Agrobacterium

Cell Mol Biol Lett, 2002, 7(2B), 709 - 19
Reproducible transformation in two grain legumes--soybean and azuki bean--using different systems; El-Shemy H et al.; Two plasmid vectors were introduced into soybean (Glycine max (L.) Merr.) and azuki bean (Vigna angularis Willd . Ohwi and Ohashi) using different transformation systems . Azuki bean epicotyl explants were prepared from etiolated seedlings and co-cultivated with Agrobacterium tumefaciens for 2 days . Adventitious shoots were developed from the callus of the explants on a regeneration medium containing hygromycin, and the shoots were excised and transferred to a rooting medium containing hygromycin at the same concentration . Rooting shoots were transferred to soil and grown in a glass-house to produce viable seeds . PCR analysis confirmed clearly the presence of the hpt gene in most of the azuki beans regenerated under hygromycin selection . A soybean embryogenic suspension culture was generated from immature cotyledons, and used for the introduction of plasmids by particle bombardment . Hygromycin-resistant embryogenic clones were isolated after 8 weeks of hygromycin selection, and then the green clones were matured on the differentiation medium . After desiccation, the embryos were germinated on the rooting medium, and the plants were transferred to soil in a glass-house . More than 50% of the regenerated soybean plants tolerant to hygromycin yielded the hpt fragment on PCR analysis . The azuki bean transformants were obtained more rapidly and with higher efficiency than the soybean transformants.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Aug, 22(8), 736 - 8
{Construction of plant expression vectors containing the gene encoding cholera toxin B subunit}; Peng ZQ et al.; OBJECTIVE: To construct the plant expression vector containing the nucleotide sequence encoding cholera toxin B (CTB) subunits . METHOD: Using high-fidelity PCR, we amplified CTB genes that were then subcloned into the transition vector pRTL2 . Following confirmation of the CTB nucleotide sequence, the vector was subcloned into the plant vector pBI121 that was subsequently transferred into Agrobacterium tumefaciens LBA4404 by electroporation . RESULTS: CTB DNA that was ligated into the transition vectors resulted in the 2 vectors designated as pRCTB and pRCTBK . After the 2 vectors were ligated into the plant binary vector pBI121 respectively, new plant binary vectors, namely pBI-CTB and pBI-CTBK, were produced . Analysis with restriction endonucleases confirmed successful transfer of pBI-CTB and pBI-CTBK into Agrobacterium tumefaciens LBA4404 . CONCLUSION: With appropriate technological strategy, the plant binary expression vectors encoding CTB have been constructed, which facilitates further investigation of CTB protein expressions in transgenic plant.

Arch Microbiol, 2002 Nov, 178(5), 338 - 43 Epub 2002 Aug 07.
Identification of Agrobacterium spp . present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures; Weller SA et al.; A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp . The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains . Seedlings were grown from seven lots of surface-sterilised Brassica napus seed . Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2 . Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp . in one of the seed samples . Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays . None of the strains possessed a plasmid . This is the first report of Agrobacterium sp . present within the seed of B . napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.

Scand J Infect Dis, 2002, 34(9), 693 - 4
Central venous catheter-related infection due to Agrobacterium radiobacter: a report of 2 cases; Landron C et al.; We report 2 cases of Agrobacterium radiobacter bacteremia in immunocompromised patients . Removal of the central venous catheter and administration of antimicrobial therapy led to favorable outcomes in both patients . Infections due to A . radiobacter are rare and usually occur in patients with predisposing factors.

Plant Mol Biol, 2002 Nov, 50(4-5), 757 - 71
Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems; Buzeli RA et al.; The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER . In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation . We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5' flanking sequences were fused to beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation . Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems . Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development . While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (-358 to -211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (-211 to -80) . Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays . These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter.

Plant Mol Biol, 2002 Oct, 50(3), 333 - 44
Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature; Huang T et al.; Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation . The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expressed in transformed plants . Thermal hysteresis activity (indicating the presence of active AFPs) was present in protein extracts from plants expressing both proteins and was also detected in leaf apoplast fluid from plants expressing AFPs with the signal peptide . Transgenic lines did not demonstrate improved ability to survive freezing when compared to wild-type . However, when cooled under four different regimes, transgenic lines with AFPs in the apoplast fluid froze at significantly lower temperatures than did wild-type, especially in the absence of extrinsic nucleation events.

Protein Eng, 2002 Aug, 15(8), 689 - 95
Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution; Oh KH et al.; N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability . We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling . In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized . The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme . Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions . The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme . DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A . The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme . Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect . V40A and G75S gave rise to an increase only in oxidative stability . The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp . KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.

Plant Cell, 1989 Jul, 1(7), 691 - 698
cis-Acting Elements for Light Regulation of Pea Ferredoxin I Gene Expression Are Located within Transcribed Sequences; Elliott RC et al.; An intact pea gene encoding ferredoxin I (Fed-1) and several chimeric constructs containing portions of Fed-1 were introduced into tobacco plants by Agrobacterium-mediated transformation . The intact gene was correctly transcribed and translated to produce a protein that was imported into the chloroplast and processed to its mature size . Fed-1 mRNA accumulation in these plants was strongly light-dependent, as it is in pea leaves . In chimeric constructs, the Fed-1 promoter was active but no light responses were seen, even when as much as 2 kilobases of 5{prime} -flanking sequence were included . We also failed to observe clear light responses with a construct containing 3{prime} -flanking sequences from Fed-1 attached to a {beta}-glucuronidase gene driven by the cauliflower mosaic virus 35S promoter . However, the transcribed portion of Fed-1 conveyed normal light responsiveness when driven by the 35S promoter . The results are discussed in terms of the hypothesis that light determines Fed-1 mRNA abundance by affecting RNA stability rather than by affecting transcription.

Plant Cell, 1989 Apr, 1(4), 403 - 413
Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene; Medford JI et al.; Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development . We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70) . The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants . Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5{prime}-monophosphate twofold . At the control temperature zeatin riboside and zeatin riboside 5{prime}-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants . This uninduced cytokinin increase affected various aspects of development . In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system . In Arabidopsis, reduction of root growth was also found . However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering . Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state . The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors . Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation . Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

Plant Cell, 1989 Nov, 1(11), 1043 - 1050
Trichome Development in Arabidopsis thaliana . I . T-DNA Tagging of the GLABROUS1 Gene; Marks MD et al.; Progeny from a transformed Arabidopsis plant (produced by the Agrobacterium-mediated seed transformation procedure) were found to be segregating for an altered trichome phenotype . The mutant plants have normal leaf trichomes but completely lack trichomes usually found on the stem . The mutation is tightly linked to a T-DNA insert . Complementation analysis with genetically characterized trichome mutants revealed that the new mutation is an allele of the GL1 locus . The new trichome mutant has been designated gl1-43 . DNA gel blot analysis indicated that the insert site contains a complex array of at least four tandemly linked T-DNA units oriented as both direct and inverted repeats . A genomic library, constructed using DNA from gl1-43 plants, was used to clone DNA that flanks the left end of the T-DNA insert . The availability of DNA from the region interrupted by the insert has allowed initial characterization of the wild-type GL1 gene and will permit the eventual cloning and sequencing of this developmentally interesting gene.

Biochim Biophys Acta, 2002 Sep 27, 1577(3), 452 - 6
Isolating the promoter of a stress-induced gene encoding betaine aldehyde dehydrogenase from the halophyte Atriplex centralasiatica Iljin; Yin X et al.; The betaine aldehyde dehydrogenase (AcBADH) gene of the halophyte Atriplex centralasiatica Iljin is induced by drought, salinity, cold stress and abscisic acid, in parallel with an increase in betaine level . In order to study the molecular basis of its expression and to obtain an effective stress-induced promoter, the 5' flanking region of betaine aldehyde dehydrogenase gene (about 1.2 kb) was isolated from the halophyte A . centralasiatica Iljin by screening the genomic library . The transcription start site, which localized at 84 bases upstream of the start ATG, was determined by primer extension and 5'-RACE method . To investigate the molecular mechanism of the stress-induced gene regulation, the AcBADH promoter-beta-glucuronidase chimeric gene constructs containing six deletions were introduced into tobacco by Agrobacterium-mediated transformation . The AcBADH 5'-flanking region, a promoter strongly induced by salt stress, contains two salt-responsive enhancer regions localized between -1115 and -890, -462 and -230 and one silencer region between -890 and -641.

Plant Cell, 1990 May, 2(5), 379 - 392
Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types; Koes RE et al.; Chalcone synthase (CHS) catalyzes the first step in the biosynthesis of flavonoids that function in flower pigmentation, protection against stress, and induction of nodulation . The petunia genome contains eight complete chs genes, of which four are differentially expressed in floral tissues and UV-light-induced seedlings . The 5{prime}-flanking regions of these four chs genes were fused to the {beta}-glucuronidase (GUS) reporter gene and introduced into petunia plants by Agrobacterium-mediated transformation . We show that expression of each construct is identical to the expression of the authentic chs gene, implying that the differences in expression pattern between these chs genes are caused at least in part by their promoters . Histochemical analyses of GUS expression show that chs promoters are not only active in pigmented cell types (epidermal cells of the flower corolla and tube and {sub} epidermal cells of the flower stem) but also in a number of unpigmented cell types (mesophylic cells of the corolla, several cell types in the ovary and the seed coat) . Comparison of chs-GUS expression and flavonoid accumulation patterns in anthers suggests that intercellular transport of flavonoids and enzymes occurs in this organ . Analysis of the flavonoids accumulated in tissues from mutant lines shows that only a subset of the genes that control flavonoid biosynthesis in the flower operates in the ovary and seed . This implies that (genetic) control of flavonoid biosynthesis is highly tissue specific.

Plant Cell, 1990 Oct, 2(10), 1009 - 1017
Nodulin Gene Expression and ENOD2 Localization in Effective, Nitrogen-Fixing and Ineffective, Bacteria-Free Nodules of Alfalfa; Van De Wiel C et al.; Alfalfa plants form bacteria-free nodules in response to a number of agents, including Rhizobium meliloti exo mutants, Agrobacterium tumefaciens transconjugants carrying cloned R . meliloti nodulation genes, and compounds that function as auxin transport inhibitors, N-( 1-naphthyl)phthalamic acid or 2,3,5-triiodobenzoic acid . These bacteria-free nodules contain transcripts for the nodulins Nms30 and MsENOD2; transcripts for late nodulins like leghemoglobin are not detected . In situ hybridization studies demonstrated that ENOD2 transcripts were localized in parenchyma cells at the base and along the periphery of nitrogen-fixing alfalfa root nodules . The ENOD2 gene was also expressed in a tissue-specific manner in nodules elicited by N-( 1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid . In bacteria-free nodules induced by R . meliloti exo mutants and A . tumefaciens transconjugants carrying either one or both R . meliloti symbiotic plasmids, ENOD2 transcripts were also detected but were usually localized to parenchyma cells at the base instead of along the periphery of the nodule . On the basis of the pattern of ENOD2 gene expression, we conclude that the developmental pathway of bacteria-free nodules, whether bacterially or chemically induced, is the same as that of nitrogen-fixing nodules, and, furthermore, that the auxin transport inhibitors in their action mimic some factor(s) that trigger nodule development.

BMC Biotechnol . 2002 Sep 27;2(1):18.
Genetic transformation of Vitis vinifera via organogenesis; Mezzetti B et al.; BACKGROUND: Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape . The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus . However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered . RESULTS: Transgenic grape plants (V . vinifera, table-grape cultivars Silcora and Thompson Seedless) were produced using a method based on regeneration via organogenesis . In vitro proliferating shoots were cultured in the presence of increasing concentrations of N6-benzyl adenine . The apical dome of the shoot was removed at each transplantation which, after three months, produced meristematic bulk tissue characterized by a strong capacity to differentiate adventitious shoots . Slices prepared from the meristematic bulk were used for Agrobacterium-mediated transformation of grape plants with the gene DefH9-iaaM . After rooting on kanamycin containing media and greenhouse acclimatization, transgenic plants were transferred to the field . At the end of the first year of field cultivation, DefH9-iaaM grape plants were phenotypically homogeneous and did not show any morphological alterations in vegetative growth . The expression of DefH9-iaaM gene was detected in transgenic flower buds of both cultivars . CONCLUSIONS: The phenotypic homogeneity of the regenerated plants highlights the validity of this method for both propagation and genetic transformation of table grape cultivars . Expression of the DefH9-iaaM gene takes place in young flower buds of transgenic plants from both grape cultivars.

Nature, 2002 Sep 26, 419(6905), 399 - 403
A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis; Maldonado AM et al.; Localized attack by a necrotizing pathogen induces systemic acquired resistance (SAR) to subsequent attack by a broad range of normally virulent pathogens . Salicylic acid accumulation is required for activation of local defenses, such as pathogenesis-related protein accumulation, at the initial site of attack, and for subsequent expression of SAR upon secondary, distant challenge . Although salicylic acid moves through the plant, it is apparently not an essential mobile signal . We screened Agrobacterium tumefaciens transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants specifically compromized in SAR . Here we show that Defective in induced resistance 1-1 (dir1-1) exhibits wild-type local resistance to avirulent and virulent Pseudomonas syringae, but that pathogenesis-related gene expression is abolished in uninoculated distant leaves and dir1-1 fails to develop SAR to virulent Pseudomonas or Peronospora parasitica . Petiole exudate experiments indicate that dir1-1 is defective in the production or transmission from the inoculated leaf of an essential mobile signal . DIR1 encodes a putative apoplastic lipid transfer protein and we propose that DIR1 interacts with a lipid-derived molecule to promote long distance signalling.

Plant Cell, 1991 Jul, 3(7), 647 - 656
Delayed Leaf Senescence in Tobacco Plants Transformed with tmr, a Gene for Cytokinin Production in Agrobacterium; Smart CM et al.; The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence . Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871 . This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis . After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20{deg}C, rose and remained high for up to 8 hours . The levels of cytokinin and tmr mRNA were substantially lower by 24 hours . Transformed plants grown at 20{deg}C were shorter, had larger side shoots, and remained green for longer than untransformed plants . The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves . Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.

Plant Cell, 1991 Feb, 3(2), 149 - 157
Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis; Errampalli D et al.; T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development . We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens . Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis . The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers . Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families . Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene . Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process . DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion . A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.

Plant Cell, 1991 Nov, 3(11), 1167 - 1175
An Auxin-Responsive Promoter Is Differentially Induced by Auxin Gradients during Tropisms; Li Y et al.; We constructed a chimeric gene consisting of a soybean small auxin up RNA (SAUR) promoter and leader sequence fused to an Escherichia coli {beta}-glucuronidase (GUS) open reading frame and a 3{prime} untranslated nopaline synthase sequence from Agrobacterium tumefaciens . This chimeric gene was used to transform tobacco by Agrobacterium-mediated transformation . In R2 etiolated transgenic tobacco seedlings, GUS expression occurred primarily in elongation regions of hypocotyls and roots . In green plants, GUS was expressed primarily in the epidermis and cortex of stems and petioles, as well as in elongation regions of anther filaments in developing flowers . GUS expression was responsive to exogenous auxin in the range of 10-8 to 10-3 M . During gravitropism and phototropism, the GUS activity became greater on the more rapidly elongating side of tobacco stems . Auxin transport inhibitors and other manipulations that blocked gravitropism also blocked the asymmetric distribution of GUS activity in gravistimulated stems . Light treatment of dark-grown seedlings resulted in a rapid decrease in GUS activity . Light-induced decay in GUS activity was fully reversed by application of auxin . Taken together, our results add support for the formation of an asymmetric distribution of auxin at sites of action during tropism.

Plant Cell, 1992 May, 4(5), 513 - 524
Regulation of the Osmotin Gene Promoter; Kononowicz AK et al.; By introducing a chimeric gene fusion of the osmotin promoter and {beta}-glucuronidase into tobacco by Agrobacterium-mediated transformation, we have demonstrated a very specific pattern of temporal and spatial regulation of the osmotin promoter during normal plant development and after adaptation to NaCl . We have found that the osmotin promoter has a very high natural level of activity in mature pollen grains during anther dehiscence and in pericarp tissue at the final, desiccating stages of fruit development . GUS activity was rapidly lost after pollen germination . The osmotin promoter thus appears to be unique among active pollen promoters described to date in that it is active only in dehydrated pollen . The osmotin promoter was also active in corolla tissue at the onset of senescence . Adaptation of plants to NaCl highly stimulated osmotin promoter activity in epidermal and cortex parenchyma cells in the root elongation zone; in epidermis and xylem parenchyma cells in stem internodes; and in epidermis, mesophyll, and xylem parenchyma cells in developed leaves . The spatial and temporal expression pattern of the osmotin gene appears consistent with both osmotic and pathogen defense functions of the gene.

Plant Cell, 1992 Mar, 4(3), 319 - 332
Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants; Tovar J et al.; Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants . We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco . Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats . As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells . The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis . Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings) . Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell . Low dose {gamma} irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants . Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection . DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.

Plant Cell, 1992 Feb, 4(2), 119 - 128
Cloning the Arabidopsis GA1 Locus by Genomic Subtraction; Sun T et al.; Arabidopsis thaliana ga1 mutants are gibberellin-responsive dwarfs . We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive ga1 deletion mutant, ga1-3 . The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome . Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus . First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus . Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation . Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3 . One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus . The three other lesions are all single base changes located within the two most distal exons . RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA . We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.

Plant Cell, 1992 Jan, 4(1), 7 - 16
Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer; Schlappi M et al.; Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen . The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection . There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection . Agroinfection frequency was highest in the embryogenic line A188 . All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection . The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer . This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation.

J Bacteriol, 2002 Oct, 184(20), 5572 - 82
A novel cytology-based, two-hybrid screen for bacteria applied to protein-protein interaction studies of a type IV secretion system; Ding Z et al.; DivIVA of Bacillus subtilis and FtsZ of Escherichia coli were used to target heterologous protein complexes to cell division sites of E . coli and Agrobacterium tumefaciens . DivIVA and FtsZ that were fused to the dimerizing leucine zipper (LZ) domain of the yeast transcription activator GCN4 directed the green fluorescent protein (GFP) that was fused to an LZ domain to E . coli division sites, resulting in fluorescence patterns identical to those observed with DivIVA::GFP and FtsZ::GFP . These cell division proteins also targeted the VirE1 chaperone and VirE2 secretion substrate complex to division sites of E . coli and A . tumefaciens . Coproduction of the native VirE1 or VirE2 proteins inhibited the dihybrid interaction in both species, as judged by loss of GFP targeting to division sites . The VirE1 chaperone bound independently to N- and C-terminal regions of VirE2, with a requirement for residues 84 to 147 and 331 to 405 for these interactions, as shown by dihybrid studies with VirE1::GFP and DivIVA fused to N- and C-terminal VirE2 fragments . DivIVA also targeted homo- and heterotypic complexes of VirB8 and VirB10, two bitopic inner membrane subunits of the A . tumefaciens T-DNA transfer system, in E . coli and homotypic complexes of VirB10 in A . tumefaciens . VirB10 self-association in bacteria was mediated by the C-terminal periplasmic domain, as shown by dihybrid studies with fusions to VirB10 truncation derivatives . Together, our findings establish a proof-of-concept for the use of cell-location-specific proteins for studies of interactions among cytosolic and membrane proteins in diverse bacterial species.

J Virol Methods, 2002 Sep, 105(2), 343 - 8
Agroinfection as a rapid method for propagating Cowpea mosaic virus-based constructs; Liu L et al.; To increase the efficiency of infections with Cowpea mosaic virus (CPMV)-based constructs, clones suitable for agroinfection were constructed . Full-length copies of RNA-1 and RNA-2 were inserted between the sequence of a Cauliflower mosaic virus (CaMV) 35S promoter and a nos terminator and were introduced into the Agrobacterium tumefaciens plasmid, pBINPLUS . Infiltration of leaves of either Nicotiana benthamiana or cowpea (Vigna unguiculata) with a bacterial suspension containing a mixture of the RNA-1- and RNA-2-based plasmids resulted in the plants developing typical CPMV symptoms . To confirm the utility of this approach for use with CPMV-based vectors, a GFP construct based on RNA-2 was adapted for agroinfection . Infiltration of N . benthamiana leaves with a mixture of Agrobacteria containing this construct and the RNA-1 plasmid resulted in high levels of GFP expression . The results demonstrate that agroinfection is a suitable method for the propagation of CPMV-based derivatives.

Plant Cell, 1994 Feb, 6(2), 215 - 225
Morphogenetic Rescue of Rhizobium meliloti Nodulation Mutants by trans-Zeatin Secretion; Cooper JB et al.; The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria . We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37 . This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures . Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation . This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses . Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.

Plant Cell, 1994 Jan, 6(1), 43 - 52
Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression; Kuipers A et al.; The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS) . GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants . Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose . Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained . Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers . In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule . Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule . This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules . The size of this zone is suggested to be dependent on the GBSS protein level . During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose . RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes . Antisense RNA could only be detected in leaf tissue of the transgenic plants.

Mol Gen Mikrobiol Virusol, 2002, (3), 40 - 1
{Search for conjugative plasmids in Azospirillum brasilense and Azospirillum lipoferum associated with plants}; Katsy EI; The conjugative plasmids in Azospirillum brasilense strains S17 . Sp107, Sp245, SpBr14, JM6B2, JM82Al, UQ1794, UQI796 and in Azospirillum lipoferum strain RG20 were prove to exist for the first time in connection with their potency to mobilize a non-conjugative IncQ-plasmid pVZ361 from IncQ-group (ori RSF1010, KmR, SuR . 11.4 kb) for conjugated transfer to aplasmid strains Agrobacterium tumefaciens and Pseudomonas putida at high frequencies.

Zhongguo Zhong Yao Za Zhi, 1999 Mar, 24(3), 140 - 2, 190
{Factors affecting Agrobacterium-mediated genetic transformation of Poncirus trifoliata (L.) Raf.}; He H et al.; OBJECTIVE: Establishing an effective system for the Agrobacterium-mediated genetic transformation of Poncirus trifoliata, so as to lay a foundation for improving breeds and introducing foreign genes . METHOD: The explants used for the genetic transformation were the epicotyls from Poncirus trifoliata . The Agrobacterium tumefaciens strain was EHA101, containing vector plasmid PGA482GG . The GUS gene and NPT II gene were introduced into the transformation plasmid . RESULTS: 20-day epicotyls were suitable for transformation . 2-day precultivation enhanced shoot regeneration . Shoot regeneration frequency was high when the cocultivation lasted 2-3 days . With the presence of acetosyringone during cocultivation, the efficiency of transformation could be greatly enhanced, and the frequency of shoot regeneration rose as high as 27.5% . After the explants were cocultivated with Agrobacterium, 2-day delayed selection could stimulate shoot regeneration . CONCLUSION: An effective genetic transformation system has been established for Poncirus trifoliata on the basis of factors affecting Agrobacterium-mediated genetic transformation.

J Ethnopharmacol, 2002 Oct, 82(2-3), 117 - 25
Biological activity of common mullein, a medicinal plant; Turker AU et al.; Common Mullein (Verbascum thapsus L., Scrophulariaceae) is a medicinal plant that has been used for the treatment of inflammatory diseases, asthma, spasmodic coughs, diarrhea and other pulmonary problems . The objective of this study was to assess the biological activity of Common Mullein extracts and commercial Mullein products using selected bench top bioassays, including antibacterial, antitumor, and two toxicity assays--brine shrimp and radish seed . Extracts were prepared in water, ethanol and methanol . Antibacterial activity (especially the water extract) was observed with Klebsiella pneumonia, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli . Agrobacterium tumefaciens-induced tumors in potato disc tissue were inhibited by all extracts . Toxicity to Brine Shrimp and to radish seed germination and growth was observed at higher concentrations of the extracts.

Plant Cell, 1997 Dec, 9(12), 2135 - 2142
Transfer and Integration of T-DNA without Cell Injury in the Host Plant; Escudero J et al.; Agrobacterium colonizes plant cells via a gene transfer mechanism that results in plant tumorigenesis . Virulence (vir) genes are transcriptionally activated in the bacteria by plant metabolites released from the wound site . Hence, it is believed that agrobacteria use injuries to facilitate their entrance into the host plant and that the wounded state is required for plant cell competence for Agrobacterium-mediated gene delivery . However, our experiments using vir gene-activated bacteria sprayed onto tobacco plantlets demonstrated that cells in unwounded plants could also be efficiently transformed . The condition of the plant cells was monitored using {beta}-glucuronidase under the control of a wound-inducible promoter . Infection of leaf tissue is light dependent, and it is drastically reduced when abscisic acid is exogenously applied to the plant . Under these experimental conditions, stomatal opening seems to be used by Agrobacterium to circumvent the physical barrier of the cuticle . These results thus show that the proposed cellular responses evoked by wounding in higher plants are not essential for Agrobacterium-mediated transformation.

