Plasmid pTiA6 Research Articles

Functional role of the Ti plasmid-encoded catabolic mannopine cyclase in mannityl opine catabolism by Agrobacterium spp.

Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactonizes MOP to agropine (AGR). The gene of the octopine-type Ti plasmid pTi15955 encoding the catabolic MOP cyclase enzyme previously was localized to a 1.6-kb segment within a cosmid clone, pYDH208. A subclone containing only this region complemented the AGR catabolism-negative phenotype conferred by a derivative of the octopine-type plasmid pTiB6S3 containing a Tn7 insertion in the region encoding the MOP cyclase enzyme. Uptake assays of strains harboring pRiA4 or pArA4a, along with complementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transport system. To determine the requirement for MOP cyclase in opine catabolism unequivocally, a site-specific, nonpolar deletion mutation abolishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA). Strains harboring this MOP cyclase-negative mutant clone, pYDPH208, did not utilize AGR but continued to utilize MOP. Growth on AGR was restored in this strain upon introduction of clones encoding the pTi15955-derived catabolic or anabolic MOP cyclase genes. The induction pattern of MOA catabolism shown by strain NT1 harboring the MOP cyclase-deficient pYDPH208 suggests that AGR is converted into MOP by MOP cyclase and that MOP, but not AGR, induces catabolism of MOA. Genetic and biochemical analyses of MOP and AGR metabolism suggest that only the conversion of AGR to MOP is directly involved in catabolism of AGR, even though the reaction catalyzed by MOP cyclase predominantly lies in the lactonization of MOP to AGR.

A novel gene tag for identifying microorganisms released into the environment

A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the environment. Pseudomonas fluorescens 1855.344, a plant-growth-promoting rhizosphere bacterium, was chosen as the organism in which to develop and test the system. moc genes carried by pYDH208, a cosmid clone containing a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P. fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy. Modified P. fluorescens strains containing moc or moc::nptII inserted into a chromosomal site were constructed by marker exchange. One such modified strain, PF5MT12, utilized AGR as a sole carbon source and contained detectable levels of mannopine cyclase, an easily assayable enzyme encoded by the moc region. Catabolism of AGR could be used to recover selectively the marked strain from mixed populations containing a large excess of closely related bacteria. Nucleic acid-based detection strategies were developed on the basis of the unique fusion region between Agrobacterium DNA and Pseudomonas DNA in strain PF5MT12. The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fused DNA region by using PCR. The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseudomonas, or Escherichia coli DNA. This gene tag strategy gives a method for direct selection and enumeration of the marked strain from mixtures containing a large excess of closely related bacteria and a sensitive and highly specific system for detection by PCR amplification of the target fragment even in the presence of large amounts of DNA from related or unrelated organisms.

Chromosomal and pTi genotypes of agrobacterium strains isolated from Populus tumors in two nurseries

Agrobacterium tumefaciens strains isolated from aspen tumors have been characterized in order to study Ti plasmid stability in different chromosomal backgrounds under natural conditions. Chromosomal and pTi genotypes were characterized by DNA-DNA hybridization. Single tumors contained one to four strains of A. tumefaciens as characterized by their chromosomal genotypes and one or two different nopaline-type pTi genotypes. Genotypes of pTi were associated with identical chromosomal backgrounds in different tree nurseries, suggesting that pTi plasmids are stably maintained in the small set of strains causing the present aspen crown gall outbreaks.

Chromosomal and pTi genotypes of agrobacterium strains isolated from Populus tumors in two nurseries

Agrobacterium tumefaciens strains isolated from aspen tumors have been characterized in order to study Ti plasmid stability in different chromosomal backgrounds under natural conditions. Chromosomal and pTi genotypes were characterized by DNA-DNA hybridization. Single tumors contained one to four strains of A. tumefaciens as characterized by their chromosomal genotypes and one or two different nopaline-type pTi genotypes. Genotypes of pTi were associated with identical chromosomal backgrounds in different tree nurseries, suggesting that pTi plasmids are stably maintained in the small set of strains causing the present aspen crown gall outbreaks.

The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants.

Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence. In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used. An A. tumefaciens strain, A348 delta D, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed. Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers. As expected, deletion of the virD operon led to an avirulent phenotype. The virulence of this strain could be restored by providing virD1, virD2, and virD4 in trans. No requirement for virD3 in tumor formation was observed in these assays.

Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6.

Neoplastic crown gall tumors incited by Agrobacterium tumefaciens release novel amino acid or sugar derivatives known as opines, whose synthesis is directed by genes transferred to plant cells. Agrobacterium cells can transport and catabolize these compounds as sources of carbon and nitrogen. This article describes a region of the pTiA6 plasmid which is required for catabolism of the opine octopine and whose transcription is induced by octopine. This region of the plasmid contains four open reading frames, occQ, occM, occP, and occJ, which show homology to the family of so-called shock-sensitive permeases. TnphoA mutagenesis demonstrated that the OccJ and OccM proteins lie fully or partly in the periplasmic space. The OccJ protein was identified by electrophoresis and found to be fully localized in the periplasmic space. When these proteins were expressed in Escherichia coli, radiolabeled octopine became cell-associated.

