Modification of rhizobacterial populations by engineering bacterium utilization of a novel plant-produced resource.

The ability to catabolize distinct nutrients produced by a plant may be a factor in the successful colonization of that host by a bacterium when in competition with other rhizosphere microorganisms. We tested this hypothesis by examining the influence of a novel substrate produced by a transgenic plant on root colonization by near-isogenic bacteria, differing only in their ability to use the resource. When inoculated alone, both bacteria colonized the roots of the normal and transgenic plants with equal kinetics and to indistinguishable levels. When the two bacteria were coinoculated, the catabolizer reached a population density significantly higher than that of the noncatabolizer on the roots of the resource-producing plant. No such advantage was observed on the roots of normal plants. These results support the theory that resources produced and exuded by a plant host can confer a selective advantage to microorganisms that use the substrate.

Attempts to detect Agrobacterium tumefaciens and bacteriophage PS8 DNA in crown gall tumors by DNA-DNA-filter hybridization.

A systematic study of the DNA-DNA-filter reaction is presented which measures its ability to detect small amounts of simple DNA (bacterial or bacteriophage) in model mixtures of DNA immobilized on filters. Saturation curves show qualitatively that significant binding occurs when there is 10% Agrobacterium tumefaciens DNA on the filter but not 1%. PS8 bacteriophage DNA is detectable at a level of 0.1%. True saturation is not attained in the bacterial DNA reaction : radioactivity bound represents only 3% of the theoretical saturation value. The bacteriophage DNA reactions attain 15-30% of the expected saturation value. When crown gall tumor DNA filters were tested for the presence of A. tumefaciens or PS8 bacteriophage DNA by saturation reactions, an apparently significant amount of binding was observed compared with usual background levels for heterologous DNA filters. However thermal dissociation profiles revealed that no well-matched duplexes were formed. Normal tobacco callus DNA filters exhibited the same type of binding of labeled DNA to a similar extent (50-100% as much as tumor DNA filters). Both types of DNA-filters bound Bacillus subtilis and bacteriophage T4 DNA as efficiently as A. tumefaciens and PS8 DNA. The high non-specific background binding of labeled DNA by filters containing DNA isolated from plant tissue culture materials is ascribed to low single strand molecular weight of the filterbound DNA. This study provides no evidence for foreign DNA in crown gall tumors, and raises objections to the interpretation of the data of earlier investigators (Quetier, F., Huguet, T. and Guille, E. (1969) Biochem, Biophys. Res, Commun. 34, 128-133 and Srivastava, B.I.S. (1970) Life Sci. 9, 889-892) who claimed to detect A. tumefaciens DNA in crown gall tumors by DNA-DNA-filter hybridization.

RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.

Opine catabolic loci from Agrobacterium plasmids confer chemotaxis to their cognate substrates.

Opines are carbon compounds produced by crown galls and hairy roots induced by Agrobacterium tumefaciens and A. rhizogenes, respectively. These novel condensation products of plant metabolic intermediates are utilized as nutritional sources by the Agrobacterium strains that induced the growths. Thus, opines are thought to favor the propagation of agrobacteria in the tumorsphere. Certain Agrobacterium strains were chemoattracted to opines. The chemotactic activities to octopine, to nopaline, to mannopine, and to agrocinopines A + B were dependent on the type of the Ti plasmid present in the bacterium. The determinants for chemotaxis to these opines were localized to the regions of the octopine- and nopaline-type Ti plasmids coding for transport and catabolism of that opine. An insertion in accA, which encodes the periplasmic binding protein for agrocinopines A + B, abolished chemotaxis while an insertion in accC, which encodes a component of the transport system, and an insertion in accF, which encodes a function required for agrocinopine catabolism, did not affect chemotaxis to this opine. Thus, transport and catabolism of these opines are not required for the chemotactic activity. Analyses of subclones of the acc region confirmed that accA is the only gene required from the Ti plasmid for chemotaxis to agrocinopines A + B.

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.

Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance.

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria.

Many gram-negative bacteria regulate expression of specialized gene sets in response to population density. This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer. In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL). Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain. We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12. The traG::lacZ fusion reporter from the A. tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four. Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants. Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions. Only a few isolates of Xanthomonas produced a detectable signal. We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography. Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL. However, a few of the erwinias, and the P. fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards. The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL. Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals. Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains. Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.

Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens.

pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1. NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR. In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine. Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine. When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited. In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58. These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly. The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours. Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium. Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain. Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form. MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms. Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition. Genetic and physiological analyses indicate that the mutations are located on pRE10 and abolish uptake of the opine.

Translocation and exudation of tumor metabolites in crown galled plants.

Crown gall tumors are induced on susceptible plants by pathogenic strains of Agrobacterium. These neoplastic plants cells produce metabolites, called opines, which provide a source of nutrients to colonizing agrobacteria. Opine production previously has been shown to influence microbial communities in the immediate vicinity of the tumor. We have obtained evidence for opine translocation to and exudation from distal uninfected regions of the plant host. Grafted plants made from an opine-producing transgenic scion with a wild-type stock, or with a wild-type scion and an opine-producing stock accumulate opines in the wild-type portions of the plant. Moreover, opines were detected in root, stem, and leaf tissues of nontransgenic plants on which stem crown galls had been induced by pathogenic strains of Agrobacterium. These plants exuded opines from their roots as a component of their root exudates. Translocation of opines from the tumor to other parts of the plant, and their exudation from roots, indicates that these biologically active compounds are available to opine-catabolizing microbes that have not induced the tumors but are present in the rhizosphere or on portions of the plant distant from the site of the gall.

An R plasmid of broad host-range, coding for resistance to nine antimicrobial agents endemic in Gram-negative nosocomial isolates.

Of 3952 clinical isolates of Enterobacteriaceae, 246 exhibited resistance to at least carbenicillin, gentamicin and tobramycin. All these isolates, representing eight genera, were resistant to at least nine antimicrobial agents in common, including the three key antibiotics and streptomycin, kanamycin, sisomycin, ampicillin, cephalothin and sulphonamide. The strains could be subdivided into seven groups depending upon additional resistance traits and some were resistant to as many as 15 antibiotics. When mated with a standard strain of Escherichia coli, 85% of 123 randomly selected donors transferred resistance to at least the nine core antibiotics. Some donors occasionally transferred resistance to two additional antibiotics, neomycin and tetracycline, while one Citrobacter freundi donor always transferred linked resistance to all 11 drugs. Although many donors were found to harbour more than one species of plasmid DNA, all except a strain of C. freundi contained at least a plasmid of mol. wt 89 x 10(6). Analysis of E. coli transconjugants showed this plasmid to be responsible for transferable resistance to the nine core antibiotics. Restriction-endonuclease analysis indicates that the 89 x 10(6) plasmids originating from different isolates were essentially identical with each other. These results show that a particular R plasmid has established itself among the Enterobacteriaceae at Hines VA Hospital. This R plasmid appears to be the predominant genetic element responsible for linked resistance to carbenicillin, gentamicin and tobramycin among these hospital-associated bacteria.

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.

The replicator of the nopaline-type Ti plasmid pTiC58 is a member of the repABC family and is influenced by the TraR-dependent quorum-sensing regulatory system.

The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034-1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, and repC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids from Rhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region between traI and repA contained two copies of the tra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from the tra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal rep plasmid increased five- to sevenfold in strains expressing traR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.

Quorum sensing but not autoinduction of Ti plasmid conjugal transfer requires control by the opine regulon and the antiactivator TraM.

Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is controlled by autoinduction via the transcriptional activator TraR and the acyl-homoserine lactone ligand, Agrobacterium autoinducer (AAI). This control process is itself regulated by opines, which are small carbon compounds produced by the crown gall tumors that are induced by the bacteria. Opines control autoinduction by regulating the expression of traR. Transfer of pTiC58 from donors grown with agrocinopines A and B, the conjugal opines for this Ti plasmid, was detected only after the donors had reached a population level of 10(7) cells per cm(2). Donors incubated with the opines and AAI transferred their Ti plasmids at population levels about 10-fold lower than those incubated with opines only. Transcription of the tra regulon, as assessed by monitoring a traA::lacZ reporter, showed a similar dependence on the density of the donor population. However, even in cultures at low population densities that were induced with opines and AAI, there was a temporal lag of between 15 and 20 h in the development of conjugal competence. Moreover, even after this latent period, maximal transfer frequencies required several hours to develop. This lag period was independent of the population density of the donors but could be reduced somewhat by addition of exogenous AAI. Quorum-dependent development of conjugal competence required control by the opine regulon; donors harboring a mutant of pTiC58 deleted for the master opine responsive repressor accR transferred the Ti plasmid at maximum frequencies at very low population densities. Similarly, an otherwise wild-type derivative of pTiC58 lacking traM, which codes for an antiactivator that inhibits TraR activity, transferred at high frequency in a population-independent manner in the absence of the conjugal opines. Thus, while quorum sensing is dependent upon autoinduction, the two phenomena are not synonymous. We conclude that conjugal transfer of pTiC58 is regulated in a quorum-dependent fashion but that supercontrol of the TraR-AAI system by opines and by TraM results in a complex control process that requires not only the accumulation of AAI but also the expression of TraR and the synthesis of this protein at levels that overcome the inhibitory activity of TraM.