Biochem Biophys Res Commun, 2002 Sep 20, 297(2), 282 - 7
Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues; Roger Chien HC et al.; N-Carbamoyl D-amino acid amidohydrolase (D-NCAase) that catalyzes the stereospecific hydrolysis of N-carbamoyl D-amino acids to their corresponding D-amino acids is valuable in pharmaceutical industry . Agrobacterium radiobacter D-NCAase is sensitive to oxidative damage by hydrogen peroxide . To investigate the role of methionine residues in oxidative inactivation, each of the nine methionine residues in A . radiobacter D-NCAase was substituted with leucine, respectively, by site-directed mutagenesis . Except for two mutants (Met5Leu and Met31Leu) with similar activities, seven mutants (Met73Leu, Met167Leu/Met169Leu, Met184Leu, Met220Leu, Met239Leu, Met244Leu, and Met239Leu/Met244Leu) were found to have reduced activities . In the presence of H(2)O(2), three mutants (Met239Leu, Met244Leu, and Met239Leu/Met244Leu) with substitution of highly solvent-accessible methionines by leucines retained their activities . The other mutants were also considerably resistant to chemical oxidation than was the wild-type enzyme . Thus, substitution of solvent-accessible methionine residues with leucine to enhance oxidative stability of D-NCAase is practical but might be with compromised activity.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 956 - 62
The rolB-like part of the Agrobacterium rhizogenes orf8 gene inhibits sucrose export in tobacco; Umber M et al.; Many Agrobacterium T-DNA genes belong to the highly diverse rolB family . The mode of action of most of these genes is still unknown . rolB-like sequences also are present at the 5' ends of the T-DNA-located iaaM genes and the iaaM homolog orf8, whereas iaaM genes from Pseudomonas and Erwinia spp . lack such sequences . iaaM genes encode tryptophan monooxygenases; these enzymes convert tryptophan into indole-3-acetamide, a precursor of indole-3-acetic acid . Tobacco plants expressing the rolB-like part of the A4 orf8 gene (2x35S-A4-Norf8 plants) accumulate glucose, fructose, sucrose, and starch and resemble sucrose transporter (NtSUT1) antisense plants . Different lines of evidence indicate that 2x35S-A4-Norf8 plants export less sucrose from source leaves . Glucose, fructose, sucrose, and starch accumulate in source leaves during sink-source transition, whereas sink tissues like petioles and midveins contain lower levels than normal . Petiole exudation experiments demonstrate a significant decrease in export of label after 14C-sucrose infiltration and after 14CO2 labeling . Grafting of stunted homozygous 2x35S-A4-Norf8 plants onto wild-type rootstocks restores growth, indicating that unloading is not affected . Growth of 2x35S-A4-Norf8 seedlings is inhibited on naphthalene acetic acid-containing media, suggesting a link between sucrose transport and auxin sensitivity.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 866 - 74
Engineering bacterial competitiveness and persistence in the phytosphere; Savka MA et al.; Several tactics exist to improve the survival of an introduced microorganism of interest in the plant environment . One, derived from studies on the Agrobacterium-plant interaction and the role of opines in this interaction, proposes to promote growth of the inoculant in the plant environment via the establishment of a bias in the rhizosphere . It is supported by the occurrence of natural biases, such as those generated by opine-like molecules, by calestegins, or by mimosine . Opine-mediated biases have allowed several investigators to favor the growth of opine-degrading bacteria or communities under sterile or axenic environments or in microcosms mimicking near field conditions . Another way to favor a given microbe consists in impeding growth of competing microorganisms . Experiments performed using detergent or bacteriostatic agents as amendments under field or near field conditions yielded promising results . Research perspectives for engineering plant-microbe interactions also include specific engineering of predation and strategies designed to interfere with some of the signals perceived by the microbes, provided these signals control the expression of functions central to microbial fitness . In this respect, quorum-sensing signal molecules, such as N-acyl-homoserine lactones, may be valuable targets for the development of biocontrol agents and procedures.

Plant Physiol, 1994 Nov, 106(3), 1179 - 1185
Metabolic Control of Anaerobic Glycolysis (Overexpression of Lactate Dehydrogenase in Transgenic Tomato Roots Supports the Davies-Roberts Hypothesis and Points to a Critical Role for Lactate Secretion; Rivoal J et al.; Roots of all plants examined so far have the potential for both ethanol and lactate fermentation . A short burst of lactate fermentation usually occurs when plant tissues are transferred from normoxic to anoxic conditions . According to the Davies-Roberts hypothesis, the consequent pH drop both initiates ethanol fermentation and blocks further production of lactate by inhibiting lactate dehydrogenase (LDH) . However, the role of LDH in this pH control mechanism is still a matter of debate . To perturb the control system in a defined way, a barley LDH cDNA under the control of the cauliflower mosaic virus 35S promoter was introduced into tomato (Lycopersicon esculentum Mill . cv VFMT) using Agrobacterium rhizogenes . The transgenic root clones expressed up to 50 times the LDH activity of controls . The fermentative metabolism of these clones was compared using roots grown previously in normoxic conditions or roots given a 3-d hypoxic pretreatment . During the transition from normoxia to anoxia, lactate accumulation was no faster and no more extensive in transgenic roots than in controls . Similarly, during prolonged anoxia the flux of 14C from {U-14C} glucose to lactate and ethanol was not modified by the expression of the transgene . However, in both transgenic and control roots, hypoxic pretreatment increased the flux to lactate and promoted lactate export to the medium . These results show that LDH has a very low flux control coefficient for lactate fermentation, consistent with the Davies-Roberts hypothesis . Moreover, they suggest that lactate secretion exerts major control over long-term lactate glycolysis in vivo.

Plant Physiol, 1994 Jun, 105(2), 563 - 569
The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration; Delbarre A et al.; Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J . Estruch, J . Schell, A . Spena {1991} EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone) . Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin . Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels . Accumulation of free 1-{3H}-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-{beta}-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts . Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level . Essentially similar results were obtained with indoleacetic acid . Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.

Plant Physiol, 1994 Jun, 105(2), 491 - 496
Complementation of the Tomato anthocyanin without (aw) Mutant Using the Dihydroflavonol 4-Reductase Gene; Goldsbrough A et al.; We isolated the dihydroflavonol 4-reductase (DFR) gene from tomato (Lycopersicon esculentum) using a previously characterized cDNA as probe . Earlier studies had indicated that the DFR gene is present in tomato as a single gene located on chromosome 2 near the locus anthocyanin without (aw) . Mutant alleles of the aw locus result in the complete absence of anthocyanin pigmentation throughout all stages of plant development . When the genomic DFR clone was introduced by Agrobacterium-mediated transformation into plants bearing the aw mutation, primary transgenic seedlings accumulated anthocyanins that could be observed while the plants were still in tissue culture and which continued to be observed as the plants matured . Progeny of self pollinated and backcrossed transgenic plants segregated for anthocyanin pigmentation, and Southern hybridization analyses indicated the presence of the DFR transgene exclusively in those plants with pigmentation . These data indicate that the aw locus likely corresponds to the structural gene for DFR and that DFR can be used as a visual, nondestructive, plant-derived marker gene for tomato.

Plant Physiol, 1994 May, 105(1), 81 - 88
Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.); Khan M et al.; We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery . Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis . Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer . DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, {beta}-glucuronidase (GUS), neomycin phosphotransferase, and an {alpha}-amylase inhibitor were used to transform subterranean clover . Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin . Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed . Southern analysis revealed the integration of T-DNA into the subterranean clover genome . The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses . Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions . In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.

Plant Physiol, 1993 Jun, 102(2), 363 - 371
Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rhizogenes TL-DNA; Nilsson O et al.; Transgenic tobacco (Nicotiana tabacum L . cv Wisconsin 38) plants expressing the Agrobacterium rhizogenes rolC gene under the control of the cauliflower mosaic virus 35S RNA promoter were constructed . These plants displayed several morphological alterations reminiscent of changes in indole-3-acetic acid (IAA), cytokinin, and gibberellin (GA) content . However, investigations showed that neither the IAA pool size nor its rate of turnover were altered significantly in the rolC plants . The biggest difference between rolC and wild-type plants was in the concentrations of the cytokinin, isopentenyladenosine (iPA) and the gibberellin GA19 . Radio-immunoassay and liquid chromatography-mass spectrometry measurements revealed a drastic reduction in rolC plants of iPA as well as in several other cytokinins tested, suggesting a possible reduction in the synthesis rate of cytokinins . Furthermore, gas chromatography-mass spectrometry quantifications of GA19 showed a 5- to 6-fold increase in rolC plants compared with wild-type plants, indicating a reduced activity of the GA19 oxidase, a proposed regulatory step in the gibberellin biosynthesis . Thus, we conclude that RolC activity in transgenic plants leads to major alterations in the metabolism of cytokinins and gibberellins.

Plant Physiol, 1993 May, 102(1), 287 - 293
The Anti-nptII Gene (A Potential Negative Selectable Marker for Plants); Xiang C et al.; An efficient negative selection procedure is crucial to the isolation of rare homologous recombinants in gene targeting . Although gene targeting is a common practice in lower eukaryotes and is becoming routine in mammals, its application to plants has not been achieved . In this report, we have evaluated an antisense construct against the neomycin phosphotransferase gene (nptII) as a negative selectable marker . The anti-nptII gene construct was able to suppress nptII expression both transiently and in transformed tobacco (Nicotiana tabacum) calli . A construct was made which includes both a hygromycin-resistance gene and the sense plus antisense genes for neomycin phosphotransferase . Hygromy-cin-resistant calli were obtained after Agrobacterium-mediated transformation . Subsequently, hygromycin-resistant calli were tested for kanamycin sensitivity . The growth on kanamycin medium of calli harboring both the sense and antisense gene constructs was retarded, whereas that of control calli transformed with only the sense nptII gene was not inhibited . Southern blot analysis confirmed the presence of both nptII and anti-nptII genes . Northern blot analyses revealed that antisense transcripts of the nptII gene were made and that the level of sense transcripts was greatly reduced in transgenic calli . These results suggest that the anti-nptII gene could potentially be used as a negative selectable marker for gene targeting in plants.

Plant Physiol, 1993 Mar, 101(3), 751 - 757
Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.); Schroeder HE et al.; A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds . A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures . Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin . Transgenic peas were raised in the glasshouse to produce flowers and viable seeds . The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern . Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses . Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.

Plant Physiol, 1993 Jan, 101(1), 313 - 320
Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants, and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry; Sitbon F et al.; Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA . At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X) . The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates . Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose . IAAsp and IAGlu were also identified as endogenous substances in wild-type plants . In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 {plus or minus} 7%, SD) than in old leaves (36 {plus or minus} 8%), whereas there was no difference between young (73 {plus or minus} 8%) and old internodes (70 {plus or minus} 9%) . In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 {plus or minus} 10%) for both leaves and internodes, independent of the total IAA level or tissue age . These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA . The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.

Plant Physiol, 1993 Jan, 101(1), 201 - 208
Abolition of an Inducible Highly Anionic Peroxidase Activity in Transgenic Tomato; Sherf BA et al.; Locally induced expression of a highly anionic peroxidase has previously been correlated temporally and spatially with suberization of tissues responding to pathogen assault, wounding, or exogenously applied abscisic acid or fungal elicitors . DNA sequences corresponding to the 5{prime} regions of two tomato (Lycopersicon esculentum) genes encoding homologous anionic peroxidases were fused, inserted into a pTi-based plasmid designed to express a composite antisense transcript, and introduced into tomato via Agrobacterium-mediated transformation . RNA gel-blot analyses showed high expression of the antisense transcript in most transgenic plants and no detectable induction of native anionic peroxidase transcripts in wounded or abscisic acid or pathogen-treated tissues . Plants and fruits expressing the antisense transcript appeared normal in all respects . Electrophoretic analysis of anionic proteins from selected transgenic plants showed no detectable anionic peroxidase protein or activity . Depolymerization of polymeric material from the wound periderm of transgenic tomato fruits and analysis of the aliphatic products by gas-liquid chromatography/mass spectrometry showed that the content and composition of C16/C18 {omega}-hydroxy and dicarboxylic acids, characteristic of suberin, were not affected by the absence of the anionic peroxidase . Autofluorescence generated from cell wall phenolics at the wound lesion was also not affected by the absence of the highly anionic peroxidase.

Plant Physiol, 1995 Dec, 109(4), 1179 - 1189
Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium tumefaciens T-DNA Indoleacetic Acid-Biosynthetic Genes; Tuominen H et al.; A key regulator of cambial growth is the plant hormone indoleacetic acid (IAA) . Here we report on altered wood characteristics and growth patterns in transgenic hybrid aspen (Populus tremula L . x Populus tremuloides Michx.) expressing Agrobacterium tumefaciens T-DNA IAA-biosynthetic iaaM and iaaH genes . Eighteen lines simultaneously expressing both genes were regenerated . Of these, four lines, verified to be transgenic by northern blot analysis, were selected and raised under controlled growth conditions . All four lines were affected in their growth patterns, including alterations in height and stem diameter growth, internode elongation, leaf enlargement, and degree of apical dominance . Two transgenic lines, showing the most distinct phenotypic deviation from the wild type, were characterized in more detail for free and conjugated IAA levels and for wood characteristics . Both lines showed an altered IAA balance, particularly in mature leaves and roots where IAA levels were elevated . They also exhibited changes in wood anatomy, most notably a reduction in vessel size, an increase in vessel density, and changes in ray development . Thus, the recent development of techniques for gene transfer to forest trees enabled us to investigate the influence of an altered IAA balance on xylem development in an intact experimental system . In addition, the results demonstrate the possibility of manipulating wood properties in a forest tree through controlled changes of IAA concentration and distribution.

Plant Physiol, 1995 Nov, 109(3), 761 - 770
Expression of the Hevea brasiliensis (H.B.K.) Mull . Arg . 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction; Schaller H et al.; A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull . Arg . (M.-L . Chye, C.-T . Tan, N.-H . Chua {1992} Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L . cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis . Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern . Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene . In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues . Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies . These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.

Plant Physiol, 1995 Sep, 109(1), 63 - 71
Increased Putrescine Biosynthesis through Transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis; Bastola DR et al.; Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3{prime}-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter . A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin . Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells . Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells . Spermidine, however, remained unaffected . Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid . These cells acquired tolerance to {alpha}-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells.

Plant Physiol, 1995 Apr, 107(4), 1233 - 1239
Bean {alpha}-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.); Schroeder HE et al.; Bruchid larvae cause major losses of grain legume crops through-out the world . Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds . Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field . We transferred the cDNA encoding the {alpha}-amylase inhibitor ({alpha}-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation . Expression was driven by the promoter of phytohemagglutinin, another bean seed protein . The {alpha}-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and {alpha}-AI accumulated in the seeds up to 3% of soluble protein . This level is somewhat higher than that normally found in beans, which contain 1 to 2% {alpha}-AI . In the T5 seed generation the development of pea weevil larvae was blocked at an early stage . Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants . These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach . Although {alpha}-AI also inhibits human {alpha}-amylase, cooked peas should not have a negative impact on human energy metabolism.

Plant Physiol, 1995 Mar, 107(3), 807 - 814
Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta; Jacobs JJ et al.; Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts . Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T . erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type . The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants . Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta . These experiments also showed that one mutant is deficient in demethylation of the monothiophene.

Plant Physiol, 1995 Feb, 107(2), 469 - 477
Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L . cv Desiree) Plants); Hofgen R et al.; Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides . To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum tuberosum L . cv Desiree), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35S promoter, and created transgenic potato plants through Agrobacterium tumefaciens-mediated gene transfer . Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides . Antisense gene expression decreased the steady-state level of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85% . This reduction was sufficient to generate plants almost inviable without amino acid supplementation . In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions . Thus, antisense inhibition of ALS generated an in vivo model of herbicide action . Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.

Plant Physiol, 2002 Sep, 130(1), 164 - 78
Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots; Park SW et al.; Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step . In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana) . The protein was purified by anion- and cation-exchange chromatography . PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8 . Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity . Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells . PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction . Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi . In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi . We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

Plant Physiol, 1996 Oct, 112(2), 493 - 502
Expression of the Agrobacterium rhizogenes rolC Gene in a Deciduous Forest Tree Alters Growth and Development and Leads to Stem Fasciation; Nilsson O et al.; We have altered the growth and development of a deciduous forest tree by transforming hybrid aspen (Populus tremula x Populus tremuloides) with the Agrobacterium rhizogenes rolC gene expressed under the strong cauliflower mosaic virus 35S promoter . We demonstrate that the genetically manipulated perennial plants, after a period of dormancy, maintain the induced phenotypical changes during the second growing period . Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several gibberellins on one transgenic line correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism . We also show that the presence of the RolC protein increases the levels of the free cytokinins, but not by a process involving hydrolysis of the inactive cytokinin conjugates.

Plant Physiol, 1996 Sep, 112(1), 115 - 120
Insect Control and Dosage Effects in Transgenic Canola Containing a Synthetic Bacillus thuringiensis cryIAc Gene; Stewart Jr CN et al.; Zygotic hypocotyls of canola (Brassica napus L.) cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation . Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes . A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein . The Brassica specialists, the diamondback month (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni Hubner), were completely controlled by low-, medium-, and high-expressing lines . Whereas control of the generalist lepidopteran, the corn earworm (Helicoverpa zea Boddie), was nearly complete, the other generalist caterpillar tested, the beet armyworm (Spodoptera exigua Hubner), showed a dose response that had a negative association between defoliation and cry expression . These plants were produced as models for an ecological research assessment of the risk involved in the field release of naturalized transgenic plants harboring a gene (Bt) that confers higher relative fitness under herbivore-feeding pressure.

Plant Physiol, 1996 May, 111(1), 259 - 267
Attenuation of the Phenotype Caused by the Root-Inducing, Left-Hand, Transferred DNA and Its rolA Gene (Correlations with Changes in Polyamine Metabolism and DNA Methylation); Martin-Tanguy J et al.; We present four examples of attenuation of the transformed phenotype caused by the root-inducing, left-hand, transferred DNA from Agrobacterium rhizogenes in tobacco (Nicotiana tabacum) . The first was associated with a genetic variable (homozygosity for the T-DNA), and the second was induced at the physiological level by putrescine and tyramine, suggesting that the transformed phenotype depends on defective polyamine metabolism . Physiological attenuation is further illustrated in the third example, in which the inhibition of flowering caused by P35S-rolA, a gene from the root-inducing, left-hand, transferred DNA driven by a strong viral promoter, was attenuated by grafting the transformed shoot onto non-transformed rootstock that had been induced to flower . Infertility in the resulting flowers was corrected by a mixture of putrescine and tyramine, indicating that P35S-rolA inhibited flowering through interference with polyamine conjugation and that tyramine was essential to fertility . A fourth example of attenuation of the transformed phenotype occurred in lateral branches of plants expressing rolA under the control of its native promoter . In these branches, reduction in the accumulation of rolA transcripts was correlated with the methylation of a site 3{prime} to the rolA coding sequence; thus, the transformed plant seems capable of recognizing and repressing a gene that interferes with flowering.

Plant Physiol, 1996 Feb, 110(2), 431 - 438
Transfer RNA Is the Source of Extracellular Isopentenyladenine in a Ti-Plasmidless Strain of Agrobacterium tumefaciens; Gray J et al.; Even in the absence of the classical Ti plasmid-encoded cytokinin biosynthetic genes ipt and tzs, Agrobacterium tumefaciens strains still release significant amounts of the cytokinin isopentenyladenine (iP) into the culture medium (R.W . Kaiss-Chapman and R.O . Morris {1977} Biochem Biophys Res Commun 76: 453-459) . A potential source of the iP is isopentenylated transfer RNA (tRNA), which, in turn, is synthesized by the activity of tRNA:isopentenyltransferase encoded by the bacterial miaA gene . To determine whether secreted iP had its origin in isopentenylated tRNA, a miaA- deletion/insertion mutant was prepared and reconstructed in Agrobacterium tumefaciens in vivo . The mutant no longer possessed tRNA:isopentenylation activity and no longer released iP into the extracellular medium . Transfer RNA therefore makes a small but significant contribution to the total amount of cytokinin normally secreted by Agrobacterium strains . tRNA-mediated synthesis may also account for cytokinin production by other plant-associated bacteria, such as Rhizobia, that have been reported to secrete similarly low levels of nonhydroxylated cytokinins.

Plant Physiol, 1997 Nov, 115(3), 971 - 980
Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens; Cheng M et al.; A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants . The explants were inoculated with a disarmed A . tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the {beta}-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene . Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium . The inoculated immature embryos or embryogenic calli were selected on G418-containing media . Transgenic plants were regenerated from all three types of explants . The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months . So far, more than 100 transgenic events have been produced . Almost all transformants were morphologically normal . Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis . One to five copies of the transgene were integrated into the wheat genome without rearrangement . Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events . Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.

Plant Physiol, 1997 Oct, 115(2), 577 - 585
A Radial Concentration Gradient of Indole-3-Acetic Acid Is Related to Secondary Xylem Development in Hybrid Aspen; Tuominen H et al.; The radial distribution pattern of indole-3-acetic acid (IAA) was determined across the developing tissues of the cambial region in the stem of hybrid aspen (Populus tremula L . x Populus tremuloides Michx) . IAA content was measured in consecutive tangential cryo-sections using a microscale mass spectrometry technique . Analysis was performed with wild-type and transgenic trees with an ectopic expression of Agrobacterium tumefaciens IAA-biosynthetic genes . In all tested trees IAA was distributed as a steep concentration gradient across the developing tissues of the cambial region . The peak level of IAA was within the cambial zone, where cell division takes place . Low levels were reached in the region where secondary wall formation was initiated . The transgenic trees displayed a lower peak level and a wider radial gradient of IAA compared with the wild type . This alteration was related to a lower rate of cambial cell division and a longer duration of xylem cell expansion in the transgenic trees, resulting in a decreased xylem production and a larger fiber lumen area . The results indicate that IAA has a role in regulating not only the rate of physiological processes such as cell division, but also the duration of developmental processes such as xylem fiber expansion, suggesting that IAA functions as a morphogen, conveying positional information during xylem development.

J Microbiol Methods, 2002 Nov, 51(3), 387 - 92
An internal control for the diagnosis of crown gall by PCR; Cubero J et al.; The addition of an internal control (IC) at the appropriate concentration enables recognize of false negatives in the detection of Agrobacterium tumefaciens and improves the reliability of PCR for crown gall diagnosis . Co-amplification of the IC and target sequence from A . tumefaciens ensures the attainment of at least one amplification product in every PCR reaction if the DNA extracted is of high enough quality to be amplified .

FEBS Lett, 2002 Sep 11, 527(1-3), 315 - 8
The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5'-phosphate from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate and AMP; Krall L et al.; The plant pathogen Agrobacterium tumefaciens produces cytokinins upon induction of the virulence genes by secondary metabolites from wounded plants, and these hormones are believed to stimulate the infection process . To study the biosynthetic pathway, the tzs gene, encoding the Tzs (trans-zeatin-synthesizing) protein from A . tumefaciens, was cloned and the protein was overproduced and purified . Analysis of its reactivity with radioactively labeled substrate demonstrated conversion of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate, a product of the deoxyxylulose phosphate pathway, with AMP to zeatin riboside 5'-phosphate . This suggests that A . tumefaciens uses an alternative pathway of cytokinin biosynthesis, which had previously been hypothesized to operate in plants.

Genes Genet Syst, 2002 Jun, 77(3), 137 - 46
Gene cluster for ferric iron uptake in Agrobacterium tumefaciens MAFF301001; Sonoda H et al.; Agrobacterium tumefaciens harboring a Ti plasmid causes crown gall disease in dicot plants by transferring its T-DNA into plant chromosomes . Iron acquisition plays an important role for pathogenicity in animal pathogens and several phytopathogens and for growth in the rhizosphere and on plant surfaces . Under iron-limiting condition, bacteria produce various iron-chelating agents called siderophores . Agrobacterium strains have the diversity in producing siderophores and a certain strain produces a typical catechol-type siderophore, called agrobactin, although its biosynthesis genes have not been analyzed yet . Here we describe the cloning and characterization of a functional gene cluster involved in ferric iron uptake in A . tumefaciens strain MAFF301001 . Four complete open reading frames (ORFs) were found in 5-kb region of a genomic library clone 1A3 . We named these genes agb, after agrobactin . agbC, agbE, agbB and agbA genes were identified in this order, and narrow intergenic spaces suggested that these genes constitute an operon . Predicted agb gene products and their phylogenetic analysis showed sequence similarity with enzymes which are involved in ferric iron uptake in other bacteria . Southern hybridization analysis clearly indicated the location of agb genes on the linear chromosome in strain MAFF301001 but the complete lack in another A . tumefaciens strain C58 . Mutation analysis of agbB revealed that it is essential for growth and production of catechol compounds in iron-limiting medium.

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12369 - 74 Epub 2002 Sep 06.
A global pH sensor: Agrobacterium sensor protein ChvG regulates acid-inducible genes on its two chromosomes and Ti plasmid; Li L et al.; A sensor protein ChvG is part of a chromosomally encoded two-component regulatory system ChvG/ChvI that is important for the virulence of Agrobacterium tumefaciens . However, it is not clear what genes ChvG regulates or what signal(s) it senses . In this communication, we demonstrate that ChvG is involved in the regulation of acid-inducible genes, including aopB and katA, residing on the circular and linear chromosomes, respectively, and the tumor-inducing (Ti)-plasmid-harbored vir genes, virB and virE . ChvG was absolutely required for the expression of aopB and very important for the expression of virB and virE . However, it was responsible only for the responsiveness of katA and, to a limited extent, the vir genes to acidic pH . ChvG appears to play a role in katA expression by repressing katA at neutral pH . ChvG had no effect on the expression of two genes that were not acid-inducible . Because ChvG regulates unlinked acid-inducible genes encoding different functions in different ways, we hypothesize that ChvG is a global sensor protein that can directly or indirectly sense extracellular acidity . We also analyzed the re-sequenced chvG and found that ChvG is more homologous to its Sinorhizobium meliloti counterpart ExoS than was previously thought . Full-length ChvG is conserved in members of the alpha-proteobacteria, whereas only the C-terminal kinase domain is conserved in other bacteria . Sensing acidity appears to enable Agrobacterium to coordinate its coping with the environment of wounded plants to cause tumors.

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12375 - 80 Epub 2002 Sep 06.
The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae; Guzman-Verri C et al.; The Brucella BvrR/BvrS two-component regulatory system is homologous to the ChvI/ChvG systems of Sinorhizobium meliloti and Agrobacterium tumefaciens necessary for endosymbiosis and pathogenicity in plants . BvrR/BvrS controls cell invasion and intracellular survival . Probing the surface of bvrR and bvrS transposon mutants with monoclonal antibodies showed all described major outer membrane proteins (Omps) but Omp25, a protein known to be involved in Brucella virulence . Absence of Omp25 expression was confirmed by two-dimensional electrophoresis of envelope fractions and by gene reporter studies . The electrophoretic analysis also revealed reduction or absence in the mutants of a second set of protein spots that by matrix-assisted laser desorption ionization MS and peptide mass mapping were identified as a non-previously described Omp (Omp3b) . Because bvrR and bvrS mutants are also altered in cell-surface hydrophobicity, permeability, and sensitivity to surface-targeted bactericidal peptides, it is proposed that BvrR/BvrS controls cell envelope changes necessary to transit between extracellular and intracellular environments . A genomic search revealed that Omp25 (Omp3a) and Omp3b belong to a family of Omps of plant and animal cell-associated alpha-Proteobacteria, which includes Rhizobium leguminosarum RopB and A . tumefaciens AopB . Previous work has shown that RopB is not expressed in bacteroids, that AopB is involved in tumorigenesis, and that dysfunction of A . tumefaciens ChvI/ChvG alters surface properties . It is thus proposed that the BvrR/BvrS and Omp3 homologues of the cell-associated alpha-Proteobacteria play a role in bacterial surface control and host cell interactions.

Microbiology, 2002 Sep, 148(Pt 9), 2847 - 56
Molecular characterization of functional modules of plasmid pWKS1 of Paracoccus pantotrophus DSM 11072; Bartosik D et al.; The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined . The G+C content of the sequence of this plasmid was 62 mol% . Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44.1 and 37.8 kDa, respectively . Mutational analysis showed that ORF2 was crucial for plasmid replication . The translational product of ORF2 shared local homologies with replication proteins of several theta-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts . An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV) . ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria . A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli . MOB modules of pWKS1 and pBBR1 were functionally complementary to each other . Hybridization analysis revealed that only plasmid pSOV1 (6.5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1 . Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E . coli.

