Biomedical subjects

S K Farrand

Publications and source records attributed to S K Farrand.

At least 55 records · Page 3Linked to original sources

PubMed1996

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.

Agrobacterium tumefaciens↗

PubMed1996

Purification and characterization of catabolic mannopine cyclase encoded by the Agrobacterium tumefaciens Ti plasmid pTi15955.

Catabolic mannopine (MOP) cyclase encoded by certain Agrobacterium Ti and Ri plasmids lactonizes MOP to agropine (AGR). The enzyme, purified to homogeneity from a recombinant clone, has a molecular mass of 45 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme catalyzed the lactonization of MOP to AGR without the need for any cofactors. The enzyme also converted AGR to MOP with the lactonizing activity being predominant over the reverse reaction. MOP cyclase is specific for imine conjugates of D-hexose and L-glutamine and was not inhibited by sugars or amino acids. The enzyme lactonized deoxyfructosyl glutamine, a natural intermediate of MOP synthesis and catabolism, to a product indistinguishable from chrysopine, a newly discovered crown gall opine. The enzyme also lactonized N-l-(1,2-dideoxy-D-mannityl)-L-glutamine, indicating that a hydroxyl group at carbon atom 2 of the sugar moiety is not required for the enzymatic reaction.

Agrobacterium tumefaciens↗

PubMed1995

Expression of multiple eukaryotic genes from a single promoter in Nicotiana.

We engineered an expression unit composed of three eukaryotic genes driven by a single plant-active promoter and demonstrated functional expression in planta. The individual genes were linked as translational fusions to produce a polyprotein using spacer sequences encoding specific heptapeptide cleavage recognition sites for NIa protease of tobacco vein mottling virus (TVMV). The NIa gene itself was included as the second gene of the multi-gene unit. The first and third genes, obtained from the TR region of pTi15955, encoded enzymatic functions associated with the mannityl opine biosynthetic pathway. The mannityl opine conjugase gene (mas2) was the first unit of the construct and provided the native plant-active promoter and 5' untranslated regulatory sequence. The third gene (mas1), encoding the mannityl opine reductase, furnished the native 3' untranslated region. Cis-processing of the polyprotein by the NIa protease domain was demonstrated in vitro using rabbit reticulocyte lysate and wheat germ cell-free translation systems. Tobacco plant cells transformed with the multi-gene unit produced detectable levels of mannopine, mannopinic acid, and their biosynthetic intermediates, deoxyfructosyl-glutamate and deoxyfructosyl-glutamine. This indicates that the polygene construct results in a set of functional enzymatic activities that constitute a complete metabolic pathway.

Animals↗

PubMed1995

Altered Epiphytic Colonization of Mannityl Opine-Producing Transgenic Tobacco Plants by a Mannityl Opine-Catabolizing Strain of Pseudomonas syringae.

The plasmid pYDH208, which confers the ability to catabolize the mannityl opines mannopine and agropine, was mobilized into the nonpathogenic Pseudomonas syringae strain Cit7. The growth of the mannityl opine-catabolizing strain Cit7(pYDH208) was compared with that of the near-isogenic non-opine-catabolizing strain Cit7xylE on leaves of wild-type tobacco (Nicotiana tabacum cv. Xanthi) and transgenic mannityl opine-producing tobacco plants (N. tabacum cv. Xanthi, line 2-26). The population size of Cit7(pYDH208) was significantly greater on the lower leaves of transgenic plants than on middle or upper leaves of those plants. The population size of Cit7(pYDH208) on lower leaves of transgenic plants was also significantly greater than the population size of Cit7xylE on similar leaves of wild-type plants. High-voltage paper electrophoresis demonstrated higher levels of mannityl opines in washings from lower- and mid-level leaves than in washings from upper-level leaves. The ability of Cit7(pYDH208) to catabolize mannityl opines in the carbon-limited phyllosphere increased the carrying capacity of the lower leaves of transgenic plants for Cit7(pYDH208). In coinoculations, the increase in the ratio of population sizes of Cit7(pYDH208) to Cit7xylE on transgenic plants was apparently due to a subtle difference in the growth rates of the two strains and to the difference in final population sizes. An ability to utilize additional carbon sources on the transgenic plants also enabled Cit7(pYDH208) to achieve a higher degree of coexistence with Cit7xylE on transgenic plants than on wild-type plants. This supports the hypothesis that the level of coexistence between epiphytic bacterial populations can be altered through nutritional resource partitioning.

Journal Article↗

PubMed1995

Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer.

N-Acylhomoserine lactone (acyl-HSL)-mediated gene expression, also called autoinduction, is conserved among diverse gram-negative bacteria. In the paradigm Vibrio fischeri system, bioluminescence is autoinducible, and the lux operon requires the transcriptional activator LuxR and the acyl-HSL autoinducer for expression. The production of the acyl-HSL signal molecule is conferred by the luxI gene, and luxR encodes the transcriptional regulator. We show here that Erwinia stewartii, the etiological agent of Stewart's wilt of sweet corn, synthesizes an acyl-HSL. Mass spectral analysis identified the signal molecule as N-(-3-oxohexanoyl)-L-homoserine lactone, which is identical to the V. fischeri autoinducer. We have cloned and sequenced the gene that confers acyl-HSL biosynthesis, called esaI, and the linked gene, esaR, that encodes a gene regulator. The two genes are convergently transcribed and show an unusual overlap of 31 bp at their 3' ends. Sequence analysis indicates that EsaI and EsaR are homologs of LuxI and LuxR, respectively. EsaR can repress its own expression but seems not to regulate the expression of esaI. The untranslated 5' region of esaR contains an inverted repeat with similarity to the lux box-like elements located in the promoter regions of other gene systems regulated by autoinduction. However, unlike the other systems, in which the inverted repeats are located upstream of the -35 promoter elements, the esaR-associated repeat overlaps a putative -10 element. We mutagenized the esaI gene in E. stewartii by gene replacement. The mutant no longer produced detectable levels of the acyl-HSL signal, leading to a concomitant loss of extracellular polysaccharide capsule production and pathogenicity. Both phenotypes were restored by complementation with esal or by exogenous addition of the acyl-HSL.

