloned genes.

The role of the catabolic plasmid pAtC58 in conjugal transfer. In addition to the well-studied Ti plasmids, most isolates of Agrobacterium spp. harbor other extrachromosomal elements. These replicons usually are very large, but little is known concerning traits they confer on their bacterial hosts. A. tumefaciens C58 harbors at least one such plasmid, pAtC58, with a size variously estimated at 450 to 550 kb (1, 12, 39, 40). This plasmid, which codes for catabolism of a set of Amadori compounds produced by rotting vegetation and also by some crown gall tumors (52), is self-conjugal (51) and can mobilize an RSF1010 derivative at low but detectable frequency (14). Our results suggest that components of the transfer system of pAtC58 can substitute for certain of the trb functions of pTiC58. Thus, NT1, which harbors pAtC58, transfers most of our trb mutants at low frequency, while these same mutant plasmids fail to transfer from UIA5, a strain that lacks pAtC58 but otherwise is nearly isogenic to NT1 (Table 4). However, our oriT-tra plasmids such as pFRtra and pDCtra-5 are not mobilized from NT1 in the absence of a functional Ti plasmid trb system (15). Moreover, Ti plasmid trb mutants derived by insertion of Tn3HoHo1, which can exert strong polarity, fail to transfer from an NT1 donor (42). These observations suggest that the Mpf of pAtC58 is not itself able to substitute for that of the Ti plasmid. Rather, we propose that Mpf components of pAtC58 can replace some but not all of those coded for by the trb operon of pTiC58 to form a functional chimeric conjugal transporter. In this regard, only the trbJ mutant of pTiC58 failed to transfer at detectable frequencies from a donor harboring pAtC58 (Table 4). This observation suggests that the function coded for by this gene is an essential component of, and highly specific to, the Ti plasmid transporter and cannot be replaced by the corresponding Mpf component of pAtC58. Such a dependence on the Ti plasmid trbJ product for transfer from the Ti plasmid relaxosome might explain why the Mpf of pAtC58 cannot substitute for that of pTiC58. Interestingly, pPH1JI, which codes for a trb system closely related to that of pTiC58 (42), apparently does not complement any mutations in the Ti plasmid trb operon. Our nonpolar trb mutants do not transfer from a donor harboring pPH1JI at frequencies any higher than from donors lacking this IncP1 plasmid (data not shown). This is reminiscent of our observation that although the Dtr components of pTiC58 and RSF1010 are related, the Ti plasmid will not mobilize the IncQ plasmid (14). Determining the functional and phylogenetic interrelationships of these type IV systems and the points at which specificity is conferred should aid us in understanding how these transporters recognize and translocate their substrates.