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.     

Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific D-carbamoylase guarantee the total conversion from D,L-5-monosubstituted hydantoins with a low velocity of racemization to optically pure D-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55 degrees C with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni(2+) and Co(2+) and strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with Mn(2+), EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Construction of an Agrobacterium tumefaciens C58 recA mutant.

Clones encoding the recA gene of Agrobacterium tumefaciens C58 were isolated from a cosmid bank by complementation of an Escherichia coli recA mutation. Subcloning and mutagenesis with the lacZ fusion transposon Tn3HoHo1 located the Agrobacterium recA gene to a 1.3-kilobase segment of DNA. beta-Galactosidase expression from the fusions established the direction in which the gene was transcribed. The gene restored homologous recombination as well as DNA repair functions in E. coli recA mutants. Similar complementation of DNA repair functions was observed in the UV-induced Rec- Agrobacterium mutant, LBA4301. The Agrobacterium recA gene was disrupted by insertion of a cassette encoding resistance to erythromycin, and the mutated gene was marker exchanged into the chromosome of strain NT-1. The resulting strain, called UIA143, was sensitive to UV irradiation and methanesulfonic acid methyl ester and unable to carry out homologous recombination functions. The mutation was stable and had no effect on other genetic properties of the Agrobacterium strain, including transformability and proficiency as a conjugal donor or recipient. Furthermore, strain UIA143 became tumorigenic upon introduction of a Ti plasmid, indicating that tumor induction is independent of recA functions. Sequence homology was detected between the recA genes of strain C58 and E. coli as well as with DNA isolated from agrobacteria representing the three major biochemically differentiated biovars of this genus. In some cases, biovar-specific restriction fragment length polymorphisms were apparent at the recA locus.     

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.     

Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Molecular analysis of the recA gene of Agrobacterium tumefaciens C58.

The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined. A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation. However, unlike its Escherichia coli equivalent, A. tumefaciens recA lacks the upstream 'SOS box', suggesting a different mechanism of regulation for this gene.     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants.

Agrobacterium tumefaciens Tn5 mutants deficient in chemotaxis to root exudates were used to study the significance of chemotaxis in crown gall pathogenesis. Mutants deficient in motility and in chemotaxis were fully virulent when inoculated by direct immersion in inoculum, followed by growth for 2 weeks in moist growth pouches. Ability of mutant bacteria to move through soil to infect roots was tested by planting wounded seedlings into air-dried soil or sand that had been infested with inoculum. Mutant bacteria were almost as virulent as the parent on plants grown in sand but were avirulent on soil-grown plants.     

Indoleacetic acid production: a plasmid function of Agrobacterium tumefaciens C58.

Indoleacetic acid (IAA) production is regulated by the 117 Mdalton pTi plasmid that is harbored by the crown gall bacterium $\text{Agrobacterium tumefaciens}$ strain C-58. In the presence of tyrosine, about 5- to 10-fold higher IAA production (0.8–0.9 μg/ml) occurs in strain C-58 than in the plasmidless avirulent derivative strain NT1. The high level of IAA production is restored when the plasmid is reintroduced into strain NT1.     

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k_cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K_m) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k_cat = 1.9 × 10² s⁻¹ on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.Aldohexuronate catabolism in bacteria is reported to involve two different pathways, one initiating with an isomerization step and the other with an oxidation step. In the isomerization pathway, aldohexuronate (glucuronate and galacturonate) is isomerized to ketohexuronate by uronate isomerase and ultimately degraded to pyruvate and 3-phosphoglyceraldehyde. The isomerization pathway has been previously reported to occur in bacteria, including Escherichia coli (7), Erwinia carotovora (18), Erwinia chrysanthemi (15), Klebsiella pneumoniae (9, 23), and Serratia marcescens (28). In the oxidation pathway, aldohexuronate is oxidized to aldohexarate by uronate dehydrogenase (Udh) and further catabolized to pyruvate (2, 5, 7, 9, 18, 19, 24). Uronate dehydrogenase, the key enzyme of this pathway, has been investigated in two plant pathogen bacteria, Pseudomonas syringae and Agrobacterium tumefaciens. To date, only limited studies pertaining to the properties of Udh have been reported in the literature (3, 6, 38, 43), and no sequence has yet been identified. Udh is classified as an NAD-linked oxidoreductase (EC 1.1.1.203), with a total molecular weight of about 60,000. It is a homodimer composed of two subunits with molecular weights of about 30,000 each (38). Udh is a thermally unstable, reversible enzyme, with an optimum pH of about 8.0 (3, 6, 38).In E. coli MG1655 that has the isomerization pathway for aldohexuronate catabolism, glucuronate is transported by an aldohexuronate transporter encoded by exuT and converted to fructuronate by uronate isomerase, encoded by uxaC (22, 30) (Fig. ​(Fig.1).1). Fructuronate is transferred to the Entner-Doudoroff pathway to be utilized as an energy source via 2-keto-3-deoxy-6-phospho-gluconate (7, 27, 31, 32). Therefore, E. coli MG1655 with a uxaC deletion cannot use glucuronate as a carbon source. In this strain, glucarate is converted to 5-keto-4-deoxy-d-glucarate by d-glucarate dehydratase, encoded by gudD, and then transferred to glycolysis via pyruvate or 2-phosphoglycerate (27, 33). Recently, a number of bacterial genome sequences have been published, including those of the Udh-containing P. syringae pv. tomato strain DC3000 and A. tumefaciens strain C58 (4, 10). A shotgun library of P. syringae was constructed to identify the gene encoding Udh. Screening for Udh was conducted in E. coli MG1655 ΔuxaC. Since uronate dehydrogenase converts glucuronate to glucarate, uxaC deletion strains of E. coli harboring the shotgun library of P. syringae that can grow in a minimal medium containing glucuronate as a sole carbon source may carry the gene encoding Udh (Fig. ​(Fig.1).1). Once an initial Udh is identified from P. syringae, a BLAST homology search may lead to the identification of Udhs from other bacteria.Open in a separate windowFIG. 1.Catabolism of glucuronate and glucarate in bacteria. Glucuronate consumption is prevented by knockout of the uxaC gene. The presence of uronate dehydrogenase in a uxaC knockout enables growth of E. coli on glucuronate.     

