Modification of T-DNA of nopaline Ti plasmid by intermediary vector and utilization of agrocin 84 sensitivity as simple criterion of conjugation transfer of modified Ti plasmid

The chloramphenicol resistance gene from pSa was introduced into T-DNA of pTi T37 of Agrobacterium tumefaciens by cointegration with intermediary plasmid based on pBR322. The resulting intermediary vector was mobilized to A. tumefaciens T37 by conjugative plasmid pRK2. The RK2 plasmid also forms contegrates with pTi due to the Tn3 transposon which was used for the mobilization of modified pTi into plasmid-less A. tumefaciens strain. Transconjugants were selected on the basis of their antibiotic resistance markers and tested for agrocin sensitivity as proof of Ti plasmid transfer. Agrocin sensitivity of tranconjugants together with chloramphenicol resistance was shown to be a sufficient and simple criterion of transfer of modified Ti plasmids. Agrobacterium strains with modified Ti plasmids showed decreased virulence in consequence of the presence of additional borderline sequence inside their T-DNA.     

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.     

Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium tumefaciens

We have identified a new insertion sequence, IS866, located in the auxin synthesis gene TA iaaH of Tm4, a wide host range biotype III octopine/cucumopine type Agrobacterium tumefaciens strain with two T regions on its tumor-inducing (Ti) plasmid, TA, and TB. IS866 is 2716 bp long, has inverted repeats of 27 bp with three mismatches, and generates 8-bp direct repeats upon integration. In addition to IS866, pTiTm4 carries two copies of a related element, IS867, associated with TA and TB, respectively. A systematic study of 92 virulent Agrobacterium strains has shown that among the three biotypes all octopine/cucumopine and vitopine biotype III isolates contain IS866-like elements. The various octopine/cucumopine Ti plasmids always carry IS866 and IS867 at the same position as in pTiTm4. The chromosomes of the bacteria which contain these Ti plasmids also carry IS866 and IS867 copies but in varying numbers and locations.     

Transfer of the Ti plasmid from Agrobacterium tumefaciens into Escherichia coli cells

We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.     

Homologous Recombination as the Main Mechanism for DNA Integration and Cause of Rearrangements in the Filamentous Ascomycete Ashbya Gossypii

A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resistance gene expressed from a strong A. gossypii promoter. Transformations with circular plasmids yielded slowly and irregularly growing geneticin-resistant mycelia in which 1% of nuclei contained plasmid sequences. Occasionally, fast growing sectors appeared which were shown to be initiated by homologous integration of the transforming DNA. Transformants obtained with plasmids linearized within the homology region immediately exhibited fast radial growth. In all 28 transformants analyzed plasmid DNA was integrated homologously. Such apparent lack of nonhomologous recombination has so far not been observed in filamentous ascomycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, mainly in the older mycelium: excision of whole plasmid copies and rearrangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lacking sequence repetitions were constructed and used for stable one-step gene replacement in A. gossypii.     

Plasmid maintenance of derivatives of oriP of Epstein-Barr virus.

oriP is the origin of plasmid replication of Epstein-Barr virus. Replication from oriP requires both the cis-acting elements (the family of repeats and the dyad symmetry element) and the viral origin-binding protein, EBNA-1. The ability of plasmids containing oriP to be maintained stably in EBNA-1-positive cells reflects the efficiency both of their replication and of their segregation each cell cycle. The efficiency of plasmid maintenance was determined for plasmids containing derivatives of oriP with one copy of the dyad symmetry element and two copies of the family of repeats by measuring the rate at which they were lost from cells in the absence of selection. These measurements demonstrated that plasmids with derivatives of oriP with two copies of the family of repeats in one orientation are maintained only slightly less efficiently than is wild-type oriP. To determine whether plasmid maintenance could be affected by reinitiation at the dyad symmetry element (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989), plasmids containing derivatives of oriP with two copies of the dyad symmetry element and one copy of the family of repeats were compared with plasmids containing wild-type oriP in EBNA-1-positive cells. These measurements showed that plasmids containing a derivative of oriP with two copies of the dyad symmetry element are maintained as efficiently as is wild-type oriP and are not amplified relative to wild-type oriP. These observations indicate that the trans-acting factors that regulate DNA to replicate once per S phase are insensitive to multiple cis-acting regulatory sites within a replicon.     

