In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR,and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.     

Opine-regulated promoters and LysR-type regulators in the nopaline (noc) and octopine (occ) catabolic regions of Ti plasmids of Agrobacterium tumefaciens.

Essential steps in the uptake and catabolism of the plant tumor metabolites nopaline and octopine in Agrobacterium spp. are performed by proteins encoded in the nopaline catabolic (noc) and octopine catabolic (occ) regions of Ti plasmids. We investigated the opine activation of the genes by using (i) promoter studies of Agrobacterium spp. and (ii) analysis of the promoter interaction with the regulatory proteins NocR (noc) and OccR (occ). The noc region contained two nopaline-induced promoters (Pi1[noc] and Pi2[noc]) and one autogenously regulated promoter (Pr [control of NocR expression]). Pi2 and Pr overlapped and were divergently oriented (Pi2 [noc]). DNA binding studies and DNase I footprints indicated that NocR bound specifically to single binding sites in Pi1[noc] and Pi2/Pr[noc] and that Pi2 and Pr were regulated from the same binding site. The binding was independent of the inducer nopaline, and nopaline caused small changes in the footprint. The promoters in the noc and occ regions shared sequence motif and contained the sequence T-N11-A, which is characteristic for LysR-type-regulated promoters. The occ region contained one octopine-induced and one autogenously regulated promoter (Pi/Pr[occ]) in the same arrangement as Pi2/Pr[noc] in the noc region. Promoter deletions indicated that sequences flanking the OccR binding site determined the extent of induction, although they did not bind OccR. The promoter bound OccR in the absence and presence of octopine. The opine caused a change in the mobility of the DNA-protein complex with the complete promoter. The resected fragments did not reveal this opine-induced shift, and it was also not detectable with the DNA-NocR complexes with the two promoters of the noc region.     

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.     

Expression of the nopaline synthase gene in Escherichia coli

The Agrobacterium tumor-inducing (Ti) plasmid pTiT37 encodes nopaline synthase (NOS) gene (nos) with eukaryotic promoter elements that is expressed in transformed plant cells but not in the bacterial host. We have fused the nos gene to the Escherichia coli trp promoter, and observed synthesis of NOS in E. coli. The nopaline produced by this enzyme is excreted into the culture medium. NOS is enzymatically active at 30 degrees C but not 37 degrees C, as based on nopaline production. NOS protein is produced at both temperatures, based on production in minicells.     

Cloning and nucleotide sequence of the tzs gene from Agrobacterium tumefaciens strain T37.

The trans-zeatin secretion locus (tzs), from the nopaline Ti plasmid of Agrobacterium tumefaciens strain T37, was cloned and the nucleotide sequence determined. This gene is located in the virulence region of pTiT37. The tzs gene is responsible for the secretion of trans-zeatin into bacterial culture medium and in addition has the cytokinin biosynthetic activity, dimethylallylpyrophosphate:AMP dimethylallyltransferase. Sequence analysis showed an open reading frame of 729 nucleotides, capable of encoding a protein of 27,545 daltons. A single new labelled protein of 27,200 daltons was detected in Escherichia coli maxicells expressing the cloned tzs gene. Significant sequence homology was observed between the tzs and the published tmr sequence from pTiT37.     

Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA,and transmission of T-DNA to R1 progeny

Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the “rooty” locus. Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA. Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings. Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform. The disarming of pTiT37 by insertions at the “rooty” locus thus appears to produce a useful gene vector for higher plants.     

Transformation ofBrassica juncea byAgrobacterium tumefaciens harbouring plasmid pTiT37 and its ‘rooty’ mutant pTiT37.14a/a

Indian mustard (Brassica juncea Linn., Czern and Coss) plants were inoculated withAgrobacterium tumefaciens strain A208 harbouring either plasmid pTiT37 or pTiT37.14a/a. The latter carries an insertion at the ‘rooty’ locus (gene 4 orcyt) of the T-DNA governing cytokinin biosynthesis. The tumours induced by pTiT37.14a/a were small and formed many roots. Forin vitro culture, these transformed cells required supplementation of the basal medium with IAA and kinetin in the initial stages though after six sub-cultures they were also hormone autotrophic.In vitro cultures showed profuse rooting like the inplanta tumours. Unlike transformed tobacco and carrot cells,Brassica juncea cells transformed by pTiT37.14a/a could not be induced to differentiate into shoots. In contrast, the cells transformed by the wild type pTiT37 were hormone autotrophic and occasionally differentiated into shoots but could not be induced to produce roots. These results demonstrate how a single gene mutation affecting cytokinin biosynthesis can alter the inplanta as well as thein vitro response of a transformed cell of the same genotype.     

The construction of a nopaline-inducible marker system for rapid detection of Agrobacterium strains

Summary An inducible marker system suitable for Agrobacterium tumefaciens was constructed to enable detection and enumeration of specific bacterial cells introduced into soil. A BamHI cassette carrying the catechol 2,3-dipxygenase (C230) gene, tdnC, fused to the nopaline-inducible Agrobacterium promoter Pi 2[noc] was constructed. This cassette was introduced into the broad host range vector pDSK5019 resulting in plasmid pTVNC2. Inducible C230 activity was observed in an A. tumefaciens strain carrying the plasmid pTVNC2 when nopaline was present. Colonies of bacteria tagged with the system could easily be identified by spraying agar plates containing nopaline with catechol, which is converted to the bright yellow compound 2-hydroxymuconic semialdehyde. The inducible tdnC cassette has also been introduced into the BamHI site of the transposon Tn5 carried by the pSUP1011 suicide vector which can be used as a delivery system for the stable introduction of the inducible marker into the chromosome of target cells.