| Edited by Fujio Kawamura. Katsunori Suzuki: Corresponding author. E-mail: ksuzuki@hiroshima-u.ac.jp |
Agrobacterium tumefaciens causes crown gall disease in dicot plants. The tumor-inducing plasmid (Ti) is indispensable for the phytopathogenicity of this bacterium, because it harbors oncogenic genes in its T-DNA region and many virulence genes for infection and T-DNA transport functions. The plasmids are stably maintained at a low copy number in the bacterial cells (at an equimolar ratio with the bacterial linear and circular chromosomes) (Suzuki et al., 2001). The stability of Ti plasmids still differs depending on the strain (Holsters et al., 1982). The stability difference is likely to exert a serious effect on Ti plasmid evolution. The repABC locus is sufficient for autonomous replication of the plasmids (Tabata et al., 1989; Li and Farrand, 2000). To date, the nucleotide sequence of the repABC region of Ti has been determined in an octopine-type plasmid, pTiB6S3 (Tabata et al., 1989), and two nopaline-type plasmids, pTi-SAKURA and pTiC58 (Suzuki et al., 1998; Li and Farrand, 2000).
Rhizobiaceae bacteria commonly possess repABC plasmids (Rigottier-Gois et al., 1998). The repABC region consists of three clustered genes (repA, repB and repC) and a replication origin (oriV) required for replication and partitioning. The RepA and RepB proteins are involved in plasmid partitioning and copy number control, which are important for stable inheritance. RepC is an indispensable protein for replication initiation. These genes are organized as an operon (Ramírez-Romero et al., 2000). Thus, the repABC region contains factors for autonomous replication and stable inheritance. Some strains of Rhizobiaceae bacteria possess more than one repABC replicon. For instance, A. tumefaciens strain C58 has three repABC replicons: pTiC58, pAtC58 and the linear chromosome, each belonging to three different incompatibility groups of the repABC replicon in this strain (Goodner et al., 2001). It is well known that most Ti plasmids belong to the same incompatibility group, whereas most Ri plasmids belong to another incompatibility group (White and Nester, 1980).
Incompatibility has been defined as a situation where two plasmids harboring a related replication and/or partitioning system are unable to exist in a cell simultaneously without external selection. The incompatibility phenomenon is considered to be caused by competition for a limited number of components for replication, a partitioning mechanism or by disturbance in plasmid copy number correction (Novick, 1987; Austin and Nordström, 1990). Mini-Ti and -Ri plasmids carrying repABC genes confer their respective incompatibility characteristics (Nishiguchi et al., 1987, Tabata et al., 1989) and two sites in the repABC locus were defined as incompatibility-determining regions in Rhizobium etli p42d (Ramírez-Romero et al., 2000).
Ti plasmids have been practically categorized according to their production and catabolism of opines. pTiC58 and pTi-SAKURA are nopaline-type Ti plasmids. The frequency of ejection of pTi-SAKURA in MAFF301001 by transformation via conjugation of the strain with an incompatible small repABC plasmid is much lower than that of pTiC58 in the C58 strain (Uraji et al., 2002). The two Ti plasmids are almost identical along 60% of their length (200 kbp), but the remaining regions exhibit no or very low levels of homology. We named the largest nonhomologous region VAR (large variable region) (Suzuki et al., 2000). Because VAR contains many genes whose functions are unidentified (Hattori et al., 2000), we hypothesized that the factor causing the difference of the ejection frequency between the two Ti plasmids is contained within VAR. In the present study, we localized and characterized the factor within VAR of pTi-SAKURA which can enhance incompatibility in small repABC plasmids and increase the stability of pTiC58.
Bacterial strains and plasmids used in this study are listed in Table 1. A. tumefaciens cells were cultured at 28°C in LB medium. Escherichia coli cells were cultured at 37°C in LB. When necessary, antibiotics were added to the medium at the following final concentrations: gentamicin (45 μg/ml), kanamycin (50 μg/ml), rifampicin (40 μg/ml), tetracycline (5 μg/ml) and chloramphenicol (35 μg/ml). A. tumefaciens cells were also grown on AB minimal salts medium (Chilton et al., 1974) supplemented with nopaline as the sole source of carbon instead of glucose.
