Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.     

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.     

Fingerprints of Agrobacterium Ti plasmids

Many crown gall-inducing Agrobacterium tumefaciens strains have multiple plasmids, only one of which, the tumor-inducing (Ti) plasmid, is essential for oncogenicity. For comparison of Ti plasmids, single-plasmid-containing transconjugant or transformant derivatives were used as sources of pure Ti-plasmid DNA. Fingerprinting was undertaken using the restriction endonuclease SmaI because it produced a relatively simple cleavage pattern. Three groups of Ti plasmids are discernible based upon both their genetic characteristics and their SmaI fingerprints: (1) Octopine-type Ti plasmids, which confer oncogenicity and octopine utilization on the bacterium. Tumors incited produce octopine. This group of plasmids is highly conservative; fingerprints of all members were identical except for two minor variations. (2) Nopaline-type Ti plasmids, which confer oncogenicity and nopaline utilization on the bacterium. Tumors incited may or may not produce nopaline; these plasmids have fingerprints that suggest variable degrees of relationship, including one that appears unrelated to the rest (3) Null-type plasmids, which confer oncogenicity but neither utilization trait on the bacterium. Tumors incited by this class of strains produce neither octopine nor nopaline. Only one member of this group has been examined thus far. Fingerprints of plasmids from several nononcogenic strains examined bore no resemblance to fingerprints of any of the Ti plasmids.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

Rapid divergence of Agrobacterium vitis octopine-cucumopine Ti plasmids from a recent common ancestor

The octopine/cucumopine (o/c) Ti plasmids of the grapevine-associated Agrobacterium vitis strains constitute a family of related DNA molecules. Restriction maps were established of two limited-host-range o/c Ti plasmids, pTiAg57 and pTiAB3, and of the wide-host-range o/c Ti plasmid pTiHml. Together with the previously obtained map of the wide-host-range o/c Ti plasmid pTiTm4, about 1000 kb were mapped with a resolution of 0.2 kb, allowing a detailed comparison of the various structures. One region of the o/c Ti plasmids is highly conserved and differs mainly by the presence or absence of relatively small DNA fragments (0.9–2.7 kb); the other region has been modified more extensively and carries large sequences specific for each Ti plasmid type. The sequence similarity within large conserved regions shows that these plasmids have diverged recently and that their evolution was driven by large-scale genetic events rather than single nucleotide changes. These results have important implications for studies on bacterial evolution.     

Wound-Released Chemical Signals May Elicit Multiple Responses from an Agrobacterium tumefaciens Strain Containing an Octopine-Type Ti Plasmid

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.     

Construction and application of R prime plasmids, carrying different segments of an octopine Ti plasmid from Agrobacterium tumefaciens, for complementation of vir genes

Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with Ti plasmids. They were used to study genetic complementation of Ti plasmid insertion mutants, outside the T-DNA region, which affected oncogenicity. Complementation was observed in both recombination-proficient and -deficient strains. The complementation in trans indicates that certain functions essential for tumor formation outside the T-DNA region are probably expressed in the bacterium. Therefore, the authors proposed to make a distinction between virulence (Vir) functions and oncogenic (Onc) functions of the octopine Ti plasmid of Agrobacterium tumefaciens. A large R prime was obtained, carrying the whole Ti plasmid, except a 7-Mdalton segment, containing the Ti plasmid replicator region. Strains harboring this plasmid induced normal tumors, showing that the replicator region of the octopine Ti plasmid is dispensible for tumor induction.     

The use of transposons to introduce well-defined deletions in plasmids: Possibilities for in vivo cloning

A method for obtaining well-defined deletions in an octopine Ti plasmid was developed. It was based on the assumption that deletions would occur between two directly repeated transposons, when both are temporarily present in one plasmid molecule. To obtain such a situation, recombination has been forced between Agrobacterium tumefaciens Ti plasmids, each carrying the transposon Tn1831 at a different position. In a number of cases, most probably when the transposons are directly repeated, deletion formation indeed occurred and at high frequency. Mutants were isolated carrying Ti plasmids with one copy of Tn1831, and the region of DNA, between the positions of the transposons in the original plasmid, deleted. Moreover, in the case that the segment of DNA, enclosed by the two transposons, harbors the requirements for autonomous replication of an R plasmid, it is shown that in vivo cloning of such a segment of the Ti plasmid on the R plasmid can be accomplished.     

