Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization.

The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.     

Ti plasmid and chromosomal ornithine catabolism genes of Agrobacterium tumefaciens C58.

The pTiC58 plasmid noc genes of Agrobacterium tumefaciens C58 code for nopaline oxidase (nocC), nopaline permease (nocP), the inducible periplasmic protein n1 (nocB), and a function(s) required for ornithine catabolism (nocA). In addition, strains C58 and Ach-5 of A. tumefaciens have chromosomal ornithine catabolism genes. The chromosomal orc gene codes for ornithine dehydrogenase. Strain C58 is normally orc, but orc+ mutants can be selected. We have characterized both chromosomal orc and pTiC58 nocA plasmid genes. Complementation of most chromosomal orc mutants by pTiC58 restored growth on both nopaline and L-ornithine but did not restore ornithine dehydrogenase activity. We conclude that ornithine is an intermediate of nopaline degradation and that the Ti plasmid and chromosome both code for ornithine-degradative enzymes. A model for nopaline catabolism is presented.     

Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens.

The occ and noc regions of octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of octopine and nopaline, respectively. Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regions contained additional DNA stretches which were under the control of opine-inducible promoters, but the functions were unknown. We investigated these stretches by DNA sequence and functional analyses. The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction. The occ and noc region genes are related, but the arrangement of the genes is different. The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria. The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein. We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled octopine showed that the putative transport genes in the occ region code for octopine uptake proteins. The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region.     

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.     

Characteristics of the nopaline catabolic plasmid in Agrobacterium strains K84 and K1026 used for biological control of crown gall disease

Wild-type Agrobacterium radiobacter strain 84 and its Tra- derivative K1026, used for biological control of crown gall disease, each contain the plasmid pAtK84b. It confers incompatibility to tumor-inducing (Ti) plasmids of pathogenic A. tumefaciens, thus preventing transfer of Ti plasmids into K84 and K1026, and the consequent development of pathogens resistant to the specific antibiotic, agrocin 84 produced by K84 and K1026. pAtK84b also resembles one group of Ti plasmids in its capacity for directing nopaline catabolism. A study of the DNA homology among pAtK84b, pTiC58, and pTiAch5 was carried out. pAtK84b was transferred by conjugation to a plasmidless recipient and, after isolation, was hybridized with Ti plasmid DNA. Areas of DNA homology were located on published maps of pTiC58 and pTiAch5, a restriction enzyme map of pAtK84b was constructed, and areas of homology with DNA of known genetic function were located on the map. Strong and extensive (over 50%) homology was found between pAtK84b and pTiC58 (nopaline catabolic, Noc), but much less between pAtK84b and pTiAch5 (octopine catabolic). There was no detectable homology between pAtK84b and the oncogenic T-DNA and virulence (Vir) regions of either Ti plasmid. The size of pAtK84b was 173 kb and the orientation of regions of identified gene function (Noc, incompatability/origin of replication, and conjugal transfer) on pTiC58 was matched by the locations of homologous areas on pAtK84b. It is concluded that pAtK84b may be a deletion product of a pTiC58-type plasmid which has been disarmed in the oncogenic T-DNA and Vir regions.     

Physical map of the vitopine Ti plasmid pTiS4.

Within the Agrobacterium vitis group the vitopine strains represent a special subclass. Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped. Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHRI identified several regions of homology. The origin of replication was localized to within 2.5 kb.     

Analysis of Agrobacterium tumefaciens plasmid pTiC58 replication region with a novel high-copy-number derivative.

The origin of replication, ori, of the nopaline tumor-inducing plasmid, pTiC58, mapped in a region that shares sequence homology with octopine plasmids pTiAch5 and pTiB6. Within this region, the minimum amount of DNA necessary for maintaining autonomous replication was a 2.6-kilobase region, which also comprised the incompatibility function inc. pTiC58 derivatives containing inc were incompatible with Agrobacterium tumefaciens plasmids pTiC58, pTiD1439, pTiAch5, pTi15955, and pTiA5 and were compatible with A. rhizogenes plasmid pRi12. Situated adjacent to the origin region was a 1.5-kilobase par segment involved in stable inheritance of pTiC58 under nonselective growth conditions. When par was present, plasmid maintenance approached that of the wild-type pTiC58. Rapid loss from the cell population was observed for plasmids not containing this locus. Another 1.5-kilobase region, cop, positively regulated pTiC58 copy number, enabling certain pTiC58 derivatives to exist at a copy number up to 80 times higher than that of wild-type pTiC58. Deletions within the cop locus resulted in reduced copy number. The ori/inc regions were flanked on either side by the par and cop loci.     

Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.     

Restriction map of an agropine-type Ri plasmid and its homologies with Ti plasmids

The Ri plasmid of an agropine-type Agrobacterium rhizogenes, strain HRI, was cloned in a cosmid and mapped with the restriction endonucleases BamHI, EcoRI, KpnI, SmaI, and XbaI. This plasmid is almost identical to pRi1855 and pRiA₄b. A study by Southern hybridizations of the homologies with octopine pTiB₆806 and nopaline pTiC58 makes it possible to propose the localization of certain functions on this plasmid, such as virulence, agropine catabolism, agropine synthesis, and the origin of replication.     

