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At least 19 recordsLinked to original sources

PubMed1992

Marine star-shaped-aggregate-forming bacteria: Agrobacterium atlanticum sp. nov.; Agrobacterium meteori sp. nov.; Agrobacterium ferrugineum sp. nov., nom. rev.; Agrobacterium gelatinovorum sp. nov., nom. rev.; and Agrobacterium stellulatum sp. nov., nom. rev.

Two new species of aerobic, gram-negative, peritrichously flagellated or nonmotile marine bacteria usually forming star-shaped aggregates were isolated from northeastern Atlantic Ocean bottom sediments. These organisms resembled eight star-shaped-aggregate-forming bacterial species from the Baltic Sea originally ascribed to the genus Agrobacterium but not included on the Approved Lists of Bacterial Names because of their questionable relationships to true agrobacteria. These two sets of star-shaped-aggregate-forming bacteria were compared by means of phenotypic data, DNA base compositions, DNA-DNA relatedness, and one-dimensional electrophoretic analysis of low-molecular-weight RNAs (5S rRNA and tRNA). According to the results of genotyping, the northeastern Atlantic Ocean isolates and three of the Baltic Sea species formed a group of closely related bacteria that could not be excluded from the genus Agrobacterium with certainty. Until more genotypic data are available, these five marine species are regarded as a distinct subdivision of the genus Agrobacterium consisting of Agrobacterium atlanticum sp. nov. (type strain, 1480T = DSM 5823T), A. meteori sp. nov. (type strain, 1513T = DSM 5824T), A. ferrugineum sp. nov. nom. rev. emend. (type strain, ATCC 25652T), A. gelatinovorum sp. nov. nom. rev. emend. (type strain, ATCC 25655T), and A. stellulatum sp. nov. nom. rev. emend. (type strain, ATCC 15215T). "A. aggregatum" proved to be a later subjective synonym of A. stellulatum, which had priority. The remaining four Baltic Sea species, "A. agile," "A. kieliense," "A. luteum," and "A. sanguineum," could not be placed in the new subdivision of Agrobacterium.

DNA, Bacterial↗

PubMed1992

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

Agrobacterium tumefaciens↗

PubMed2026

Development of an Efficient Regeneration and Agrobacterium-Mediated Transformation Protocol for Hosta 'Light Star' Using the RUBY Reporter Gene.

Hosta plantaginea is a perennial shade-tolerant herb of the Liliaceae family, with high ornamental and urban greening value. Hosta 'Light Star' is a newly developed ornamental cultivar with yellow-margined leaves and lilac flowers, but no efficient in vitro regeneration or genetic transformation system has been established for this cultivar to date. In this study, we established a highly efficient in vitro regeneration system for Hosta 'Light Star,' and developed an Agrobacterium-mediated genetic transformation protocol using the RUBY visual reporter gene for non-invasive screening of positive transformants. The optimal callus induction medium was MS + 2 mg/L 6-BA + 0.3 mg/L NAA + 0.05 mg/L 2, 4-D, with a callus induction rate of 53.33% for leaf explants (the optimal explant for sterile seedlings). The optimal adventitious bud proliferation medium was MS + 2 mg/L 6-BA + 0.1 mg/L NAA, with a proliferation coefficient of 5.87. The optimal rooting medium was 1/2 MS + 0.5 mg/L NAA + 0.5 mg/L IBA, with a 100% rooting rate. The optimal transplant substrate was perlite:vermiculite = 2:1, with a 100% transplant survival rate after acclimatization. For Agrobacterium-mediated transformation, the optimal infection parameters were as follows: Agrobacterium suspension OD600 = 0.6, infection time of 10 min, and 200 μM acetosyringone; the optimal selection conditions were 300 mg/L cefotaxime for bacteriostasis and 30 mg/L hygromycin for transformant screening. The final stable transformation efficiency was 2.50% (95% CI 1.23-3.77%), with an escape rate of 16.13%. Transgenic plants showed distinct purplish-red coloration in roots, stems, and leaves, with significantly higher betacyanin accumulation than wild-type plants (p < 0.05). Stable integration and expression of the RUBY gene were confirmed by PCR, RT-PCR, and RT-qPCR. This study establishes the first efficient regeneration and Agrobacterium-mediated transformation system for Hosta 'Light Star,' and validates the feasibility of the RUBY reporter gene as a visual marker for Hosta transformation. This system provides a solid technical platform for functional genomic studies, CRISPR/Cas9-mediated gene editing, and molecular breeding of ornamental traits in Hosta.

Transformation, Genetic↗

PubMed1992

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders.

Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.