Cloning of F DNA fragments carrying the origin of transfer oriT and the fertility inhibition gene finP

The two SalI fragments derived from the F transfer region that are bounded at one end by the SalI cleavage site in traM were cloned into pBR322. From these, smaller (540 bases) SalI-BglII fragments were subcloned to give plasmids containing the origin of transfer oriT (pED806) and finP (pED812), respectively, but no entire tra genes. All four plasmids were characterized by genetic tests and by restriction endonuclease analysis. pED806 could not be used to search for an F oriT-related “relaxation complex” because of its unexpected instability in the presence of Flac, and extensive efforts to prepare such a complex using other oriT⁺ plasmids were unsuccessful. We therefore suggest that a cell-free F relaxation complex does not exist. Protein synthesis directed by pED812 in minicells allowed the finP product to be tentatively identified as a 4000 M_r, protein.     

Seasonal Fluctuations and Long-Term Persistence of Pathogenic Populations of Agrobacterium spp. in Soils

Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 × 10⁵ pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.     

Novel Toxin-Antitoxin System Composed of Serine Protease and AAA-ATPase Homologues Determines the High Level of Stability and Incompatibility of the Tumor-Inducing Plasmid pTiC58

Stability of plant tumor-inducing (Ti) plasmids differs among strains. A high level of stability prevents basic and applied studies including the development of useful strains. The nopaline type Ti plasmid pTiC58 significantly reduces the transconjugant efficiency for incoming incompatible plasmids relative to the other type, such as octopine-type plasmids. In this study we identified a region that increases the incompatibility and stability of the plasmid. This region was located on a 4.3-kbp segment about 38 kbp downstream of the replication locus, repABC. We named two open reading frames in the segment, ietA and ietS, both of which were essential for the high level of incompatibility and stability. Plasmid stabilization by ietAS was accomplished by a toxin-antitoxin (TA) mechanism, where IetS is the toxin and IetA is the antitoxin. A database search revealed that putative IetA and IetS proteins are highly similar to AAA-ATPases and subtilisin-like serine proteases, respectively. Amino acid substitution experiments in each of the highly conserved characteristic residues, in both putative enzymes, suggested that the protease activity is essential and that ATP binding activity is important for the operation of the TA system. The ietAS-containing repABC plasmids expelled Ti plasmids even in strains which were tolerant to conventional Ti-curing treatments.Agrobacterium tumefaciens strains bearing a tumor-inducing (Ti) plasmid are the etiological agents of crown gall disease. Most genes required for pathogenicity are located on the plasmids (17, 33). Ti plasmids are kept stable at a low copy number equivalent to that of the chromosomal DNA in the bacterial cells (32) due to the repABC locus (16, 30, 34). The stability of Ti plasmids differs among strains (11).Many genes for keeping plasmids stable have been reported in eubacteria, and these are divided into three categories based on their mechanism: multimer resolution systems, active partitioning systems, and toxin-antitoxin (TA) systems (15). Multimer resolution systems increase the number of plasmid molecules by resolving a multimer plasmid into monomers, resulting in a higher probability of plasmid distribution to daughter cells during cell division even when plasmid distribution occurs randomly (29). Active partitioning systems deliver plasmid copies to each progeny cell at cell division (21). In the repABC locus, the RepA and RepB proteins and parS site(s) ensure stable plasmid inheritance by the active partitioning system (2). TA systems contribute to plasmid maintenance in cell populations by initiating growth inhibition or death of plasmid-free cells and are widely distributed among eubacterial and archaeal plasmids as well as their chromosomes (9). Generally, the TA module consists of two genes which encode toxin and antitoxin. The antitoxin neutralizes the action of a cognate toxin by interaction with the toxin or its target molecules. When a plasmid harboring the TA module is lost from a host cell, the antitoxin molecules decrease to an ineffective level because the antitoxin is degraded quickly or diluted by cell division (15). Thereafter, the toxin exerts its toxicity and inhibits the host cell growth. RNA antitoxins can suppress toxin expression by binding to the toxin mRNA as an antisense RNA or repress toxicity effects by an unknown mechanism (6, 4). In pTi-SAKURA, the Ti plasmid in the A. tumefaciens strain MAFF301001, it was shown that the tiorf24 and tiorf25 module increased plasmid stability by the TA mechanism (40; also S. Yamamoto, unpublished data).Differences in Ti plasmid stability are critical for plasmid engineering and evolution (33). However, little is known about the stability factors of Ti plasmids other than the repABC locus. In our previous study (40), tiorf24 and tiorf25 were shown to increase the segregational stability and incompatibility of Ti plasmids and reduce the efficiency of transconjugants by the introduction of incompatible plasmids into host cells. The two genes are located 2.5-kbp downstream of repABC (8). Generally, incompatibility has been defined as a situation where two plasmids contain a related replication and/or partitioning system and are unable to exist in a cell simultaneously without external selection (1). A. tumefaciens strain C58, which contains a Ti plasmid pTiC58, allows entry of an incompatible repABC plasmid into the cell 60-fold less efficiently than a derivative of C58. The derivative of C58 harbors a small repABC vector instead of pTiC58 (40). This suggests the presence of incompatibility-enhancing genes on pTiC58.In this study, we located the responsible genes in pTiC58 and found that the novel genes ietA and ietS enhance the incompatibility and stability of the plasmid by the TA mechanism.     

