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At least 19 recordsLinked to original sources
PubMed1991

Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids.

The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone. Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic. Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency. Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies. The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ. The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751. This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants.

Base Sequence↗
PubMed1992

A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation.

The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.

Agrobacterium tumefaciens↗
PubMed1979

Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.

The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a.

Conjugation, Genetic↗
PubMed1991

A DNA transformation-competent Arabidopsis genomic library in Agrobacterium.

We have constructed a nuclear genomic library from the cruciferous plant Arabidopsis thaliana ecotype Columbia in a cosmid vector, pLZO3, and a host organism, Agrobacterium tumefaciens AGL1, which can directly DNA-transform the parent organism, Arabidopsis. The broad host range cosmid pLZO3 carries a gentamicin acetyltransferase gene as bacterial selective marker and tandem, chimeric neomycin and streptomycin phosphotransferase genes as plant selective markers. Agrobacterium AGL1 carries the hypervirulent, attenuated tumor-inducing plasmid pTiBo542 from which T-region DNA sequences have been precisely deleted, allowing optimal DNA transformation of many dicotyledonous plants. Agrobacterium AGL1 also carries an insertion mutation in its recA general recombination gene, which stabilizes the recombinant plasmids. The Arabidopsis genomic library consists of some 21,600 clones gridded onto 96-well microtiter dishes and, if random, carries at least three genomic equivalents. When probed for the presence of several Arabidopsis low copy-number genes, the genomic library seems representative. As with the unicellular organisms Escherichia coli and Saccharomyces cerevisiae, this DNA transformation competent genomic library should expedite gene isolation, by gene rescue, in multicellular organisms like Arabidopsis.

Agrobacterium tumefaciens↗
PubMed1990

A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a beta-galactosidase fusion protein into tobacco nuclei.

Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA to the genome of higher plants. Of the virulence region-encoded proteins of the tumor-inducing (Ti) plasmid of A. tumefaciens, the VirD1 and VirD2 proteins are essential for T-DNA transfer to plant cells. These two proteins have been shown to be directly responsible for the formation of T-strands. VirD2 was also shown to be firmly attached to the 5' termini of T-strands; these facts have led to its postulation as a pilot protein in the T-DNA transfer process and as a nucleus-targeting signal in plants. We have constructed a chimeric gene by fusing the virD2 gene and the Escherichia coli lacZ gene. Cell fractionation and electron microscopy studies with transgenic tobacco plants containing the VirD2-LacZ fusion protein indicate that the first 292 amino acids of VirD2 are able to direct the cytoplasmic protein beta-galactosidase to the plant nucleus. This provides an example of cross-kingdom nuclear localization between two free-living organisms: a bacterial peptide is capable of acting as a eukaryotic (plant) nuclear targeting signal.

Bacterial Proteins↗
PubMed1992

Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism.

During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.

Agrobacterium tumefaciens↗
PubMed1990

The virD genes from the vir region of the Ti plasmid: T-region border dependent processing steps in different rec mutants of Escherichia coli.

We evaluated the substrate requirements for virD-mediated T-circle formation in an in vivo binary test system in Escherichia coli. Two copies of the 25-bp sequence which defines the right border of the T-DNA (transferred DNA) are sufficient, and the right and the left copy of the border are equivalent in function in this system. Experiments with different rec mutants show that the occurrence and frequency of circular double-stranded and single-stranded T-DNA equivalents strongly depend on rec functions of the host. These results are discussed in the context of processing of the tumor-inducing Ti plasmid preceding the T-DNA transfer from agrobacteria to plants.

Blotting, Southern↗
PubMed1989

Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens.

Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinF1 and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinF1) and 46,740 (pinF2). A comparison of the amino acid sequences of pinF1 and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes.

Amino Acid Sequence↗
PubMed1990

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.

Bacterial Proteins↗
PubMed1989

Putative start codon TTG for the regulatory protein VirG of the hairy-root-inducing plasmid pRiA4.

The nucleotide sequence of the virG gene for a transcriptional activator on the agropine-type hairy-root-inducing plasmid pRiA4 was determined. The sequence contained one possible open reading frame. The gene product with a molecular size of 26.5 kDa was identified by an Escherichia coli coupled-transcription-translation system using cloned virG plasmids as templates. However, neither an ATG nor a GTG start codon which could give rise to such a protein was identified in the nucleotide sequence. Instead, TTG was found as a candidate for the start codon. This TTG was preceded, like most other TTG start codons, by both a Shine-Dalgarno (SD) sequence and a T signal which are respectively complementary to the 3'-end region of 16S rRNA and the T psi loop of initiator tRNA. Further evidence for the start at TTG was obtained by gene fusion experiments. When the E. coli lacZ gene, whose expression entirely depends on the transcription and translation from upstream regions, was connected in-phase with virG either directly upstream or downstream of the TTG sequence, only the latter fused gene expressed the beta-galactosidase activity in Agrobacterium cells in response to a plant phenolic compound, acetosyringone. The TTG codon preceded by an SD sequence and a T signal is also conserved in the virG sequences from other three tumor-inducing plasmids previously reported.

Amino Acid Sequence↗
PubMed1977

Reversal of the neoplastic state in plants.