Transgenic Res, 2002 Aug, 11(4), 411 - 23
Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas); Khanna HK et al.; Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic or Agrobacterium-mediated approaches . A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used . Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system . Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher (approximately 9%) for Agrobacterium . An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes . Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium . In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert . Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India . Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays . However, there was an occasional white head even among such plants when assayed at the reproductive stage . Results are discussed in the light of resistance management strategies for deployment of Bt-rice.

Transgenic Res, 2002 Aug, 11(4), 381 - 96
High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system; Miller M et al.; For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize . We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system . Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the beta-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment . Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment . A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment . For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0 plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment . A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0 events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1 generation . Of these R0 events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1 generation . A total of 64.4% of the R0 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1 progeny . A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0 plants displaying both transgenic phenotypes makes this method less efficient . Molecular analyses were then used to confirm that the observed segregation of R1 phenotypes were highly correlated to genetic segregation of the bar and GUS genes . A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.

Mol Microbiol, 2002 Sep, 45(5), 1325 - 35
Agrobacterium type IV secretion is a two-step process in which export substrates associate with the virulence protein VirJ in the periplasm; Pantoja M et al.; Type IV secretion systems are virulence determinants in many bacteria and share extensive homology with many conjugal transfer systems . Although type IV systems and their homologues have been studied widely, the mechanism by which substrates are secreted remains unclear . In Agrobacterium, we show that type IV secretion substrates that lack signal peptides form a soluble complex in the periplasm with the virulence protein VirJ . Additionally, these proteins co-precipitate with constituents of the type IV transporter: the VirB pilus and the VirD4 protein . Our findings suggest that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ, and suggest a two-step process for type IV secretion in Agrobacterium . Our analyses of protein-protein interactions in a variety of mutant backgrounds indicate that substrates are probably secreted independently of one another.

Physiol Plant, 2002 Sep, 116(1), 79 - 86
In vitro regeneration and genetic transformation of the berberine-producing plant, Thalictrum flavum ssp . glaucum; Samanani N et al.; Protocols have been developed for the in vitro regeneration and Agrobacterium-mediated genetic transformation of meadow rue, Thalictrum flavum ssp . glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days . The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l-1 sucrose, 2 mg l-1 kinetin, and 3 g l-1 Gelrite) containing 25 mg l-1 hygromycin B as the selection agent and 250 mg l-1 timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A . tumefaciens strains A281 and C58 produced transgenic T . flavum callus tissues . A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants . Transgenic callus was subcultured on the shoot induction medium every 2 weeks . After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l-1 hygromycin B, 250 mg l-1 timentin, and 3 g l-1 Gelrite) . Detection of the beta-glucuronidase (GUS) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants . The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.

Eur J Biochem, 2002 Sep, 269(17), 4226 - 37
Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin; Kragol G et al.; Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein . Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge . A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment . While fluorescein-labeled versions of the native peptides entered E . coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells . These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells . By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule . Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated . Mutational analysis on a synthetic E . coli DnaK fragment identified a possible binding surface for pyrrhocoricin.

Plant Cell Physiol, 2002 Aug, 43(8), 939 - 50
Resistance of transgenic tobacco seedlings expressing the Agrobacterium tumefaciens C58-6b gene, to growth-inhibitory levels of cytokinin is associated with elevated IAA levels and activation of phenylpropanoid metabolism; Galis I et al.; We previously reported that the Agrobacterium tumefaciens C58-6b gene confers resistance to growth-inhibitory levels of exogenously applied N(6)-benzyladenine (BA, cytokinin) in transgenic tobacco (Nicotiana tabacum) seedlings . Here, we found that intracellular levels of indoleacetic acid (IAA, auxin) increased in transgenics but declined in wild-type seedlings upon BA treatment . Since exogenously supplied 1-naphthalene acetic acid (NAA), a stable synthetic auxin, counteracted the growth inhibition of wild-type seedlings by BA, we suggest that BA-induced growth inhibition in wild-type seedlings occurs, at least in part, as a result of intracellular IAA deficiency . Further HPLC analysis of cell extracts from BA-treated seedlings revealed that a fluorescent compound, later identified as the phenylpropanoid, scopolin, and the major phenolic compound, chlorogenic acid, accumulated earlier in transgenics than in wild-type seedlings . Gene transcripts encoding phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase, which are responsible for the early steps of phenylpropanoid biosynthesis, accumulated earlier and to higher levels in transgenics than in wild-type seedlings as determined by Northern hybridization analysis, thus accounting for the early accumulation of scopolin and chlorogenic acid in transgenics . As some phenolic compounds, including chlorogenic acid and scopoletin (aglycon of scopolin) are suggested to inhibit IAA catabolism, we further propose that C58-6b gene expression protects IAA from degradation by inducing the early phenylpropanoid pathway.

EMBO J, 2002 Sep 2, 21(17), 4393 - 401
The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA; Vannini A et al.; The quorum sensing system allows bacteria to sense their cell density and initiate an altered pattern of gene expression after a sufficient quorum of cells has accumulated . In Agrobacterium tumefaciens, quorum sensing controls conjugal transfer of the tumour- inducing plasmid, responsible for plant crown gall disease . The core components of this system are the transcriptional regulator TraR and its inducing ligand N-(3-oxo-octanoyl)-L-homoserine lactone . This complex binds DNA and activates gene expression . We have determined the crystal structure of TraR in complex with its autoinducer and target DNA (PDB code 1h0m) . The protein is dimeric, with each monomer composed of an N-terminal domain, which binds the ligand in an enclosed cavity far from the dimerization region, and a C-terminal domain, which binds DNA via a helix-turn-helix motif . The structure reveals an asymmetric homodimer, with one monomer longer than the other . The N-terminal domain resembles GAF/PAS domains, normally fused to catalytic signalling domains . In TraR, the gene fusion is between a GAF/PAS domain and a DNA-binding domain, resulting in a specific transcriptional regulator involved in quorum sensing.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 323 - 6
{Callus induction and regeneration from mature seeds of indica rice minghui 63 and anti-fungal assay of transgenic rice plants}; Wang LJ et al.; A large number of callus from mature seeds of indica rice minghui 63 were obtained through pre-induction on medium with 2 mg/L 2,4-D but without inorganic and organic components for 9 days . Trichosanthin gene was transferred into indica rice minghui 63 by using agrobacterium with the help of bombardment and the transgenic plants were obtained by inducing regeneration . Southern and Western blot analysis showed that the trichosanthin gene had been transferred into genome of minghui 63 and expressed in rice plants . The anti-fungal assay suggested that transgenic rice plants enhanced resistance to infection of Pyricularia oryzae.

Proc Natl Acad Sci U S A, 2002 Sep 3, 99(18), 11628 - 33 Epub 2002 Aug 19.
Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site; Lamparter T et al.; Phytochromes are photochromic photoreceptors with a bilin chromophore that are found in plants and bacteria . The soil bacterium Agrobacterium tumefaciens contains two genes that code for phytochrome-homologous proteins, termed Agrobacterium phytochrome 1 and 2 (Agp1 and Agp2) . To analyze its biochemical and spectral properties, Agp1 was purified from the clone of an E . coli overexpressor . The protein was assembled with the chromophores phycocyanobilin and biliverdin, which is the putative natural chromophore, to photoactive holoprotein species . Like other bacterial phytochromes, Agp1 acts as light-regulated His kinase . The biliverdin adduct of Agp1 represents a previously uncharacterized type of phytochrome photoreceptor, because photoreversion from the far-red absorbing form to the red-absorbing form is very inefficient, a feature that is combined with a rapid dark reversion . Biliverdin bound covalently to the protein; blocking experiments and site-directed mutagenesis identified a Cys at position 20 as the binding site . This particular position is outside the region where plant and some cyanobacterial phytochromes attach their chromophore and thus represents a previously uncharacterized binding site . Sequence comparisons imply that the region around Cys-20 is a ring D binding motif in phytochromes.

Yi Chuan Xue Bao, 2002, 29(3), 260 - 5
{Factors affecting Agrobacterium tumefaciens-mediated transformation of wheat (Triticum aestivum L.)}; Wang YQ et al.; Immature embryos and embryo-derived calli from two cultivars of winter wheat (Triticum aestivum L.), BAU146 and BAU170, were transformed with three strains of Agrobacterium tumefaciens, AGL-1, EHA105 and LBA4404 harboring expression vector p3301 or pBTAaB . Both vectors contained bar gene and p3301 contained also gus gene with an intron . The highest explant survival rate and transformation efficiency was obtained when the bacterial cell density was OD600 1.0 with 1 h of infection incubation . Higher osmotic treatment of the explants before inoculation had a positive effect on transformation, while addition of acetosyringone showed ambiguous one, depending on the explant types and bacterium strains . The efficiencies of transformation and transgenic plant regeneration were varied greatly with the bacterium strain, receptor genotype, explant type and its age and physiological state . After optimizing these factors, a large number of PPT-resistant calli and some of PPT-resistant plants were obtained . The resistant plantlet tested and 50% to 60% of the resistant calli were GUS-positive . The integration of foreign DNA into the genome of transgenic plants (3 out 6) was further confirmed by PCR and Southern Blot analysis.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11405 - 10 Epub 2002 Aug 12.
Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens; Krall L et al.; The VirB/D4 type IV secretion system of Agrobacterium tumefaciens translocates virulence factors (VirE2, VirF, and the VirD2-T-DNA complex) to plant cells . The membrane-bound translocation machinery consists of 12 proteins (VirB1-11 and VirD4) required for substrate translocation . Protein-protein interactions in the membranes were analyzed after extraction with the mild detergent dodecyl-beta-d-maltoside followed by separation under native conditions . Incubation of the membranes with increasing concentrations of the detergent differentially extracted virulence proteins . Separation of the solubilized proteins by blue native electrophoresis revealed cofractionation between two classes of protein complexes containing VirB7 . The first class, consisting of major T-pilus component VirB2 and associated proteins VirB5 and VirB7, comigrated in the low molecular mass portion of the gel of about 100 kDa . The second class contains putative translocation complex core components VirB8, VirB9, and VirB10 in the high molecular mass portion of the gel larger than 232 kDa, as well as VirB7 . Solubilized proteins were characterized further by gel filtration chromatography . This procedure separated T-pilus-associated proteins VirB2, VirB5, and VirB7 in the low molecular mass range from the other components of the translocation machinery and the substrates VirE2 and VirD2 . Fractionation of VirB7-containing complexes (VirB7-VirB7 homodimers and VirB7-VirB9 heterodimers) suggested that they may link the T-pilus components to the core of the translocation machinery . Based on previously described VirB protein interactions and biochemical analysis of C58 wild type as well as of virB5 and virB6 deletion mutants, a model of T-pilus assembly in A . tumefaciens is suggested.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11493 - 500 Epub 2002 Aug 12.
Peptide linkage mapping of the Agrobacterium tumefaciens vir-encoded type IV secretion system reveals protein subassemblies; Ward DV et al.; Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells . Here, evidence for interactions among components of the Agrobacterium tumefaciens vir-encoded T4SS is presented . The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions . The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins . We established interactions between VirB11 (an inner membrane pore-forming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component) . We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A . tumefaciens envelope and including a potential pore protein . In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS) . VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components . The identification of these interactions suggests a model for assembly of the T4SS.

Curr Genet, 2002 Jun, 41(3), 183 - 8
T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker; Hanif M et al.; The T-DNA of Agrobacterium tumefaciens can be transferred to plants, yeasts, fungi and human cells . Using this system, dikaryotic mycelium of the ectomycorrhizal fungus Suillus bovinus was transformed with recombinant hygromycin B phosphotransferase (hph)and enhanced green fluorescent protein (EGFP) genes fused with a heterologous fungal promoter and CaMV35S terminator . Transformation resulted in hygromycin B-resistant clones, which were mitotically stable . Putative transformants were analysed for the presence of hph and EGFP genes by PCR and Southern analysis . The latter analysis proved both multiple- and single-copy integrations of the genes in the S . bovinus genome . A . tumeficiens transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for S . bovinus.

Mol Genet Genomics, 2002 Jul, 267(5), 577 - 86 Epub 2002 Jun 15.
Functional diversity and mutational analysis of Agrobacterium 6B oncoproteins; Helfer A et al.; Many Agrobacterium T-DNA genes belong to a diverse family of T-DNA genes, the rolB family . These genes cause various growth abnormalities but their modes of action remain largely unknown . So far, none of the RolB-like proteins has been subjected to mutational analysis . The RolB-like oncoprotein 6B, which induces tumours on species such as Nicotiana glauca and Kalanchoe tubiflora, was chosen to investigate the role of the most conserved amino acid residues within the RolB family . We first determined which of the natural 6B variants had the strongest oncogenic activity; to this end, six 6b coding sequences (A- 6b, AB- 6b, C- 6b, CG- 6b, S- 6b and T- 6b) were placed under the control of the strong constitutive 2x35S promoter and compared for tumour induction on N . glauca, N . tabacum and K . daigremontiana . Oncogenicity increased in the order C- 6b/CG- 6b, A- 6b/AB- 6b, and S- 6b/T- 6b . The most conserved amino acid residues in the strongly oncogenic T-6B protein were mutated and shown to be required for oncogenicity and accumulation of the T-6B protein in planta but not in bacteria . Hybrids between T-6B and the weakly oncogenic A-6B protein revealed an additional oncogenic determinant required for the formation of large tumours.

J Med Microbiol, 2002 Aug, 51(8), 661 - 71
Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation; Rosinha GM et al.; To identify antigens of Brucella spp . that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes . One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised . The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively . Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome . B . abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP) . To demonstrate the functional activity of Brucella GAPDH, E . coli gap mutants were transformed with a Brucella pMAL-gap construct . Genetic complementation was achieved and as a result E . coli mutants were able to grow on glucose or other carbon source medium . The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised . In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH . In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B . abortus S19 were able to produce gamma-interferon and tumour necrosis factor-a but not interleukin (IL)-4 . Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

J Bacteriol, 2002 Sep, 184(17), 4838 - 45
A new type IV secretion system promotes conjugal transfer in Agrobacterium tumefaciens; Chen L et al.; Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens . The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium . Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae . We have designated this system avhB for Agrobacterium virulence homologue virB . The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid . However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium . This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems . Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system . The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively . These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.

Plant J, 2002 Mar, 29(6), 797 - 808
Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants; Zubko E et al.; Cytokinins (CKs) are phytohormones that play an important role in plant growth and development . Although the first naturally produced CK, zeatin, was isolated almost four decades ago, no endogenous gene has been shown to produce active CKs in planta . In an activation tagging experiment we have identified a petunia line that showed CK-specific effects including enhanced shooting, reduced apical dominance and delayed senescence and flowering . This phenotype correlated with the enhanced expression of a gene we labelled Sho (Shooting) . Sho, which encodes a protein with homology to isopentenyl transferases (IPTs), also causes CK-specific effects when expressed in other plant species . In contrast to the ipt gene from Agrobacterium, which primarily increases zeatin levels, Sho expression in petunia and tobacco especially enhances the levels of certain N6-(delta2-isopentenyl) adenosine (2iP) derivatives . Our data suggest that Sho encodes a plant enzyme whose activity is sufficient to produce active CKs in plants.

Plant J, 2002 Mar, 29(6), 783 - 96
An approximately 400 kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4; Rivas S et al.; Despite sharing more than 91% sequence identity, the tomato Cf-4 and Cf-9 proteins discriminate between two Cladosporium-encoded avirulence determinants, Avr4 and Avr9 . Comparative studies between Cf-4 and Cf-9 are thus of particular interest . To investigate Cf-4 protein function in initiating defence signalling, we established transgenic tobacco lines and derived cell suspension cultures expressing c-myc-tagged Cf-4 . Cf-4:myc encodes a membrane-localized glycoprotein of approximately 145 kDa, which confers recognition of Avr4 . Elicitation of Cf-4:myc and Cf-9:myc tobacco cell cultures with Avr4 and Avr9, respectively, triggered the synthesis of active oxygen species and MAP kinase activation . Additionally, an Agrobacterium-mediated transient assay was used to express Cf-4:myc and a newly engineered fusion protein Cf-4:TAP . Both transiently expressed proteins were found to be functional in an in vivo assay, conferring a hypersensitive response (HR) to Avr4 . Consistent with previous observations that Cf-9 is present in a protein complex, gel filtration analysis of microsomal fractions solubilized with octylglucoside revealed that epitope-tagged Cf-4 proteins migrated at a molecular mass of 350-475 kDa . Using blue native gel electrophoresis, the molecular size was confirmed to be approximately 400 kDa . Significantly, this complex appeared to contain only one Cf-4 molecule, supporting the idea that, as previously described for Cf-9, additional glycoprotein partners participate with Cf-4 in the perception of the Avr4 protein . Intriguingly, Cf proteins and Clavata2 (CLV2) of Arabidopsis are highly similar in structure, and the molecular mass of Cf-4 and CLV complexes is also very similar (400 and 450 kDa, respectively) . However, extensive characterization of the Cf-4 complex revealed essentially identical characteristics to the Cf-9 complex and significant differences from the CLV2 complex.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(2), 149 - 54
{Cloning, sequencing and high expression in Escherichia coli of D-hydantoinase gene from Burkholderia pickettii}; Xu Z et al.; A strain, MMR003, used for D-p-HPG production in industry was classified as Burkholderia pickettii by morphological observation and biochemical characterization . The gene encoding the D-hydantoinase enzyme was cloned, sequenced and expressed in Escherichia coli . The nucleotide sequence of the 5.0 kb insert of subclone pXZ-total was determined . One open reading frame of 1374 bp was found and predicted to encode a polypeptide consisting of 458 amino acids in size of 50 kD . The amino acid sequence alignment of D-hydantoinase from Burkholderia pickettii shows the 85% homologous with the corresponding enzyme from Agrobacterium radiobacter NRRL B11291 . The D-hydantoinase gene (dha) harboured in the plasmid pXZPH2 in E . coli BL21(DE3) was highly expressed by IPTG induction . The D-hydantoinase activity for D, L-p-hydroxyphenylhydantion is 0.66 u/mL broth, which is 2-fold increase compared to the parent strain Burkholderia pickettii.

Annu Rev Phytopathol, 2002, 40, 169 - 89 Epub 2002 May 13.
Comparative genomic analysis of plant-associated bacteria; Van Sluys MA et al.; This review deals with a comparative analysis of seven genome sequences from plant-associated bacteria . These are the genomes of Agrobacterium tumefaciens, Mesorhizobium loti, Sinorhizobium meliloti, Xanthomonas campestris pv campestris, Xanthomonas axonopodis pv citri, Xylella fastidiosa, and Ralstonia solanacearum . Genome structure and the metabolism pathways available highlight the compromise between the genome size and lifestyle . Despite the recognized importance of the type III secretion system in controlling host compatibility, its presence is not universal in all necrogenic pathogens . Hemolysins, hemagglutinins, and some adhesins, previously reported only for mammalian pathogens, are present in most organisms discussed . Different numbers and combinations of cell wall degrading enzymes and genes to overcome the oxidative burst generally induced by the plant host are characterized in these genomes . A total of 19 genes not involved in housekeeping functions were found common to all these bacteria.

OMICS, 2002, 6(2), 163 - 74
Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis; Krysan PJ et al.; A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome . A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen . One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations . We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches . To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV . Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes . We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV . In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines . 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes . For comparison, coding sequences account for 44% of the Arabidopsis genome . Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions . However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.

J Bacteriol, 2002 Aug, 184(16), 4510 - 9
N-acyl-homoserine lactone inhibition of rhizobial growth is mediated by two quorum-sensing genes that regulate plasmid transfer; Wilkinson A et al.; The growth of some strains of Rhizobium leguminosarum bv . viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-L-homoserine lactone (3OH-C(14:1)-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL) . Tn5-induced mutants of R . leguminosarum bv . viciae resistant to 3OH-C(14:1)-HSL were isolated, and mutations in two genes were identified . These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI . The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR . Mutations in bisR, triR, traI, or trbL blocked plasmid transfer . Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C(14:1)-HSL . In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer . bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C(14:1)-HSL . The cloned bisR and triR genes conferred 3OH-C(14:1)-HSL sensitivity to strains of R . leguminosarum bv . viciae normally resistant to this AHL . Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R . leguminosarum bv . viciae strains producing 3OH-C(14:1)-HSL . Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C(14:1)-HSL, the presence of other AHLs such as N-octanoyl-L-homoserine lactone and/or N-(3-oxo-octanoyl)-L-homoserine lactone . In an attempt to identify the causes of growth inhibition, a strain of R . leguminosarum bv . viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C(14:1)-HSL . N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts.

Plant Mol Biol, 2002 Sep, 50(1), 17 - 27
A set of modular plant transformation vectors allowing flexible insertion of up to six expression units; Goderis IJ et al.; We have constructed a binary vector for Agrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13 hexanucleotide restriction sites, 6 octanucleotide restriction sites and 5 homing endonuclease sites . The homing endonuclease sites have the advantages to be extremely rare in natural sequences and to allow unidirectional cloning . We have also constructed a set of auxiliary vectors allowing the assembly of expression cassettes flanked by homing endonuclease sites . The expression cassettes assembled in these auxiliary vectors can be transferred into the binary vector with virtually no risk of cutting the vector within previously introduced sequences . This vector set is ideally suited for the construction of plant transformation vectors containing multiple expression cassettes and/or other elements such as matrix attachment regions . With this modular vector system, six different expression units were constructed in as many auxiliary vectors and assembled together in one plant transformation vector . The transgenic nature of Arabidopsis thaliana plants, transformed with this plant transformation vector, was assessed and the expression of each of the six genes was demonstrated.

Anal Sci, 2002 Jul, 18(7), 779 - 83
Monitoring of the pesticide diazinon in soil, stem and surface water of rice fields; Ghassempour A et al.; Diazinon is an organophosphorus insecticide (OPP) that is used as a pesticide for Chilo suppressalis (WLK) (Lep., Pyralidae) in rice fields . The extraction of diazinon from soil and the stems of rice plants has been carried out by microwave-assisted extraction (MAE) and the results compared with ultrasonic extraction (USE) . The best parameters for MAE are hexane-acetone (8:2 v/v) as a solvent, a 2.5 min extraction time, and 20 ml of the solvent volume . Also, surface-water samples of the rice fields were extracted by solid phase extraction (SPE) using a C18 disc . The optimum conditions of SPE were a sample volume of 750 ml, a pH of 7 and high ionic strength of water . The extracted samples were analyzed by gas chromatography-mass spectrometry (GC-MS) . The relative standard deviation (RSD) and regression coefficients related to the linearity were <3.5% (n = 5) and 0.99, respectively . The limit of detection (LOD) is 0.1 ng ml(-1) with selected ion monitoring (SIM) at 137 m/z . The average recoveries of diazinon in soil and stem samples by MAE and surface-water by SPE were 98% (+/-3), 94% (+/-5) and 87% (+/-3), respectively . In June, the concentration of diazinon in soil and stem samples of the rice plants in Guilan province is high (55 ng ml(-1)) and in September is low (2 ng ml(-1)) . In surface-water samples, the results are converse . In November, diazinon can not be detected in soil, stem or surface-water samples . Diazinon is degraded to diethylthiophosphoric acid . Also, three microorganism genera (Pseudomonas sp, Flavobacterium sp and Agrobacterium sp) have been found to degrade diazinon in soil and surface water.

Acta Crystallogr D Biol Crystallogr, 2002 Aug, 58(Pt 8), 1362 - 4 Epub 2002 Jul 20.
Crystallization and preliminary X-ray diffraction studies of the transcriptional regulator TraR bound to its cofactor and to a specific DNA sequence; Vannini A et al.; TraR is an Agrobacterium tumefaciens transcriptional regulator which binds the pheromone N-3-oxooctanoyl-L-homoserine lactone (AAI) in response to the bacterial population density . The TraR-AAI complex dimerizes and interacts with a specific 18-base-pair DNA sequence (TraBox), activating promoters containing this site . TraR was overexpressed and purified from Escherichia coli . Crystals of the ternary complex, in which dimeric TraR-AAI is bound to the TraBox sequence, have been obtained by the vapour-diffusion method . The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 66.99, b = 94.67, c = 209.66 A, with two (TraR-AAI)(2)-TraBox complexes in the asymmetric unit . A three-wavelength MAD data set for the seleno-L-methionine-substituted form has been collected to a resolution of 3 A . 20 of the 24 crystallographically independent selenium sites were located as part of the MAD-phasing process.

Biochemistry, 2002 Jul 30, 41(30), 9431 - 7
Characterization of chimeric ADPglucose pyrophosphorylases of Escherichia coli and Agrobacterium tumefaciens . Importance of the C-terminus on the selectivity for allosteric regulators; Ballicora MA et al.; ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis . The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway . Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate . Little is known about the regions determining the specificity for the allosteric regulator . To study the function of different domains, two chimeric enzymes were constructed . "AE" contains the N-terminus (271 amino acids) of the A . tumefaciens ADPglucose pyrophosphorylase and the C-terminus (153 residues) of the E . coli enzyme, and "EA", the inverse construction . Expression of the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid . Characterization of the purified chimeric enzymes showed that the C-terminus of the E . coli enzyme is relevant for the selectivity by FBP . However, this region seems to be less important for the specificity by F6P in the A . tumefaciens enzyme . The chimeric enzyme AE is activated by both FBP and F6P, neither of which affect EA . Pyruvate activates EA with higher apparent affinity than AE, suggesting that the C-terminus of the A . tumefaciens enzyme plays a role in the binding of this effector . The allosteric inhibitor site is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes.

Phytochemistry, 2002 Aug, 60(7), 683 - 9
Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase; Landtag J et al.; As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides . Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response . To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation . While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds . The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses . Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.

Ann Bot (Lond), 2002 Jul, 90(1), 31 - 6
Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A(b)-transformed rice; Jin WW et al.; The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH) . Eight of the nine transgenic lines tested showed hybridization signals . Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26.2 (near the centromere) to 95.2 (distal regions) . No signal was found on regions where the fraction length was less than 26.2, while six of the nine signals detected were located on regions with FL values of 75.3 or over . This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration . The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH . Results show that the transformed DNA sequences remained linked in the recipient genome . The relationship between integration position and transgene silencing, known as the 'position effect', is discussed.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10435 - 40 Epub 2002 Jul 17.
Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1; Tzfira T et al.; Agrobacterium is a unique model system as well as a major biotechnological tool for genetic manipulation of plant cells . It is still unknown, however, whether host cellular factors exist that are limiting for infection, and whether their overexpression in plant cells can increase the efficiency of the infection . Here, we examined the effect of overexpression in tobacco plants of an Arabidopsis gene, VIP1, which encodes a recently discovered cellular protein required for Agrobacterium infection . Our results indicate that VIP1 is imported into the plant cell nucleus via the karyopherin alphadependent pathway and that elevated intracellular levels of VIP1 render the host plants significantly more susceptible to transient and stable genetic transformation by Agrobacterium, probably because of the increased nuclear import of the transferred-DNA.