Diversity of determinants encoding carbenicillin, gentamicin, and tobramycin resistance in nosocomial Pseudomonas aeruginosa.

Plasmid pFMH1010, an 89-megadalton R plasmid, is endemic among members of the family Enterobacteriaceae at Hines Veterans Administration Hospital, Hines, Ill. It encodes resistance to nine antibiotics, including resistance to carbenicillin (Cb), gentamicin (Gm), and tobramycin (Tm). Pseudomonas aeruginosa strains resistant to carbenicillin, gentamicin, and tobramycin were isolated from five patients at Hines Veterans Administration Hospital from whom Serratia marcescens strains harboring pFMH1010 were also obtained. The P. aeruginosa strains were investigated to determine whether their Cb, Gm, and Tm characteristics derived from pFMH1010. One of the isolates, Ps559, was shown by Southern hybridization to contain approximately 76% of pFMH1010. Several lines of evidence suggested that the pFMH1010 sequences in Ps559 are integrated in the chromosome. Southern hybridization also demonstrated that the beta-lactam resistance of pFMH1010 is most probably due to the presence of sequences homologous with Tn3 and that these sequences are retained in Ps559. In two other Pseudomonas isolates, resistance to carbenicillin, gentamicin, tobramycin, and kanamycin was encoded by R plasmids unrelated to pFMH1010. In the last two isolates, resistance to gentamicin and tobramycin and several other antibiotics appeared to be chromosomally encoded, and it was rescuable from one of these strains by RP4-mediated mobilization.

Characterization and mapping of the agrocinopine-agrocin 84 locus on the nopaline Ti plasmid pTiC58.

Overlapping segments of pTiC58 inserted into cosmid vectors were used to characterize the agrocinopine-agrocin 84 locus from the nopaline/agrocinopine A and B Agrobacterium tumefaciens strain C58. All of the clones conferring agrocin 84 sensitivity on agrobacteria also conferred uptake of agrocin 84 and agrocinopines A and B. Transposon Tn3-HoHo1 insertion mutations of one such clone were generated that simultaneously abolished agrocin 84 sensitivity and transport of agrocinopines A and B and agrocin 84. Such insertions were found to cluster within a 4.4-kilobase region. Analysis of beta-galactosidase activity in these insertion mutants suggested a single transcriptional unit regulated at the transcriptional level by agrocinopines A and B. The smallest DNA fragment subcloned from the region to confer all three activities was 8.5 kilobases long. This subclone was still properly regulated, indicating that the regulatory gene is closely linked to the locus. The data are consistent with a single operon encoding catabolism of agrocinopines A and B and conferring sensitivity to agrocin 84. Based on these results, we support the locus name acc, for agrocinopine catabolism.

Characterization of transposon Tn5-facilitated donor strains and development of a chromosomal linkage map for Agrobacterium tumefaciens.

We have further characterized the transposon Tn5-facilitated chromosomal gene transfer system developed for Agrobacterium tumefaciens 15955. Using a strain whose chromosome contained Tn5, we compared the chromosome-mobilizing ability of plasmid pDP37 (containing Tn5) with that of its parent plasmid R68.45. For R68.45, we observed nonpolar transmission from multiple origins. For pDP37 we found polarized transmission from a single origin near ilv. When we examined the transmission gradients of a number of pDP37-containing donor strains each differing at the site of the chromosomal insertion we found just two classes. One set of donors transmitted markers with a gradient of Ilv+ greater than Rifr greater than His+ greater than Met+, whereas the second set transferred His+ greater than Rifr greater than Ilv+ greater than Met+. Using representatives from each transmission class of donor strains, we conducted matings to measure the degree of linkage between pairs of adjacent donor markers. From this information we developed a map of the A. tumefaciens 15955 chromosome. Attempts to isolate R-prime plasmids or Hfr-like donors were unsuccessful.

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.