Agrobacterium tumefaciens↗

PubMed2026

Rapid Agrobacterium-mediated transformation and high-efficiency regeneration of finger millet (Eleusine coracana) for crop improvement.

Finger millet (Eleusine coracana) is a nutritionally important and climate-resilient cereal cultivated in rainfed regions of India and Eastern Africa, yet its genetic improvement has been limited by the lack of efficient and reproducible transformation systems. In this study, we developed a rapid and efficient Agrobacterium tumefaciens-mediated transformation and regeneration system using shoot apical meristem (SAM) explants, enabling direct, callus-free shoot organogenesis. Optimal regeneration and shoot elongation were achieved on Murashige and Skoog (MS) medium supplemented with 3.5 mg L⁻1 6-benzylaminopurine (BAP), 1.5 mg L⁻1 kinetin, 0.1 mg L⁻1 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg L⁻1 gibberellic acid (GA₃). Genotype-dependent responses were observed, with PR-202 requiring 2 mg L⁻1 AgNO3 to reduce phenolic browning, whereas VL-376 regenerated efficiently without AgNO3. Transformation efficiencies of 30-32% were achieved in PR-202 and VL-376, respectively, by optimising infection and co-cultivation conditions, including reduced MS salt strength and pre-incubation of Agrobacterium. Molecular analyses, including PCR and Southern blot hybridisation, confirmed stable T-DNA integration in independent lines, while segregation analysis of T₁ progenies demonstrated Mendelian inheritance of the transgene. In addition, CRISPR/Cas9 constructs targeting EcCKX2 were successfully introduced via Agrobacterium, demonstrating the suitability of this system for genome engineering applications. Overall, this optimised SAM-based protocol provides a rapid (45-50 days), efficient, and reproducible platform for stable genetic transformation in finger millet and establishes a strong foundation for transgenic research and future genome editing studies in this underutilized crop.

Eleusine↗

PubMed1991

A DNA transformation-competent Arabidopsis genomic library in Agrobacterium.

We have constructed a nuclear genomic library from the cruciferous plant Arabidopsis thaliana ecotype Columbia in a cosmid vector, pLZO3, and a host organism, Agrobacterium tumefaciens AGL1, which can directly DNA-transform the parent organism, Arabidopsis. The broad host range cosmid pLZO3 carries a gentamicin acetyltransferase gene as bacterial selective marker and tandem, chimeric neomycin and streptomycin phosphotransferase genes as plant selective markers. Agrobacterium AGL1 carries the hypervirulent, attenuated tumor-inducing plasmid pTiBo542 from which T-region DNA sequences have been precisely deleted, allowing optimal DNA transformation of many dicotyledonous plants. Agrobacterium AGL1 also carries an insertion mutation in its recA general recombination gene, which stabilizes the recombinant plasmids. The Arabidopsis genomic library consists of some 21,600 clones gridded onto 96-well microtiter dishes and, if random, carries at least three genomic equivalents. When probed for the presence of several Arabidopsis low copy-number genes, the genomic library seems representative. As with the unicellular organisms Escherichia coli and Saccharomyces cerevisiae, this DNA transformation competent genomic library should expedite gene isolation, by gene rescue, in multicellular organisms like Arabidopsis.

Agrobacterium tumefaciens↗

PubMed2026

Establishment of an efficient Agrobacterium-mediated genetic transformation protocol for Saccharum officinarum using Black Cheribon as a model genotype.

Efficient Agrobacterium-mediated transformation (AMT) is vital for the biotechnological improvement of sugarcane (Saccharum spp.). Saccharum officinarum is the main ancestor of all modern cultivars, yet little research has been conducted on its AMT system. In this work, an efficient AMT protocol for S. officinarum was developed, with Black Cheribon as the model genotype owing to its superior tissue culture performance and regeneration capacity. The optimized agro-infection protocol comprised the following main parameters: concentration of acetosyringone (AS) in Agrobacterium culture, concentration of AS for infection, Agrobacterium concentration at OD600 = 0.4, infection time of 30 minutes, vacuum infiltration time of 10 minutes and co-cultivation time of 3 days. To further improve transformation efficiency, 0.5 mg/L thidiazuron and 200 mg/L citric acid were added to the regeneration medium, which enhanced the regeneration of shoots. A modified stage-dependent selection strategy (FlexII) was established by using glufosinate-ammonium at concentrations of 2.0, 1.0, and 0.75 mg/L in the callus proliferation, shoot regeneration, and rooting stages, respectively. This strategy was more successful than the minimum inhibitory concentration-based strategy in S. officinarum transformation. The optimized protocol further boosted the transformation efficiency of Black Cheribon from 1.12% to 7.17%. The resulting transgenic lines were confirmed by PCR amplification of T-DNA regions and immunochromatographic detection of Bar protein expression in primary transformants, respectively. These results provide a sound technical foundation for the functional genomics and biotechnological optimization of S. officinarum germplasm, and may serve as a reference for future transformation studies in other sugarcane germplasm.