Expression of Bean Storage Protein Minigene in Tobacco Seeds: Introns are not Required for Seed Specific Expression

Summary A native and mini (intronless) phaseolin gene were inserted into a mutant derivative of Agrobacterium tumefaciens plasmid pTi15955, in which the oncogenic tms2 and tmr loci were deleted. This pTi vector is referred to as a rooty-shooty mutant, pTiRS 2 014. The vector pTiRS 2 014 was used for the transfer and integration of phaseolin genes into the genome of tobacco followed by the regeneration of phenotypically normal tobacco plants. The presence of phaseolin polypeptides in R 1 and R 2 seeds was confirmed by immunological methods. These results demonstrate that native and mini-phaseolin genes are integrated stably into the respective tobacco plant lines and that the absence of the phaseolin introns did not affect its developmental expression.

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5′ and 3′ flanking sequences. The mas1′ and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2′ proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.

Transcription of the Octopine Catabolism Operon of theAgrobacteriumTumor-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.

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.

Localization and Characterization of the Region Encoding Catabolism of Mannopinic Acid from the Octopine-Type Ti Plasmid pTi15955

The region of the octopine-type tumor-inducing (Ti) plasmid pTi15955 encoding catabolism of mannopinic acid was localized to an 18-kilobase (kb) segment mapping between Ti plasmid coordinates 70 and 88. A subclone containing only this region normally did not allow utilization of any other mannityl opine. However, spontaneous mutants of the plasmid were isolated that conferred catabolism of agropinic acid. While the mutants remained regulated for mannopinic acid utilization, induction by this opine resulted in increased activity associated with agropinic acid catabolism. Respirometric studies and results from growth assays on mannopinic acid analogues indicated that the genes encoded on the 18-kb subclone were regulated in a wild-type fashion. By means of these analogues, spontaneous constitutive mutants were isolated. In several cases, the mutant phenotype was associated with an insertion event mapping within a 300-base pair region of the subclone insert. Although constitutive for catabolism of mannopinic acid, these mutants remained unable to catabolize any of the other mannityl opines. Segregation studies and genetic reconstitution experiments showed that one of the subclones isolated directly in Agrobacterium was actually composed of two complementing recombinant plasmids contained in the same cell. This indicated that the genes encoding mannopinic acid catabolism were organized into at least two complementation groups. These results point to a high degree of complexity in the organization and regulation of Ti plasmid genes encoding catabolism of a relatively simple carbon source.

Agrobacterium-mediated Transformation of Peach Cells Derived from Mature Plants that were Propagated in Vitro

Abstract Stem explants of in vitro-propagated peach (Prunus persica L. Batsch.) cultivars Compact Redhaven, Jerseyqueen, Rio Oso Gem, and Suncrest and peach rootstock Nemaguard were infected with several strains of Agrobacterium tumefaciens. Tumor tissue was produced on all cultivars infected with strain tms328::Tn5, which carries a mutated octopine Ti plasmid pTiA6. The tumor tissues were cytokinin-independent and all tissues, except those on ‘Rio Oso Gem’, produced octopine. A DNA probe specific for the T-DNA region of the Ti plasmid hybridized to the DNA extracted from these tumors. No tumor tissue was produced in response to inoculation of the stem explants with the Ti-plasmidless strain A136 or with uninoculated controls. These results demonstrate the transformation of peach cells derived from mature plants and the potential for using A. tumefaciens to transfer economically important genes to peach.

Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.

A simple, efficient method for the Agrobacterium-mediated transformation of carrot callus cells

A simple, efficient system for Agrobacterium-mediated transformation of carrot callus cells is described. Transformation of callus cells with Agrobacterium tumefaciens strains containing a non-oncogenic helper plasmid, and a binary vector plasmid yielded transgenic callus clones containing multiple insertions of the T-DNA from the binary vector; these clones have been regenerated into plants. A. tumefaciens strains containing the oncogenic plasmid pTiA6 and a binary vector plasmid yielded transgenic callus clones containing multiple insertions of both the oncogenic plasmid T-DNA and the T-DNA from the binary vector.