 View Details | Table 1. Bacterial strains and plasmids used in this work |
E. coli S17-1 λpir was used as a host strain for plasmid construction and conjugal transfer of mobilizable plasmids (Simon et al., 1983; Penfold and Pemberton, 1992). Fosmid clones containing portions of pTi-SAKURA were selected from an A. tumefaciens MAFF301001 genomic DNA library (De Costa et al., 2001) by colony hybridization using purified pTi-SAKURA DNA as a probe. Among the 14 recombinant fosmids selected, four clones were chosen for the present study. In order to allow the four plasmids to move into, replicate in and be selected in Agrobacterium cells, repABC of pTi-SAKURA, oriT and Kmr genes were inserted into the four fosmids by homologous recombination in vivo between plasmids in the recA+ E. coli strain LE392. Two fosmid clones, 2K1 and 1F23, contained repABC (Fig. 1). They were fused with pJP5603, which contains Kmr and oriT. The resultant plasmids were named pfK and pfF. The other two fosmids, 1M3 and 2I19, had no repABC. They were fused with pJP5603rep, which contains repABC in addition to Kmr and oriT. The resultant plasmids were named pfM and pfI. pfKΔAscI was constructed by AscI digestion of pfK and subsequent self-ligation. pK18msr is a pK18mobsacB plasmid carrying a 4.9-kbp HindIII fragment of pMGTrep1 (Uraji et al., 2002); the HindIII fragment contains repABC. pN2, pN5, pN7 and pN12 were constructed by inserting each NheI fragment of pfK into pK18msr (see Fig. 3). A derivative of pN2, pN2ΔNspV, was constructed by NspV digestion of pN2 and subsequent self-ligation. pHRPN2 and pBINN2 were constructed by inserting a 2.6-kbp NheI fragment of pN2 into pHRP311 and pBIN19, respectively. pBINTc was constructed by cloning into pBIN19 a 2.1-kbp BamHI fragment containing a Tcr cassette excised from pHP45Ω-Tc. pN2T-r was constructed by digestion of pN2 with HindIII followed by self-ligation and insertion of a 1.7-kbp BglII fragment of pTiC58 T-DNA. pTiC58px is a fusion plasmid formed in vivo in C58 cells between pN2T-r and pTiC58. pN2G-r was constructed by digestion of pN2ΔNspV with HindIII followed by self-ligation and insertion of a 2.8-kbp AccI-NspV fragment containing a Gmr cassette from pUC19Gm into a unique NspV site. pTi-SAKURAmx is pTi-SAKURA with a tiorf25::Gmr mutation, which was formed in vivo in MAFF301001rif cells by replacement of tiorf25+ with tiorf25::Gmr of pN2G-r via double crossover between pTi-SAKURA and pN2G-r.
DNA manipulations for plasmid construction were performed using standard procedures (Sambrook et al., 1989).
Plasmids were delivered from E. coli to A. tumefaciens by conjugal transfer as described elsewhere with some modifications (Uraji et al., 2002). Conjugation efficiency was calculated by dividing the number of transconjugant colonies by the number of donor colonies. Conjugal transfer of plasmids from E. coli to E. coli was carried out as follows. Donor E. coli S17-1 λpir cells harboring a plasmid and recipient E. coli Sy327 cells were cultured in LB medium until the cell density reached an OD600 of 1. Approximately 1.0 × 108 donor cells were washed with 0.9% (w/v) NaCl and then mixed with the same number of recipient cells in 5 ml of LB medium. After incubation at 37°C for 1 hour, 50-μl aliquots were plated onto LB agar supplemented with rifampicin and other antibiotics that were selective for recipient cells that had acquired incoming plasmid.
Electroporation was performed as described by Wise et al. (2006) using a Cell-Porator (BRL, Gaithersburg U.S.A) with Bio-Rad Pulser cubettes ( 1 mm electrode gap ) under the following conditions: voltage, 300 V; capacitance, 330 μF; impedance, low ohms; charge rate, fast; voltage boost resistance, 4 K. After electroporation the cells were immediately re-suspended in 1 ml of SOC medium, and incubated at 28°C with gentle shaking for 1 hour.