Agrobacterium↗

PubMed1979

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

DNA Restriction Enzymes↗

PubMed1975

Characterization of different plaque-forming and defective temperate phages in Agrobacterium.

Four Agrobacterium tumefaciens temperate phages (PB2A, PB6(omega), PV-1(LV-1) and PS8), were shown to have the same genome size. Moreover hybridization experiments by the heteroduplex method and electron microscopy showed a 100% homology between these four phage genomes. Indications for lysogeny were found by direct means for the Agrobacterum timefaciens strain 396, Agrobacterium radiobacter strain 8149 and Agrobacterium species 0362 and by the electron microscope negative staining technique for the A.tumefaciens strains b6-806,b6-6,b6s3,b2as,cv-1,4452,11156,11158,396, and 925; for A.radiobacter strains tr-1 and 8149, the latter being bi-lysogenic, and for the A. species 0362. These isolated phage particles, most of which appear to be defective, could be grouped into different classes. No particles could be detected in the lysates of A. tumefaciens RV3, A. radiobacter strains 4718 and S1005, and A. species 0363. Further characterization by genome size was carried out for the defective temperate phages PB6-806, P4452,P8149 and P0362. No evidence for homology between PB6-806 and PB6 omega could be found. The defective phages PB6-806 and P4452 showed the same morphology but a different genome size, whereas the two phages P0362 and P8149 had a very different morphology and genome size.

Bacteriolysis↗

PubMed1976

Role of Agrobacterium cell envelope lipopolysaccharide in infection site attachment.

Lipopolysaccharide (LPS) isolated from Agrobacterium tumefaciens inhibited tumor induction by virulent bacteria. LPS from site-binding strains was not effective if added to the plant wound shortly after the bacteria, and LPS from avirulent, non-site-binding strains of Agrobacterium was not inhibitory regardless of the order of addition. However, LPS and whole cells of avirulent strains NT1 and IIBNV6, which lack of Agrobacterim virulence plasmid, were inhibitory. Chromosomal deoxyribonucleic acid thus determines specificity of this essential component of the Agrobacterium infection process.

Cell Adhesion↗

PubMed1976

Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium.

Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.

Chromosomes, Bacterial↗

PubMed1977

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.

Arginine↗

PubMed1977

Plasmid content and tumor initiation complementation by Agrobacterium tumefaciens IIBNV6.

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.

Plant Diseases↗

PubMed2026

Embryophyte-wide detection of natural Agrobacterium-mediated horizontal gene transfer reveals an ancient role for mini T-DNAs.

Agrobacterium transfers DNA into plant cells, leading to tumors, hairy roots (HR), and natural genetically modified organisms (nGMOs). Transferred DNAs (T-DNAs) from agrobacteria and T-DNA-derived cellular T-DNAs (cT-DNAs) from nGMOs vary considerably and may carry up to 15 different genes. Among these, opine synthase (ops) genes encode the synthesis of opines used as nutrients by the agrobacteria. Earlier studies predicted large numbers of naturally transformed plant species, but only few have been identified and studied so far. We therefore developed a general method to detect cT-DNAs in all publicly available whole genome sequences (WGS) and Sequence Read Archive (SRA) data from land plants. To avoid false positives, we only retained DNA sequences coding for T-DNA proteins. A total of 2614 nGMO species were identified, most are eudicots. However, cT-DNAs were also found in 82 mosses and 75 ferns, showing that Agrobacterium can also generate natural transformants among the early land plants. Analysis of 149 cT-DNA maps revealed different types of T-DNAs. Most notably, these included small T-DNAs (mini T-DNAs) with a single opine synthase gene. Mini T-DNAs are not expected to induce tumors or HRs. The predominance of mini cT-DNAs in mosses and ferns, and the presence of more complex cT-DNAs in spermatophytes, indicate that mini T-DNAs represent the earliest types of T-DNA. Our study also detected unusual T-DNA integration patterns, with multiple copies spread out over several hundreds of kilobases.

DNA, Bacterial↗

PubMed2026

4D-DIA proteomics reveals distinct proteolytic landscapes induced by mechanical stress, Agrobacterium, and a viral capsid precursor.

