Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

Klebsiella pneumoniae nif-lac fusions are expressed in Agrobacterium tumefaciens C58

Summary Plasmids containing hybrid genes, in which different Klebsiella pneumoniae nif (nitrogen-fixation) promoters were fused with the structural part of the Escherichia coli lac operon, were introduced into a double auxotrophic derivative of Agrobacterium tumefaciens C58. A study of their expression in the new host was made simple by the inherent inability of A. tumefaciens C58 to produce -galactosidase unless provided with the wild-type lac operon of E. coli. As shown by quantitative measurements of the enzyme, all K. pneumoniae promoters were expressed well in A. tumefaciens C58, even under conditions known to repress them. It also has been shown that the activity of K. pneumoniae nif A is essential for the expression of nifHDK even when introduced into A. tumefaciens. After entering the new host the plasmids, the nif genes and the fusion alleles contained in them, remained stable. Possible mechanisms responsible for the constitutive behaviour of nif promoters in A. tumefaciens are discussed.     

A functional map of the nopaline catabolism genes on the Ti plasmid of Agrobacterium tumefaciens C58

Summary The nopaline catabolism (noc) genes are located in a 14.4 kb region on the pTiC58 plasmid of A. tumefaciens C58. These genes permit the bacterium to grow on nopaline N²-(1,3-dicarboxylpropyl) arginine, a substrate produced in plant tumors initiated by strain C58. The functions of the noc genes include the use of nopaline and L-ornithine as sole carbon and nitrogen sources. Using Tn5 insertional mutants, we have identified and mapped the positions of the genes that are responsible for nopaline catabolism (NopC), ornithine catabolism (OrnC) and nopaline uptake (NopU). A polar relationship was found between these phenotypes, which extended leftward over the noc region to the T-region. The NopC mutants were also deficient in nopaline oxidase, an enzyme that liberates free arginine from nopaline.The noc region also encodes the synthesis of a periplasmic protein, n1 that was induced by nopaline. Tn5 insertional mutations and molecular cloning were used to map the n1 production locus. The recombinant plasmids, pSa4480 and pSa4481, containing the 8.9 kb right-hand end of the noc region, conferred n1 production when introduced into a pTi-free strain of A. tumefaciens. Production of n1 by the strains carrying these plasmids required nopaline induction.We have identified in toto three noc loci: nocB, nocC, and nocA, which confer n1 production, nopaline oxidase production and ornithine catabolism respectively. A model is proposed whereby the noc genes of pTiC58 are contained on a leftward reading operon in the order nocB, nocC, and nocA.     

The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Summary Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis (Engstromm et al. 1987) that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.     

T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefaciens C58 derivatives

Summary The detailed structural organization of DNA sequences transferred to the plant genome via Agrobacterium tumefaciens has been determined in 11 transgenic tomato plants that carry the transferred DNA (T-DNA) at a single genetic locus. The majority (seven) of these plants were found to carry multiple copies of T-DNA arranged in inverted repeat structures. Such a high frequency of inverted repeats among transgenotes has not been previously reported and appears to be characteristic of transformation events caused by C58/pGV3850 strains of Agrobacterium. The inverted repeats were found to be centered on either the left or the right T-DNA boundary and both types were observed at similar frequency. In several plants both types of inverted repeat were found to coexist in the same linear array of elements. Direct repeats were observed in two plants, each time at the end of an array of inverted repeat elements, and at a lower frequency than inverted repeats. The junctions between T-DNA elements and plant DNA sequences and the junctions between adjacent T-DNA elements were mapped in the same 11 plants, allowing the determination of the distribution of junction points at each end for both types of junction. Based on a total of 17 distinct junctions at the right end of T-DNA and 19 at the left end, the distribution of junction points was found to be much more homogeneous at the right end than at the left end. Left end junctions were found to be distributed over a 3 kb region of T-DNA with two thirds of the junctions within 217 bp of the left repeat. Two thirds of the right end junctions were found to lie within 11 bp of the right repeat with the rest more than 39 bp from the right repeat. T-DNA::plant DNA junctions and T-DNA::T-DNA inverted repeat junctions showed similar distributions of junction points at both right and left ends. The possibilities that T-DNA inverted repeats are unstable in plants and refractory to cloning in wild type Escherichia coli is discussed. Two distinct types of mechanisms for inverted repeat formation are contrasted, replication and ligation mechanisms.     

Behavior of Inc-Q plasmids in Agrobacterium tumefaciens

Inc-Q plasmids were introduced into Agrobacterium tumefaciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results suggest that these plasmids may be used in genetic complementation studies of Ti plasmid mutants in A. tumefaciens.     

