Agrobacterium

Mol Gen Mikrobiol Virusol, 2000, (3), 26 - 31
{Formation of extracellular structures in conjugated cultures of agrobacteria}; Kurbanova IV et al.; Electron microscopy of noncentrifugated agrobacterial cells on a nitrocellulose membrane labeled with colloid gold-conjugated antibodies to VirB1 showed that the labeled complex bound to acetosyringone (AS)-induced cells but failed to form red-colored stains during incubation with Ti aplasmid cells . Supramembrane structures of AS-treated A . tumefaciens cells were for the first time visualized by transmission electron microscopy . Colloid gold labeling of VirB2-specific antibodies showed that VirB2 proteins produce long thin pilus structures emerging at the poles of AS-induced agrobacterial cells but never on the surface untreated with AS and Ti-plasmid-free agrobacterial cells . As a rule, one (or rarely two) thread-like connections and bridges were observed between the cells at the primary contact stage . The bridges were not destroyed by SDS, did not react with VirB2-specific antibodies, and remained visible at 30 degrees C . Visible close contacts between mating bacteria did not cease after SDS treatment . SDS pretreatment of donor cells or a mating cell suspension significantly modified the efficiency of pTd33 plasmid transfer from donor to recipient agrobacterial cells . In the presence of AS the optimal temperature for transfer was 25 degrees C . The frequency of plasmid pTd33 transfer from A . tumefaciens via vir-dependent pathway decreased 2-4-fold due to increase of temperature from 19.25 to 31 degrees C.

Plant J, 2000 Sep, 23(5), 687 - 95
Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector; Liu YG et al.; Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv . Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens . The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses . The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents . Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library) . In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp . TAC clones containing gene(s) of interest were identified by the pooled PCR technique . Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system . Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family) . TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.

Chromosoma, 2000 Jul, 109(4), 287 - 97
Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event; Vergunst AC et al.; Cre recombinase was used to mediate recombination between a chromosomally introduced loxP sequence in Arabidopsis thaliana (35S-lox-cre) and transferred DNA (T-DNA) originating from Agrobacterium tumefaciens (plox-npt), carrying a single loxP sequence . Constructs were designed for specific Cre-mediated recombination between the two lox sites, resulting in restoration of neomycin phosphotransferase (nptII) expression at the target locus . Kanamycin resistant (Km(r)) recombinants were obtained with an efficiency of about 1% compared with random integration . Molecular analyses confirmed that these were indeed due to recombination between the lox sites of the target and introduced T-DNA . However, polymerase chain reaction analysis revealed that these reflected site-specific integration events only in a minority (4%) . The other events were classified as translocations/inversions (71%) or deletions (25%), and were probably caused by site-specific recombination between a randomly integrated T-DNA and the original target locus . We studied some of these events in detail, including a Cre-mediated balanced translocation event, which was characterized by a combination of molecular, genetic and cytogenetic experiments (fluorescence in situ hybridization to spread pollen mother cells at meiotic prophase I) . Our data clearly demonstrate that Agrobacterium-mediated transfer of a targeting T-DNA with a single lox site allows the isolation of multiple chromosomal rearrangements, including translocation and deletion events . Given that the complete sequence of the Arabidopsis genome will have been determined shortly this method has significant potential for applications in functional genomics.

Plant Cell Physiol, 2000 Jul, 41(7), 831 - 9
Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris; Shoji T et al.; Nicotine alkaloids are synthesized in the root of Nicotiana species, and their synthesis increases after insect attack, wounding and jasmonate treatment of the leaf . Putrescine N-methyltransferase (PMT) catalyzes the first committed step in nicotine biosynthesis . The expression patterns of the three Nicotiana sylvestris PMT genes (NsPMT1, NsPMT2, and NsPMT3) are reported in this study . Transcripts of the NsPMT genes were detected only in the root, and were up-regulated by methyl jasmonate treatment . When the 5'-flanking regions of NsPMT1, NsPMT2, and NsPMT3 were fused independently to beta-glucuronidase reporter gene and introduced into N . sylvestris by Agrobacterium-mediated transformation, all introduced transgenes were expressed in the cortex, endodermis, and xylem in the root, as well as upregulated by methyl jasmonate treatment . These qualitatively similar patterns of expression for the NsPMT genes are achieved with only 0.25 kb of their conserved 5'-flanking regions, which contained no known jasmonate-responsive elements.