In order to determine the ejection ratio of the resident plasmid after introduction of another plasmid, 100 transconjugant colonies selected for incoming plasmid markers were streaked onto LB agar plates with antibiotics to which the resident and incoming plasmids conferred resistance. When the resident plasmid was a Ti plasmid, growth on AB minimal agar with nopaline (2 mg/ml) as the sole source of carbon was used instead of examination of antibiotic resistance, because pTi-SAKURA and pTiC58 code for nopaline utilization genes. After incubation at 28°C for 2 days, growth on the selective agar media was examined to distinguish transconjugants that still retained the resident plasmid from resident-plasmid-less transconjugants.
Agrobacterium cells were cultured overnight in LB at 37°C. Then, 1 × 108 cells were inoculated into fresh LB to repeat the overnight culture. The serial cultivation was repeated 5 times in total. The final culture was diluted and spread on LB agar to establish single colonies. The presence and absence of Ti plasmid in the colonies were checked by colony hybridization using virB gene fragment as a probe.
Searches for DNA and protein homologs in the DNA databases were carried out using the FASTA program (http://www.ddbj.nig.ac.jp/search/fasta-j.html). Motif/domain search was performed using the MOTIF program (http://motif.genome.jp/) and the Pfam program (http://www.sanger.ac.uk/Software/Pfam/).
Logarithmically growing cells were lysed by vortexing with glass beads in the presence of SDS, phenol and chloroform principally as described elsewhere (Suzuki et al., 1989). RNA was purified from the cell extract by treatment with phenol/chloroform, then treatment with DNase and proteinase followed by ethanol precipitation. For cDNA synthesis, 10 μg of the total RNA were used as a template. cDNA synthesis was carried out using reverse transcriptase ReverTra Ace (Toyobo, Osaka) in accordance with the manufacturer’s instructions. An oligonucleotide primer (5’-tcgaagctcatcgtgccacatct-3’) complementary to the 3’ end region of the tiorf25 open reading frame (ORF) was used as a primer for the reverse transcription (Fig. 3B). Subsequent PCR amplification was performed using KOD Dash DNA polymerase (Toyobo, Osaka) in accordance with the standard method. The gene-specific primer and a forward primer (5’-tatgcggaggcgcatccgaagg-3’), the corresponding sequence of which is located in the middle of tiorf24, were used for the PCR amplification (Fig. 3B).
The large variable region (VAR) was thought to contain a region that makes pTi-SAKURA more resistant to interference by incoming incompatible repABC replicons than pTiC58. In order to localize the responsible region, we constructed five repABC plasmids. As shown in Fig. 1, pfK and pfF contained a portion of the pTi-SAKURA VAR region and pfM harbored another portion of VAR, whereas pfI carried the vir region without VAR, and a small vector plasmid, pK18msr, possessed only repABC (negative control). We used pTi-SAKURA as a positive control in the next experiment.
 View Details | Fig. 1. Structure of pTi-SAKURA and the four derivative fosmid clones used in this study. The gray region indicates the repABC operon. The hatched region indicates the large variable region (VAR). Arches outside of the Ti plasmid circle indicate the regions that are contained in each fosmid clone. Shown in parentheses are the fosmids with oriT and Kmr genes. repABC was also added in pfI and pfM. |
The mini Ti plasmids and the vector were introduced into a Ti-less strain, MNS-1. Then, the incoming repABC plasmid, pJP5608rep, was introduced by conjugation, in order to evaluate the five plasmids. As shown in Fig. 2A, MNS-1 harboring pfK or pfF exhibited low transconjugant efficiency, like that harboring pTi-SAKURA. In contrast, strains possessing pfM or pfI exhibited high efficiency, which was as high as that of MNS-1 containing the vector plasmid. The difference in efficiency between the two plasmid groups was greater than about 20-fold.