An enhancer-like element present in the promoter of a T-DNA gene from the Ti plasmid of Agrobacterium tumefaciens

The promoter of the 780 gene of T-right [Thomashow, M., Nutter, R., Montoya, A., Gordon, M. & Nester, E. (1980) Cell 19, 729-739] from Agrobacterium tumefaciens Ti plasmid (pTi15955) was shown to contain an upstream cis-acting element (activator) having enhancer-like properties. To characterize the properties of this promoter element, it was placed in both polarities, upstream and downstream of a Delta-37 deletion mutant of the 780 gene. The Delta-37 deletion contains the entire 780 gene with the 5' flanking sequences deleted upstream of TATA to -37. The effect of the activator on in vivo transcriptional activity was assessed by S1 nuclease mapping utilizing a homologous reference gene as an internal standard. Transcript levels from sunflower crown gall tumors were between 127% and 90% of the wild-type 780 gene depending on the polarity of the activator element when placed directly upstream to the 780 gene Delta-37 promoter. Repositioning the activator element 613 base pairs further upstream increased transcription by 2-fold regardless of polarity. However, the activator element did not promote transcription when placed in either polarity approximately 200 base pairs downstream of the poly(A) addition site. Upstream promoter fragments (TATA-distal) from the octopine synthase (ocs) and agropine synthase (ags) genes were also tested for activation of the Delta-37 construction. The ocs sequences activated transcription of the Delta-37 deletion to 14% of wild-type levels when placed upstream in the B (reverse) orientation. All other constructions with the ocs and ags sequences showed no enhancement of promoter activity.

Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6.

The virE operon of the Agrobacterium tumefaciens Ti plasmid pTiA6 encodes at least one trans-acting protein involved in the expression of virulence. Two open reading frames designated virE1 and virE2 code for polypeptides of 7 and 60 kilodaltons (kDa), respectively, that can be visualized after expression in Escherichia coli minicells. To determine which virE sequences are required for virulence, a strain deleted for the entire locus [strain KE1(pTiA6 delta E)] was constructed and tested for the ability to be complemented by subclones with and without site-directed mutations in the virE operon. One subclone containing only virE1 and virE2 as well as upstream promoter sequences was sufficient to restore full virulence on the host plant Kalanchoe daigremontiana. However, some other virulence locus representing a host range determinant appeared to be deleted from strain KE1(pTiA6 delta E), since virE1 and virE2 were not sufficient to fully restore virulence on wounded tomato plants. virE operon constructs with specific lesions in either virE1 or virE2 were impaired for complementation of pTiA6 delta E. Several mutations specific for the promoter-proximal virE1 locus appeared to have a polar effect on expression of the virE2-encoded 60-kDa protein. However, virE2::lacZ fusion constructs suggest that this effect is not at the level of transcription or translation. Collectively, these data indicate that both the 7- and the 60-kDa polypeptides are virulence determinants for the Ti plasmid pTiA6 and suggest that the 60-kDa protein may be less stable in the absence of the 7-kDa protein.

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified by the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.

Double-stranded cleavage of T-DNA and generation of single-stranded T-DNA molecules in Escherichia coli by a virD-encoded border-specific endonuclease from Agrobacterium tumefaciens

The virD locus of Agrobacterium tumefaciens Ti plasmid pTiA6 was sequenced. Computer analysis of the sequence indicated five possible open reading frames (ORFs) within this locus. Two additional ORFs were identified distal to this locus. However, only two polypeptides of apparent molecular masses 16 and 56 kilodaltons, the products of ORFs 1 and 2, were detected in Escherichia coli, both in vivo and in an in vitro coupled transcription-translation system. The virD locus was cloned in expression vector pKK223.3 under control of a tac promoter and introduced into an E. coli strain harboring mini-Ti plasmid pAL1050. When induced with isopropyl-beta-D-thiogalactopyranoside, the virD gene products exhibited double-stranded T-DNA border-specific endonuclease activity. Deletion analysis demonstrated that this activity is encoded within the 5'-proximal 1.7-kilobase-pair portion of the virD locus that carries ORF 1 and most of ORF 2. Neither ORF 1 nor ORF 2 independently showed endonuclease activity; complementation studies indicated that the products of ORFs 1 and 2 together have this activity. The expression of this 1.7-kilobase-pair region of the virD locus caused double-stranded cleavage of the T-DNA at or near the borders and generated single-stranded T-DNA molecules with approximately equal frequencies in E. coli.

Factors Affecting the Level of Kanamycin Resistance in Transformed Sunflower Cells

A 230 base pair DNA segment containing the sequences 5' to the 700 to 750 nucleotide (nt) transcript 7' (ORF 3; RF Barker, KB Idler, DV Thompson, JD Kemp 1983 Plant Mol Biol 2: 335-350) of the octopine tumor inducing plasmid pTiA6 has been isolated. This region has (a) 180 base pairs of DNA upstream of the TATA box, (b) the start of RNA synthesis, and (c) the entire 5' untranslated region of the gene. We have fused this presumed promoter fragment to the neomycin phosphotransferase II (NPTII) gene from Tn5 in a plant expression cassette. After recombination into a tumor inducing plasmid delivery plasmid, this cassette confers selectable kanamycin resistance to transformed sunflower cells. Removal of the out-of-frame ATG in the 5' leader sequence of the NPTII gene by two different modifications increased both the levels of NPTII enzyme activity and the ID(50) for kanamycin in the tumor cells. The promoter region of the transcript 7 gene gives levels of kanamycin resistance equivalent to the nopaline synthase promoter and octopine synthase promoter when used in the same constructions and assayed in the same tissues.