Plant Cell, 2002 Jul, 14(7), 1497 - 508
A surveillance system regulates selective entry of RNA into the shoot apex; Foster TM et al.; Phloem-mobile endogenous RNA is trafficked selectively into the shoot apex . In contrast, most viruses and long-distance post-transcriptional gene silencing (PTGS) signals are excluded from the shoot apex . These observations suggest the operation of an underlying regulatory mechanism . To examine this possibility, a potexvirus movement protein, known to modify cell-to-cell trafficking and PTGS, was expressed ectopically in transgenic plants . These plants were found to be compromised in their capacity to exclude both viral RNA and silencing signals from the shoot apex . The transgenic plants also displayed various degrees of abnormal leaf polarity depending on transgene expression level . Normal patterns of organ development were restored by either virus- or Agrobacterium tumefaciens-mediated induction of PTGS . This revealed the presence of an RNA signal surveillance system that acts to allow the selective entry of RNA into the shoot apex . We propose that this surveillance system regulates signaling and protects the shoot apex, in particular the cells that give rise to reproductive structures, from viral invasion.

Mol Plant Microbe Interact, 2002 Jul, 15(7), 637 - 46
The xanthomonas type III effector protein AvrBs3 modulates plant gene expression and induces cell hypertrophy in the susceptible host; Marois E et al.; Xanthomonas campestris pv . vesicatoria bacteria expressing the type III effector protein AvrBs3 induce a hypersensitive response in pepper plants carrying the resistance gene Bs3 . Here, we report that infection of susceptible pepper and tomato plants leads to an AvrBs3-dependent hypertrophy of the mesophyll tissue . Agrobacterium-mediated transient expression of the avrBs3 gene in tobacco and potato plants resulted in a similar phenotype . Induction of hypertrophy was shown to depend on the repeat region, nuclear localization signals, and acidic transcription activation domain (AAD) of AvrBs3, suggesting that the effector modulates the host's transcriptome . To search for host genes regulated by AvrBs3 in an AAD-dependent manner, we performed a cDNA-amplified fragment length polymorphism analysis of pepper mRNA populations . Thirteen AvrBs3-induced transcripts were identified and confirmed by reverse transcriptase-polymerase chain reaction . Sequence analysis revealed homologies to auxin-induced and expansinlike genes, which play a role in cell enlargement . These results suggest that some of the AvrBs3-induced genes may be involved in hypertrophy development and that xanthomonads possess type III effectors that steer host gene expression.

Transgenic Res, 2002 Jun, 11(3), 269 - 78
Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development; Sa G et al.; Isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens T-DNA was placed under the control of a TA29 promoter which expresses specifically in anther . The chimeric TA29-ipt gene was transferred to tobacco plants . During flowering, mRNA of the ipt gene in the anthers of the transgenic plants accumulated and the level of iPA + iPs increased 3-4-fold in the leaves, petals, pistils, and stamens compared with those in the wild type plants . This cytokinin increase affected various aspects in development indicating that the alterations of endogenous cytokinin level by using anther-specific expression of the TA29-ipt gene affected morphology, floral organ systems and reproductivity of the transgenic plants.

Transgenic Res, 2002 Jun, 11(3), 249 - 56
Expression of an antisense GIGANTEA (GI) gene fragment in transgenic radish causes delayed bolting and flowering; Curtis IS et al.; A late-flowering transgenic radish has been produced by the expression of an antisense GIGANTEA (GI) gene fragment using a floral-dip method . Twenty-five plants were dipped into a suspension of Agrobacterium carrying a 2.5 kb antisense GI gene fragment from Arabidopsis, along with the gusA and bar reporter genes, all under the control of a CaMV 35S promoter . From a total of 1462 seeds harvested from these floral-dipped plants, 16 Basta-resistant T1 plants were found to have GUS activity (transformation efficiency of 1.1%) . Southern analysis confirmed the integration of one or two copies of the gusA gene in these herbicide-resistant plants . Expression of the GI gene in T1 plants was much reduced compared to both wildtype plants and plants transformed with pCAMBIA3301 (positive control) . In the progenies of eleven T1 plants analysed (T2 generation), all lines showed a significant delay in both bolting and flowering times compared to wildtype and positive control plants, and that, the level of GI transcript was inversely proportional to the time of bolting and flowering . At a maximum, bolting and flowering times were delayed by 17 and 18 days respectively, compared to wildtype plants (in positive control plants, the delay was 23 and 26 days, respectively) . Ten of the 11 lines exhibited a significant reduction in plant height compared to wildtype and positive control plants . This study provides evidence that down-regulation of the GI gene by co-suppression could delay bolting in a cold-sensitive long-day (LD) plant . Production of late-flowering germplasms of radish may allow this important crop to be cultivated over an extended period and also provide further food to the famine countries of S/E Asia.

Arch Pharm (Weinheim), 2002 Apr, 335(4), 143 - 51
Heterologous expression of a bacterial homospermidine synthase gene in transgenic tobacco: effects on the polyamine pathway; Kaiser A et al.; Homospermidine synthase (HSS) is a branch-point enzyme that links the secondary pathway (pyrrolizidine alkaloids) to primary metabolism (polyamines) . Since the diamine putrescine is a precursor of homospermidine and nicotine in tobacco, we performed heterologous expression of a bacterial homospermidine synthase gene (hss)in Nicotiana tabacum and determined the effect on free and conjugated polyamine levels . The hss gene from Rhodopseudomonas viridis was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in Agrobacterium tumefaciens Ti-plasmid in sense and antisense orientation and both hss constructs were transformed into tobacco plants . Expression of the hss gene was verified by "Northern" and "Southern Blot" analysis . 2 transgenic sense lines were generated from 1000 calli which showed weak expression of homospermidine synthase, i.e . 50 pktal/mg protein and 45 pktal/mg protein.These transgenic sense plants showed a significantly decreased content of free spermidine while the pool of conjugated spermidine was not affected.The 2 sense plants exhibited a range of abnormal phenotypes such as dwarfness and stunted growth . Homospermidine was sporadically detectable in wild type tobacco . To our knowledge, this is the first biotechnological approach to express a prokaryotic homospermidine synthase gene in tobacco plants.

Planta, 2002 Jul, 215(3), 494 - 501 Epub 2002 May 29.
Oligogalacturonides inhibit the induction of late but not of early auxin-responsive genes in tobacco; Mauro ML et al.; Oligogalacturonides (OGs) released from the plant cell wall regulate several defense responses, as well as various aspects of plant growth and development . In these latter effects, OGs exhibit auxin-antagonist activity . To shed light on the mechanism by which OGs antagonise auxin, we analysed the ability of these oligosaccharides to inhibit the activity of four auxin-up-regulated promoters {pGm-GH3 of soybean (Glycine max L . Merr.), pNt114 of tobacco (Nicotiana tabacum L.), and prolB and prolD of Agrobacterium rhizogenes} driving the expression of the beta-glucuronidase reporter gene (GUS) in transgenic tobacco seedlings . Our results indicate that OGs at submicromolar concentrations inhibit the activation by auxin of pNt114, prolB and prolD, but not that of pGm-GH3 . Comparative analysis of the kinetics of activation of the four promoters in response to the hormone shows that, while pGm-GH3 is rapidly activated, the other three promoters exhibit a delayed activation, with a lag of at least 4 h before the appearance of GUS activity . The lack of effect of the OGs on early auxin-responsive genes was confirmed by RNA gel blot analysis of the tobacco genes Nt-GH3 and Nt-iaa2.3/2.5 . Our results suggest that the auxin-antagonist action of OGs affects the expression of late but not of early auxin-responsive genes.

J Bacteriol, 2002 Aug, 184(15), 4296 - 300
The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation; Rogers EJ et al.; Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991) . The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible . Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.

J Bacteriol, 2002 Aug, 184(15), 4114 - 23
Cloning and characterization of the phosphatidylserine synthase gene of Agrobacterium sp . strain ATCC 31749 and effect of its inactivation on production of high-molecular-mass (1-->3)-beta-D-glucan (curdlan); Karnezis T et al.; Genes involved in the production of the extracellular (1-->3)-beta-glucan, curdlan, by Agrobacterium sp . strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999) . To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe . One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pss(AG), encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted . The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased . In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the Pss(AG) protein at its first step . Moreover, PC can be produced in a medium lacking choline . When the pss(AG)::TnphoA mutation was complemented by the intact pss(AG) gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics . The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.

Comp Biochem Physiol C Toxicol Pharmacol, 2002 Jun, 132(2), 261 - 8
Demonstration of antifungal and anti-human immunodeficiency virus reverse transcriptase activities of 6-methoxy-2-benzoxazolinone and antibacterial activity of the pineal indole 5-methoxyindole-3-acetic acid; Wang HX et al.; 6-methoxy-2-benzoxazolinone (6-MBOA), a naturally occurring progonadal compound present in grasses with structural resemblance to melatonin, was tested for antifungal activity against Fusarium oxysporum, Rhizoctonia solani and Coprinus comatus . A variety of pineal products was also examined for the sake of comparison, including 5-methoxytryptamine, melatonin, 5-methoxytryptophol, 5-hydroxytryptamine, 5-methoxyindole-3-acetic acid and 5-hydroxytryptophol . The assay for antifungal activity was carried out in Petri plates containing potato dextrose agar . It was found that 6-MBOA most potently inhibited the growth of C . comatus, R . solani and F . oxysporum . When 6-MBOA and pineal indoles were tested for antibacterial activity against the bacterium Agrobacterium tumefaciens, 5-methoxyindole-3-acetic acid was found to be the most potent . 6-MBOA most potently inhibited human immunodeficiency virus-1 reverse transcriptase.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1137 - 9
Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens; Fujisawa H et al.; The structural gene for NADP+-dependent serine dehydrogenase {EC 1.1.1.-} from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed . The gene encodes a polypeptide containing 249 amino acid residues . The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.

Shokuhin Eiseigaku Zasshi, 2002 Apr, 43(2), 68 - 73
Increased digestibility of two products in genetically modified food (CP4-EPSPS and Cry1Ab) after preheating; Okunuki H et al.; We performed experiments on in vitro digestion of newly expressed proteins by SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) to assess the allergenicity of food components derived from biotechnological modification . For newly expressed proteins, we chose CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp . strain CP4) and Cry1Ab derived from Bacillus thuringiensis subsp . kurstaki strain HD-1 . The former is expressed in GM-soybeans and the latter is expressed in GM-corns . Firstly, we examined the digestibility of purified CP4-EPSPS and Cry1Ab by SGF . Both proteins were rapidly digested within 60 sec . After preheating, the digestibility by SGF was slightly increased . Secondly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SGF . The digestion time of both proteins by SGF was almost the same as that of the purified proteins . Thirdly, the digestibility of CP4-EPSPS and Cry1Ab by SIF was examined . The digestion time of these proteins was 240 min or more . However, digestibility of these proteins by SIF was dramatically increased by preheating, and the digestion time was less than 5 sec . Fourthly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SIF . Digestion time of both proteins by SIF was almost the same as that of the purified proteins . From these results, we concluded that the digestibility of both CP4-EPSPS and Cry1Ab by SGF and SIF was increased by preheating . Therefore, we suggest that the allergenicity of both proteins should be extremely low because of the easy digestibility of these proteins by SGF and also by SIF with preheating.

Appl Environ Microbiol, 2002 Jul, 68(7), 3582 - 7
Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene; Bosma T et al.; Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP) . This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin . Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment . The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol . We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp . m15-3 (DhaA) . A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase . Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe . The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source . These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.

Appl Environ Microbiol, 2002 Jul, 68(7), 3371 - 6
Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate; Horne I et al.; We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides . A gene encoding a protein involved in OP hydrolysis was cloned from A . radiobacter P230 and sequenced . This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp . strain ATCC 27551 and Brevundimonas diminuta MG . Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A . radiobacter P230 . The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed . Neither protein was able to hydrolyze the aliphatic OP malathion . The kinetics of the two proteins for diethyl OPs were comparable . For dimethyl OPs, OpdA had a higher k(cat) than OPH . It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.

Appl Environ Microbiol, 2002 Jul, 68(7), 3358 - 65
Seasonal fluctuations and long-term persistence of pathogenic populations of Agrobacterium spp . in soils; Krimi Z et al.; Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence . No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 x 10(5) pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer . PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter . The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines . The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils . An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil . In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids . In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains . These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.

Trends Microbiol, 2002 Jun, 10(6), 269 - 75
Do plant and human pathogens have a common pathogenicity strategy?
Kempf VA, Hitziger N, Riess T, Autenrieth IB.
Recently, a novel 'two-step' model of pathogenicity has been described that suggests host-cell-derived vasculoproliferative factors play a crucial role in the pathogenesis of bacillary angiomatosis, a disease caused by the human pathogenic bacterium Bartonella henselae . The resulting proliferation of endothelial cells could be interpreted as bacterial pathogens triggering the promotion of their own habitat: the host cell . Similar disease mechanisms are well known in the plant pathogen Agrobacterium tumefaciens, which causes crown gall disease . There are notable similarities between the pathogenicity of A . tumefaciens leading to tumourous disease in plants and to the B . henselae-triggered proliferation of endothelial cells in humans . Here, we hypothesize that this pathogenicity strategy might be common to several bacterial species in different hosts owing to shared pathogenicity factors.

Virus Genes, 2002 Jun, 24(3), 231 - 4
Phosphorylation of the movement protein of cucumber mosaic virus in transgenic tobacco plants; Matsushita Y et al.; The 3a protein of Cucumber mosaic virus is essential for the cell-to-cell movement of the viral RNA through plasmodesmata . We have introduced an epitope peptide before the stop codon of the 3a protein and cloned the tagged ORF into a binary vector for Agrobacterium-mediated transformation . The established transgenic tobacco lines produced the 3a protein, which was specifically detected with anti-3a and anti-epitope antisera . Metabolic labeling and subsequent immunoprecipitation revealed that {32P}-orthophosphate was incorporated into the 3a protein . The phosphoamino acid analysis indicated that the 3a protein contained phosphoserine but not phosphothreonine or phosphotyrosine . This is the first demonstration of the 3a protein phosphorylation in planta.

J Biotechnol, 2002 Aug 28, 97(3), 213 - 21
Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes; Bulgakov VP et al.; It has been suggested that the rol genes of Agrobacterium rhizogenes could play an essential role in the activation of secondary metabolite production in plant transformed cultures . This study investigated whether the content of anthraquinone phytoalexins was changed in callus cultures of Rubia cordifolia transgenic for the 35S-rolB and 35S-rolC genes in comparison with a non-transformed callus culture . The anthraquinone content was shown to be significantly increased in transgenic cultures, thus providing further evidence that the rol-gene transformation can be used for the activation of secondary metabolism in plant cells . Methyl jasmonate and salicylic acid strongly increased anthraquinone accumulation in both transgenic and non-transgenic R . cordifolia calluses, whereas ethephon did not . A treatment of the cultures by cantharidin, the protein phosphatase 2A inhibitor, resulted in massive induction of anthraquinone accumulation in the transgenic cultures only . We suggest the involvement of a cantharidin-sensitive protein phosphorylation mechanism in anthraquinone biosynthesis in transgenic cultures.

Environ Microbiol, 2002 Jun, 4(6), 361 - 73
Stratification and seasonal stability of diverse bacterial communities in a Pinus merkusii (pine) forest soil in central Java, Indonesia; Krave AS et al.; In Java, Indonesia, many nutrient-poor soils are intensively reforested with Pinus merkusii (pine) . Information on nutrient cycles and microorganisms involved in these cycles will benefit the management of these important forests . Here, seasonal effects on the stratification of bacterial community structure in the soil profile of a tropical pine forest are described, and differences in bacterial communities are related to chemical and physical soil parameters . Culture-independent community profiles of litter, fragmented litter and mineral soil layers were made by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA-specific polymerase chain reaction (PCR) fragments . The community profiles of the different soil layers clustered separately, correlating with significant differences in organic matter content between the three layers . The bacterial communities appeared to be stable during the wet season of 1998 . The drought in 1997, caused by the El Nino climatic effect, did not influence the bacterial communities in fragmentation and mineral soil, although moisture content and other soil parameters were markedly lower than in the wet season . However, communities in litter were influenced by drought . In the litter layer, the moisture content was significantly lower than in the fragmentation and mineral layers during the dry season . A clone library was made from a litter sample taken during the wet season . Partial sequencing of 74 clones and linking the DGGE banding positions of these clones to bands in the DGGE profile of the sample from which the clone library was derived showed considerable bacterial diversity . Alpha-proteobacteria (40.5% of the clones, of which 57% belonged to the Rhizobium-Agrobacterium group) and high-G+C content, Gram-positive bacteria (36.5%) dominated the clone library.

Eur J Biochem, 2002 Jun, 269(12), 2885 - 8
Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens, strain DSM 30205; De Castro C et al.; Agrobacterium tumefaciens is a Gram-negative, phytopathogenic bacterium and is characterized by an unique mode of action on dicotyledonous plants: it is able to genetically modify the host, and because of this feature, it is used as a tool for transgenic plants . Many experiments have demonstrated that lipopolysaccharides (LPSs) play an important role for the disease development, as they are involved in the adhesion process of the bacterium on the plant cell wall . Despite the wealth of information on the role of LPS on phytopathogenesis, the present paper appears as the first report on the molecular primary structure of the O-chain produced from Agrobacterium . Its repeating unit was determined by means of chemical and spectroscopical analysis, and has the following structure: (3)-alpha-D-Araf-(1-->3)-alpha-l-Fucp-(1-->.

Protein Expr Purif, 2002 Jun, 25(1), 195 - 202
Expression and affinity purification of recombinant proteins from plants; Desai UA et al.; With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses . However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy . In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag . In the proposed system, CaM is fused to GUS . In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column . When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein . The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein . Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system .

Protein Expr Purif, 2002 Jun, 25(1), 160 - 5
Expression and production of recombinant human interleukin-2 in potato plants; Park Y et al.; Interleukin-2 is a pharmacologically important cytokine secreted by T lymphocytes . Recombinant interleukin-2 has been produced and found to be useful for many medical applications . Mass production of recombinant interleukin-2 will be prerequisite for a wider application of this molecule . In this study we investigated the possibility of using potato tubers for the production of recombinant human interleukin-2 in large quantity . A binary vector carrying the human interleukin-2 gene under a potato tuber-specific promoter (patatin promoter) was constructed . Several potato transformants expressing the human interleukin-2 gene were generated by Agrobacterium-mediated transformation . Expression of the human interleukin-2 gene was confirmed by Northern blotting and the protein level was determined by Western blot analyses . A bioassay revealed that human interleukin-2 expressed in the potato tuber supported proliferation of interleukin-2-dependent cells, CTLL-2 . We found that the recombinant protein in the 2-week-old microtuber has the highest activity (115 units per gram of microtuber) and estimated that an average yield for a potato (average 200 g per potato) was 23,000 units of rhIL-2 activity . The results suggest that the potato tuber is an excellent system for the mass production of biologically active human interleukin-2 .

Arch Microbiol, 2002 Jul, 178(1), 45 - 52 Epub 2002 Apr 24.
Characterization of Porphyrobacter sanguineus sp . nov., an aerobic bacteriochlorophyll-containing bacterium capable of degrading biphenyl and dibenzofuran; Hiraishi A et al.; Three strains of " Agrobacterium sanguineum", an aerobic marine bacterial species described previously, were re-characterized from phylogenetic and taxonomic viewpoints . 16S rDNA sequence comparisons showed that the " A . sanguineum" strains belong to the alpha-4 subgroup of alpha-Proteobacteria, with members of the genera Erythromicrobium and Porphyrobacter as their closest relatives . DNA-DNA hybridization studies indicated that the " A . sanguineum" strains were distinguishable from any previously known species of these genera . Bacteriochlorophyll a, monosaccharide-type glycosphingolipids, 2-OH fatty acids of C14:0, C15:0, C16:0, and C16:1, and ubiquinone-10 were detected in the " A . sanguineum" strains . The G+C of the DNA was 63.8-64.0 mol% . Two of the " A . sanguineum" strains, IAM 12620 (=ATCC 25659) and ATCC 25661, were able to grow with biphenyl and dibenzofuran as sole carbon source in the presence of 0.05% yeast extract . The medium in these cultures turned yellowish-orange at the exponential phase of growth due to the release of soluble chromogenic metabolites . The remaining " A . sanguineum" strain, ATCC 25660, and all test strains of Erythromicrobium and Porphyrobacter neither grew nor produced yellow-orange pigment with biphenyl or dibenzofuran . In PCR experiments, bphA1 gene, coding for the large subunit protein of biphenyl dioxygenase, was detected in " A . sanguineum" IAM 12620 and ATCC 25661 . Based on these results, we propose classifying " A . sanguineum" IAM 12620 and ATCC 25661 as a new species of the genus Porphyrobacter with the name Porphyrobacter sanguineus sp . nov.

Z Naturforsch {C}, 2002 Mar-Apr, 57(3-4), 307 - 12
Inability of Agrobacterium tumefaciens ribosomes to translate in vivo mRNAs containing non-Shine-Dalgarno translational initiators; Golshani A et al.; Numerous data accumulated during the last decade have shown that the Shine-Dalgarno (SD) sequence is not a unique initiator of translation for Escherichia coli . Several other sequences, mostly of viral origin, have demonstrated their capability of either enhancing or initiating translation in vivo . A phage T7 gene 10 sequence, called "epsilon" (epsilon), has shown its high enhancing activity on translation in both Escherichia coli and Agrobacterium tumefaciens cells . In this study the epsilon, together with three other nucleotide sequences derived from the 5' non-translated regions of tobacco mosaic virus (TMV), papaya mosaic virus (PMV) and clover yellow mosaic virus (CYMV) RNAs are tested for translation initiation activity in A . tumefaciens cells . The obtained results indicate that none of them was capable of initiating translation in vivo of chloramphenicol acetyltransferase (CAT) mRNA . To determine whether their inactivity was related with structural differences in the ribosomal protein S1, the rpsA gene (coding for S1 protein in E . coli) was co-expressed in A . tumefaciens together with the cat gene placed under the translational control of the above sequences . Our results showed that the rpsA gene product did not make any of the four viral enhancers active in A . tumefaciens cells . The inability of A . tumefaciens ribosomes to translate mRNAs devoid of SD sequences indicates for a substantial difference in the ribosome structure of the two Gram negative bacteria E . coli and A . tumefaciens.

Physiol Plant, 2002 Jun, 115(2), 284 - 290
Inhibitory effects of elevated endogenous cytokinins on nitrate reductase in ipt-expressing tobacco are eliminated by short-term exposure to benzyladenine; Lexa M et al.; Using a novel system for expressing ipt gene from Agrobacterium tumefaciens in tobacco (Nicotiana tabacum L., cv . Petit Havana SR1), we were able to grow seedlings and teratoma-like tissue with increased content of cytokinins . This material enabled us to investigate new regulatory aspects of nitrate reduction . We grew control plants and plants with elevated cytokinins on MS media, with or without nitrate and benzyladenine (BA) . We determined in vitro nitrate reductase (EC 1.6.6.1) activity (NRA) in this plant material . Initially, we found that ipt-expressing plants always displayed lowered levels of NRA when compared to wild-type SR1 plants . We determined that long-term exposure of tobacco plants and tissue to cytokinins caused up to 60% decrease in NRA . Exposure to 40 mM nitrate was able to induce the activity in such plants 3-fold, increasing the activity in SR1 plants more than 5-fold . We were able to restore wild-type levels of NRA in ipt-expressing plants by simultaneous induction of NR with BA and nitrate . Our results suggest that regulation of NR by nitrate and cytokinin is a result of overlaying cytokinin-driven regulatory processes, with those acting in the short-term having a positive effect on NRA, and those acting over extended periods of time having inhibitory effects on NRA.

J Bacteriol, 2002 Jul, 184(13), 3560 - 8
The Rhizobium etli cyaC product: characterization of a novel adenylate cyclase class; Tellez-Sosa J et al.; Adenylate cyclases (ACs) catalyze the formation of 3',5'-cyclic AMP (cAMP) from ATP . A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant . The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E . coli cya mutant . Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E . coli cya mutant . CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function . Thus, CyaC represents the first member of a novel class of ACs (class VI) . Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens . The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides . The physiological performance of an R . etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis.

J Bacteriol, 2002 Jul, 184(13), 3466 - 75
Identification of two quorum-sensing systems in Sinorhizobium meliloti; Marketon MM et al.; Sinorhizobium meliloti is a free-living soil bacterium which is capable of establishing a symbiotic relationship with the alfalfa plant (Medicago sativa) . This symbiosis involves a network of bacterium-host signaling, as well as the potential for bacterium-bacterium communication, such as quorum sensing . In this study, we characterized the production of N-acyl homoserine lactones (AHLs) by two commonly used S . meliloti strains, AK631 and Rm1021 . We found that AK631 produces at least nine different AHLs, while Rm1021 produces only a subset of these molecules . To address the difference in AHL patterns between the strains, we developed a novel screening method to identify the genes affecting AHL synthesis . With this screening method, chromosomal groEL (groELc) was shown to be required for synthesis of the AHLs that are unique to AK631 but not for synthesis of the AHLs that are made by both AK631 and Rm1021 . We then used the screening procedure to identify a mutation in a gene homologous to traM of Agrobacterium tumefaciens, which was able to suppress the phenotype of the groELc mutation . A traR homolog was identified immediately upstream of traM, and we propose that its gene product requires a functional groELc for activity and is also responsible for inducing the synthesis of the AHLs that are unique to AK631 . We show that the traR/traM locus is part of a quorum-sensing system unique to AK631 and propose that this locus is involved in regulating conjugal plasmid transfer . We also present evidence for the existence of a second quorum-sensing system, sinR/sinI, which is present in both AK631 and Rm1021.