产生无标记农杆菌突变体方法的建立及优化

农杆菌已经用作许多生物过程研究的模型细菌,为了解析这些生物过程的分子机理,对农杆菌的某些基因进行突变就显得非常重要．以自杀性基因sacB作为反向可选择性标记基因,利用同源重组的原理,建立了一种可对农杆菌基因进行准确插入、删除和位点置换的突变方法,所获突变体不带任何不需要的外源DNA序列．通过详细研究同源序列的长度对农杆菌同源重组效率和突变体产生概率的影响,以及对农杆菌中的同源重组机理的分析,提出了优化该突变体产生方法的方案,即通过设计不等长的上下游同源序列和选择其中一种类型的单交换重组体来筛选二次交换重组体的方法,可以显著地提高理想突变体的产生概率．研究结果对如何提高突变体的产生概率和减少突变体筛选的工作量具重要的参考价值．利用该方法成功地获得了两个基因被同时删除而且不含抗性标记的农杆菌突变株．     

Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome.     

A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1.

Several autonomously replicating sequences of Hansenula polymorpha DL-1 (HARSs) with the characteristics of tandem integration were cloned by an enrichment procedure and analyzed for their functional elements to elucidate the mechanism of multiple integration in tandem repeats. All plasmids harboring newly cloned HARSs showed a high frequency of transformation and were maintained episomally before stabilization. After stabilization, the transforming DNA was stably integrated into the chromosome. HARS36 was selected for its high efficiency of transformation and tendency for integration. Several tandemly repeated copies of the transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end. Bal 31 digestion of the total DNA from the integrants followed by Southern blotting generated progressive shortening of the hybridization signal, indicating the telomeric localization of the transforming plasmids on the chromosome. The minimum region of HARS36 required for its HARS activity was analyzed by deletion analyses. Three important regions, A, B, and C, for episomal replication and integration were detected. Analysis of the DNA sequences of regions A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology with the ARS core consensus sequence of Saccharomyces cerevisiae. Region B contained two directly repeated sequences which were predicted to form a bent DNA structure. Deletion of the AT-rich core in region A resulted in a complete loss of ARS activity, and deletion of the repeated sequences in region B greatly reduced the stability of the transforming plasmid and resulted in retarded cell growth. Region C was required for the facilitated chromosomal integration of transforming plasmids.     

Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions.

The control of expression of the Bacillus subtilis spoIIA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIIA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglII, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by beta-galactosidase activity. The results indicate that spoIIA was under positive control by a moiety present at about five copies per chromosome. Spore formation was not affected by amplification, so spoIIA-lacZ reiteration did not sequester a molecule required elsewhere for sporulation.     

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.     

Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.     

Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens

Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.     

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogenous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.     

A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens

Ti (Tumor inducing) plasmids in Agrobacterium tumefaciens can transfer their T-DNA region into dicotyledonous plants, in which the expression of T-DNA genes causes plant tumors and the production of bacterial nutrients, e.g., opines such as nopaline. Naturally occurring Ti plasmids (pTi) are difficult to cure by conventional curing methods because of their high stability. Here, we developed a novel curing method based on plasmid incompatibility. For this, a curing plasmid, pMGTrep1, was newly constructed and subsequently introduced into A. tumefaciens strains harboring pTi by conjugation with Escherichia coli harboring pMGTrep1. The conjugation yielded 32-99% nopaline non-utilizing agrobacterial transconjugants in which pMGTrep1 replaced pTi due to incompatibility. Then, pMGTrep1-less derivatives of the transconjugants are easily selected in the presence of sucrose because pMGTrep1 contains a sucrose-sensitive sacB gene. This efficient method is directly applicable for curing plasmids with the same incompatibility group and shoud also applicable to other types of plasmids in Agrobacterium groups, including A. rhizogenes, by replacing the rep gene region of the curing plasmid with that of the corresponding incompatibility.     

New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.