Nicotiana benthamiana is a widely used platform for plant molecular farming, yet recombinant protein yields are frequently compromised by the host's innate defense mechanisms, particularly proteolytic degradation. While the general effects of Agroinfiltration are known, the distinct contributions of mechanical injury, bacterial perception, and product-specific stress remain poorly resolved. Here we utilized high-depth 4D-DIA proteomics to dissect the host response across three dimensions: physical stress (buffer infiltration), pathogen-associated stress (Agrobacterium), and product-associated stress (GFP vs. the FMDV capsid precursor P1_2A). We demonstrate that buffer infiltration is not a neutral event but an independent inducer of cell wall remodeling and oxidative stress. By filtering out these background effects, we defined a core Agrobacterium-responsive proteome characterized by a growth-defense trade-off. We also expanded the known protease repertoire of N. benthamiana to 1,505 enzymes through improved genomic annotation. We found that the expression of the FMDV capsid precursor P1_2A was associated with a distinct and more pronounced protease profile compared to soluble GFP, characterized by the upregulation of subtilases and cysteine proteases. These findings suggest that host proteolytic responses vary with the recombinant cargo, a factor worth considering when designing engineering strategies for the production of complex biopharmaceuticals in plants.

Proteomics↗

PubMed1990

Oxygen-dependent desulphation of monomethyl sulphate by Agrobacterium sp. M3C.

Agrobacterium sp. M3C, previously isolated from canal-water for its ability to grow on monomethyl sulphate, degraded this ester with stoichiometric liberation of inorganic sulphate. In contrast with the biodegradation of monomethyl sulphate in Hyphomicrobium sp., and of other longer-chain alkyl sulphates in Pseudomonas spp., the pathway in Agrobacterium appeared not to involve a sulphatase enzyme capable of catalysing ester-bond hydrolysis. No such sulphatase was detectable under a range of conditions of bacterial culture, or using various methods for preparing cell-extracts, or different assay conditions. There was no incorporation of 18O-label from H2(18O) into the liberated inorganic sulphate. No methanol was detectable during biodegradation, and the organism was incapable of growth on methanol, and did not produce methanol dehydrogenase activity when grown on monomethyl sulphate. Tracer studies using mono[14C]-methyl sulphate indicated that formate serine and glycine were produced during the biodegradation. The presence of these amino acids, together with high activity of hydroxypyruvate reductase, indicated the operation of the serine pathway common in methylotrophs. Use of an oxygen electrode in conjunction with monomethyl[35S]sulphate showed that release of 35SO2(-4) was dependent on availability of O2, and that there was equimolar stoichiometry among monomethyl sulphate degraded, O2 consumed and 35SO2(-4) released. A proposed pathway for the degradation involved an initial mono-oxygenation to methanediol monosulphate with subsequent elimination of SO2(-4) and concomitant formation of formaldehyde. The pathway was compared with degradation mechanisms for other C1 compounds and for other sulphate esters.

Biodegradation, Environmental↗

PubMed1975

Agrocin 84 sensitivity: a plasmid determined property in Agrobacterium tumefaciens.

It was shown for some oncogenic Agrobacterium tumefaciens strains that agrocin 84 sensitivity is determined by the presence of a large closed circular DNA plasmid, called the Ti-plasmid. Whereas wild-type strain C58 is agrocin 84 sensitive, all Ti-plasmid cured derivatives were found to be fully resistant. Moreover all independently isolated agrocin 84 resistant colonies were stably non-oncogenic and plasmid negative. In a growth experiment carried out at 37 degrees C it was shown that the kinetics of appearance of non-oncogenic cells on the one hand and of agrocin 84 resistant cells on the other were identical. The fact that not all oncogenic, plasmid harbouring, Agrobacterium tumefaciens strains are sensitive to agrocin 84, points to the possibility that the genes determining agrocin 84 sensitivity are not essential for tumor-inducing ability.

Adenine Nucleotides↗

PubMed1976

L-Sorbose metabolism in Agrobacterium tumefaciens.

The pathway of L-sorbose metabolism in Agrobacterium tumefaciens strain B6 was determined to be: L-sorbose leads to D-glucitol (sorbitol) leads to D-fructose leads to D-fructose-6-phosphate leads to D-glucose-6-phosphate. The reduction of L-sorbose and the oxidation of D-glucitol were mediated by NADPH- and NAD+-linked oxidoreductases, respectively. The intermediates, D-glucitol and D-fructose, were isolated from in vitro reaction mixtures by column chromatography on Dowex 1-borate, and identified enzymatically. D-Fructose was identified chemically by its 1H-NMR spectrum and the IR spectrum and the melting point of the fructosazone. D-Glucitol was characterized chemically by the melting point and the IR spectrum of its hexaacetate. A. tumefaciens ICPB TT111, a representative of another genetic race of Agrobacterium, lacked L-sorbose reductase and therefore failed to grow on L-sorbose; it grew normally on D-glucitol.

Alcohol Oxidoreductases↗