Microbiology, 2002 Jun, 148(Pt 6), 1923 - 9
Evolutionary relationship of phototrophic bacteria in the alpha-Proteobacteria based on farnesyl diphosphate synthase; Cantera JJ et al.; Partial sequences of farnesyl diphosphate (FPP) synthase genes derived from the Rhodobacter-Rhodovulum group and from the Rhodopseudomonas palustris-Bradyrhizobium japonicum group of the alpha-Proteobacteria were subjected to phylogenetic analysis to investigate the relationships of phototrophic and non-phototrophic bacteria in the alpha-Proteobacteria . The four Rhodovulum species formed a monophyletic group within the Rhodobacter cluster, and Agrobacterium ferrugineum IAM 12616(T) intermingled with the Rhodobacter species . This topology is in good agreement with the 16S rRNA phylogeny, although the FPP synthase gene was more divergent than the 16S rRNA . On the other hand, strains of the phototrophic Rps . palustris formed a cluster far from that of the non-phototrophic Bradyrhizobium japonicum strains . Moreover, Rps . palustris strains were differentiated from the nodule-forming B . japonicum, Mezorhizobium loti MAFF 303099 and Sinorhizobium sp . NGR 234 in the FPP synthase phylogeny . This relationship does not agree with the 16S rRNA phylogeny, wherein Rps . palustris was more closely related to B . japonicum than to strains of the Rhodobacter-Rhodovulum group . These results suggest that the FPP synthase gene of Rps . palustris diverged from that of B . japonicum.

J Org Chem, 2002 Jun 14, 67(12), 4143 - 9
Solid-phase oligosaccharide and glycopeptide synthesis using glycosynthases; Tolborg JF et al.; Enzymatic approaches for the preparation of oligosaccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for protecting groups . The use of solid-phase techniques offers easy workup procedures and the prospect of automatability . Here, we report the first application of glycosynthases to solid-phase oligosaccharide synthesis by use of the 51 kDa serine and glycine mutants of Agrobacterium sp . beta-glucosidase, Abg E358S and E358G . Acceptors were linked to PEGA resin through a backbone amide linker (BAL), and using these mutated enzymes, a galactose moiety was transferred from a donor sugar, alpha-D-galactosyl fluoride, with high efficiency (>90%) together with excellent recovery of material . Furthermore, it was demonstrated that a resin-bound model glycopeptide was also an acceptor for the glycosynthase.

Transgenic Res, 2002 Apr, 11(2), 215 - 9
The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus; Shin R et al.; Transgenic pepper plants coexpressing coat proteins (CPs) of cucumber mosaic virus (CMV-Kor) and tomato mosaic virus (ToMV) were produced by Agrobacterium-mediated transformation . To facilitate selection for positive transformants in transgenic peppers carrying an L gene, we developed a simple and effective screening procedure using hypersensitive response upon ToMV challenge inoculation . In this procedure, positive transformants could be clearly differentiated from the nontransformed plants . Transgenic pepper plants expressing the CP genes of both viruses were tested for resistance against CMV-Kor and pepper mild mottle virus (PMMV) . In most transgenic plants, viral propagation was substantially retarded when compared to the nontransgenic plants . These experiments demonstrate that our transgenic pepper plants might be a useful marker system for the transgene screening and useful for classical breeding programs of developing virus resistant hot pepper plants.

Transgenic Res, 2002 Apr, 11(2), 161 - 73
The expression of a mammalian proteinase inhibitor, bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae; Christeller JT et al.; The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation . Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven . SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide . Both were retained on a trypsin-agarose affinity gel and had inhibitory activity . Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present . Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring . The stability of SI was shown by the presence of protein at high levels in completely senescent leaves . Modifications to the cDNA (3' and 5' changes and minor codon changes) resulted in a 20-fold variation in expression . Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar . In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased . Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered . The use of SI as an insect resistance factor is discussed.

Biotechnol Prog, 2002 May-Jun, 18(3), 413 - 7
Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability; Oh KH et al.; Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability . A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine) . Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability . Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability . The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased . DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3 . From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability . Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.

Yi Chuan Xue Bao, 2002 May, 29(5), 458 - 60
{Characterization of an ultra-violet inducible gene that encodes glutathione S-transferase in Arabidopsis thaliana}; Liu XF et al.; Glutathione S-transterased (GSTs) are a highly divergent gene family . They play important roles not only in many stress responses, but also in plant growth and development . To understand whether the GST can protect plant from the damage of ultra-violet (UV) radiation, a UV inducible GST gene was isolated from an Arabidopsis cDNA library . The plant expression vector containing the GST cDNA was constructed and transferred into Arabidopsis thaliana plants by the Agrobacterium-mediated vacuum method . Molecular genetic analyses showed that the overexpression of the UV inducible GST could increase the tolerance of the transgenic plants to UV radiation.

Plant Cell Physiol, 2002 May, 43(5), 555 - 62
Synthesis of a novel class of polyhydroxyalkanoates in Arabidopsis peroxisomes, and their use in monitoring short-chain-length intermediates of beta-oxidation; Arai Y et al.; The poly{(R)-3-hydroxyalkanoate} (PHA) synthase gene (phaC(Ac)) of Aeromonas caviae FA440 was modified by adding a peroxisome targeting signal encoding the last 10 amino acids at the carboxyl-terminal of spinach glycolate oxidase . The modified gene was introduced into Arabidopsis thaliana plants by Agrobacterium-mediated transformation . The transgenic Arabidopsis plant expressed the introduced gene and its protein, and it accumulated PHA in its tissues . Gas chromatography-mass spectrometry analysis demonstrated the accumulation of a novel type of PHA, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) . This strongly suggests that short-chain-length (R)-3-hydroxyacyl-CoAs were generated from intermediates of peroxisomal beta-oxidation . It was revealed by using this transgenic plant that Tween-20 can activate peroxisomal beta-oxidation of short-chain-length fatty acids.

Genes Genet Syst, 2002 Feb, 77(1), 1 - 9
A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens; Uraji M et al.; Ti (Tumor inducing) plasmids in Agrobacterium tumefaciens can transfer their T-DNA region into dicotyledonous plants, in which the expression of T-DNA genes causes plant tumors and the production of bacterial nutrients, e.g., opines such as nopaline . Naturally occurring Ti plasmids (pTi) are difficult to cure by conventional curing methods because of their high stability . Here, we developed a novel curing method based on plasmid incompatibility . For this, a curing plasmid, pMGTrep1, was newly constructed and subsequently introduced into A . tumefaciens strains harboring pTi by conjugation with Escherichia coli harboring pMGTrep1 . The conjugation yielded 32-99% nopaline non-utilizing agrobacterial transconjugants in which pMGTrep1 replaced pTi due to incompatibility . Then, pMGTrep1-less derivatives of the transconjugants are easily selected in the presence of sucrose because pMGTrep1 contains a sucrose-sensitive sacB gene . This efficient method is directly applicable for curing plasmids with the same incompatibility group and shoud also applicable to other types of plasmids in Agrobacterium groups, including A . rhizogenes, by replacing the rep gene region of the curing plasmid with that of the corresponding incompatibility.

J Interferon Cytokine Res, 2002 Mar, 22(3), 371 - 8
Expression of biologically active human tumor necrosis factor-alpha in transgenic potato plant; Ohya K et al.; We report the successful insertion of the cDNA of human tumor necrosis factor-alpha (HuTNF-alpha) into the genome of potato plant species, Solanum tuberosum, using Agrobacterium tumefacience-mediated transformation . HuTNF-alpha is a known and essential cytokine mediating host defense against tumors and infectious diseases and an immunomodulating agent . To enhance the accumulation of foreign gene product expression in plant cells, the molecular design of the constructed HuTNF-alpha is presented . Transcription and translation of TNF-alpha in transformants were confirmed by Northern blot, RT-PCR, ELISA, and Western blot, respectively . Expression of the bioactive HuTNF-alpha in plant cells was confirmed by way of the cytotoxic effect of the extract obtained from the transformants against murine L929 cells . We think that the expression level of HuTNF-alpha (15 microg/g potato plant tissue) obtained in the present study may be sufficient to induce responses/effects similar to those generated by TNF-alpha in human milk administered orally . We believe that the TNF-alpha expressed in edible potato plants has tremendous potential for clinical use in the areas of medicine and veterinary science . Its usefulness and applicability, therefore, need to be fully explored.

Microb Ecol, 2001 Feb, 41(4), 369 - 383
Analysis of Endophytic Bacterial Communities of Potato by Plating and Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA Based PCR Fragments; Garbeva P et al.; The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desiree) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants . The culturable endophytic bacterial communities detected in potato stem bases as well as in roots were in most cases on the order 103 to 105 CFU g?1 of fresh plant tissue . Dilution plating revealed that a range of bacterial types dominated these populations . Dominant isolates fell into the a and g subgroups of the Proteobacteria, as well as in the Flavobacterium/Cytophaga group . Different representatives of the Firmicutes were also found . The most frequently isolated strains (>5% of the total) were characterized as different Pseudomonas spp . (including P . aureofaciens, P . corrugata, and P . putida), Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resinovorans, using fatty acid methyl ester (FAME) analysis and/or sequencing of their partial 16S ribosomal RNA genes . Other Proteobacteria or Firmicutes were also found, albeit infrequently, and mainly in potato stem tissue . The fate of three putative potato endophytes, Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis, was monitored following their release into potato plants via injection, via root dipping, or via the soil . Following stem injection, the S . maltophilia and Bacillus inoculants could be tracked over time periods of, respectively, 22 and 1 day(s) by dilution plating as well as via PCR-DGGE . However, only S . maltophilia was able to colonize, and persist in, plant tissue from soil or dipped roots . S . paucimobilis was never recovered from the plant irrespective of the mode of introduction . The diversity of the indigenous bacterial flora associated with potato was then monitored via PCR-DGGE . The patterns obtained revealed the existence of bacterial communities of limited complexity, with communities from potato stems typically differing from those from stem peel and roots . Evidence was obtained for the endophytic occurrence of a range of organisms falling into the a, b, and g subgroups of the Proteobacteria as well as in the Firmicutes . Several of the sequences found matched those from isolates, suggesting that the molecular evidence reported culturable organisms . However, a number of sequences did not have matching sequences from isolates, suggesting that non-culturable or as-yet-uncultured endophytic organisms were being detected.

FEMS Microbiol Lett, 2002 Apr 23, 210(1), 111 - 4
The Agrobacterium tumefaciens T pilus composed of cyclic T pilin is highly resilient to extreme environments; Lai EM et al.; Agrobacterium tumefaciens T pili are long semi-rigid, flexuous filaments of 10 nm diameter that are primarily composed of T pilin cyclized protein subunits . The cyclic character of T pilin apparently confers a high level of structural stability on the T pilus . Purified T pili subjected to extreme environmental conditions such as acid and alkali, including glycerol remained relatively unaffected morphologically . T pili lost their semi-rigidity when subjected to high temperatures and high pH, and dissociated into donut shaped subunits when exposed to Triton X-100 . Sodium dodecyl sulfate increased the uptake of uranyl acetate exposing a 2 nm wide lumen running the length of the T pilus filament.

J Biol Chem, 2002 Aug 9, 277(32), 28959 - 71 Epub 2002 May 17.
Expression cloning and characterization of the C28 acyltransferase of lipid A biosynthesis in Rhizobium leguminosarum; Basu SS et al.; An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety . An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, and Sinorhizobium meliloti but not Escherichia coli . The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2 ((3-deoxy-d-manno-octulosonic acid)2)-lipid IV(A) . We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening approximately 4000 lysates of individual colonies of an R . leguminosarum 3841 genomic DNA library in the host strain S . meliloti 1021 . In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced . Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous . Nine other open reading frames around lpxXL were also sequenced . Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes . AcpXL purified from S . meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate . Expression of lpxXL in E . coli behind a T7 promoter results in overproduction in vitro of the expected R . leguminosarum acyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-d-manno-octulosonic acid)2-lipid IV(A) as the acceptor . These findings confirm that lpxXL is the structural gene for the C28 acyltransferase . LpxXL is distantly related to the lauroyltransferase (LpxL) of E . coli lipid A biosynthesis, but highly significant LpxXL orthologues are present in Agrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms.

Mol Cells, 2002 Apr 30, 13(2), 209 - 20
Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants; Wi SJ et al.; The amount of polyamines (such as putrescine, spermidine, and spermine) increased under environmental stress conditions . We used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants . Because there is a metabolic competition for S-adenosylmethionine as a precursor between polyamine (PA) and ethylene biosyntheses, it was expected that the antisense-expression of ethylene biosynthetic genes could result in an increase in PA biosynthesis . Antisense constructs of cDNAs for senescence-related 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CAS) and ACC oxidase (CAO) were isolated from carnation flowers that were introduced into tobacco by Agrobacterium-mediated transformation . Several transgenic lines showed higher PA contents than wild-type plants . The number and weight of seeds also increased . Stress-induced senescence was attenuated in these transgenic plants in terms of total chlorophyll loss and phenotypic changes after oxidative stress with hydrogen peroxide (H2O2), high salinity, acid stress (pH 3.0), and ABA treatment . These results suggest that the transgenic plants with antisense CAS and CAO cDNAs are more tolerant to abiotic stresses than wild-type plants . This shows a positive correlation between PA content and stress tolerance in plants.

Indian J Exp Biol, 2001 Dec, 39(12), 1263 - 7
Genetic transformation and hairy root induction in Holostemma ada-kodien K . Schum--a vulnerable medicinal plant; Karmarkar SH; Hairy roots were induced from shoot buds and seedling hypocotyls of Holostemma by infection with agropine type Agrobacterium rhizogenes strains . Type of explant, Agrobacterium rhizogenes strain used for infection, co-culture time and photoperiod influenced the transformation frequencies . Hairy roots were induced from seedling hypocotyls and shoot bud explants upon infection with agropine type Agrobacterium rhizogenes strains . The hairy roots were thin, whitish in colour and showed negatively geotropic growth . The transformed nature of hairy roots was confirmed by opine analysis.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 1129 - 39
Optimum conditions for transformed Panax ginseng hairy roots in flask culture; Jeong GT et al.; Panax ginseng hairy roots were transformed by Agrobacterium rhizogenes KTCT 2744 . They showed an active branching pattern and fast growth in hormone-free medium, and good growth at 23 degrees C, pH 5.8, 1/2 MS medium, and 3% sucrose . Sucrose provided the highest growth among seven carbon sources tested . Six complex media were also tested . In the combined sugar study, hairy roots grew better on sucrose without glucose or fructose than with glucose or fructose . In the 1/2 MS basal medium, 30 mM in nitrogen and 0.62 mM phosphate salt concentration was the optimum . The growth ratio was maximal at an inoculum size of 0.4% (w/v) . Crude saponin and polysaccharide levels were also measured.

Appl Biochem Biotechnol, 2002 Spring, 98-100, 1115 - 27
Studies on mass production of transformed Panax ginseng hairy roots in bioreactor; Jeong GT et al.; The growth properties of Panax ginseng hairy roots transformed by Agrobacterium rhizogenes were compared between flask and aerated column or stirred bioreactor . In flask cultures, sucrose, initially 30 g/L, was nearly exhausted after 45 d of culture . The pH of the medium dropped from 5.5 to 4.96 after 10 d, but afterward it gradually increased to 6.4 . After 45 d, hairy roots grew about 16-folds . The growth rate of hairy roots in air-bubble column or stirred bioreactor cultures was 1.13 (1.11) to 1.23 (1.20) g fresh wt (dry wt)/(g of cells x d), respectively . For both bioreactors, growth was about three times as high as in the flask cultivation.

Yao Xue Xue Bao, 1998 Nov, 33(11), 869 - 72
{Anthraquinone production and analysis in the hairy root cultures of Rheum palmatum L.}; Chang Z et al.; Hairy root culture of the medicinal plant Rheum palmatum L . was established by genetic transformation with Agrobacterium rhizogenes . The effects of various media with different pH on growth of the hairy roots and biosynthesis of free anthraquinones were investigated . The experimental results showed that MS agar medium with pH 5.5-5.8 is suitable for growth of the hairy roots . Dark condition is favourable and 62.5-fold increase in fresh weight was reached within a culture period of 25 days . Auxin (0.1 mg.L-1 IAA) activated the hairy root growth but inhibited the biosynthesis of free anthraquinones . About 28% of the total free anthraquinones was released into the liquid medium from the rhubarb hairy roots.

Planta, 2002 May, 215(1), 79 - 89 Epub 2002 Jan 23.
4-Hydroxycinnamoyl-CoA hydratase/lyase, an enzyme of phenylpropanoid cleavage from Pseudomonas, causes formation of C(6)-C(1) acid and alcohol glucose conjugates when expressed in hairy roots of Datura stramonium L; Mitra A et al.; 4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L . established by transformation with Agrobacterium rhizogenes . The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside . In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass . No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected . There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates . There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol . The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.

Plant Physiol, 2002 May, 129(1), 169 - 80
A plant gene up-regulated at rust infection sites; Ayliffe MA et al.; Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection . Infection of transgenic plants containing a beta-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites . The level of induction reflects the extent of fungal growth . In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression . Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection . Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment . Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm . Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%-82%) to the FIS1 protein . The Arabidopsis homologue has been reported to encode a delta1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate . RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust . The rust-induced genes homologous to fis1 are present in many plants . The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.

Plant Physiol, 2002 May, 129(1), 13 - 22
Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system; Frame BR et al.; We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system . Immature zygotic embryos of the hybrid line Hi II were infected with A . tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine . Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation . The average stable transformation efficiency (no . of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5% . Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events . To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A . tumefaciens standard binary vector system.

J Bacteriol, 2002 Jun, 184(11), 3126 - 9
Roles of internal cysteines in the function, localization, and reactivity of the TraV outer membrane lipoprotein encoded by the F plasmid; Harris RL et al.; We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein . Each was mutated to a serine separately and together to yield three mutant traV genes: traV(C10S), traV(C18S), and traV(C10S/C18S) . All three cysteine mutations complemented a traV mutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of the traV(C18S) and, especially, traV(C10S/C18S) mutant strains were significantly less than those of the traV(+) and traV(C10S) strains . Thus, unlike the Agrobacterium tumefaciens T-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity . By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane . However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraV(C18S) mutant were shown to form mixed disulfides with numerous cell envelope proteins . This was not observed with the TraV(C10S) or TraV(C10S/C18S) proteins . Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation . Finally, whereas the TraV(C10S) and TraV(C18S) proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraV(C10S/C18S) protein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions . Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.

J Bacteriol, 2002 Jun, 184(11), 3086 - 95
Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A; Sullivan JT et al.; The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts . It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element . We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M . loti strain MAFF303099 . The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains . The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer . Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb . The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon . The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island . The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes . Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.

Biotechnol Bioeng, 2002 Jun 30, 78(7), 779 - 93
Modeling and kinetic analysis of the reaction system using whole cells with separately and co-expressed D-hydantoinase and N-carbamoylase; Park JH et al.; We developed a kinetic model that describes a heterogeneous reaction system for the production of D-p-hydroxyphenylglycine from D,L-p-hydroxyphenyl-hydantoin using D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoylase of Agrobacterium tumefaciens NRRL B11291 . As a biocatalyst, whole cells with separately or co-expressed enzymes were used . The reaction system involves dissolution of substrate particles, enzymatic conversion, racemization of the L-form substrate, and transfer of the dissolved substrate, intermediate, and product through the cell membrane . Because the two enzymes have different pH optimum, kinetic parameters were evaluated at different pH for the reaction systems . The model was simulated using the kinetic parameters and compared with experimental data, and it was found that the kinetic model well describes the behavior of the reaction systems using whole cells with separately and co-expressed enzymes . Factors affecting the kinetics of the reaction systems were analyzed on the basis of the kinetic model . In the reaction system with separately expressed enzymes, racemization rate and transport of the reaction intermediate (N-carbamoyl-D-p-hydroxyphenylglycine) were revealed to be the limiting factors at neutral pH, resulting in accumulation of intermediate in the reaction medium . At alkaline condition, on the other hand, inhibition of N-carbamoylase by ammonia was severe, and thereby the reaction rate significantly reduced . In the co-expressed enzyme system, accumulation of intermediate was negligible in the reaction medium, and the improved performance was observed compared to that with separately expressed enzymes . The present model might be applied for the optimization and development of the reaction system using two sequential enzymes .

Trends Plant Sci, 2002 May, 7(5), 193 - 5
GATEWAY vectors for Agrobacterium-mediated plant transformation; Karimi M et al.; Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species . Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E . coli and Agrobacterium . Because these vectors are generally large, cloning can be time-consuming and laborious . Recently, the GATEWAY conversion technology has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids.

Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 69 - 73
{Hairy-root culture of Polygonium multiflorum Thunb . and the production of its active constituents-anthraquinones}; Wang L et al.; Transformed hairy roots of Polygonium multiflorum Thunb . were obtained by the transformation of Agrobacterium rhizogenes LBA9402 . It was clearly demonstrated that T-DNA of A . Rhizogenes Ri plasmid was integrated into the cells of hairy roots by the experiments of PCR and Southern hybridization . Through the screen of basic medium and the research of growth curve of hairy roots, the optimum inoculum time was selected in 30 days or so in the optimum condition-MS medium . On the condition, the content of rhein was 2.85 folds higher in hairy roots than that of natural plants by means of HPLC.

J Exp Bot, 2002 May, 53(371), 1143 - 54
Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis; Pavlovkin J et al.; Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles . The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays . To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (E(m)) in cross-sections were mapped . The measured cells were identified by injected Lucifer Yellow . Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to E(m) values of about -170 mV, which suggest high plasma membrane proton pump activities . Rapidly dividing cells of cambia or small tumour parenchyma cells had low E(m) . The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential . The lowest values were recorded in stem pith cells . Cell K(+) concentrations largely matched the respective E(m) . The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements . The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells . TTPs were inhibited by addition of NaN(3), CN(-) plus SHAM or N(2) gas in the xylem perfusion solution and by external N(2) flushing . The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.

FEBS Lett, 2002 Apr 10, 516(1-3), 179 - 82
Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types; Selth LA et al.; We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA . However, the reproducibility of this observation with other geminiviruses has been questioned . Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium . Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present . Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid . These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E . coli.

Appl Microbiol Biotechnol, 2002 Mar, 58(4), 446 - 53
High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system; Chao YP et al.; The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system . We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter . As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock . In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C . Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case . Using this approach, the recombinant protein accounted for 20% of total protein content of the cell . These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability . In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow . By applying the two-step temperature change . a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained . This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale . Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.

Mol Microbiol, 2002 Mar, 43(6), 1523 - 32
Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4; Kumar RB et al.; Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants . VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore . To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus . In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes . In this study, we demonstrate that VirD4 has a unique cellular location . In immunofluorescence microscopy, cells probed with anti-VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole . Polar location of VirD4 was not dependent on T-DNA processing, the formation of the transport apparatus and the presence of other Vir proteins . VirD4 is an integral membrane protein with one periplasmic domain . The large cytoplasmic region contains a nucleotide-binding domain . To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer . A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer . Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide-binding domains are required for polar localization of VirD4 . The periplasmic domain mutant VirD4Delta36-61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles . Polar location of VirD4 suggests a role for the cell pole in DNA transfer.

Mol Plant Microbe Interact, 2002 Mar, 15(3), 292 - 300
Molecular determinants required for the avirulence function of AvrPphB in bean and other plants; Tampakaki AP et al.; The avirulence gene avrPphB from Pseudomonas syringae pv . phaseolicola determines incompatibility, manifested as a hypersensitive reaction (HR), on bean cultivars carrying the R3 resistance gene and also confers avirulence on other plants . The AvrPphB protein carries an embedded consensus myristoylation motif and is cleaved in bacteria and certain plants to yield fragments of about 6 and 28 kDa . We investigated plant recognition and type III translocation determinants in AvrPphB by constructing three N-terminally truncated and two site-directed mutants carrying substitutions in the conserved G63 residue of the myristoylation motif, which lies adjacent to the proteolytic cleavage site . The peptides were either delivered to plant cells by pseudomonads or were expressed transiently in planta via the Agrobacterium tumefaciens or Potato virus X . The 63 amino terminal residues were required for type III-mediated translocation from Pseudomonas strains to the plant, but were partially dispensable for effector recognition following in planta expression . Substitution of the G63 residue resulted in differential HR phenotypes in two different R3 cultivars of bean and abolished effector processing in Pseudomonas strains . Agrobacterium-mediated expression of the mutant proteins elicited HR in resistant bean hosts and in tomato but elicited no reaction in Nicotiana species.

Plant Physiol, 2002 Apr, 128(4), 1291 - 302
Ocatin . A novel tuber storage protein from the andean tuber crop oca with antibacterial and antifungal activities; Flores T et al.; The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized . Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8 . This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops . Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus) . Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function . Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family . Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens.

FEBS Lett, 1971 May 10, 14(4), 245 - 250

Daussant J, Roussaux J, Manigault P.
Two auxin oxidases with distinct antigenic specificities have been identified in extracts of plant tumor induced in Datura stramonium by Agrobacterium tumefaciens . Auxin oxidase and peroxidase activities are demonstrated for both enzymes . The adaptation of an immunochemical method allows the quantitative evaluation of both enzymes in different plant tissue extracts . The amount of these enzymes which is low in the organ of the healthy plant increases during tumorisation, healing and in vitro culture of healthy tissues.

FEBS Lett, 2002 Mar 27, 515(1-3), 114 - 8
Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant; Merle C et al.; Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule . In continuation of our previous work {Ruggiero et al . (2000) FEBS Lett . 469, 132-136}, here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H) . This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through co-expression with an animal cell-derived modifying enzyme . We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37 degrees C.

Cell Mol Biol Lett, 2002, 7(1), 19 - 29
The production of transgenic potato plants expressing human alpha-interferon using lipofectin-mediated transformation; Sawahel WA; The lipofectin system was used to transform potato protoplasts with plasmid DNA (pIG3031), which contains human alpha-interferon cDNA and codes for the selectable neomycin phosphotransferase II gene (NPT II) . Both genes are under the control of the bi-directional plant active transcriptional promoter from Agrobacterium tumefaciens T-DNA . The criteria of phenotype stability after selective pressure removal, in vitro activity assay of NPT II, the biological analysis of alpha-interferon activity, Southern blot analysis and RNA slot blot were used to confirm the mitotic stability of the foreign gene and its expression and stable integration into the host genome . These studies demonstrate that human alpha-interferon cDNA can be correctly expressed in potato cells.

Biotechnol Prog, 2002 Mar-Apr, 18(2), 394 - 400
Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter; Chao YP et al.; The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter . To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter . Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol . This protein yield accounts for 30% of total cell protein content . In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome . In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis . In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase . Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase . As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale . Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.