Elucidation of the O-chain structure from the lipopolysaccharide of Agrobacterium tumefaciens strain C58

A linear homopolysaccharide built of 3-alpha-L-6dTalp residues, randomly acetylated at position C-4, is described for the O-specific polysaccharide of Agrobacterium tumefaciens strain C58. This structure, determined by spectroscopical and chemical methods, is strictly correlated to that of Rhizobium loti strain NZP2213, which differs for the degree and the position of O-acetylation.     

Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome.

Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons, used as probes, hybridize with different restriction fragments, showing that these replicons are two independent genetic entities. A 16S rRNA probe and genes encoding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature. In PFGE, megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome always generated an intense band, while the 3-Mb band was barely visible. After linearization of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of the 2.1-Mb remained constant. This suggests that the 3-Mb chromosome is circular and that the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA present. The plug was then recovered, and the remaining DNA was digested with either PacI or SwaI and then separated by PFGE. The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained, further proof of the linear nature of the 2.1-Mb chromosome.     

Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule.

Induction of Agrobacterium tumefaciens virulence genes by plant phenolic compounds is essential for successful T-DNA transfer to a host plant. In Douglas fir needles, the major virulence region inducer is the glycoside coniferin (J. W. Morris and R. O. Morris, Proc. Natl. Acad. Sci. USA 87:3612-3618, 1990). Agrobacterium strains with high beta-glucosidase activity respond to coniferin and infect Douglas fir seedlings, whereas most strains with low beta-glucosidase activity fail to respond to coniferin and are avirulent on this host. We have cloned two beta-glucosidase genes from A. tumefaciens B3/73 and sequenced one of them, cbg1. It appears to be part of a polycistronic unit and shows a high bias for GC-rich codons. When expressed in Escherichia coli, Cbg1 beta-glucosidase hydrolyzes coniferin but not cellobiose. The 88-kDa predicted product of cbg1 is highly similar to one other bacterial beta-glucosidase and several fungal beta-glucosidases. There is little homology between Cbg1 and other bacterial beta-glucosidases, including an Agrobacterium cellobiase.     

Promotion of somatic embryo production from embryogenic calluses of monocotyledonous and dicotyledonous plants by heavy-ion beam irradiation

Effect of heavy-ion beam irradiation on the growth and development of embryogenic calluses was examined in the liliaceous monocotyledon Tricyrtis hirta and the umbelliferous dicotyledon Daucus carota. Embryogenic calluses of T. hirta were irradiated with 5, 10, 20 or 50 Gy of ¹²C⁶⁺, ¹⁴N⁷⁺ or ²⁰Ne¹⁰⁺ ions, and those of D. carota were irradiated with 5, 10, 20 or 50 Gy of ¹⁴N⁷⁺ ions. In both species, irradiation at 10–50 Gy inhibited growth of embryogenic calluses, and callus growth rate decreased as irradiation dose increased. Interestingly irradiation at low doses greatly promoted somatic embryo production from embryogenic calluses in both species. In T. hirta, calluses irradiated with 5 and 10 Gy ¹²C⁶⁺ ions, 10 Gy ¹⁴N⁷⁺ ions, and 5 Gy ²⁰Ne¹⁰⁺ ions produced more than twice as many embryos as the control, non-irradiated calluses. In D. carota, embryogenic calluses irradiated with 5 Gy ¹⁴N⁷⁺ ions produced more than one and a half times as many embryos as the control. Somatic embryo production in both species was inhibited by irradiation at 20 and 50 Gy.     

Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction

For the first time, the complete structure of the lipid A from the lipopolysaccharide of an Agrobacterium species is here reported. In particular, the structure of the lipid A from A. tumefaciens strain C58, a soil pathogen bacterium strictly related to Rhizobiaceae, was determined. The structural study, carried out by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy, revealed that lipid A fraction consisted of a mixture of species all sharing the bis-phosphorylated glucosamine disaccharide backbone that could be designated in two main structural motifs, according to the acylation pattern. The main species was a penta-acylated lipid A bearing two unsubstituted 14:0 (3-OH) fatty acids in ester linkage and two 16:0 (3-OH) in amide linkage; the one on GlcN II was O-acylated by a long chain fatty acid, 28:0 (27-OH). This in turn was esterified by a 3-hydroxy-butyroyl residue at its hydroxy group. The second species, in lesser amounts, was identified as a tetra-acylated lipid A and lacked the 14:0 (3-OH) residue on GlcN I. Other species deriving from these two lacked a phosphate group or 3-hydroxy-butyroyl residue or otherwise carried a 26:0 (25-OH) as long chain fatty acid. The lipid A structure of phytopathogen A. tumefaciens strain C58 presents deep structural analogies with lipid A of symbiotic Rhizobium, and the hypothesis is advanced that it can be a strategy of the bacterium to escape or attenuate the plant response.     

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.     

Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.