 View Details | Fig. 2. Frequency of transconjugant formation induced by incompatible plasmids. (A) Agrobacterium tumefaciens MNS-1 harboring various resident plasmids was used as recipient for conjugation with donor E. coli S17-1 λpir (pJP5608rep). Each value is the average of at least three independent experiments. Thin vertical bars represent standard deviations. (B) Introduction of pMGTrep1 into the C58C1 harboring plasmid with or without the 2.6-kbp NheI fragment by electroporation. (C) Results of reciprocal experiments. A. tumefaciens MAFF301001rif strain was used as conjugal recipient, E. coli S17-1 λpir harboring plasmid with or without 2.6-kbp NheI fragment was used as conjugal donor. + and – in parenthesis indicate presence and absence of the 2.6-kbp NheI fragment, respectively. |
Using the cells with mini Ti as a recipient, all of the transconjugant colonies examined (100 colonies) were sensitive to kanamycin, suggesting that the resident mini Ti plasmid, which determines kanamycin resistance, was excluded in every transconjugant (data not shown).
pfK and pfF share a 39-kbp segment of pTi-SAKURA (Fig. 1). Thus, the common overlapping region shared by the two plasmids should contain the genes responsible for the effect described above. We localized the effective region to a 2.6-kbp NheI fragment using a series of deletion plasmids and subclone plasmids, as shown in Fig. 3A.
 View Details | Fig. 3. Transconjugant efficiency of cells harboring a VAR portion of pTi-SAKURA. Left panel, physical map; right panel, efficiency of transconjugant colony formation. (A) Recombinant fosmid clones and subclone plasmids. Gray arrows indicate the repABC operon. Hatched boxes indicate the region shared by pfF and pfK. Restriction site: N, NheI; A, AscI. (B) Enlarged physical map of the 2.6-kbp NheI fragment. Two small arrows indicate the locations and directions of the two oligonucleotide primers used for RT-PCR (Fig. 4). |
To discriminate whether the suppression takes place during the conjugational entry process or vegetative replication process after the conjugation, we introduced the incoming plasmid by electroporation in place of conjugation. As shown in Fig. 2B, the 2.6-kbp NheI fragment decreased the efficiency of transformation via electroporation 7.4-fold. This result indicates that the effect by the 2.6-kbp NheI fragment is not due to inefficient cell-cell interaction during conjugation.
In order to know whether the 2.6-kbp NheI fragment affects conjugation efficiency when the fragment is present in the incoming plasmid in the conjugation assay, pN2 was introduced into the MAFF301001rif strain by conjugation. The transconjugant efficiency of pN2 was about ten times higher than that of vector plasmid (Fig. 2C). Thus, the fragment is effective even when it is on the incoming plasmid.
On examination of the nucleotide sequence of pTi-SAKURA (Hattori et al., 2000; Suzuki et al., 2000), the 2.6-kbp NheI fragment was found to cover nucleotide positions 23587 to 26207 in pTi-SAKURA. The fragment codes for two complete ORFs (tiorf24 and tiorf25) (Fig. 3B). In order to confirm that the two ORFs are responsible for suppressing repABC plasmid incursion, deletion mutants were constructed (Fig. 3B). Cells with a plasmid lacking either one of the two ORFs (pN2ΔNspV, pK18mssr) allowed entry of the incoming plasmid with similar efficiency to that observed in cells with the vector plasmid (Fig. 2A and Fig. 3B).
The 2.6-kbp NheI fragment affected whole Ti plasmids in a manner different from the effect on the small plasmids. C58rif, which harbors pTiC58, allowed introduction of a repABC plasmid as efficiently as MAFF301001rif, which harbors pTi-SAKURA (Table 2). However, the ejection ratio of pTiC58 in transconjugants (47%) was higher than that of pTi-SAKURA in transconjugants (6%) (Table 2). Here the ejection ratio was defined as the percentage of transconjugant cells which lost the resident plasmid. Addition of the 2.6-kbp NheI fragment to pTiC58 (see pTiC58px in Table 2) decreased the ejection ratio of pTiC58 in transconjugants down to 1%, although the addition had only a negligible effect on the transconjugant efficiency. Furthermore, a similar phenomenon was also observed in MAFF301001rif. A tiorf25 mutant, pTi-SAKURAmx, was ejected more frequently (53%) than wild-type pTi-SAKURA (6%). As shown in Table 2, the ejection ratio of the tiorf25::Gmr mutant (pTi-SAKURAmx) of pTi-SAKURA was as high as that of pTiC58. The mutation had only a negligible effect on suppression of the entry of repABC plasmids. These results indicate that the 2.6-kbp NheI fragment is responsible for the lower ejection ratio of pTi-SAKURA than of pTiC58. In contrast to the intact Ti plasmids, pN2, the small plasmid harboring the 2.6-kbp NheI fragment showed a significantly higher ejection ratio (4%) than pTi-SAKURA (data not shown).
