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.     

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.     

D-glucaric acid and galactaric acid catabolism by Agrobacterium tumefaciens

Cell-free extract (crude extract) of Agrobacterium tumefaciens grown on d-glucuronate or d-glucarate converts d-glucarate and galactarate to a mixture of 2-keto-3-deoxy- and 4-deoxy-5-keto-d-glucarate. These compounds are then converted by partially purified crude extract to an intermediate tentatively identified as 2,5-diketoadipate. The same enzyme preparation further decarboxylates this intermediate to alpha-ketoglutarate semialdehyde, which is subsequently oxidized in a nicotinamide adenine dinucleotide-dependent reaction to alpha-ketoglutaric acid. Since A. tumefaciens converts d-glucuronic acid to d-glucarate, a pathway from d-glucuronate to alpha-ketoglutarate in A. tumefaciens was determined.     

Chemotaxis to plant phenolic inducers of virulence genes is constitutively expressed in the absence of the Ti plasmid in Agrobacterium tumefaciens.

The virulence (vir) genes are required in the early stages of plant tumor formation and are located together on the tumor-inducing (Ti) plasmid in Agrobacterium tumefaciens. Five of the vir genes are expressed inducibly in response to the following monocyclic phenolic compounds: acetosyringone, catechol, gallate, beta-resorcylate, protocatechuate, p-hydroxybenzoate, and vanillin. Of these compounds, only the latter six, excluding vanillin [corrected] served as chemoattractants and only the latter three served as growth substrates for A. tumefaciens A348. Strain A136, isogenic except for lack of the Ti plasmid, demonstrated chemotactic behavior and nutritional capabilities similar to those of strain A348. The chemotactic response to the vir gene inducers was expressed constitutively.     

Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955

Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.     

Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes.

The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide.     

vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.     

A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity.     

Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens

Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.     

Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor.

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.     

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid.

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2- to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.     

Positive selection for mutations affecting bioconversion of aromatic compounds in Agrobacterium tumefaciens: analysis of spontaneous mutations in the protocatechuate 3,4-dioxygenase gene

A positive selection method for mutations affecting bioconversion of aromatic compounds was applied to a mutant strain of Agrobacterium tumefaciens A348. The nucleotide sequence of the A348 pcaHGB genes, which encode protocatechuate 3,4-dioxygenase (PcaHG) and beta-carboxy-cis,cis-muconate cycloisomerase (PcaB) for the first two steps in catabolism of the diphenolic protocatechuate, was determined. An omega element was introduced into the pcaB gene of A348, creating strain ADO2077. In the presence of phenolic compounds that can serve as carbon sources, growth of ADO2077 is inhibited due to accumulation of the tricarboxylate intermediate. The toxic effect, previously described for Acinetobacter sp., affords a powerful selection for suppressor mutations in genes required for upstream catabolic steps. By monitoring loss of the marker in pcaB, it was possible to determine that the formation of deletions was minimal compared to results obtained with Acinetobacter sp. Thus, the tricarboxylic acid trick in and of itself does not appear to select for large deletion mutations. The power of the selection was demonstrated by targeting the pcaHG genes of A. tumefaciens for spontaneous mutation. Sixteen strains carrying putative second-site mutations in pcaH or -G were subjected to sequence analysis. All single-site events, their mutations revealed no particular bias toward multibase deletions or unusual patterns: five (-1) frameshifts, one (+1) frameshift, one tandem duplication of 88 bp, one deletion of 92 bp, one nonsense mutation, and seven missense mutations. PcaHG is considered to be the prototypical ferric intradiol dioxygenase. The missense mutations served to corroborate the significance of active site amino acid residues deduced from crystal structures of PcaHG from Pseudomonas putida and Acinetobacter sp. as well as of residues in other parts of the enzyme.     

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens

Summary Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10⁸ cells·ml^–1), cocultivated for 48–96 h and placed on Murashige and Skoog (MS) medium with 5.0 M each of 2,4-D and BA, 50 mg·l^–1 kanamycin and 500 mg·l^–1 carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 M 2,4-D/BA, 50 mg·l^–1 kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 M NAA/BA and 50 mg·l^–1 kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyl-aminopurine - CaMV cauliflower mosaic virus - NAA naphthaleneacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction     

Genes required for cellulose synthesis in Agrobacterium tumefaciens.

A region of the chromosome of Agrobacterium tumefaciens 11 kb long containing two operons required for cellulose synthesis and a part of a gene homologous to the fixR gene of Bradyrhizobium japonicum has been sequenced. One of the cellulose synthesis operons contained a gene (celA) homologous to the cellulose synthase (bscA) gene of Acetobacter xylinum. The same operon also contained a gene (celC) homologous to endoglucanase genes from A. xylinum, Cellulomonas uda, and Erwinia chrysanthemi. The middle gene of this operon (celB) and both the genes of the other operon required for cellulose synthesis (celDE) showed no significant homology to genes contained in the databases. Transposon insertions showed that at least the last gene of each of these operons (celC and celE) was required for cellulose synthesis in A. tumefaciens.     

Ferrisiderophore reductase activity in Agrobacterium tumefaciens.

Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant. Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory. This ferrisiderophore reductase activity may be a part of the iron assimilation process in A. tumefaciens.