T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivatives

Summary We have previously described substantial variation in the level of expression of two linked genes which were introduced into transgenic petunia plants using Agrobacterium tumefaciens. These genes were (i) nopaline synthase (nos) and (ii) a chimeric chlorophyll a/b binding protein/octopine synthase (cab/ocs) gene. In this report we analyze the relationship between the level of expression of the introduced genes and T-DNA structure and copy number in 40 transgenic petunia plants derived from 26 transformed calli. Multiple shoots were regenerated from 8 of these calli and in only 6 cases were multiple regenerated shoots from each callus genotypically identical to each other. Many genotypes showed no nos gene expression (22/28). Most of the plants (16/22) which lacked nos gene expression did contain nos-encoding DNA with the expected restriction enzyme map. Similarly, amongst the genotypes showing no cab/ocs gene expression, the majority (11/28) did not show any alterations in restriction fragments corresponding to the expected cab/ocs coding sequences (10/11). Approximately half of the plants carried multiple copies of T-DNA in inverted repeats about the left or right T-DNA boundaries. No positive correlation was observed between the copy number of the introduced DNA and the level of expression of the introduced genes. However, plants with high copy number complex insertions composed of multiple inverted repeats in linear arrays usually showed low levels of expression of the introduced genes.     

Tzs, a nopaline Ti plasmid gene from Agrobacterium tumefaciens associated with trans-zeatin biosynthesis

Summary The tzs gene, present in nopaline Ti plasmids, confers on Agrobacterium tumefaciens the ability to produce the phytohormone, trans-zeatin (Regier and Morris (1982) Biochem Biophys Res Comm 104:1560–1566). This gene has now been cloned from the nopaline Ti plasmid pTiC58. It occurs outside the T-DNA in a region close to that associated with virulence functions. Sequence studies indicate that tzs has substantial homology with the T-region gene, ipt, which is known to encode a dimethylallylpyrophosphate transferase, the first enzyme of the cytokinin biosynthetic pathway. As expected from its homology with ipt, tzs possesses significant DMA transferase activity but when expressed in Escherichia coli it causes secretion of trans-zeatin.     

Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis

Summary A pathway for biosynthesis of the crown-gall opine, agropine is proposed and three potential new precursors characterised. The location of genes involved in the three steps of this pathway was determined by site directed insertions and deletions in the T_R region of the octopine Ti plasmid, pTiB6Tra^c. The proposed biosynthetic pathway for agropine which involves three T-DNA genes is in contrast to the biosynthesis of octopine and nopaline where single T-DNA genes are involved.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.     

A spontaneous insertion in the agrocin sensitivity region of the Ti-plasmid of Agrobacterium tumefaciens C58

A spontaneous agrocin-resistant mutant of Agrobacterium tumefaciens strain C58 was shown to have an insertion of 1.2 kb in the agrocin-sensitivity region of pTiC58. The insertion was cloned from the Ti-plasmid, and a subclone containing only DNA internal to the insertion was used to probe the Ti-plasmid and chromosomal DNA of the wild-type strain C58. The probe showed homology to chromosomal sequences but showed no homology to wild-type pTiC58. Homology was also detected with chromosomal sequences of A. tumefaciens strains, B6, K24, and T37. These results suggest that an indigenous insertion sequence of 1.2 kb transposed from the chromosome to the agrocin-sensitivity region of the Ti-plasmid in this spontaneous mutant of C58.     

Identification of an Agrobacterium tumefaciens pTiB6S3 vir region fragment that enhances the virulence of pTiC58

Summary The Agrobacterium tumefaciens octopine strain B6S3 and the nopaline strain C58 were compared for their ability to induce opine synthesis on Kalanchoe daigremontiana stem fragments. Whereas B6S3 induced high levels of octopine synthesis, C58 induced only low levels of nopaline synthesis. However, C58-induced nopaline synthesis was greatly increased by mixed infection with B6S3. This effect (called helper-effect) was shown to be due to the activity of a 5 kb fragment from the virulence region of the B6S3 Ti plasmid, since incorporation of this fragment into the C58 plasmid enabled C58 to induce high levels of nopaline synthesis in the absence of a helper strain. The 5kb region contains the vir F locus, as defined earlier (Hooykaas et al. 1984b). A possible correlation between the helper function and vir F is discussed. Our results show that large differences in virulence on particular host plants exist between natural Agrobacterium strains and can be overcome by mixed infections.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens

Summary Host range variations were noted when 23 wildtype strains of Agrobacterium tumefaciens were tested on 27 different plant species. Because we have shown previously that host range specificity is conferred by the pTi plasmid, these variations in host specificity implicated genetic differences among p Ti plasmids within the A. tumefaciens population that was tested. Host specificity was independent of the type of opine utilized and biotype of the strain used. These data suggested that separate genetic determinants operate for host specificity. This hypothesis was confirmed by Tn5 mutagenesis of the pTi plasmid, which generated mutants affected in host specificity. The regions of host specifying genes were located by displacement analysis of mutant pTi-plasmid-DNA restriction fragments. There are at least two sites on the pTiC58 plasmid: one within the T-region and the other about 75–77 kb to the right of this region. Mutations within the T-region were chemically complemented by indoleacetic acid, which restored the host range of the mutants. Such complementations were not observed with mutants outside the T-region.     

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

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

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

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