Tumour genes in plants: T-DNA encoded cytokinin biosynthesis

Gene 4 from the T-region of Ti plasmids is responsible for cytokinin effects in crown gall cells; we investigated whether it codes for an enzyme of hormone biosynthesis. In a first set of experiments, gene 4 from octopine plasmid pTiAch5 and nopaline plasmid pTiC58 was expressed in Escherichia coli, and the gene products were identified by reaction with antiserum raised against a decapeptide derived from the DNA sequence of the gene. Extracts from cells expressing the gene contained high isopentenyl-transferase activity catalyzing the formation of N6-(delta2-isopentenyl)adenosine from 5'-AMP and delta2-isopentenylpyrophosphate. The cytokinin was identified by sequential h.p.l.c. chromatography and mass spectrometry. In a second set of experiments it was shown that crown gall cells contained isopentenyltransferase activity and a protein of mol. wt. 27 000 which was identified as the product of gene 4 by reaction with the antiserum. Isopentenyltransferase activity was specifically inhibited by the antiserum. No comparable enzyme activity or immunoreactive protein was detected in cytokinin-autotrophic, T-DNA free tobacco cells. The results establish that gene 4 from the T-region of octopine and nopaline Ti plasmids codes for an enzyme of cytokinin biosynthesis.     

Relationship between Nif plasmids of fast-growing Rhizobium species and Ti plasmids of Agrobacterium tumefaciens.

By use of the Southern blot hybridization technique the extent of DNA homology was determined between the Nif plasmid of a number of fast-growing Rhizobium species and Ti plasmids of the octopine (pTiAch5) and nopaline (pTiC58) type. DNA sequences common to these plasmids were located on functional maps of the Ti plasmids. No homology between Nif plasmids and the T region of Ti plasmids was detected.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli

The vir region of Ti plasmids is responsible for the transfer of the T region from Agrobacteria to plant cells; previous experiments suggested that formation of independent T region DNA circles is one step in this process. To study this step in Escherichia coli, we developed a binary vector system. One plasmid (= substrate) contains correctly oriented right and left borders from octopine plasmid pTiAch5. A gene with a counterselectable function (galK) was cloned between these borders. The galK gene is under control of the tac promoter-operator and the lac repressor with the laci gene also in the selection cassette. This construction allows determination of substrate plasmid mutants which have lost the selectable galK function. The second component of the system is one of a set of compatible plasmids harbouring various cloned parts from the vir region of nopaline plasmid pTiC58. A 3.6-kbp segment of the vir region turned out to be necessary and sufficient for production of substrate plasmid mutants which represented the equivalent of the T region containing a complete left border. From this vir region fragment four discrete proteins were expressed in minicells. The coding regions were mapped to a part conserved in nopaline and octopine plasmids; in the latter it appears to correspond to virC/D.     

Efficient vir gene induction in Agrobacterium tumefaciens requires virA, virG, and vir box from the same Ti plasmid

The vir genes of octopine, nopaline, and L,L-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopaline vir components. The induction of an octopine virE(A6)::lacZ fusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopine virG(A6) in a nopaline strain with pSM358 did not completely restore virE(A6) induction. However, addition of the octopine virA(A6) to the above strain increased virE(A6) induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopaline virE(C58)::cat fusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) and L,L-succinamopine (A281) strains. Supplementation of nopaline virA(C58) and virG(C58) in an octopine strain (A348) harboring pUCD1553 increased induction levels of virE(C58)::cat fusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine and L,L-succinamopine VirG proteins induce the octopine virE(A6) more efficiently than they do the nopaline virE(C58). Conversely, the nopaline VirG protein induces the nopaline virE(C58) more efficiently than it does the octopine virE(A6). The ability of Bo542 virG to bring about supervirulence in tobacco is observed for an octopine vir helper (LBA4404) but not for a nopaline vir helper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids. Efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids.     

Ornithine cyclodeaminase from Ti plasmid C58: DNA sequence, enzyme properties and regulation of activity by arginine

Nopaline, an abundant opine in plant cells transformed with nopaline-type Ti plasmids, is catabolized in Agrobacterium by three Ti-plasmid-coded steps via arginine and ornithine to proline. The last enzyme, ornithine cyclodeaminase (OCD), converts ornithine directly into proline with release of ammonia. We describe the DNA sequence of the ocd gene from Ti plasmid C58, antiserum against an OCD fusion protein overexpressed in Escherichia coli, induction and identification of the gene product in Agrobacterium and enzymatic properties of the protein. The DNA sequence suggests a soluble protein with a stretch of some homology with ornithine carbamoyltransferases from other bacteria. OCD activity is subject to substrate inhibition, is stimulated by NAD+ (presumably acting as a catalytic cofactor) and is regulated by L-arginine which has pronounced effects on the optima for pH and temperature and on the Km for ornithine. The regulation of OCD activity by L-arginine is discussed as part of the mechanisms which integrate the pathway of Ti-plasmid-coded opine utilization with general metabolism in Agrobacterium.     

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.     

Characterization of the virE locus of Agrobacterium tumefaciens plasmid pTiC58.

The virE locus that is responsible for the efficiency of infection by Agrobacterium tumefaciens (T. Hirooka and C. Kado, J. Bacteriol. 168:237-243, 1986) is located next to the right boundary of the virulence (Vir) region of the nopaline plasmid pTiC58. This locus is very similar to the virE locus of octopine type Ti plasmids on the basis of nucleotide and amino acid sequence comparisons as well as genetic complementation analyses. The nucleotide sequence of virE revealed three open reading frames, arranged as an operon, with a potential coding capacity for proteins of 9, 7.1, and 63.5 kilodaltons. The promoter region of virE was analyzed by using gene fusions to promoterless cat and lux genes. Two different promoters were detected, one which operates in A. tumefaciens and one which operates in Escherichia coli. virE is transcribed from left to right toward the T region. In A. tumefaciens, the expression of virE was induced by acetosyringone and required the presence of pTiC58.