Res Microbiol, 2000 Jul-Aug, 151(6), 487 - 91
Genetic competence in Helicobacter pylori: mechanisms and biological implications; Hofreuter D et al.; Helicobacter pylori is naturally competent for genetic transformation . The comB locus, consisting of the open reading frames orf2, comB1, comB2, and comB3, is involved in natural transformation competence . Homologies of the ComB proteins with components of the type IV secretion apparatus (VirB9 and VirB10) from the Ti plasmid of Agrobacterium tumefaciens, as well as proteins involved in conjugation of plasmids RP1 and RP4, suggest a similar organization of DNA import (transformation) in H . pylori with well-known DNA export systems.

Mol Gen Genet, 2000 Jul, 263(6), 939 - 47
Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens; Toyoda-Yamamoto A et al.; The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor . VirA also responds to specific monosaccharides, which enhance vir expression . These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential . To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity . The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars . Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes . In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region . We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein . Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars.

Transgenic Res, 2000 Apr, 9(2), 103 - 13
Transgenic Trifolium repens with foliage accumulating the high sulphur protein, sunflower seed albumin; Christiansen P et al.; With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T . repens by Agrobacterium tumefaciens-mediated transformation . The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER) . Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (Assu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (Lssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T . repens cv . Haifa . Transgenic T0-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting . The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein . We observed that the promoter had a substantive effect on the level of SSA accumulation with Assu > CaMV35S > Lssu . Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited . However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci . During this work, we attempted to improve the efficiency of A . tumefaciens-mediated transformation of T . repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T . repens . T-DNA transfer was in general not enhanced by sonication compared to traditional A . tumefaciens-mediated transformation . Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A . tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event . We concluded from these results that only one plant from each A . tumefaciens-treated cotyledon should be taken to avoid transgenic clones.

Microbiol Res, 2000 Jul, 155(2), 113 - 21
Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations; Belimov AA et al.; The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp . L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2 . Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants . Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings . The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd . Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly . The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A . lipoferum 137 . Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots . The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.

Plant Cell, 2000 Aug, 12(8), 1319 - 29
Evidence for a role of the N terminus and leucine-rich repeat region of the Mi gene product in regulation of localized cell death; Hwang CF et al.; The tomato Mi gene confers resistance against root-knot nematodes and potato aphids . Chimeric constructs of the functional gene, Mi-1 . 2, with a homolog, Mi-1.1, were produced, and their phenotypes were examined in Agrobacterium rhizogenes-transformed roots . Exchange of the leucine-rich repeat (LRR) region of Mi-1.1 into Mi-1.2 resulted in the loss of ability to confer nematode resistance, as did substitution of a 6-amino acid sequence from the Mi-1.1 LRR into Mi-1.2 . Introduction of the Mi-1.2 LRR-encoding region into Mi-1.1 resulted in a lethal phenotype, as did substitution of the fragment encoding the N-terminal 161 amino acids of Mi-1.1 into Mi-1.2 . Transient expression of the latter two chimeric constructs in Nicotiana benthamiana leaves produced localized cell death . The cell death caused by the N-terminal exchange was suppressed by coinfiltration with a construct expressing the N-terminal 161 amino acids of Mi-1.2 . The phenotypes of these and other constructs indicate that the LRR region of Mi-1.2 has a role in signaling localized cell death and that the N-terminal 161 amino acids have a role in regulating this death.

J Exp Bot, 2000 Jun, 51(347), 1167 - 9
Sequence analysis of the vir-region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955; Schrammeijer B et al.; The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here . Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid . The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids . Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence.