Chembiochem, 2002 Apr 2, 3(4), 311 - 7
Ratcheting up vir gene expression in Agrobacterium tumefaciens: coiled coils in histidine kinase signal transduction; Wang Y et al.; The transmembrane histidine kinase VirA is responsible for the recognition of information from several plant-derived xenognostic signals that control gene transfer between Agrobacterium tumefaciens and its eukaryotic host . As with other histidine autokinases, VirA appears to exist as a homodimer within the inner membrane of the bacterium . In this study, we identify the putative homodimeric coiled-coil-like motifs Helix TM2 (amino acids (aa) 259-288) and Helix C (aa 293-327) within the previously assigned signal input domain . The functional importance of these coiled-coil interactions in signal-mediated VirA activation is investigated by the construction of fusion proteins with the leucine zipper domain of the transcription factor GCN4 . Replacement of the membrane-spanning and periplasmic domains of VirA with the GCN4 leucine zipper gave functional proteins with increased signal-induced vir gene expression . When the GCN4 fusion was used to conformationally bias the interface of the Helix C coiled coil, constitutively active chimeras were created . The activity of these constructs was dependent on the interface of the Helix C coiled coil, and a ratchet model is proposed in which VirA activation is achieved by signal-induced switching of the interfaces of the homodimer . Since VirA functions as a transducer and integrates various host cues indirectly, these data highlight its role as an "antenna" for the tumor-inducing (Ti) plasmid, able to monitor the host proteome so as to select for successful xenognostic signaling strategies.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 573 - 86
A mathematical method for determining genome divergence and species delineation using AFLP; Mougel C et al.; The delineation of bacterial species is presently achieved using direct DNA-DNA relatedness studies of whole genomes . It would be helpful to obtain the same genomically based delineation by indirect methods, provided that descriptions of individual genome composition of bacterial genomes are obtained and included in species descriptions . The amplified fragment length polymorphism (AFLP) technique could provide the necessary data if the nucleotides involved in restriction and amplification are fundamental to the description of genomic divergences . Firstly, in order to verify that AFLP analysis permits a realistic exploration of bacterial genome composition, the strong correspondence between predicted and experimental AFLP data was demonstrated using Agrobacterium strain C58 as a model system . Secondly, a method is proposed for determining current genome mispairing and evolutionary genome divergences between pairs of bacteria, based on arbitrary sampling of genomes by using AFLP . The measure of current genome mispairing was validated by comparison with DNA-DNA relatedness data, which itself correlates with base mispairing . The evolutionary genome divergence is the estimated rate of nucleotide substitution that has occurred since the strains diverged from a common ancestor . Current genome mispairing and evolutionary genome divergence were used to compare members of Agrobacterium, used as a model of closely related genomic species . A strong and highly significant correlation was found between calculated genome mispairing and DNA-DNA relatedness values within genomic species . The canonical 70% DNA-DNA hybridization value used to delineate genomic species was found to correspond to a range of current genome mispairing of 13-13.6% . These limits correspond to 0.097 and 0.104 nucleotide substitutions per site, respectively . In addition, experimental data showed that the large Ti and cryptic plasmids of Agrobacterium had little effect on the estimation of genome divergence . Evolutionary genome divergence was used for phylogenetic inferences . Data showed that members of the same genomic species clustered consistently, as supported by bootstrap resampling . On the basis of these results, it is proposed that the genomic delineation of bacterial species could be based, in future, on phylogenetic groups supported by bootstraps and genome descriptions of individual strains, obtained by AFLP analysis, recorded in accessible databases; this approach might eventually replace DNA-DNA hybridization studies.

Proc Natl Acad Sci U S A, 2002 Apr 2, 99(7), 4638 - 43
Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens; Zhang HB et al.; A signal turnover system is an essential component of many genetic regulatory mechanisms . The best-known example is the ubiquitin-dependent protein degradation system that exists in many organisms . We found that Agrobacterium tumefaciens adopts a unique signal turnover system to control exiting from a quorum-sensing mode . A . tumefaciens regulates Ti plasmid conjugal transfer by a quorum-sensing signal, N-3-oxo-octanoyl homoserine lactone (3OC8HSL), also known as Agrobacterium autoinducer . By using Tn5 mutagenesis and a functional cloning approach, we identified two genes that are involved in switching from a conjugal quorum-sensing mode to a nonconjugal mode at the onset of stationary phase . First, we located attJ, which codes for an IclR-type suppressor that regulates the second gene attM . The latter encodes a homologue of N-acylhomoserine lactone (AHL)-lactonase . Mass spectrometry analysis shows that the enzyme encoded by attM is an AHL-lactonase that hydrolyzes the lactone ring of 3OC8HSL . In wild-type A . tumefaciens, attM expression is initially suppressed by AttJ but significantly elevated at the stationary phase accompanied a sharp decline in 3OC8HSL . DNA gel retardation analysis shows that AttJ specifically binds to the promoter that controls AHL-lactonase expression . Mutation of attJ resulted in constitutive production of AHL-lactonase that abolishes 3OC8HSL accumulation and Ti plasmid transfer . These data suggest that A . tumefaciens has a sophisticated multicomponent quorum-sensing signal turnover system, allowing the cell to sense a change in growth and adjust cellular activities accordingly.

J Chem Ecol, 2002 Feb, 28(2), 333 - 41
Characterization and toxicity of Amanita cokeri extract; Drehmel DC et al.; The nonprotein amino acids 2-amino-3-cyclopropylbutanoic acid and 2-amino-5-chloro-4-pentenoic acid were isolated from the mushroom Amanita cokeri . The cyclopropyl amino acid is toxic to the fungus Cercospora kikuchii, the arthropod Oncopeltus fasciatus (milk weed bug), and the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Xanthomonas campestris . Toxicity to bacteria was reversible by addition of isoleucine to the medium . No toxicity was observed for 2-amino-5-chloro-4-pentenoic acid.

Planta, 2002 Feb, 214(4), 653 - 60
Mycorrhizal colonization of transgenic aspen in a field trial; Kaldorf M et al.; Mycorrhizal colonization of genetically modified hybrid aspen (Populus tremula x P . tremuloides Michx.) was investigated over 15 months in a field experiment . The aspen carried the rolC gene from Agrobacterium rhizogenes under control of either the constitutive cauliflower mosaic virus 35S promoter or the light-inducible rbcS promoter . Arbuscular mycorrhizas (AMs) were rare in all root samples, while fully developed ectomycorrhizas (EMs) were found in all samples . No significant differences in the degree of mycorrhizal colonization between aspen lines were seen with either AMs or EMs . The EM community on the release area was dominated by four fungal species that formed more than 90% of all mycorrhizas, while eleven EM types were found occasionally . Mycorrhizal diversity did not differ between transgenic and non-transgenic trees . The structure of mycorrhizal communities was similar for most aspen lines . The sole significant difference was found in the abundance and development of one of the four common EM morphotypes, which was rare and poorly developed on roots from the transgenic aspen line Esch5:35S-rolC-#5 compared with non-transgenic controls . This effect is clone specific as the formation of this EM type was not affected by the transgene expression in the other transgenic line, Esch5:35S-rolC-#1 . This is the first demonstration of a clonal effect influencing the ability of a transgenic plant to form a mycorrhizal symbiosis with a potential fungal partner.

Genes Genet Syst, 2001 Dec, 76(6), 363 - 71
Genome analysis of Agrobacterium tumefaciens: linkage map and genetic features of the left region of the linear chromosome; De Costa DM et al.; In addition to a unique tumor-inducing (Ti) plasmid, the plant pathogenic bacterium Agrobacterium tumefaciens has an unconventional chromosomal organization . Our previous studies on A . tumefaciens MAFF301001 revealed that it possesses a 2 Mb linear and a 2.8 Mb circular chromosome plus a 206.479 kbp Ti plasmid (pTi-SAKURA) . In this study, a linkage map for the left half of its linear chromosome covering a 900 kbp region was constructed and the number of potential genes existing in the region was estimated . The linkage map consists of 31 BAC and 8 lambda phage recombinants without any gaps . It confirmed the size and all the structural landmarks indicated in the corresponding region of our previously constructed physical map for the linear chromosome . Sequencing analysis of the end-regions of each linking clone led to the identification of 6 genes and another 27 potential genes or ORFs, including genes and/or gene clusters responsible for homologous recombination (ruvB), trehalose/maltose sugar transport (thuR, thuG) and alanine catabolism (dadR) . Two virulence-related gene homologues (attK and celB), previously reported in the circular chromosome of a different strain of A . tumefaciens were found in this region . These findings will provide a ready-to-use linkage map for further functional analysis of the linear chromosome.

Yeast, 2002 Apr, 19(6), 529 - 36
Insertional mutagenesis in yeasts using T-DNA from Agrobacterium tumefaciens; Bundock P et al.; Insertional mutagenesis is a powerful tool for the isolation of novel mutations . The gene delivery system of the bacterium Agrobacterium tumefaciens, which mediates transfer not only to plants but also to yeasts and fungi, could be exploited to generate collections of yeasts containing insertional mutations if there were no bias towards particular integration sites, as is the case in plants . To test this, we have analysed a small collection of Saccharomyces cerevisiae strains with T-DNA copies integrated in the S . cerevisiae genome . The position of 54 of these T-DNAs was determined . The T-DNA showed no clear preference for certain DNA sequences or genomic regions . We have isolated insertions in the coding regions of the genes YGR125w, YDR250c, YGR141w, YGR045c, YPL017c, YGR040w, YDL052c, YJL148w, YCL033c, YFL061w, YJR033c, YDR175c and YLR309c confirming that these genes are non-essential for S . cerevisiae haploid growth on minimal medium . Given the advantages of T-DNA, we propose its use as an ideal mobile DNA element for insertional mutagenesis in yeasts .

Mol Genet Genomics, 2002 Mar, 267(1), 115 - 23 Epub 2002 Feb 22.
Identification of a chromosomal tra-like region in Agrobacterium tumefaciens; Leloup L et al.; The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens . Unexpectedly, we found that a pTi-free A . tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies . This suggested that this strain may contain a homologue of the VirD4 protein . A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced . This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A . tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E . coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E . coli, TraG and VirD4 encoded by A . tumefaciens plasmid pTiC58 . The product of orf2 showed no significant similarity to any known protein . Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer . The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.

Mol Microbiol, 2002 Mar, 43(5), 1359 - 65
Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes; Baron C et al.; Type IV secretion systems (TFSS) mediate secretion or direct cell-to-cell transfer of virulence factors (proteins or protein-DNA complexes) from many Gram-negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella tribocorum, Bordetella pertussis, Brucella suis, Helicobacter pylori, Legionella pneumophila and Rickettsia prowazekii, into eukaryotic cells . Bacterial conjugation is also classified as a TFSS-like process mediating the spread of broad-host-range plasmids between Gram-negative bacteria such as RP4 and R388, which carry antibiotic resistance genes . Genetic, biochemical, cell biological and structural biology experiments led to significant progress in the understanding of several aspects of TFSS processes . X-ray crystallography revealed that homologues of the A . tumefaciens inner membrane-associated proteins VirB11 and VirD4 from H . pylori and R388, respectively, may form channels for substrate translocation or assembly of the transmembrane TFSS machinery . Biochemical and cell biological experiments revealed interactions between components of the periplasmic core components VirB8, VirB9 and VirB10, which may form the translocation channel . Analysis of A . tumefaciens virulence proteins VirE2 and VirF suggested that the periplasmic translocation route of the pertussis toxin from B . pertussis may be more generally valid than previously anticipated . Secretion and modification of toxins from H . pylori and L . pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology . Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge . TFSS-carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell-cell interactions.

Plant Cell Physiol, 2002 Mar, 43(3), 323 - 30
Involvement of the VEP1 gene in vascular strand development in Arabidopsis thaliana; Jun JH et al.; A dominant mutant line characterized by abnormal leaf venation pattern was isolated from a transgenic Arabidopsis plant pool that was generated with Agrobacterium culture harboring an Arabidopsis antisense cDNA library . In the mutant line, the phenotype was due to antisense suppression of a gene we named VEP1 (Vein Patterning) . The predicted amino acid sequence of the gene contained a motif related to the mammalian death domain that is found in the apoptotic machinery . Reduced expression of the VEP1 gene resulted in the reduced complexity of the venation pattern of the cotyledons and foliar leaves, which was mainly due to the reduced number of the minor veins and their incomplete connection . The analysis of mutant embryos indicated that the phenotype was originated, at least in part, from a defect in the procambium patterning . In the mutant, the stem and root were thinner than those in wild type . This phenotype was associated with reduced vascular development . The promoter activity of the VEP1 gene was detected preferentially in the vascular regions . We propose that the death domain-containing protein VEP1 functions as a positive element required for vascular strand development in Arabidopsis thaliana.

Appl Environ Microbiol, 2002 Apr, 68(4), 1919 - 24
Towards growth of arbuscular mycorrhizal fungi independent of a plant host; Hildebrandt U et al.; When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G . Becard and J . A . Fortin (New Phytol . 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up . These bacteria were able to support growth of the fungus on the agar plates . In the presence of P . validus, hyphae branched profusely and formed coiled structures . These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes . The presence of P . validus alone also enabled G . intraradices to form new spores, mainly at the densely packed hyphal coils . The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei . In these experiments P . validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7 . Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P . validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.

Environ Pollut, 2002, 117(2), 329 - 35
Tolerance and metabolism of phenol and chloroderivatives by hairy root cultures of Daucus carota L; Santos D et al.; Hairy root cultures are shown to be suitable experimental systems to screen higher plants for tolerance to various inorganic and organic pollutants, and for determining the role of the root matrix in the uptake and further metabolism of contaminants . A number of clones were obtained by infection of carrot tissues with Agrobacterium rhizogenes and two (the fastest and the slowest growing root clones) were chosen for further experimentation . Both clones showed a similar degree of tolerance towards phenol and its chlorinated derivatives, i.e . the growth of root biomass was maintained in concentrations of phenol equivalent to 1000 micromol/l, whilst the chlorophenols were tolerated only at concentrations 20 times lower (50 micromol/l) . Transformed carrot roots were able to remove more than 90% of the exogenous phenolic compounds from the culture medium within 120 h after treatment . Metabolism of these compounds occurred in the root tissue and was accompanied by an increase in peroxidase activity.

Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 2001, (119), 52 - 6
Isoquinoline alkaloid production by transformed cultures of Papaver somniferum; Yoshimatsu K et al.; Three clones of transformed cultures of opium poppy (Papaver somniferum L.) were established by infection with Agrobacterium rhizogenes MAFF 03-01724 . MAFF clone 1 being capable of forming somatic embryos was selected and its growth and isoquinoline alkaloid production was investigated . The illumination, temperature and nutrient medium composition greatly affected growth, cell morphology and alkaloid accumulation . The MAFF clone 1 cultured in Root Culture medium in the dark at 22 degrees C accumulated a high quantity of sanguinarine (652 micrograms/g dry weight) though the growth was poor (4.4 fold as fresh weight basis after 2 months of culture) . The MAFF clone 1 cultured in a quarter macro salt strength Woody Plant medium under 14 h/day light at 22 degrees C developed into plantlets and accumulated significant quantity of codeine (648 micrograms/g dry wt) together with papaverine, noscapine, and sanguinarine . This clone was applied to a rotating drum fermenter (2 L working volume), and ca . 0.3 mg codeine and 0.06 mg sanguinarine were obtained after 4 weeks of culture . One quarter of the codeine produced was found in the culture medium.

Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 658 - 62
{Cloning of HAL1 gene and characterization for salt tolerance tomato}; Zhang Q et al.; The HAL1 gene was cloned by PCR strategy and confirmed by sequencing . Its open read frame is 879 bp, encoding a peptide of 294 amino acids (32 kD Protein) . A chimaeric construct of HAL1 and Npt II (neomycin phosphotransferase II) was constructed and introduced into commercial cultivars of tomato (Zhong SU No . 5: Lycopersicon escullentum) by Agrobacterium tumefacien-mediated gene transformation . Transformants were selected for their ability to grow and root on media containing kanamycin . Transformation was confirmed by analysis of PCR, Southern blot and RT-PCR . The salt tolerance of transgenic tomato is evaluated by comparing the fresh weight, dry weight, Na+, K+ content of transgenic tomato and control tomato . It is concluded that the over-expressing of HAL1 in tomato could enhance the salt tolerance of the transgenic tomato.

Phytochemistry, 2002 Apr, 59(7), 697 - 702
Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene; Moyano E et al.; Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants . The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4 . The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively . The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.

Yi Chuan Xue Bao, 2002 Feb, 29(2), 166 - 74
Growth and differentiation of transgenic callus regulated by phytohormones and antibiotics in transformation of loblolly pine; Tang W et al.; Mature zygotic embryos of loblolly pine (Pinus taeda L.) were transformed by Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pBI121 which carried the selectable marker gene, neomycin phosphotransferase II (npt II) controlled by the promoter of the nopaline synthase gene, and the uidA reporter gene, encoding beta-glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter . Organogenic transgenic calli and transgenic regenerated plantlets were produced on selection medium containing 15 mg/L kanamycin, and confirmed by GUS histochemical staining, polymerase chain reaction (PCR), and southern blot analysis . Influences of phytohormone (BA/IBA) and antibiotics on growth and differentiation of organogenic transgenic calli were investigated . Of the phytohormone (BA/IBA) and antibiotics administered, 500 mg/L carbenicillin combined with 2 mg/L BA and 0.5 mg/L IBA (BA/IBA = 4) resulted in a 54.2% higher increase in the growth of transgenic calli as well as in the differentiation of transgenic calli, which was 45.7% more than that of control on the 6th week of culture . Claforan at 500 mg/L combined with 2 mg/L BA and 0.5 mg/L IBA resulted in a 40.8% increase in the growth of transgenic calli, and 38.7% increase in the frequency of transgenic calli forming adventitious shoots compared with the control . The growth and differentiation of transgenic calli of loblolly pine was reduced preferentially by higher BA/IBA (BA/IBA = 8), as well as high concentration of antibiotics (carbenicillin and claforan, 550 mg/L each) . But it was observed that 450 mg/L and 500 mg/L carbenicillin and claforan caused an increase in growth and differentiation of transgenic calli . These results suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine was also dependent on the regulation of phytohormone and antibiotic on growth and differentiation of transgenic calli . This work could be useful for the future studies of genetic transformation of conifers.

Genetika, 2002 Feb, 38(2), 274 - 7
{Transgenic tobacco (Nicotiana tabacum SR1) plants expressing the gene coding for Serratia marcescens nuclease}; Trifonova EA et al.; The gene coding for the secreted Serratia marcescens endonuclease was fused with the mannopine synthase promoter of Agrobacterium tumefaciens Ti plasmid and transferred to Nicotiana tabacum SR1 plants . The promoter is leaf- and root-specific . The resulting transgenic plants demonstrated elevated nuclease activity . The level of the transgene product was determined in the transgenic lines.

EMBO J, 1983, 2(3), 419 - 26
Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models; Dhaese P et al.; Sequences in the 3'-untranslated region of two different octopine T-DNA genes were analyzed with regard to their significance in polyadenylation . Poly(A) addition sites were localized precisely by S1 nuclease mapping with T-DNA-derived mRNAs isolated from tobacco . The gene encoding transcript 7' contains two AATAAA hexanucleotides, respectively 119 bp and 170 bp downstream of the TAA stop codon . A single poly(A) site was mapped 24-25 bp downstream of the first AATAAA . Further, we show that a mutant octopine synthase gene, which has lost part of its 3'-untranslated region by deletion, is still active . This mutant gene terminates 19 bp upstream from the major wild-type polyadenylation site . The deletion also removes the AATAAT signal preceding this site . The mutant octopine synthase gene contains a minimum of four different poly(A) sites . The most prominent of these sites is identical to the minor poly(A) site of the wild-type gene, and is preceded by a sequence AATGAATATA . Three other sites are located within the adjacent plant DNA, giving rise to hybrid T-DNA/plant DNA transcripts . The two most distal sites are probably dependent on a motif AATAAATAAA, found 29 bp away from the T-DNA/plant DNA junction.

EMBO J, 1983, 2(3), 411 - 7
Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens; Van Haute E et al.; Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium . This was accomplished by using E . coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively . For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E . coli to A . tumefaciens with a transfer frequency of 4.5 x 10(-3) . As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination . These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids . pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E . coli . The mutant clones were transmitted from an E . coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid . Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid . A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment . These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.

EMBO J, 1983, 2(3), 403 - 9
The conserved part of the T-region in Ti-plasmids expresses four proteins in bacteria; Schroder G et al.; The T-region of Ti-plasmids expresses four proteins (mol . wts . 74,000, 49,000, 28,000 and 27,000) in Escherichia coli minicells . Promoter activities are determined by sequences within the T-region, and the protein-coding regions map in that part of the T-region which is highly conserved in octopine and nopaline plasmids and which is responsible for shoot and root inhibition when expressed in plant cells . Three of the regions expressed in bacteria correlate with three regions which are transcribed in transformed plant cells; the fourth protein-coding region has no corresponding transcript in plants . At least three of the proteins synthesized in E . coli minicells are also expressed in cell-free systems prepared from E . coli and from Agrobacterium tumefaciens; the fourth protein (mol . wt . 49,000) is poorly expressed in both cell-free extracts . The possibility is discussed that the same genes are expressed in Agrobacteria and in transformed plant cells and that in both cases the gene products mediate growth regulatory effects to non-transformed plant cells.

Gene, 2002 Feb 6, 284(1-2), 113 - 24
An Agrobacterium gene involved in tumorigenesis encodes an outer membrane protein exposed on the bacterial cell surface; Jia YH et al.; A gene designated as aopB was identified which was involved in tumorigenesis of Agrobacterium tumefaciens . aopB is located on the circular chromosome as a single copy . This gene shares high homology with ropB, a Rhizobium leguminosarum gene encoding an outer membrane protein . A transposon mutant CGI1 containing a gfp-tagged transposon insertion at aopB caused attenuated tumors on plants when inoculated at a low cell concentration (5x10(7) cells/ml) . The mutation did not affect the bacterial growth on different media . A broad host range plasmid containing the wild type aopB could restore the tumor formation ability of CGI1 to the wild type level . When both aopB-gfp and aopB-phoA fusions were used to study the aopB gene expression, we found that the aopB gene was inducible by acidic pH but not by plant phenolic compound acetosyringone . aopB encodes a putative protein of 218 amino acids with a predicted molecular weight of 22.8 kDa . TnphoA transposon mutagenesis of aopB, subcellular fractionation and whole cell ELISA experiments indicated that AopB is an outer membrane protein exposed on the bacterial cell surface . It appeared that AopB was exclusively present in the outer membrane and not in other fractions . The vir gene induction assays showed that the aopB gene was not required for the expression of the Ti plasmid encoded vir genes that are essential for tumorigenesis . The C-terminal half of AopB is slightly homologous to some of the bacterial porin proteins and some of plant dehydrins . The role of AopB in Agrobacterium-plant interaction is discussed.

Int J Med Microbiol, 2002 Feb, 291(6-7), 555 - 60
Bacterial persistence within erythrocytes: a unique pathogenic strategy of Bartonella spp; Seubert A et al.; The genus Bartonella comprises human-specific and zoonotic pathogens responsible for a wide range of clinical manifestations, including Carrion's disease, trench fever, cat scratch disease, bacillary angiomatosis and peliosis, endocarditis and bacteremia . These arthropod-borne pathogens typically parasitise erythrocytes in their mammalian reservoir host(s), resulting in a long-lasting haemotropic infection . We have studied the process of Bartonella erythrocyte parasitism by tracking green fluorescent protein-expressing bacteria in the blood of experimentally infected animals . Following intravenous infection, bacteria colonise a yet enigmatic primary niche, from where they are seeded into the blood stream in regular intervals of approximately five days . Bacteria invade mature erythrocytes, replicate temporarily and persist in this unique intracellular niche for the remaining life span of the infected erythrocytes . A triggered antibody response typically results in an abrogation of bacteremia within 3 months of infection, likely by blocking new waves of bacterial invasion into erythrocytes . The recent establishment of genetic tools for Bartonella spp . permitted us to identify several putative pathogenicity determinants . Application of differential fluorescence induction technology resulted in the isolation of bacterial genes differentially expressed during infection in vitro and in vivo, including an unknown family of autotransporter proteins as well as a novel type IV secretion system homologous to the conjugation system of E . coli plasmid R388 . Mutational analysis of a previously described type IV secretion system displaying homology to the virB locus of Agrobacterium tumefaciens provided the first example of an essential pathogenicity locus in Bartonella . Though required for establishing haemotropic infection, it remains to be demonstrated if this type IV secretion system is necessary for colonisation of the primary niche or for the subsequent colonisation of erythrocytes.

Mol Microbiol, 2001 Dec, 42(5), 1337 - 48
Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8; Fischer W et al.; Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8) . The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon . To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes . Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8 . The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8 . In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction . Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI . We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI . We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.

Plant Cell, 2002 Feb, 14(2), 451 - 63
The protein encoded by oncogene 6b from Agrobacterium tumefaciens interacts with a nuclear protein of tobacco; Kitakura S et al.; The 6b gene in the T-DNA from Agrobacterium has oncogenic activity in plant cells, inducing tumor formation, the phytohormone-independent division of cells, and alterations in leaf morphology . The product of the 6b gene appears to promote some aspects of the proliferation of plant cells, but the molecular mechanism of its action remains unknown . We report here that the 6b protein associates with a nuclear protein in tobacco that we have designated NtSIP1 (for Nicotiana tabacum 6b-interacting protein 1) . NtSIP1 appears to be a transcription factor because its predicted amino acid sequence includes two regions that resemble a nuclear localization signal and a putative DNA binding motif, which is similar in terms of amino acid sequence to the triple helix motif of rice transcription factor GT-2 . Expression in tobacco cells of a fusion protein composed of the DNA binding domain of the yeast GAL4 protein and the 6b protein activated the transcription of a reporter gene that was under the control of a chimeric promoter that included the GAL4 upstream activating sequence and the 35S minimal promoter of Cauliflower mosaic virus . Furthermore, nuclear localization of green fluorescent protein-fused 6b protein was enhanced by NtSIP1 . A cluster of acidic residues in the 6b protein appeared to be essential for nuclear localization and for transactivation as well as for the hormone-independent growth of tobacco cells . Thus, it seems possible that the 6b protein might function in the proliferation of plant cells, at least in part, through an association with NtSIP1.

Planta, 2002 Mar, 214(5), 668 - 74 Epub 2001 Dec 12.
Random antisense cDNA mutagenesis as an efficient functional genomic approach in higher plants; Jun JH et al.; Most cellular processes in an organism depend on functions of expressed sequences . Thus, efficient large-scale functional assignment of expressed sequences is crucial for understanding cellular processes . Towards this goal in plants, we designed a "random antisense cDNA mutagenesis (RAM)" approach . In a pilot experiment, 1,000 transgenic plants of Arabidopsis thaliana (L.) Heynh . (ecotype Wassilevskija) expressing random antisense cDNA(s) were generated from Agrobacterium cultures harboring an Arabidopsis antisense cDNA library . We identified 104 mutant lines from the transgenic pool by visual screening . Genetic analysis suggested that 37% of the mutations were likely due to antisense effects . When the cDNA inserts were isolated from 11 mutant lines by polymerase chain reaction and reintroduced into plants to express the antisense transcripts, the original mutant phenotypes were reproduced in 7 cDNA clones . One of the cDNA clones did not generate a database match to any sequence with known functions, but did have a dramatic effect on the architecture of the inflorescence in the antisense transgenic plants . Through the RAM approach, it should be possible to assign a large number of expressed sequences to known in vivo functions in plants.

