Incompatibility between a Rhizobium Sym plasmid and a Ri plasmid of Agrobacterium

The symbiotic plasmid of Rhizobium trifolii G1008 was mobilized to other Rhizobium strains and to Agrobacterium using Tn5-Mob, a transposon that confers on a host replicon the ability to be mobilized in trans by RP4. Incompatibility was observed between pSymG1008 and the hairy-root-inducing plasmid pRi1855. Agarose gel electrophoresis revealed that pRi1855 was eliminated as an autonomous element in the presence of pSymG1008 and its absence was correlated with loss of the ability to induce hairy root disease. This indicates a close ancestral relationship between a Rhizobium symbiotic plasmid and a plant pathogenic plasmid of Agrobacterium. pSymG1008 and pRi1855 can be assigned to the IncRh-3 incompatibility group. Furthermore, pSymG1008 was mobilized at low frequency to R. phaseoli 51E and the transconjugants isolated had lost the indigenous Sym plasmid and the ability to nodulate beans.     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

T-DNA fragments of hairy root plasmid pRi8196 are distantly related to octopine and nopaline Ti plasmid T-DNA

Agrobacterium Ti (tumor-inducing) and Ri (root-inducing) plasmids transform dicot plant cells by insertion of a specific plasmid sector called T-DNA (transferred DNA) into host plant nuclear DNA. The mannopine-type Ri plasmid pRi8196 contains four BamHI fragments that encompass core T-DNA. We report Southern hybridization studies that show that these four fragments have no strong homology to octopine-, nopaline-, or agropine-type Ti plasmids. We detected and mapped very weak homology regions, most of which are assignable to opine synthase or opine catabolic functions on the Ti plasmid. We found no homology between Ri T-DNA and the region of Ti T-DNA that encodes tumor morphology functions.     

Isolation and characterization of a cryptic plasmid from mesophilic aeromonads: potential as a cloning vector

A 2.6 kb covalently closed circular plasmid has been isolated from clinical and environmental isolates of Aeromonas sobria and A. hydrophila. The possibility that the plasmid carries genetic determinants that mediate resistance to a variety of anti-microbial agents has been eliminated. The plasmid is stable at approximately 20-25 copies per chromosome equivalent which, together with its relatively small size and the presence of unique restriction sites, makes it a good candidate for development as a cloning vector.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei.

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Genome structure of Ri plasmid (2). Sequencing analysis of T-DNA and its flanking regions of pRi1724 in Japanese Agrobacterium rhizogenes.

We sequenced 42.6 kb including T-DNA and its flanking regions which corresponds to about 1/5 of entire length of a mikimopine-type Ri plasmid, pRi1724 in A. rhizogenes. We identified 37 ORFs (Open Reading Frames) including genes in total. Among them, 20 ORFs are probably new genes. Those ORFs have similarity with those in Agrobacterium and 9 ORFs of them was newly found on Ri plasmids.     

Characterization of a plasmid from moderately halophilic eubacteria.

A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.     

Map location on Agrobacterium root-inducing plasmids of homologies with the virulence region of tumor-inducing plasmids

Southern-type hybridizations were carried out in order to identify sequence homologies with the pTi vir loci, on an agropine-type plasmid (pRiHRI) and a mannopine-type plasmid (pRi8196) of Agrobacterium rhizogenes. The localization of the sequences hybridizing with subcloned fragments containing vir A, B, G, C, and D from pTiAch5 indicated a similar linear organization of the pTi vir loci and their homologies on pRiHRI and pRi8196, though no homology was detected on both pRi with a 1.1-kb internal fragment of virD. No homology was detected either with the vir E locus on pRiHRI vir region, nor with the virF locus on both pRi vir regions. As on nopaline pTiC58, fragments bearing the homologies with virC and virG are closer together on both pRi than on octopine pTiAch5. A preliminary functional map of the pRiHRI vir region is deduced from this study.     

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Construction of a vector plasmid for use in Gluconobacter oxydans

A host vector system in Gluconobacter oxydans was constructed. An Acetobacter-Escherichia coli shuttle vector was introduced with the efficiency of 10(4) transformants/microg of DNA. Next, aiming for a self-cloning vector, we found a cryptic plasmid (which we named pAG5) of 5648 bp in G. oxydans strain IFO 3171, and sequenced the nucleotides. The plasmid seemed to have only one open reading flame (ORF) for a possible replication protein. Shuttle vectors of Gluconobacter-E. coli were constructed with the plasmid pAG5 and an E. coli vector, pUC18.     

Analysis of Agrobacterium tumefaciens plasmid pTiC58 replication region with a novel high-copy-number derivative.

The origin of replication, ori, of the nopaline tumor-inducing plasmid, pTiC58, mapped in a region that shares sequence homology with octopine plasmids pTiAch5 and pTiB6. Within this region, the minimum amount of DNA necessary for maintaining autonomous replication was a 2.6-kilobase region, which also comprised the incompatibility function inc. pTiC58 derivatives containing inc were incompatible with Agrobacterium tumefaciens plasmids pTiC58, pTiD1439, pTiAch5, pTi15955, and pTiA5 and were compatible with A. rhizogenes plasmid pRi12. Situated adjacent to the origin region was a 1.5-kilobase par segment involved in stable inheritance of pTiC58 under nonselective growth conditions. When par was present, plasmid maintenance approached that of the wild-type pTiC58. Rapid loss from the cell population was observed for plasmids not containing this locus. Another 1.5-kilobase region, cop, positively regulated pTiC58 copy number, enabling certain pTiC58 derivatives to exist at a copy number up to 80 times higher than that of wild-type pTiC58. Deletions within the cop locus resulted in reduced copy number. The ori/inc regions were flanked on either side by the par and cop loci.     

Construction of promoter-probe plasmid vector

A new plasmid vector, designated pBRS188 has been constructed for cloning of promoter-containing DNA fragments. This plasmid is a derivative of the E. coli drug-resistance plasmid pBR322 in which a small region (13 base pairs long) within the Tc promoter is eliminated. As a result of the alteration pBRS188 has lost the ability to confer Tc resistance to the host strain. Cloning of foreign DNA fragments, carrying promoters for E. coli RNA polymerase, into the unique EcoRI site of pBRS188 allows to isolate the recombinant TcR transformants. Our construction required the use of new techniques, involving partial hydrolysis of DNA fragments by E. coli DNA polymerase I in the presence of one deoxyribonucleosidetriphosphate and by nuclease S1. An important feature of this method is the ability to regenerate restriction endonuclease recognition sites at junctions of DNA fragments.     

Recovery of morphologically normal transgenic tobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid

Summary Co-transformation of tobacco (Nicotiana tabacum) leaf explants with Agrobacterium rhizogenes harbouring pRi1855 and the binary vector pBin19 was achieved at a frequency of 67%. The kanamycin resistant hairy roots were cultured via a callusing phase to regenerate plants which were partially fertile when outcrossed with wild-type pollen. Phenotypic and molecular analysis of the F₁ progeny demonstrated the efficient segregation of the hairy root marker from the kanamycin resistance marker, enabling morphologically normal plants to be recovered which retained the binary vector marker gene. This co-transformation strategy provides a means of introducing non-selectable genes into plants in cases where antibiotic resistance markers are undesirable.     

Genome structure of Ri plasmid (3). Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes.

The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced. The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5. The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids. We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.