Protein trans-Splicing To Produce Herbicide-Resistant Acetolactate Synthase

Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of E. coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E. coli cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.     

Autonomously replicating fragments of mitochondrial DNA from Penicillium urticae in Saccharomyces cerevisiae

Eight fragments which cover the whole range of the mitochondrial genome of Penicillium urticae were subcloned into the yeast integration vector YIp5. Transformation of Saccharomyces cerevisiae with the constructed plasmids by the alkali cation method indicated that six plasmids are able to replicate in yeast. Both closed and open circular forms of the plasmids were detected in the DNA extracts from transformants. Distribution of the autonomously replicating sequence in the mitochondrial genome was similar to that in P. chrysogenum except for one small region.     

Secretion of trans-zeatin by Agrobacterium tumefaciens: A function determined by the nopaline Ti plasmid

Production of the plant cytokinin, trans-zeatin, by a number of strains of Agrobacterium tumefaciens was measured by a combination of traceenrichment, HPLC and radioimmunoassay and confirmed by mass spectrometry. Secretion of trans-zeatin into a culture medium is a constitutive function of those strains which harbor a nopaline Ti plasmid. Strains cured of the nopaline Ti plasmid and those which harbor octopine or agropine plasmids are non-producers. Reacquisition of nopaline plasmids by cured strains restores production.     

Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids

Summary A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the mutagenized Ti plasmid fragment cannot replicate in these cells, they can be rescued by recombination with the homologous region of the Ti plasmid. The cointegrates formed were resolved in a second recobination event, which was detected by loss of the drug resistance marker of the E. coli plasmid. Subcloning of the Ti plasmid fragments labeled with Tn5 showed that the frequency of rescue of the hybrid plasmid as a cointegrate and its segregation in agrobacteria depend on the degree of homology with the Ti plasmid. We also applied the strategy for site-directed Tn5 mutagenesis to insert specifically the replication origin of bacteriophage fd and the thymidine kinase gene from Herpes virus into the T-DNA of Ti plasmid-C58.     

Fingerprinting and sequence homology of plasmids from different virulent strains of Agrobacterium rhizogenes

Several wild-type virulent strains of Agrobacterium rhizogenes, belonging to both biotypes 1 and 2, were analyzed for plasmid content. All the strains were found to harbor at least one large plasmid, of a size comparable to that of A. tumefaciens Ti plasmids as determined by electron microscopy. The plasmids were characterized by restriction endonuclease finger-printing and compared for sequence homology by the Southern blot-hybridization technique. Despite the diversity of the restriction cleavage patterns, the plasmids of the various strains share rather extensive sequence homology. All of the biotype 1 organisms analyzed were shown to harbor one common plasmid.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system

Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new “Ti/Ri eviction plasmids,” each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.

     

Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain

Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean. We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and l,l-succinamopine utilization loci. Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542. Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization. The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved. Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence. This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.