Microbiology, 2002 Mar, 148(Pt 3), 871 - 81
Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for plasmid-encoded traits; Bartosik D et al.; The replicon of the pTAV3 megaplasmid (approx . 400 kb) of Paracoccus versutus has been localized to a 4center dot3 kb EcoRI restriction fragment and its entire nucleotide sequence determined . The G+C content of the entire sequence is 66 mol%, which is within the range (62-66 mol%) previously determined for P . versutus total DNA . ORF1 encodes a replication initiation protein Rep (47.2 kDa), which shares substantial similarity with putative proteins of the Coxiella burnetii plasmids QpH1 and QpDV, and the replication protein of Pseudomonas syringae plasmid pPS10 . ORF2, located in the opposite transcriptional orientation to ORF1, encodes a putative protein that shares similarity to a subfamily of ATPases involved in plasmid partitioning . The highest similarity was observed with homologous proteins (RepA) encoded by the repABC family of replicons found in several plasmids of Agrobacterium, Rhizobium and Paracoccus spp . The predicted product of ORF3 was similar to AcoR, Nif and NtrC transcriptional activators . A strong incompatibility determinant (inc) was localized between ORF1 (rep) and ORF2 (parA) . The origin of replication of pTAV400 contains a short A+T-rich region and several imperfect palindromic sequences . Curing experiments demonstrated that the megaplasmid bears genes required for growth in minimal media and can therefore be referred to as a mini-chromosome . Megaplasmids pTAV3 of P . versutus UW1 and pKLW2 of Paracoccus pantotrophus DSM 11073 were found to carry closely related, incompatible replicons . It has been shown that plasmid pORI6 (containing oriV of pTAV3 cloned into plasmid pABW1, which does not replicate in Paracoccus spp.) can be trans activated not only by pTAV3, but also by pKLW2 . Using pORI6, it was demonstrated that replication systems related to pTAV3 are also present in the replicons of Paracoccus alcaliphilus JCM 7364, Paracoccus thiocyanatus IAM 12816 and Paracoccus methylutens DM 12.

BMC Biotechnol . 2002;2(1):2 . Epub 2002 Mar 06.
Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes; Herlache TC et al.; BACKGROUND: Agrobacterium vitis is a causal agent of crown-gall disease . Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of alpha-proteobacteria that includes Agrobacterium and its close relatives . The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here . RESULTS: TFX was shown to be inhibitory in vitro against several A . vitis strains . TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A . vitis . F2/5, against three virulent, TFX-sensitive strains of A . vitis tested on Nicotiana glauca . F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios . F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio . When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70% . A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio . CONCLUSIONS: TFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca . This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen.

J Biol Chem, 2002 May 3, 277(18), 15773 - 80 Epub 2002 Feb 27.
Mutations in the occQ operator that decrease OccR-induced DNA bending do not cause constitutive promoter activity; Akakura R et al.; OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid . Positive control of the occ genes occurs in response to octopine, a metabolite released from plant tumors . Octopine causes DNA-bound OccR to undergo a conformational change from an inactive to an active state; this change is marked by a decrease in footprint length from 55 to 45 nucleotides as well as a relaxation of a high angle DNA bend . In this study, we first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA . We then created a series of site-directed mutations in the OccR-binding site . Some mutations were designed to lock OccR-DNA complexes into a conformation resembling the inactive conformation, whereas other mutations were designed to lock complexes into the active conformation . These mutations altered the conformation of OccR-DNA complexes and their responses to octopine in ways that we had predicted . As expected, operator mutations that locked complexes into a conformation having a long footprint and a high angle DNA bend blocked activation by octopine in vivo . Surprisingly, however, mutations that lock OccR into a short footprint and low angle DNA bend failed to cause the protein to function constitutively . Furthermore, some of the latter mutations interfered with activation by octopine . We conclude that locking OccR into a conformation having a short footprint is not sufficient to cause constitutive activation, and octopine must cause at least one additional conformational change in the protein.

Mol Biotechnol, 2002 Feb, 20(2), 131 - 6
Production of HIV-1 p24 protein in transgenic tobacco plants; Zhang GG et al.; The production of antigens for vaccines in plants has the potential as a safe and cost-effective alternative to traditional production systems . Toward the development of a plant-based expression system for the production of human immunodeficiency virus type I (HIV-1) p24 capsid protein, the p24 gene was introduced into the genome of tobacco plants using Agrobacterium tumefaciens-mediated gene transfer . Southern blot analyses confirmed the presence of the p24 coding sequence within the genome of transgenic lines . Western blot analysis of protein extracts from transgenic plants identified plant-expressed p24 protein that cross-reacted with a p24-specific monoclonal antibody, thus confirming the maintenance of antigenicity . Quantification of the p24 protein using enzyme-linked immunosorbent assay (ELISA) estimated yields of approx 3.5 mg per g of soluble leaf protein . Similar accumulation levels of p24 were also detected in T1 plants, confirming that the p24 gene is transmitted stably . Our results indicate that plant-based transgenic expression represents a viable means of producing p24 for the development of HIV vaccine and for use in HIV diagnostic procedures.

Mol Plant Microbe Interact, 2002 Feb, 15(2), 160 - 3
Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive; Bash R et al.; Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan . They are nonattaching and avirulent and show reduced motility at room temperature . At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L . esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots . The mutant bacteria also recovered wild-type motility at lower temperatures . Two other nonattaching mutants of A . tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.

J Bacteriol, 2002 Mar, 184(6), 1772 - 8
Heat shock proteome of Agrobacterium tumefaciens: evidence for new control systems; Rosen R et al.; The regulation of Agrobacterium tumefaciens heat shock genes involves a transcriptional activator (RpoH) and repressor elements (HrcA-CIRCE) . Using proteome analysis and mutants in these control elements, we show that the heat shock induction of 32 (out of 56) heat shock proteins is independent of RpoH and HrcA . These results indicate the existence of additional regulatory factors in the A . tumefaciens heat shock response.

J Bacteriol, 2002 Mar, 184(6), 1597 - 606
raiIR genes are part of a quorum-sensing network controlled by cinI and cinR in Rhizobium leguminosarum; Wisniewski-Dye F et al.; Analysis of N-acyl-L-homoserine lactones (AHLs) produced by Rhizobium leguminosarum bv . viciae indicated that there may be a network of quorum-sensing regulatory systems producing multiple AHLs in this species . Using a strain lacking a symbiosis plasmid, which carries some of the quorum-sensing genes, we isolated mutations in two genes (raiI and raiR) that are required for production of AHLs . The raiIR genes are located adjacent to dad genes (involved in D-alanine catabolism) on a large indigenous plasmid . RaiR is predicted to be a typical LuxR-type quorum-sensing regulator and is required for raiI expression . The raiR gene was expressed at a low level, possibly from a constitutive promoter, and its expression was increased under the influence of the upstream raiI promoter . Using gene fusions and analysis of AHLs produced, we showed that expression of raiI is strongly reduced in strains carrying mutations in cinI or cinR, genes which determine a higher-level quorum-sensing system that is required for normal expression of raiIR . The product of CinI, N-(3-hydroxy-7-cis tetradecenoyl) homoserine lactone, can induce raiR-dependent raiI expression, although higher levels of expression are induced by other AHLs . Expression of raiI in a strain of Agrobacterium that makes no AHLs resulted in the identification of N-(3-hydroxyoctanoyl)-L-homoserine lactone (3OH,C(8)-HSL) as the major product of RaiI, although other AHLs that comigrate with N-hexanoyl-, N-heptanoyl-, and N-octanoyl-homoserine lactones were also made at low levels . The raiI gene was strongly induced by 3OH,C(8)-HSL (the product of RaiI) but could also be induced by other AHLs, suggesting that the raiI promoter can be activated by other quorum-sensing systems within a network of regulation which also involves AHLs determined by genes on the symbiotic plasmid . Thus, the raiIR and cinIR genes are part of a complex regulatory network that influences AHL biosynthesis in R . leguminosarum.

Trends Cell Biol, 2002 Mar, 12(3), 121 - 9
Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium; Tzfira T et al.; Genetic modification of plant cells by Agrobacterium is the only known natural example of DNA transport between kingdoms . While the bacterial factors involved in Agrobacterium infection have been relatively well characterized, studies of their host cellular partners are just beginning . Here, we describe the plant cell factors that might participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process . Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the host factors participating in Agrobacterium infection might contribute to a better understanding of such basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as help expand the host range of Agrobacterium as a genetic engineering tool.

Plant Mol Biol, 2002 Feb 1, 48(3), 223 - 31
Wound-response regulation of the sweet potato sporamin gene promoter region; Wang SJ et al.; Sporamin, a tuberous storage protein of sweet potato, was systemically expressed in leaves and stems by wound stimulation . In an effort to demonstrate the regulatory mechanism of wound response on the sporamin gene, a 1.25 kb sporamin promoter was isolated for studying the wound-induced signal transduction . Two wound response-like elements, a G box-like element and a GCC core-like sequence were found in this promoter . A construct containing the sporamin promoter fused to a beta-glucuronidase (GUS) gene was transferred into tobacco plants by Agrobacterium-mediated transformation . The wound-induced high level of GUS activity was observed in stems and leaves of transgenic tobacco, but not in roots . This expression pattern was similar to that of the sporamin gene in sweet potatoes . Exogenous application of methyl jasmonate (MeJA) activated the sporamin promoter in leaves and stems of sweet potato and transgenic tobacco plants . A competitive inhibitor of ethylene (2,5-norbornadiene; NBD) down-regulated the effect of MeJA on sporamin gene expression . In contrast, salicylic acid (SA), an inhibitor of the octadecanoid pathway, strongly suppressed the sporamin promoter function that was stimulated by wound and MeJA treatments . In conclusion, wound-response expression of the sporamin gene in aerial parts of plants is regulated by the octadecanoid signal pathway.

Virology, 2002 Feb 1, 293(1), 63 - 74
Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus; Saunders K et al.; Two bipartite begomoviruses, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), have been isolated from mosaic-diseased cassava originating from central India and Sri Lanka, respectively . ICMV was transmitted with low efficiency from cassava to Nicotiana benthamiana by sap inoculation to give leaf curl symptoms . SLCMV was much more virulent in this host, producing severe stunting, leaf curl, and chlorosis . These symptoms were reproduced when their cloned genomic components (DNAs A and B) were introduced into N . benthamiana by either mechanical or Agrobacterium-mediated inoculation (agroinoculation) . SLCMV is more closely related to ICMV (DNA A, 84%; DNA B, 94% nucleotide identity) than African cassava mosaic virus (ACMV) (DNA A, 74%; DNA B, 47% nucleotide identity) . Sequence comparisons suggest that SLCMV DNA B originated from ICMV DNA B by a recombination event involving the SLCMV DNA A intergenic region . Pseudorecombinants produced by reassortment of the cloned components of ICMV and ACMV were not infectious in N . benthamiana, emphasising their status as distinct virus species . In contrast, a pseudorecombinant between ACMV DNA A and SLCMV DNA B was infectious . Consistent with these observations, iteron motifs located within the intergenic region that may be involved in the initiation of viral DNA replication are conserved between SLCMV and ACMV but not ICMV . When introduced into N . benthamiana by agroinoculation, SLCMV DNA A alone produced a severe upward leaf roll symptom, reminiscent of the phenotype associated with some monopartite begomoviruses . Furthermore, coinoculation of SLCMV DNA A and the satellite DNA beta associated with ageratum yellow vein virus (AYVV) produced severe downward leaf curl in N . glutinosa and yellow vein symptoms in Ageratum conyzoides, resembling the phenotypes associated with AYVV DNA A and DNA beta infection in these hosts . Thus, SLCMV DNA A has biological characteristics of a monopartite begomovirus, and the virus probably evolved by acquisition of a DNA B component from ICMV.

J Appl Microbiol, 2002, 92(1), 118 - 26
Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR; Weller SA et al.; AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains . METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain . One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids . The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR . CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis . The technique is therefore a rapid and cost-effective detection method . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen . The method can be used as a model system for the detection of other Agrobacterium pathogens.

J Appl Microbiol, 2002, 92(1), 32 - 40
Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis; Wenzel M et al.; AIMS: To demonstrate the occurrence of cellulolytic bacteria in the termite Zootermopsis angusticollis . METHODS AND RESULTS: Applying aerobic cultivation conditions we isolated 119 cellulolytic strains from the gut of Z . angusticollis, which were assigned to 23 groups of aerobic, facultatively anaerobic or microaerophilic cellulolytic bacteria . 16S rDNA restriction fragment pattern and partial 16S rDNA sequence analysis, as well as numerical taxonomy, were used for the assignment of the isolates . The Gram-positive bacteria of the actinomycetes branch could be assigned to the order Actinomycetales including the genera Cellulomonas/Oerskovia, Microbacterium and Kocuria . The Gram-positive bacteria from the order Bacillales belonged to the genera Bacillus, Brevibacillus and Paenibacillus . Isolates related to the genera Afipia, Agrobacterium/Rhizobium, Brucella/Ochrobactrum, Pseudomonas and Sphingomonas/Zymomonas from the alpha-proteobacteria and Spirosoma-like from the "Flexibacteriaceae" represented the Gram-negative bacteria . CONCLUSIONS: A cell titre of up to 10(7) cellulolytic bacteria per ml, determined for some isolates, indicated that they may play a role in cellulose digestion in the termite gut in addition to the cellulolytic flagellates and termite's own cellulases . SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of bacteria on cellulose degradation in the termite gut has always been a matter of debate . In the present survey we investigated the aerobic and facultatively anaerobic cellulolytic bacteria in the termite gut.

Acta Biochim Pol, 2001, 48(3), 657 - 61
Electroporated intact BY-2 tobacco culture cells as a model of transient expression study; Koscianska E et al.; Transfer of foreign genes into plant cells can be accomplished by several methods: agrobacterium-mediated, microinjection, biolistic particle bombardment and electroporation . The last one is frequently used for transfection of plant protoplasts for transient gene expression . Electroporation is a simple procedure and allows transfecting a large number of cells at one time . Square wave-modulated porators are the most efficient for introducing expression cassettes into plant protoplasts . Based on a protocol developed by Wu & Feng (Plant Cell Reports, 1999, 18, 381-386), we optimized conditions for transfection of intact Nicotiana tabacum BY-2 cells using square wave-modulated electroporator . To simplify screening for transfected gene expression we used constructs with a GFP marker gene.

Acta Biochim Pol, 2001, 48(3), 637 - 46
Effect of nuclear matrix attachment regions on transgene expression in tobacco plants; Nowak W et al.; Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains . Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants . The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer . This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121 . Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens . Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs . This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene . However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.

Acta Biochim Pol, 2001, 48(3), 623 - 35
Odyssey of agrobacterium T-DNA; Ziemienowicz A; Agrobacterium tumefaciens, a plant pathogen, is characterized by the unique feature of interkingdom DNA transfer . This soil bacterium is able to transfer a fragment of its DNA, called T-DNA (transferred DNA), to the plant cell where T-DNA is integrated into the plant genome leading to "genetic colonization" of the host . The fate of T-DNA, its processing, transfer and integration, resembles the journey of Odysseus, although our hero returns from its long trip in a slightly modified form.

Nat Prod Lett, 2001, 15(4), 229 - 35
Enhanced podophyllotoxin production from Agrobacterium rhizogenes transformed cultures of Podophyllum hexandrum; Giri A et al.; Podophyllotoxin, a potent chemotherapeutic agent is obtained from Podophyllum hexandrum Royle . Embryos of P . hexandrum were transformed using different strains of Agrobacterium rhizogenes viz . A4, 15834, K599 . Transformed nature of the calli was ascertained and the cultures were further maintained as individual clones . HPLG analysis of transformed cultures depicted a three-fold increase in podophyllotoxin content in comparison to controls.

Arch Biochem Biophys, 2002 Feb 15, 398(2), 203 - 12
Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens); Iwamoto R et al.; A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized . The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols . In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A . radiobacter as template . The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa . The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive) . The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes . Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family . The physicochemical properties of alcohol dehydrogenase from A . radiobacter were characterized as follows . (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate . (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity) . Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0 . (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1544 - 9
The Brucella suis virB operon is induced intracellularly in macrophages; Boschiroli ML et al.; A type IV secretion system similar to the VirB system of the phytopathogen Agrobacterium tumefaciens is essential for the intracellular survival and multiplication of the mammalian pathogen Brucella . Reverse transcriptase-PCR showed that the 12 genes encoding the Brucella suis VirB system form an operon . Semiquantitative measurements of virB mRNA levels by slot blotting showed that transcription of the virB operon, but not the flanking genes, is regulated by environmental factors in vitro . Flow cytometry used to measure green fluorescent protein expression from the virB promoter confirmed the data from slot blots . Fluorescence-activated cell sorter analysis and fluorescence microscopy showed that the virB promoter is induced in macrophages within 3 h after infection . Induction only occurred once the bacteria were inside the cells, and phagosome acidification was shown to be the major signal inducing intracellular expression . Because phagosome acidification is essential for the intracellular multiplication of Brucella, we suggest that it is the signal that triggers the secretion of unknown effector molecules . These effector molecules play a role in the remodeling of the phagosome to create the unique intracellular compartment in which Brucella replicates.

Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 291 - 5
Expression of virB2 protein-containing structures on Agrobacterium in mating cultures; Kurbanova IV et al.; Exocellular structures containing VirB2 proteins were, for the first time, localized on the surface of Agrobacterium by transmission electron microscopy . Using colloidal gold (CG)-labeled VirB2-specific antibodies, it was shown that VirB2 proteins enter into the composition of short surface pili, which emerge at the poles of acetosyringone (AS)-inducedAgrobacterium cells . However, cells of the Ti plasmidless A . tumefaciens strain UBAPF-2 and cells not induced with AS were incapable of pilus synthesis . In suspension, mating Agrobacterium cells were connected together by short thick bridges . It was found that these bridges did not include as part of their structure CG-labeled VirB1 and VirB2 proteins . We did not find the tetracycline-resistant transconjugants after mating of A . tumefaciens donor cells harboring binary systems with plasmid-free A . tumefaciens GM-I9023 in vir-induced and vir-uninduced conditions . However, the same strains can transfer pSUP106 plasmid via a vir-dependent way . We found that activated vir genes slightly stimulate pTd33 plasmid transfer via a tra-dependent pathway to plasmid-free strain UBAPF-2 . It seems, that vir-induced T-DNA/plasmid DNA transfer machinery is not essential for the conjugation process between agrobacterial cells but may participate in this process.

Gene, 2002 Jan 9, 282(1-2), 247 - 55
Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning; Liu YG et al.; The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc . Natl . Acad . Sci . USA 96 (1998) 6535) . To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed . The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses . Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments . Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively . The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively . Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates . A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones . A method for rapid screening of the library II by pooled colony hybridization was developed . A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation . The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.

Mol Genet Genomics, 2002 Jan, 266(5), 732 - 9 Epub 2001 Nov 08.
The transposon Tip100 from the common morning glory is an autonomous element that can transpose in tobacco plants; Ishikawa N et al.; The mutable flaked or a (flaked) (a(f)) line of the common morning glory (Ipomoea purpurea) displays white flowers with colored flakes, and the a(f) mutation is caused by the insertion of a transposable element named Tip100 into the CHS-D gene for anthocyanin biosynthesis . The 3.9-kb Tip100 element belongs to the Ac/Ds family and contains an ORF encoding a polypeptide of 808 amino acids . The frequency and timing of flower variegation vary in different a(f) lines, and a genetic element termed Modulator has been postulated to affect the variegation pattern . Since the pattern of flower variegation is determined by the frequency and timing of excision of Tip100 from the CHS-D gene, we wished to determine whether Tip100 is an autonomous element that is itself capable of transposition in a heterologous host . To do this, we introduced the element into the genome of tobacco plants by Agrobacterium-mediated transformation . The intact Tip100 element was able to excise from its original position in the chromosome and reinsert into new sites in the tobacco genome, whereas an internal deletion derivative was not . Based on these results, we conclude that Tip100 is an autonomous element . We also discuss the nature of the putative Modulator element affecting flower and leaf variegation in various mutable lines of the morning glory.

J Bacteriol, 2002 Feb, 184(4), 1121 - 31
Two opines control conjugal transfer of an Agrobacterium plasmid by regulating expression of separate copies of the quorum-sensing activator gene traR; Oger P et al.; Conjugal transfer of Ti plasmids from Agrobacterium spp . is controlled by a hierarchical regulatory system designed to sense two environmental cues . One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself . This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon . Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine . Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer . However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84 . In this study we showed that this plasmid contains two copies of traR, and each is associated with a different opine-regulated operon . One copy, traR(noc), is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B . Mutating traR(noc) abolished induction of transfer by nopaline but not by the agrocinopines . A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine . The second copy, traR(acc), is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline . Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline . pAtK84b with mutations in both traR genes was not induced for transfer by either opine . However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer . The association of traR(noc) with nox is unique, but the operon containing traR(acc) is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively . We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.

Mol Cells, 2001 Dec 31, 12(3), 407 - 11
Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea; Rho HS et al.; An effective way to study the infection mechanisms of fungal pathogens is to disrupt their genes via transformation in both targeted and random manners . This isolates the mutants that exhibit altered virulence . In this paper, we report the successful transformation of Magnaporthe grisea, the causal agent for rice blast, that is mediated by Agrobacterium tumefaciens . Employing the binary vector pBHt2, which carries the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 500 to > 1,000 hygromycin B-resistant transformants per 1 x 10(6) conidia of M . grisea . The transformation efficiency is correlated with the number of A . tumefaciens cells used, pre-treating bacterial cells with acetosyringone prior to co-cultivation with fungal spores, and the duration of co-cultivation . All of the transformants tested remained mitotically stable, maintaining their hygromycin B resistance after several generations of growth in the absence of hygromycin B . A genomic Southern blot analysis showed that over 60% of the transformants contained a single T-DNA insert on their genome . Considering the efficiency and flexibility of A . tumefaciens-mediated transformation (ATMT), this technique offers highly efficient means for characterizing the genes that are important for the pathogenicity of M . grisea.

Planta, 2001 Dec, 214(2), 196 - 205
Expression of rolB in apomictic Hieracium piloselloides Vill . causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency; Koltunow AM et al.; The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin . Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture . These ectopic meristems formed vegetative rosettes and floral plant organs . Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context . Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct . In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules . The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.

Funct Integr Genomics, 2000 May, 1(1), 25 - 34
Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis; Galbiati M et al.; In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies . A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin) . Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions . T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci . Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions . We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening.

FASEB J, 2002 Mar, 16(3), 408 - 10 Epub 2002 Jan 14.
A carcinoembryonic antigen-specific diabody produced in tobacco; Vaquero C et al.; The feasibility of using tobacco for production of a recombinant antibody (T84.66/GS8 diabody) directed against the carcinoembryonic antigen (CEA) and used for tumor imaging was investigated . Two constructs were generated for targeting the protein either to the apoplast or to the endoplasmic reticulum . Expression of the diabody in tobacco leaves after vacuum-assisted infiltration of engineered Agrobacteria (agro-infiltration) and in regenerated transgenic tobacco plants was analyzed and compared . Results in terms of protein expression and accumulation between both systems showed a good correlation . His6-tagged T84.66 diabody was readily purified from agro-infiltrated tobacco leaves and from transgenic plants by immobilized metal ion affinity chromatography . The purified protein was analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, electrospray mass spectrometry, direct and competition ELISA, electrophoretic mobility shift assay, and staining of CEA-positive colon adenocarcinoma cell line LS174T . Our results demonstrate that tobacco is a competent production system for this clinically relevant diabody.

Cell Res, 2001 Dec, 11(4), 279 - 84
Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi; Wang YM et al.; The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials . Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites . The regenerated plants from hairy root were characterized by normal leaf morphology and stem growth but a shallow and more extensive root system than normal plants . Opine synthesis, PCR and Southern blot confirmed that T-DNA had been integrated into the A . pseudoalhagi genome . Acetosyringone (AS) was found to be vital for successful transformation of A . pseudoalhagi.

Biotechnol Bioeng, 2002 Feb 15, 77(4), 462 - 6
Expression of functional mammalian P450 2E1 in hairy root cultures; Banerjee S et al.; P450 2E1 is an important mammalian liver enzyme known to metabolize a wide range of compounds including several common environmental pollutants . The medicinal plant, Atropa belladonna, was transformed with Agrobacterium rhizogenes containing a binary vector with rabbit P450 2E1 in either the sense or antisense orientation . The resulting "hairy roots" were isolated and grown in liquid medium . Production of P450 2E1 protein was verified in the roots containing the 2E1 gene in the sense orientation . Transgenic and control root cultures were dosed with the environmental pollutant, trichloroethylene (TCE), and were analyzed for the TCE metabolites, chloral and trichloroethanol . The root cultures expressing the mammalian P450 2E1 had increased levels of the metabolites compared to the levels in the control roots . This method represents a quick way to screen transformants for expression of foreign genes before regeneration of whole plants, and also as a possible source of foreign protein for purification .

Biol Sci Space, 1996 Sep, 10(2), 102 - 4
Amyloplast distribution in hairy roots induced by infection with Agrobacterium rhizogenes; Kim YS et al.; To elucidate the rapid and plagiotropic growth of hairy root induced by A . rhizogenes, a root apex was investigated with respect to it's amyloplast deposition, activity of alpha-amylase and glucose content . The amyloplasts distributed in the hairy roots were fewer than those of the adventitious root . Since auxin availability is enhanced in hairy roots, it could affect the statolith degradation by elevating alpha-amylase activity so that the energy requirement for rapid growth could be fulfilled as represented of glucose content . Consequently, it is suggested the overall decrease of starch grains could result in the lack of gravi-response in hairy roots.

Genetics, 2001 Dec, 159(4), 1751 - 63
Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana; McElver J et al.; The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development . More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation . Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations . Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants . More than 4200 mutants with a wide range of seed phenotypes were identified . Over 1700 of these mutants were analyzed in detail . The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis . Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions . The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development . Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 680 - 8
Cloning and characterization of genes from Agrobacterium sp . IP I-671 involved in hydantoin degradation; Hils M et al.; Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase . Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition . Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain . Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene . Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.

