The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon

The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine-type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR-type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans. Purified RepA bound to an approximately 70-nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di- and tri-phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.     

Identification of a megaplasmid centromere reveals genetic structural diversity within the repABC family of basic replicons

The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility (inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning (par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity.     

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability

In Agrobacterium tumefaciens, the stability of Ti plasmids differs depending on the strain. So far, little is known about genes that cause the difference in stability. The repABC operon is responsible for replication and incompatibility of Ti plasmids. We constructed recombinant plasmids carrying the repABC operon and different portions of pTi-SAKURA. Cells having the recombinant plasmids that harbored a 2.6-kbp NheI fragment of pTi-SAKURA were found to be transformed via conjugation 100-fold less frequently with a small incompatible repABC plasmid than cells having the recombinant plasmids lacking the 2.6-kbp NheI fragment. Since the phenomenon occurred only when the resident and incoming plasmids belonged to the same incompatibility group, it was suggested that the 2.6-kbp NheI fragment bears the potential enhancing incompatibility. The fragment contained an operon consisting of two open reading frames, tiorf24 and tiorf25. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a group of plasmid stability genes. Removal of the 2.6-kbp fragment from the resident pTi-SAKURA increased the resident plasmid ejection ratio by the incoming repABC plasmid, whereas addition of the fragment to pTiC58 decreased the ejection ratio, and the loss ratio during growth at 37 degrees C. These data suggest that tiorf24 and tiorf25 are responsible for the stability of pTi-SAKURA, and reduce, in the host bacterium, the frequency of ejection of the resident plasmid, presumably through an incompatibility mechanism.     

Comparison of Biolistic and Agrobacterium -mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium -mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG-CAM, into rice (BX)Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium -mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium -mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two-cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium -mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium -mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium -mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.     

Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1

The replicator region of composite plasmid pTAV1 of Paracoccus versutus (included in mini-replicon pTAV320) belongs to the family of repABC replicons commonly found in plasmids harbored by Agrobacterium and Rhizobium spp. The repABC replicons encode three genes clustered in an operon, which are involved in partitioning (repA and repB) and replication (repC). In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (inc1, located in the intergenic sequence between repB and repC; and inc2, situated downstream of the repC gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay. The protein bound only inc2, forming two complexes in a protein concentration-dependent manner. The inc2 region contains two long (14-bp) repeated sequences (R1 and R2). Disruption of these sequences completely eliminates RepB binding ability. R1 and R2 have sequence similarities with analogous repeats of another repABC replicon of plasmid pPAN1 of Paracoccus pantotrophus DSM 82.5 and with centromeric sequences of the Bacillus subtilis chromosome. Excess RepB protein resulted in destabilization of the inc2-containing plasmid in Escherichia coli. On the other hand, the inc2 region could stabilize another unstable replicon in P. versutus when RepA and RepB were delivered in trans, proving that this region has centromere-like activity. Thus, it was demonstrated that repA, repB, and inc2 constitute a functional system for active partitioning of pTAV320.     

The repABC plasmid family

repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in Rhizobiales. Some strains of this bacterial order can contain multiple repABC replicons indicating that this plasmid family includes several incompatibility groups. The replication and stable maintenance of these replicons depend on the presence of a repABC operon. The repABC operons sequenced to date share some general characteristics. All of them contain at least three protein-encoding genes: repA, repB and repC. The first two genes encode proteins involved in plasmid segregation, whereas repC encodes a protein crucial for replication. The origin of replication maps within the repC gene. In contrast, the centromere-like sequence (parS) can be located at various positions in the operon. In this review we will summarize current knowledge about this plasmid family, with special emphasis on their structural diversity and their complex genetic regulation. Finally, we will examine some ideas about their evolutionary origin and trends.     

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.     

Comparative characterization of repABC-type replicons of Paracoccus pantotrophus composite plasmids

The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon. So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmid pTAV1 (107kb) of Paracoccus versutus. We studied the distribution of repABC-type replicons within bacteria belonging to the genus Paracoccus. We found that repABC replicons occur only in the group of pTAV1-like plasmids: pKLW1, pHG16-a, pWKS2, and pPAN1, harbored by different strains of Paracoccus pantotrophus. A partial sequencing approach followed by phylogenetic analysis revealed that these replicons constitute a distinct evolutionary branch of repABC replicons. Incompatibility studies showed that they represent two incompatibility groups designated IncABC1 (pTAV1, pKLW1, and pHG16-a) and IncABC2 (pPAN1). Sequence comparison using available databases allowed the identification, within plasmid pRS241d of Rhodobacter sphaeroides 2.4.1, of an additional sequence highly homologous to the paracoccal repABC replicons, which has been included in comparative analyses.     

Polar localization of replicon origins in the multipartite genomes of Agrobacterium tumefaciens and Sinorhizobium meliloti

The origins of replication of many different bacteria have been shown to reside at specific subcellular locations, but the mechanisms underlying their positioning and segregation are still being elucidated. In particular, little is known about the replication of multipartite genomes in bacteria. We determined the cellular positions of the origins of the replicons in the alpha proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti and found that they are located at the poles of the cells. Our work demonstrates the conserved extreme polar localization of circular chromosome origins in these alpha proteobacteria and is also the first to specify the cellular location of origin regions from the repABC family. The cellular location of a derivative of the RK2 plasmid is distinct from that of the alpha proteobacterium genomic replicon origins but is conserved across bacteria. Colocalization experiments with the genomic replicons of A. tumefaciens revealed that the repABC replicons, although preferentially positioned at the cell pole, colocalize only rarely. For the repABC replicons in this organism, occupying discrete spatial locations may contribute to their coexistence and stable inheritance.     

The expression of a novel antisense gene mediates incompatibility within the large repABC family of alpha-proteobacterial plasmids

Large extrachromosomal replicons in many members of the alpha-proteobacteria encode genes that are required for plant or animal pathogenesis or symbiosis. Most of these replicons encode repABC genes that control their replication and faithful segregation during cell division. In addition to its chromosome, the plant endosymbiont Sinorhizobium meliloti also maintains the 1.4 Mb pSymA and 1.7 Mb pSymB symbiotic megaplasmids both of which are repABC-type replicons. In all repABC loci that have been characterized, an apparently untranslated intergenic region between the repB and repC genes encodes a strong incompatibility determinant (referred to as incalpha). Here we report the isolation of mutations within the incalpha regions of pSymA and pSymB that eliminate incompatibility. These mutations map to and inactivate a promoter in the intergenic region that drives the expression of an approximately 56 nucleotide untranslated RNA molecule that mediates incompatibility. This gene, that we have named incA, is transcribed antisense to the repABC genes. Our analysis suggests that the incA gene is conserved in repABC loci from a diverse spectrum of bacteria.     

Brucella abortus arginase and ornithine cyclodeaminase genes are similar to Ti plasmid arginase and ornithine cyclodeaminase

Brucella abortus arginase and ornithine cyclodeaminase genes have been cloned and sequenced. These gene sequences are located in the same operon and occur in the same order as the homologous genes in Agrobacterium tumefaciens Ti C58 plasmid. The nucleotide sequences of the two genes have 72% and 65% identity to the respective Ti plasmid genes. Both genes are present in a single copy, and expression of arginase is regulated in response to arginine.     

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86

Summary Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.     

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.