4-Butyrolactone↗

PubMed1995

A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer.

Conjugal transfer of the Agrobacterium tumefaciens nopaline-type Ti plasmid pTiC58 is induced by agrocinopines A and B, opines secreted by crown gall tumors induced by the bacterium. This regulation functions through the transcriptional repressor, AccR. However, actual transcription of the tra genes is regulated by autoinduction through the activator TraR and the substituted homoserine lactone second messenger, Agrobacterium autoinducer (AAI). We have identified a new regulatory element that modulates the response of TraR to AAI. The gene, called traM, suppresses TraR-AAI activation of transcription of tra genes carried on recombinant clones. The suppression could be relieved by increasing the expression of TraR but not by increasing AAI levels. traM is located between traR and traAF on pTiC58 and is transcribed in the clockwise direction. The 306-bp gene encodes an 11.2-kDa protein showing no significant relatedness to other proteins in the databases. Mutations in traM in pTiC58 conferred a transfer-constitutive phenotype, and strains harboring the Ti plasmid produced easily detectable amounts of AAI. These same mutations engineered into the transfer-constitutive Ti plasmid pTiC58 delta accR conferred a hyperconjugal phenotype and very high levels of AAI production. Expression of traM required TraR, indicating that transcription of the gene is regulated by the autoinduction system. TraM had no effect on the expression of traR, demonstrating that the suppressive effect is not due to repression of the gene encoding the activator. These results suggest that TraM is not a direct transcriptional regulator. Since the suppressive effect is demonstrable only when traM is overexpressed with respect to traR, we suggest that TraM functions to sequester TraR from the very small amounts of AAI produced under conditions when the agrocinopines are not present.

4-Butyrolactone↗

PubMed1994

TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer.

Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a transcriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF). CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called autoinduction. In Vibrio fischeri HSL production is conferred by the luxI gene. Homologues of this gene are responsible for HSL production by other Gram-negative bacteria. A gene that we call traI, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra3 of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that traI could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria. A second, partial open reading frame immediately downstream of traI could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transfer. Transcription of traI and of the downstream tra3 genes requires TraR and CF and initiates from the traI promoter. The results show that traI is responsible for CF production, that it is the first gene of the tra3 operon, and that expression of this operon is regulated by autoinduction.

Amino Acid Sequence↗

PubMed1994

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.

Agrobacterium tumefaciens↗

PubMed1994

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.

Genetic Complementation Test↗

PubMed1993

Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction.

Conjugal transfer of Ti plasmids from Agrobacterium donors to bacterial recipients is controlled by two types of diffusible signal molecules. Induction is mediated by novel compounds, called opines, that are secreted by crown gall tumours. These neoplasias result from infection of susceptible plants by virulent agrobacteria. The second diffusible signal, called conjugation factor, is synthesized by the donor bacteria themselves. Production of this factor is induced by the opine. Here we show that conjugation is regulated directly by a transcriptional activator, TraR, which requires conjugation factor as a coinducer to activate tra gene expression. TraR is a homologue of LuxR, the lux gene activator from Vibrio fischeri which also requires an endogenously synthesized diffusible coinducer. The two regulatory systems are related; the two activator proteins show amino-acid sequence similarities and the lux system cofactor, autoinducer, will substitute for conjugation factor in the TraR-dependent activation of Ti plasmid tra genes.

Agrobacterium tumefaciens↗

PubMed1993

Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport.

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis.

ATP-Binding Cassette Transporters↗

PubMed1993

Organization and regulation of the mannopine cyclase-associated opine catabolism genes in Agrobacterium tumefaciens 15955.

We have isolated and characterized Tn3HoHo1- and Tn5-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR). Characterization of the transposon-induced lacZ fusion mutants by beta-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines. Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus. The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements. Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid. Analysis of beta-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source. The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen. This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability.

Agrobacterium tumefaciens↗

PubMed1992

Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.

Amino Acid Sequence↗

PubMed1992

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.

Adenine Nucleotides↗

PubMed1992

Induction of Pseudoactinorhizae by the Plant Pathogen Agrobacterium rhizogenes.

Infection of Elaeagnus angustifolia cotyledonary wounds by Agrobacterium rhizogenes strain NCPPB 2659 resulted in the formation of pseudoactinorhizae on roots differentiated from callus. These pseudoactinorhizal root nodules were anatomically indistinguishable from the actinorhizae induced by the plant's microsymbiont Frankia. This unusual hairy root phenotype provides support for the concept that the genetic program for actinorhiza morphogenesis resides in the plant's genome.

Journal Article↗

PubMed1992

Mannityl opine accumulation and exudation by transgenic tobacco.

Three genes from the T(R) region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.

Journal Article↗

PubMed1992

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts.

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

Journal Article↗

PubMed1992

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders.

Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.

Agrobacterium tumefaciens↗