Crown-gall transformation involves the gradual and progressive activation of several biosynthetic capacities of the normal cell. These changes in cellular heredity, although extremely stable, are nonetheless potentially reversible and leave the cell totipotent. There is growing evidence that tumor-inducing principle is a self-replicating entity similar to a plasmid. Thus, it could be argued that tumor progression involves changes in the number or state of these entities in the cell. Studies of CDF habituation bear directly on this problem. Conversion of a cell division factor (CDF)-requiring normal cell to the CDF-autotrophic state is a key event in transformation. The fact that CDF habituation is progressive, occurs in the absence of agents of bacterial origin, and has an epigenetic basis indicates that it is not necessary to invoke either somatic mutation or the addition of foreign genes to account for tumor stability and progression in crown-gall. This conclusion provides further support for the hypothesis that, in the words of Braun,(78) "... the cancer problem is basically a problem of anomolous differentiation... Neoplastic growth, like developmental processes, stems from epigenetic modifications against a constant cellular genome."

Cell Differentiation↗
PubMed1976

[A RNA extract from oncogenic and non oncogenic strains of Agrobacterium tumefaciens is an indispensable element for the induction of tumors in Datura stramomium].

An RNA bound to the reverse transcriptase of Agrobacterium tumefaciens has been isolated and shown to be oncogenic for stem tissues of Datura stramonium grown under axenic conditions. The tumorous nature of the cellular change induced by the infectious rna was demonstrated by serial grafts of tumors on Datura stems and by cultivation of tumorous tissue in vitro on a medium without supplemental auxins and cytokinins. Active cellular proliferation within tissues of Datura stems was a prerequisite for expression of the oncogenic potential of the RNA. Further, infectious RNA was isolated from avirulent and attenuated strains of Agrobacterium tumefaciens including attenuated derivatives of strain AC58 which have been "heat-cured" of the plasmid associated with virulence. It is proposed that the infectious RNA is an essential but not the sole component of the tumor-inducing mechanism of the crown-gall bacterium.

Cell-Free System↗
PubMed1989

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.

Bacterial Typing Techniques↗
PubMed1976

Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium.

Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.

Chromosomes, Bacterial↗
PubMed1990

Characteristics of the nopaline catabolic plasmid in Agrobacterium strains K84 and K1026 used for biological control of crown gall disease.

Wild-type Agrobacterium radiobacter strain 84 and its Tra- derivative K1026, used for biological control of crown gall disease, each contain the plasmid pAtK84b. It confers incompatibility to tumor-inducing (Ti) plasmids of pathogenic A. tumefaciens, thus preventing transfer of Ti plasmids into K84 and K1026, and the consequent development of pathogens resistant to the specific antibiotic, agrocin 84 produced by K84 and K1026. pAtK84b also resembles one group of Ti plasmids in its capacity for directing nopaline catabolism. A study of the DNA homology among pAtK84b, pTiC58, and pTiAch5 was carried out. pAtK84b was transferred by conjugation to a plasmidless recipient and, after isolation, was hybridized with Ti plasmid DNA. Areas of DNA homology were located on published maps of pTiC58 and pTiAch5, a restriction enzyme map of pAtK84b was constructed, and areas of homology with DNA of known genetic function were located on the map. Strong and extensive (over 50%) homology was found between pAtK84b and pTiC58 (nopaline catabolic, Noc), but much less between pAtK84b and pTiAch5 (octopine catabolic). There was no detectable homology between pAtK84b and the oncogenic T-DNA and virulence (Vir) regions of either Ti plasmid. The size of pAtK84b was 173 kb and the orientation of regions of identified gene function (Noc, incompatability/origin of replication, and conjugal transfer) on pTiC58 was matched by the locations of homologous areas on pAtK84b. It is concluded that pAtK84b may be a deletion product of a pTiC58-type plasmid which has been disarmed in the oncogenic T-DNA and Vir regions.

Arginine↗
PubMed1979

Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines.

Total RNA isolated from three octopine-type crown gall lines contains sequences homologous to specific regions of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens strain 15955. A comparison of transcripts in these three tumor lines suggests that tumor cells transcribe various sequences within a sector of plasmid DNA of 13 x 10(6) daltons and that transcription may not be uniform across the plasmid derived sequences (T-DNA). Transcription of T-DNA by octopine-type tumors occurs at four major sites. The levels of transcription occurring at three of these sites appear to vary considerably among the three tumor lines investigated. Part of this variability may reflect differences in the organization and copy number of T-DNA. One of the transcription sites maps within a region of DNA with common sequence homology with all Ti plasmids. Varying amounts of transcript homologous to this region of T-DNA are present in all three tumor lines. It is suggested that transcription of these conserved sequences in the plant may have significance regarding the mechanism of tumorigenesis.

Arginine↗
PubMedDate not supplied

Transcription of the octopine catabolism operon of the Agrobacterium tumor-inducing plasmid pTiA6 is activated by a LysR-type regulatory protein.

Agrobacterium tumefaciens incites crown gall tumors on plant hosts by conjugally transferring a discrete fragment of oncogenic DNA. In addition to oncogenes, the transferred DNA contains genes that direct the synthesis and exudation of opines, which are used as nutrients by the bacteria. The bacterium contains one or more operons of Ti plasmid-encoded genes that are required for the internalization and utilization of opines, and transcription of these catabolic genes is induced by cognate opines. Here we localize the gene required for regulated expression of the octopine degradative operon of the pTiA6 plasmid to a 2-kb fragment of Ti plasmid DNA. The protein encoded by this DNA positively regulates the transcription of the catabolic operon in the presence of octopine. In addition, it negatively regulates its own gene in the presence or absence of octopine. The sequence of this gene was determined and analysis of the inferred protein sequence indicates that the gene encodes a member of the LysR family of prokaryotic transcriptional regulatory proteins.

Agrobacterium tumefaciens↗