Ying Yong Sheng Tai Xue Bao, 2000 Oct, 11(5), 791 - 4
{Factors affecting transformation of Agrobacterium tumefaciens and their application on cereals}; Kong Y et al.; Agrobacterium tumefaciens mediated genetic transformation is the method most widely used in plant transformation . How to improve its transformation efficiency and extend its host range to include most cereals is what people concern about . There are many factors that influence the transformation efficiency, including the wounding response of plants, attachment of bacteria, induction of virulent gene, DNA repair and replication activity of plant cells, state of explant, etc . Recent research has proved that cerelas can be transformed effectively by A . tumefaciens under suitable conditions . This paper reviewed the recent progress in the two aspects.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2037 - 48
Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia; Gaunt MW et al.; The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera . It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole . To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the alpha-Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas . The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support . There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed . There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated . rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination.

In Vitro Cell Dev Biol Plant, 1998 Oct-Dec, 34(4), 310 - 8
Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.); Egnin M et al.; Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated . An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter . Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants . The 'Valencia Select' market type cv . New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner) . The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58 . Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency . The use of cocultivation media containing high auxin {1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D} and low cytokinin {0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA} promoted higher transformation than either hormone-free or thidiazuron-containing medium . The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies . Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones . The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls . Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Acta Crystallogr D Biol Crystallogr, 2002 Jan, 58(Pt 1), 176 - 8 Epub 2001 Dec 21.
Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1; de Jong RM et al.; Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides . Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop vapour-diffusion experiments against 50-70% saturated ammonium sulfate solution at pH 6.5-7.3 . The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 104.5, c = 121.4 A, and contain two monomers in the asymmetric unit . The crystals diffract to 3.0 A resolution with X-rays from a Cu Kalpha rotating-anode generator.

J Agric Food Chem, 2001 Dec, 49(12), 6012 - 9
Sotolone production by hairy root cultures of Trigonella foenum-graecum in airlift with mesh bioreactors; Peraza-Luna F et al.; 3-Hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) and 3-amino-4,5-dimethyl-2(5H)-furanone, the postulated precursor of sotolone, were detected in hairy root cultures of Trigonella foenum-graecum (fenugreek) by GC-MS . The hairy root cultures in both conical flasks and airlift with mesh bioreactors were achieved from hypocotyl of seedling by infection with Agrobacterium rhizogenes . In flasks, the mathematical relationship between hairy root growth and conductivity was established and afterward used to evaluate the biomass evolution in bioreactor cultures due to the difficulty of obtaining direct biomass samples from the bioreactor . The GC-MS analyses of ethanolic extracts from hairy roots revealed the presence of two important compounds: sotolone (1.2% of the volatile fraction) and 3-amino-4,5-dimethyl-2(5H)-furanone (17% of the volatile fraction) . These results point out that biotechnological production of sotolone in bioreactors is possible . Additionally, these hairy root cultures offer, for the first time, an excellent biological model to study the biosynthetic pathway of sotolone in fenugreek.

Science, 2001 Dec 14, 294(5550), 2323 - 8
Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58; Goodner B et al.; Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor . Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant . Thus, A . tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology . Here we describe the genome of A . tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome . We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Science, 2001 Dec 14, 294(5550), 2317 - 23
The genome of the natural genetic engineer Agrobacterium tumefaciens C58; Wood DW et al.; The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids . Extensive orthology and nucleotide colinearity between the genomes of A . tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence . Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement . Availability of the A . tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

J Bacteriol, 2002 Jan, 184(1), 327 - 30
Biogenesis of T pili in Agrobacterium tumefaciens requires precise VirB2 propilin cleavage and cyclization; Lai EM et al.; VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens . The cleaved VirB2 protein is further cyclized to form the T pilin in A . tumefaciens but not in E . coli . Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence . No T pilus was observed in these mutants . The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed.

Hereditas, 2001, 134(2), 97 - 101
Transformation and regeneration capacities for five Nordic barley elite cultivars--evaluation of tissue culture response and transient expression; Roussy I et al.; Using both biolistic and Agrobacterium-mediated DNA delivery, we have investigated the transformation and regeneration capacity for five Nordic elite cultivars of barley . Transformation was followed as transient expression of the uidA or gfp gene in barley callus . Callus formation and regeneration of transformed callus were evaluated based on callus induction frequency, growth rate, callus appearance, and shoot formation frequency . From the accumulated results, one of the elite cultivars has been selected for our ongoing work in molecular breeding of barley.

Microbiol Mol Biol Rev, 2001 Dec, 65(4), 481 - 96, table of contents
Comparative biology of IncQ and IncQ-like plasmids; Rawlings DE et al.; Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts . These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria . These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells . IncQ plasmids are thus highly promiscuous . Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants . These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids . This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids.

Mol Microbiol, 2001 Nov, 42(3), 645 - 57
Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction; Xu XQ et al.; Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell . An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium-plant interaction . In this paper, we report a feedback regulation pathway that controls the expression of katA in A . tumefaciens cells . We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection . This represents a new regulatory factor for catalase induction in bacteria . More importantly, a feedback regulation was observed when the katA-gfp expression was studied in different genetic backgrounds . We found that introduction of a wild-type katA gene encoding a functional catalase into A . tumefaciens cells could repress the katA-gfp expression over 60-fold . The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2 . In addition, the katA-gfp expression of one bacterial cell could be repressed by other surrounding catalase-proficient bacterial cells . Furthermore, mutation at katA caused a 10-fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium . These results suggest that the endogenous H2O2 generated during A . tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria . Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA-gfp expression . The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A . tumefaciens cells.

Protein Expr Purif, 2001 Dec, 23(3), 374 - 9
Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10; Sareen D et al.; The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3) . However, almost 80% of the enzyme remained trapped in inclusion bodies . To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206 . This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain . The histidyl-tagged D-carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein .

Planta, 2001 Oct, 213(6), 981 - 9
Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens; Tang W et al.; Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration . Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine . The frequency of transformation varied among families infected with A . tumefaciens . The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo . Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos . Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03 . 8-1082 and 11-1029 . and 19 transgenic plantlets were established in soil . The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction . Southern blot, and plant DNA/T-DNA junction analysis . These results suggest that an efficient A . tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.

J Biol Chem, 2002 Feb 22, 277(8), 5866 - 74 Epub 2001 Nov 20.
Constitutive mutations of the OccR regulatory protein affect DNA bending in response to metabolites released from plant tumors; Akakura R et al.; OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid and is also an autorepressor . Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors . OccR binds to a site upstream of the occQ promoter in the presence and absence of octopine . Octopine causes prebound OccR to undergo a conformational change at the DNA binding site that causes changes in footprint length and DNA bending . To determine the roles of these conformational changes in transcriptional activation, we isolated 22 OccR mutants that were able to activate the occQ promoter in the absence of octopine . Thirteen of these mutants contained single amino acid substitutions, and nine contained two base pair changes resulting in two amino acid substitutions, which in most cases acted synergistically . These mutations spanned the entire length of the protein . Most of these mutant proteins in the absence of octopine displayed DNA binding and bending properties characteristic of transcriptionally active OccR-octopine complexes.

J Gen Virol, 2001 Dec, 82(Pt 12), 3091 - 7
Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules; Paximadis M et al.; The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa . Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato . Efforts to identify a DNA B component were unsuccessful . These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses . Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus . Abutting primer PCR amplified ca . 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A . Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.

J Virol, 2001 Dec, 75(24), 12288 - 97
Double-stranded RNA-mediated interference with plant virus infection; Tenllado F et al.; Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms . Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay . We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach . We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals . The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not single-stranded RNA . Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.

Phytochemistry, 1998 Nov 20, 49(6), 1821 - 1824
Terpenoids in transformed root culture of Tripterygium wilfordii; Nakano K et al.; Investigation of a hairy root culture of Tripterygium wilfordii var . regelii transformed with Agrobacterium rhizogenes resulted in the isolation of new diterpenoid and sesquiterpenoid together with known triterpenoids . These structures were determined by spectroscopic analyses.

Phytochemistry, 1998 Nov 20, 49(6), 1623 - 1625
Anthraquinones from hairy root cultures of Cassia obtusifolia; Guo H et al.; Hairy root cultures of Cassia obtusifolia clones transformed with Agrobacterium rhizogenes strain 9402 were established to investigate the anthraquinone production . Seven anthraquinones, together with betulinic acid, stigmasterol and sitosterol were isolated from the hairy roots . Their chemical structures were elucidated on the basis of chromatographic and spectral data . The effects of culture conditions and rare earth element Eu(3+) on the production of six free anthraquinones have also been investigated . It was found that changes of the elements in the culture medium and addition of rare earth element Eu(3+) can greatly influence the contents of free anthraquinones in the hairy roots.

Phytochemistry, 1998 Nov 20, 49(6), 1537 - 1548
vir-Gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens; Berthelot K et al.; Expression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds . Several alkylsyringamides have recently been shown to be powerful inducers of these vir-genes . These synthetic amides, and especially ethylsyringamide, are much stronger inducers than syringic acid . In this work, four alkylamides derived from ferulic or sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to induce vir-gene expression on A . tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids . Their effectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occurring amide in plants . Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or beta-alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria of the respective acid at high concentration and thereby increased the vir-inducing potential . However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-gene induction, with the exception of ethylsyringamide at concentrations above 1mM . When tested on Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more efficient than the corresponding phenolic acids but only above 100&mgr;M.

Mol Cells, 2001 Oct 31, 12(2), 221 - 6
Constitutive expression of rice MADS box gene using seed explants in hot pepper (Capsicum annuum L.); Kim S et al.; Two transgenic pepper plants were obtained from 255 seed explants that were infected with Agrobacterium LBA4404 (pGA1209) . One of them (PT2) showed morphological change, such as dwarfism and early flowering by the constitutive expression of the rice OsMADS1 gene . The in vitro condition of the plant regeneration has been optimized from hypocotyl explants on a MS medium that was supplemented with zeatin 3 mg/L, IAA 0.3 mg/L for shoot induction . The optimal rooting condition was at NAA 0.3 mg/L . The transformation frequency was 0.8% from the total hypocotyls . DNA and RNA hybridization analyses showed that the introduced gene was integrated and stably expressed in regenerated plants.

Transgenic Res, 2001 Oct, 10(5), 435 - 44
Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker; Muller A et al.; Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B . Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium . Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure . By Southern blot analysis, integration of the gfp and nptll genes was confirmed . Stable transformation efficiency was 0.1% . From 68 T1 plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene . Expression of gfp was detected in 10 T1 plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis . Expression of the nptll gene was confirmed in 13 T1 plants . The transformation system enables the rapid transfer of agronomically important genes.

EMBO J, 2001 Nov 15, 20(22), 6550 - 8
Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration; van Attikum H et al.; Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites . Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi . The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process . Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome . We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70 . T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 423 - 7
{Construction of plant expression vectors harboring a peptide antibiotic-apidaecin gene and resistance analysis of the transgenic tobacco}; Wang H et al.; Two plant expression vectors(pBinPRHbI and pBinPRSIHbI) were constructed: Firstly, apidaecin gene were fused to the signal peptide coding sequencing of a PR-protein, and cloned into a binary vector pBin438 to form pBinPRHbI . Then, the cassette consisting of 35S promoter, PR signal peptide coding sequencing and apidaecin gene was cut off from pBinPRHbI and inserted into another plant expression vector pBinPRSI to produce a bivalent plant expression vector pBinPRSIHbI . pBinPRSI was constructed previously in our lab and contained PR signal peptide and Shiva-I fusion gene under control of 35S promoter . The three plant expression vectors were introduced into tobacco by Agrobacterium-mediated transformation . The positive rate of PCR was 95% in all putative transgenic plants . Results from Southern blot indicated that foreign genes were integrated into tobacco genome and RT-PCR analysis proved that the foreign gene was transcribed in transgenic tobacco . The transgenic tobacco showed higher resistance to P . syringae pv tabaci, the causal agent of tobacco wild fire disease, than their original cultivars . From the disease index, the transgenic plants carrying apidaecin and Shiva-I genes had highest resistance among three kinds of transgenic plants, and the plants carrying Shiva-I gene alone had lowest resistance.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 380 - 4
{A genetically modified japonica restorer line, C418-Xa21, and its hybrid rice with bacterial blight resistance}; Li XB et al.; The cloned bacterial blight (BB) resistance gene Xa21 was transferred into C418, a major restorer line of japonica hybrid rice in China, using an Agrobacterium-mediated system . The integrated single copy of transgene displayed a 3:1 segregation ratio in T1 generation in PCR and resistance analyses . The transgenic homozygous C418-Xa21 lines were selected in T2 generation through PCR and resistance analyses . The selected transgenic restorer lines were then crossed with a commonly used sterile line, TijinA, to produce Xa21 transgenic hybrid rice . Molecular analysis revealed that the produced hybrid rice, named as Tiyou418-Xa21, inherited the transgene . Both C418-Xa21 and Tiyou418-Xa21 plants displayed high resistance with a broad spectrum to Xoo races and maintained their normal elite agronomic characters . We also observed that the resistance level of Tiyou418-Xa21 was obviously higher than that of C418-Xa21 which may be attributed to their differences in genetic background . The propagation of this BB resistant hybrid variety with the transgene Xa21 with extend hybrid rice production in north China.

Arch Microbiol, 2001 Nov, 176(5), 315 - 22
Ti- and cryptic-plasmid-borne virulence of wild-type Agrobacterium tumefaciens strain CNI5 isolated from chrysanthemum (Dendranthema grandiflora Tzvelev); Ogawa Y et al.; The genetic similarity between pTiBo542 and pTiCNI5, which are harbored, respectively by the supervirulent Agrobacterium tumefaciens strain A281 and by the highly tumorigenic wild-type strain CNI5 isolated from chrysanthemum was investigated by Southern hybridization . pTiCNI5 and pTiBo542 exhibited highly similar hybridization patterns in both BamHI- and EcoRI-digested plasmids by using four vir-region-specific probes, whereas similarity in these two plasmids was not observed by probing with five TL-DNA-specific probes . The characteristics related to tumor formation of cryptic plasmids carried by strain CNI5 were investigated by using single-plasmid-cured derivatives . pTiCNI5-cured derivatives predictably failed to utilize agropine and mannopine and failed to induce tumors on chrysanthemum and tobacco leaf explants, while pAtCNI5a-, pAtCNI5c- and pAtCNI5d-cured derivatives could utilize these opines similar to the parent strain CNI5 . Interestingly, pAtCNI5c- and pAtCNI5d-cured derivatives showed low tumorigenicity in comparison with strain CNI5 or with the pAtCNI5a-cured derivative . These results suggest that the highly virulent phenotype of strain CNI5 may be due to one or more vir genes, which exhibit cartographic similarity to those of pTiBo542 . The results also suggest that the gene(s) related to tumor formation of strain CNI5 may exist not only on pTiCNI5 but also on cryptic plasmids pAtCNI5c and pAtCNI5d.

Annu Rev Phytopathol, 1999, 37, 53 - 80
CROWN GALL OF GRAPE: Biology and Disease Management; Burr TJ et al.; Not until 1973 was it reported that strains of Agrobacterium that cause crown gall disease of grape form a specific group (later characterized as Agrobacterium vitis) . Tumorigenic and nontumorigenic A . vitis have since been isolated from infected and symptomless grapes worldwide . Research on the genetic makeup of A . vitis has led to an improved understanding of pathogen biology and bacterial evolution . In addition, the identification of significant gene sequences has facilitated the development of PCR and RFLP-based identification procedures that continue to improve the detection of A . vitis in plants and soil . Current control practices rely on the use of disease-resistant cultivars, cultural practices that minimize plant injury, and the production of pathogen-free vines . Promising future controls include employment of biological control agents and development of crown gall-resistant transgenic grapevines.

Microbiology, 2001 Nov, 147(Pt 11), 3027 - 35
Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and in the chromosome; Novak KF et al.; Nine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745 . Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively . Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745 . Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA . Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities . Wild-type mating efficiency was 10(-6) transconjugants per donor, while the mutant strain yielded no transconjugants . Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10(-6), while VT747 was unable to mobilize this plasmid . These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport . However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747 . While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both ACT: actinomycetemcomitans strains VT745 and VT747.

Arch Virol, 2001, 146(9), 1811 - 9
Complementation of bipartite begomovirus movement functions by topocuviruses and curtoviruses; Briddon RW et al.; The DNA A components of bipartite species of the genus Begomovirus (family Geminiviridae) encode all viral functions required for replication and encapsidation but require gene products encoded on DNA B for cell-to-cell movement in plants . We demonstrate that geminiviruses of the genera Curtovirus and Topocuvirus are able to trans-complement efficient movement of DNA A for two bipartite begomoviruses in the absence of their corresponding DNA B . It previously has been shown that DNA A can, at low efficiency and without inducing symptoms, spread in the absence of DNA B . The spread of DNA A independent of DNA B, following Agrobacterium-mediated inoculation, is shown to require coat protein whereas trans-complemented spread of DNA A can occur independent of the coat protein encoded on DNA A . The significance of these findings to our understanding of the geminiviral infection cycle is discussed.

J Bacteriol, 2001 Dec, 183(23), 6852 - 61
Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains; Baron C et al.; That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years . Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells . The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect . T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C . Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C . Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins . Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C . Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures . Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins . However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next . To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A . tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C . Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5 . Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 43 - 9
Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens; Hartley CJ et al.; While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli . This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions . Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase . In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium . Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock) . Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated . Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock . Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13437 - 42 Epub 2001 Oct 30.
RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis; Escobar MA et al.; Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide . A . tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors . Specifically, the iaaM and ipt oncogenes, which show approximately 90% DNA sequence identity across studied A . tumefaciens strains, are required for tumor formation . By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development . In in vitro root inoculation bioassays with two biovar I strains of A . tumefaciens, transgenic Arabidopsis lines averaged 0.0-1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis . Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A . tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis . This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor-ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable . If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks.

J Biol Chem, 2001 Dec 28, 276(52), 49449 - 58 Epub 2001 Oct 30.
Inhibition of the Agrobacterium tumefaciens TraR quorum-sensing regulator . Interactions with the TraM anti-activator; Swiderska A et al.; The Agrobacterium tumefaciens quorum-sensing transcriptional regulator TraR and its inducing ligand 3-oxo-octanoyl-l-homoserine lactone control conjugal transfer of the tumor-inducing plasmid, the primary virulence factor responsible for crown gall disease of plants . This regulatory system enables A . tumefaciens to express its conjugal transfer regulon preferentially at high population densities . TraR activity is antagonized by a second tumor-inducing plasmid-encoded protein designated TraM . TraM and TraR are thought to form an anti-activation complex that prevents TraR from recognizing its target DNA-binding sites . The formation and inhibitory function of the TraM-TraR anti-activation complex was analyzed using several different assays for protein-protein interaction, including surface plasmon resonance . The TraR-TraM complex forms readily in solution and is extremely stable (K(D) of 1-4 x 10(-9) m) . Directed mutational analysis of TraM identified a number of amino acids that play important roles in the inhibition of TraR, clustering in two regions of the protein . Interestingly, several mutants were identified that proficiently bound TraR but were unable to inhibit its activity . This observation suggests a mechanistic separation between the initial assembly of the complex and conversion of TraR to an inactive form.

FEMS Microbiol Lett, 2001 Oct 16, 204(1), 163 - 7
Genetic data indicate that proteins containing the GGDEF domain possess diguanylate cyclase activity; Ausmees N et al.; A conserved domain, called GGDEF (referring to a conserved central sequence pattern), is detected in many procaryotic proteins, often in various combinations with putative sensory-regulatory components . Most sequenced bacterial genomes contain several different GGDEF proteins . The function of this domain has so far not been experimentally shown . Through genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common, we present genetic data indicating (a) that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins, and (b) that the activity of cellulose synthase in Rhizobium leguminosarum bv . trifolii and Agrobacterium tumefaciens is regulated by cyclic di-GMP as in Acetobacter xylinum.

Curr Genet, 2001 Sep, 40(2), 152 - 5
Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii; Malonek S et al.; Conidia of the phytopathogenic fungus Calonectria morganii were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, governed by a heterologous fungal promoter and the Aspergillus nidulans trpC terminator . Agrobacterium tumefaciens-mediated transformation yielded stable hygromycin B-resistant clones (average number (106 per 10(7)) {corrected} conidia) . Putative transformants appeared to be mitotically and meiotically stable . The presence of the hph gene was checked by PCR . In four randomly chosen transformants, single-copy integrations of the marker gene at different chromosomal sites were proven by Southern analysis.

J Biol Chem, 2002 Jan 4, 277(1), 462 - 8 Epub 2001 Oct 24.
The cin quorum sensing locus of Rhizobium etli CNPAF512 affects growth and symbiotic nitrogen fixation; Daniels R et al.; Rhizobium etli CNPAF512 produces an autoinducer that inhibits growth of Rhizobium leguminosarum bv . viciae 248 and activates the Agrobacterium tumefaciens tra reporter system . Production of this compound in R . etli is dependent on two genes, named cinR and cinI, postulated to code for a transcriptional regulator and an autoinducer synthase, respectively . NMR analysis of the purified molecule indicates that the R . etli autoinducer produced by CinI is a saturated long chain 3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL . Using cin-gusA fusions, expression of cinI and cinR was shown to be growth phase-dependent . Deletion analysis of the cinI promoter region indicates that a regulatory element negatively controls cinI expression . Mutational analysis revealed that expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex . Besides 3OH-(slc)-HSL, R . etli produces at least six other autoinducer molecules, for which the structures have not yet been revealed, and of which the synthesis requires the previously identified raiI and raiR genes . At least three different autoinducers, including a compound co-migrating with 3OH-(slc)-HSL, are produced in R . etli bacteroids isolated from bean nodules . This is further substantiated by the observation that cinI and cinR are both expressed under symbiotic conditions . Acetylene reduction activity of nodules induced by the cin mutants was reduced with 60-70% compared with wild-type nodules, indicating that the R . etli 3OH-(slc)-HSL is involved in the symbiotic process . This was further confirmed by transmission electron microscopy of nodules induced by the wild type and the cinI mutant . Symbiosomes carrying cinI mutant bacteroids did not fully differentiate compared with wild-type symbiosomes . Finally, it was observed that the cinR gene and raiR control growth of R . etli.

Electrophoresis, 2001 Oct, 22(16), 3413 - 7
Application of capillary electrophoresis to monitor populations of Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 in mixed culture; Yamada K et al.; A bacterial cell mixture ot Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 was analyzed by capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) . Both pherograms showed two discrete peaks . The cells in the peaks were collected, Gram stained, and examined with a microscope . The cells of the two strains were well separated by OGE, and each OGE peak consisted almost entirely of cells of one strain (greater than 98% purity), whereas each CZE peak contained cells of both strains (about 90% purity) . In the concentration range of 1.0 x 10(10) to 1.0 x 10(12) cells/mL, the area of CGE peaks was proportional to the amount of cells . The growth of the two strains in mixed culture was measured by OGE . The OGE quantification data were in good agreement with those obtained using fluorescence in situ hybridization . The CGE analyses were accomplished in 1 h, using a relatively uncomplicated procedure . Thus, OGE exhibited great advantages in accuracy, rapidity, and simplicity.

Cell Res, 2001 Sep, 11(3), 237 - 43
Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.); Tang W; This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.) . Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) . Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated . The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot . The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

Cell Res, 2001 Sep, 11(3), 231 - 6
Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene; He YK et al.; Mercury pollution is a major environmental problem accompanying industrial activities . Most of the mercury released ends up and retained in the soil as complexes of the toxic ionic mercury (Hg2+), which then can be converted by microbes into the even more toxic methylmercury which tends to bioaccumulate . Mercury detoxification of the soil can also occur by microbes converting the ionic mercury into the least toxic metallic mercury (Hg0) form, which then evaporates . The remediation potential of transgenic plants carrying the MerA gene from E . coli encoding mercuric ion reductase could be evaluated . A modified version of the gene, optimized for plant codon preferences (merApe9, Rugh et al . 1996), was introduced into tobacco by Agrobacterium-mediated leaf disk transformation . Transgenic seeds were resistant to HgCl2 at 50 microM, and some of them (10-20% ) could germinate on media containing as much as 350 microM HgCl2, while the control plants were fully inhibited or died on 50 microM HgCl2 . The rate of elemental mercury evolution from Hg2+ (added as HgCl2) was 5-8 times higher for transgenic plants than the control . Mercury volatilization by isolated organs standardized for fresh weight was higher (up to 5 times) in the roots than in shoots or the leaves . The data suggest that it is the root system of the transgenic plants that volatilizes most of the reduced mercury (Hg0) . It also suggests that much of the mercury need not enter the vascular system to be transported to the leaves for volatilization . Transgenic plants with the merApe9 gene may be used to mercury detoxification for environmental improvement in mercury-contaminated regions more efficiently than it had been predicted based on data on volatilization of whole plants via the upper parts only (Rugh et al . 1996).

Genetika, 2001 Sep, 37(9), 1251 - 7
{Hormone-dependent insertional Arabidopsis thaliana mutants with decreased viability and fertility}; Tomilova NB et al.; We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thaliana having drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant) . The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds . The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A . thaliana by exogenous hormones.

Genetika, 2001 Sep, 37(9), 1225 - 32
{Plasmid pSym1-32 from Rhizobium leguminosarum bv . viceae, controlling nitrogen-fixing activity, effectiveness of symbiosis, competitiveness, and acid tolerance}; Kurchak ON et al.; The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarum bv . viceae 1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mob into the plasmidless Agrobacterium tumefaciens Gm1-9023 strain . Plasmid pSym1-32 was transferred into R . leguminosarum bv . viceae strains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen) . Transconjugants formed Fix+ nodules on roots of V . villosa and had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains . Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected . Moreover, the donor strain R . leguminosarum bv . viceae 1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain . Both these properties were transmitted upon transfer of pSym1-32 into the recipient . Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R . leguminosarum bv . viceae.

Genetika, 2001 Sep, 37(9), 1188 - 97
{Tumor formation in plants}; Matveeva TV et al.; The data on genetic tumors in plant species and interspecific hybrids, as well as the problems of Agrobacterium-induced tumors are reviewed . The role of the horizontal gene transfer in the induction of genetic tumors is discussed.

Genetika, 2001 Aug, 37(8), 1081 - 7
{Creation of a collection of morphological insertional mutants of Arabidopsis thaliana}; Ogarkova OA et al.; A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation . The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4 . Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A . thaliana genes . Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis . Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects . Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 714 - 8
Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium; Kobayashi M et al.; The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium . The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A . tumefaciens . The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass . The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron . The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM.

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