 View Details | Table 2. Influence of addition and deletion of the 2.6-kbp NheI fragment to Ti plasmids |
In order to examine whether the addition of the 2.6-kbp NheI fragment affects the segregational stability of pTiC58, we measured the loss ratio of pTiC58 with or without the fragment after serial cultivation. After 5 repetitions of culturing at 37°C, 90% of the colonies lost pTiC58. In contrast, no colony lost pTiC58px under the same experimental conditions (Table 2). This result shows that the 2.6-kbp NheI fragment increased the segregational stability.
Transcription of tiorf24 and tiorf25 in vivo was investigated by RT-PCR experiments. RNA isolated from cells harboring various plasmids was subjected to reverse transcription as described in MATERIALS AND METHODS. The oligonucleotide primers used for PCR were located at the 3’-end of tiorf25 and in the middle of tiorf24, respectively (Fig. 3B). As shown in Fig. 4, a PCR product of about 550-bp in size was observed when total RNA extracted from MAFF301001rif and MNS-1 harboring pN2 or pBINN2 was used, whereas no such fragment was produced using total RNA from MNS-1 without the plasmids. Increased expression was observed in cells harboring pBINN2, which contained the 2.6-kbp NheI fragment on a multicopy-type vector. These data indicate that tiorf24 and tiorf25 are transcribed as a single mRNA.
 View Details | Fig. 4. RT-PCR analysis of transcription from tiorf24 and tiorf25 genes. RNAs extracted from A. tumefaciens MNS-1 harboring a plasmid were used as templates for the reverse transcriptase reaction. PCR amplification was carried out using the oligonucleotide primers shown in Fig. 3B and MATERIALS AND METHODS. |
In order to determine whether the tiorf24 and tiorf25 genes can act on plasmids not only in a cis but also in a trans manner, an incQ-type replicon, pHRPN2, which has the 2.6-kbp NheI fragment, was introduced into MNS-1 harboring a repABC vector plasmid, pK18msr. MNS-1 harboring pHRPN2 and pK18msr allowed entry of incoming repABC plasmid at the same level as cells with pK18msr only (Fig. 2A). These results show that tiorf24 and tiorf25 are able to act effectively only in a cis manner. In addition, we investigated whether the 2.6-kbp segment of pTi-SAKURA was effective even if the incoming and resident plasmids were of different replication types. MNS-1 harboring pN2 allowed entry of an incQ-type replicon, pHRP311, and an incP-type replicon, pBINTc, with the same efficiency as MNS-1 and MAFF301001rif (Fig. 5A and B). This observation indicates that the resident and incoming plasmids must have the same type of replicon for the two ORFs to exert their effect. This confirmed that the effect of the 2.6-kbp segment was caused by incompatibility enhancement.
 View Details | Fig. 5. Efficiency of introduction of incQ and incP replicons into A. tumefaciens and E. coli. Cells harboring a resident plasmid were subjected to conjugation with donor E. coli S17-1 λpir harboring an incoming plasmid shown in the upper part of each panel. A. tumefaciens MNS-1 (panels A, B and C) and E. coli Sy327 λpir (panel D) harboring a resident plasmid as shown underneath each bar were used as recipient. |
We next examined whether the incompatibility-enhancing effect of tiorf24 and tiorf25 takes place even when the two genes are located on replicons other than the repABC type or even when the two genes are contained in E. coli. The 2.6-kbp NheI fragment on the incP vector pBIN19 (see pBINN2) suppressed the entry of another incP-type replicon pBINTc into MNS-1 16-fold. However, such suppression was not detected in E. coli (Fig. 5C and D). These results show that the 2.6-kbp NheI fragment confers incompatibility-enhancing effect to the resident plasmid when the same type replicon is introduced in Agrobacterium cells, but that the fragment does not contain an incompatibility-determining region itself.
Homology analysis using the FASTA program indicated that tiorf24 is an orphan ORF, having no homologous ORF in the databases searched, and that tiorf25 is a homolog of stbB, which is a part of the stbABCD gene cluster. stbABCD is a plasmid stability locus in Pseudomonas syringae pv. tomato (Hanekamp et al., 1997). Neither Pseudomonas stbB nor any stbB homologues (Table 3) have been characterized functionally yet. No matching motif was detected by MOTIF analysis. However, domain searching using the Pfam program detected the PIN (twitching motility protein PilT N-terminus) domain in the Tiorf25 protein (score = 23.0 bits, E value = 8e–07).
 View Details | Table 3. Homologues of the Tiorf25 protein |
This study showed that the region which is responsible for plasmid stability as well as incompatibility-enhancement in pTi-SAKURA is located on a 2.6-kbp NheI fragment, which contains tiorf24 and tiorf25. The 2.6-kbp NheI fragment has the ability to inhibit transformation by an incoming plasmid only when the resident and incoming plasmids belong to the same incompatibility group. This inhibition occurred regardless of the plasmid introduction procedure (conjugation or electroporation). Thus, inhibition should occur by a mechanism that operates after cell-cell contact. Because the 2.6-kbp NheI fragment was effective on repABC replicons and an incP replicon (Fig. 2A and Fig. 5C), its effect is not directly involved in replicon-specific replication, stabilization, or incompatibility mechanisms. Since two different replication type plasmids containing the 2.6-kbp NheI fragment (pN2 and pHRPN2) can replicate independently in the same Agrobacterium cell (data not shown), it is unlikely that this region is an incompatibility-determining site, for example one that interacts directly with cellular centromere-like components. Addition of the 2.6-kbp NheI fragment to pTiC58 also decreased the loss ratio during growth at 37°C. The 2.6-kbp NheI fragment determines higher plasmid stability, and plasmid incompatibility enhancement may be reflected by increasing segregational stability.
The 2.6-kbp NheI fragment contains two complete ORFs (tiorf24 and tiorf25). Our RT-PCR experiment showed that the two ORFs are transcribed as a single mRNA. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a plasmid stabilization protein (Table 3). The putative plasmid stabilizing genes listed in Table 3 are homologues of stbB on the B-plasmid of Pseudomonas syringae pv. tomato (Hanekamp et al., 1997). It has been reported that the stbB gene is encoded in the plasmid stabilization cluster (stbABCD), but its function is still unknown. These data also suggest that the incompatibility-enhancing effect of tiorf24 and tiorf25 is caused by a plasmid stabilization mechanism. Our domain search using the Pfam program detected the PIN (twitching motility protein PilT N-terminus) domain in Tiorf25. PIN is a compact domain of about 100 amino acid residues, with two conserved aspartate residues. We also detected the PIN domain in all proteins listed in Table 3. Leptospira interrogans gene vapC has a PIN domain and is homologous to the putative stabilizer genes listed in Table 3. The vapC gene encodes a toxic protein of the toxin-antitoxin system (TA system), which also involves the antitoxin gene vapB (Zhang et al., 2004). These genes are closely located, suggesting an operonic structure, as generally found in TA systems. Although the Leptospira TA system is located in the chromosome, the region has the ability to stabilize an unstable plasmid when added to the plasmid. Because tiorf25 also contains a PIN domain and together with adjacent tiorf24 comprises an operon, the Ti plasmid stabilizing effect of two ORFs might be caused by a TA mechanism.
The difference in the plasmid ejection ratio in cells containing pTi-SAKURA and pTiC58 in response to introduction of incompatible repABC replicons is contingent on tiorf24 and tiorf25. Furthermore, differences in exclusion and permissiveness for repABC plasmid incursion were significant between intact Ti plasmids and smaller plasmids such as pN2 (data not shown). It is likely that an additional stabilization factor causes the difference in permissiveness between intact Ti and small repABC plasmids. An alternative explanation is the contribution of plasmid size differences, since the stability of yeast artificial chromosomes depends on their size (Zakian et al., 1986), and repABC replicons are generally huge plasmids or chromosomes, such as the linear chromosome of A. tumefaciens C58 (about 2,000 kbp), pSymb of Sinorhizobium meliloti (about 1,700 kbp) and Ti and Ri plasmids (about 200 kbp) (Galibert et al., 2001; Goodner et al., 2001; Moriguchi et al., 2001).
The authors are grateful to Mrs. J. Bautista-Zapanta for her helpful comments.
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