J Exp Bot, 2000 Jun, 51(347), 1005 - 16
Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum l., and California poppy, Eschscholzia californica cham., root cultures; Park SU et al.; An efficient protocol for the establishment of transgenic opium poppy (Papaver somniferum L.) and California poppy (Eschscholzia californica Cham.) root cultures using A . grobacterium rhizogenes is reported . Five strains of A . rhizogenes were tested for their ability to produce hairy roots on wounded opium poppy seedlings and California poppy embryogenic calli . Three of the strains induced hairy root formation on both species, whereas two others either caused the growth of tumorigenic calli or produced no response . To characterize the putative transgenic roots further, explant tissues were co-cultivated with the most effective A: rhizogenes strain (R1000) carrying the pBI121 binary vector . Except for the co-cultivation medium, all formulations included 50 mg l(-1) paromomycin to select for transformants and 200 mg l(-1) timentin to eliminate the Agrobacterium . Four weeks after infection, paromomycin-resistant roots appeared on 92-98% of explants maintained on hormone-free medium . Isolated hairy roots were propagated in liquid medium containing 1.0 mg l(-1) indole-3-acetic acid to promote rapid growth . Detection of the neomycin phosphotransferase gene, high levels of beta-glucuronidase (GUS) transcripts and enzyme activity, and GUS histochemical localization confirmed the integrative transformation of root cultures . Transgenic roots grew faster than wild-type roots, and California poppy roots grew more rapidly than those of opium poppy . With the exception of a less compact arrangement of epidermal cells and more root hairs, transformed roots of both species displayed anatomical features and benzylisoquinoline alkaloid profiles that were virtually identical to those of wild-type roots . Transgenic root cultures of opium poppy and California poppy are a simple, reliable and well-defined model system to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the genetic engineering potential of these important medicinal plants.

J Food Prot, 2000 Aug, 63(8), 1123 - 32
Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds; Ray P et al.; A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product . This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators . By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identified as members of the genus Bacillus, possibly a variant of Bacillus subtilis . While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identified for production in broth . The compound(s) was heat stable (121 degrees C for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E . The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae . The inhibitory compound(s) could possibly serve as a food biopreservative.

Plant Cell Physiol, 2000 Jun, 41(6), 811 - 6
A rapid and efficient system of Agrobacterium infection-mediated transient gene expression in rice Oc cells and its application for analysis of the expression and antisense suppression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene; Yang H et al.; A rapid and efficient system for Agrobacterium infection-mediated transient gene expression in rice has been developed . Using this system, transient expression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene was analyzed . The results suggest that the Agrobacterium infection-mediated transient gene expression system is as efficient in rice Oc cells as in tobacco BY-2 cells and might be useful for rapid analysis not only of foreign gene expression, but also of antisense gene suppression.

Plant Cell Physiol, 2000 Jun, 41(6), 743 - 9
Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen; Lee HJ et al.; Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides . However, Bacillus subtilis Protox is known to be resistant to the herbicides . In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B . subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation . The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots . Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies . The expression levels of B . subtilis Protox mRNA appeared to correlate with the copy number . Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines . The transgenic plants expressing the B . subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation . The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm . In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.

Plant Cell Physiol, 2000 Jun, 41(6), 733 - 42
Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants; Nakagawa Y et al.; Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer . Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter . In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis . The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination . The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other . In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested . Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site . The transgenic rice obtained in this study can be used for functional genomics of rice.

J Bacteriol, 2000 Sep, 182(17), 4849 - 55
A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication; Sieira R et al.; As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems . The B . abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs) . Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found . Gene reporter studies demonstrated that the B . abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth . A B . abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication . Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus . Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B . abortus virulence . It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B . abortus to an endoplasmic reticulum-related replication compartment.

Phytochemistry, 2000 Jun, 54(5), 517 - 23
Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography; Mateus L et al.; Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A . tumefaciens strain C58C1pRTGus104 . The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis . Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry . Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages . Significant differences appeared between the alkaloid contents of the different clones . In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively . The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine . The aerial parts of H . muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.

Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 1999, (117), 148 - 54
High production of ginsenosides by transformed root cultures of Panax ginseng: effect of basal medium and Agrobacterium rhizogenes strains; Shu W et al.; Successful transformation of Panax ginseng was achieved when petiole segments were infected with Agrobacterium rhizogenes ATCC 15834 and MAFF 03-01724 . Transformed roots were obtained after galls developed at infected sites . The root morphology, growth and ginsenoside productivity of roots transformed with different bacterial strains differed, and the roots from A . rhizogenes ATCC 15834 grew better and produced much more ginsenosides . Using the ATCC transformed root clone, various liquid culture media were tested to determine the optimum culture medium for ginsenoside production . The root growth was optimum in phytohormone-free Gamborg B5 liquid medium, however highest content of ginsenosides (a total of five ginsenosides 1.88% dry weight) was obtained when the roots were cultured in half-macro-salt strength Gamborg B5 liquid medium . Growth of the roots over a period of 8 weeks showed that their fresh and dry weight continued to increase . The ginsenoside Rb1 content was optimum after 5 weeks of culture . Ginsenoside Rc content began to decrease slightly after the third week of culture . Ginsenosides Rd and Rg1 contents fluctuated, while ginsenoside Re content continued to rise throughout the 8 weeks of culture . Ginsenoside production, however, did not peak within the 8 weeks of culture.

Mol Cell Biol, 2000 Sep, 20(17), 6317 - 22
Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro; Ziemienowicz A et al.; Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species . In the plant cell, the transferred DNA (T-DNA) is integrated into the genome . An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process . The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro . We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system . The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts . This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase . We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction . Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target . T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.

J Biotechnol, 2000 Jul 28, 81(1), 45 - 53
Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn; Grichko VP et al.; Transgenic tomato plants Lycopersicon esculentum (Solanaceae) cv . Heinz 902 expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35S cauliflower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (root specific expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgenic tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate these metals . Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoots and roots; root and shoot development; and leaf chlorophyll content . In general, transgenic tomato plants expressing ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within the plant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgenic plants.

Plant Sci, 2000 Jul 28, 156(2), 235 - 244
Analysis of transgenic grapevine (Vitis rupestris) and Nicotiana benthamiana plants expressing an Arabis mosaic virus coat protein gene; Spielmann A et al.; A disarmed LBA4404 strain of Agrobacterium tumefaciens harboring a binary vector which contained chimeric genes encoding the neomycin phosphotransferase (npt II) and the coat protein (CP) of Arabis mosaic nepovirus (ArMV) was used in co-cultivation experiments with leaf discs of Nicotiana benthamiana and somatic embryos of the grapevine rootstock cultivar Vitis rupestris . Transgenic N . benthamiana expressing the ArMV CP gene were regenerated and six independent lines were characterized . Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue demonstrated the accumulation of the ArMV CP in five of the six lines analyzed . Immunosorbent electron microscopy (ISEM) studies revealed the presence of virion-like isometric particles (VLPs) reacting to a rabbit antiserum specific to ArMV virions . ArMV-CP expressing transgenic N . benthamiana lines showed protection against ArMV expressed as a delay in infection and a reduction of the percentage of infected plants . Four independent transgenic lines of V . rupestris transformed with the ArMV CP gene were regenerated and characterized . In contrast to N . benthamiana, transgenic V . rupestris did not accumulate the ArMV CP at levels detectable by ELISA and no VLPs could be observed by ISEM . Northern blot analysis showed that the ArMV CP mRNA was expressed at lower level in V . rupestris compared with N . benthamiana . The reason for this difference in transgene expression and/or mRNA stability between grapevine and N . benthamiana is unclear, but the genetic state of the transgene(s) (homozygous in N . benthamiana versus hemizygous in V . rupestris) may have an effect on gene expression.

Mol Biotechnol, 1999 Dec 1, 13(2), 165 - 70
T-DNA transfer to maize plants; Shen WH et al.; Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants . Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize . Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.

Mol Biotechnol, 1999 Nov, 13(1), 67 - 72
Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment; Maenpaa P et al.; Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues . For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported . In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.

Biotechnol Prog, 2000 Jul-Aug, 16(4), 564 - 70
Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase; Park JH et al.; We developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino acid from 5'-monosubstituted hydantoin . For the comparison of the reaction systems using two sequential enzymes, D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell . A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B . stearothermophilus SD1 . The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system . In the case of employing separately expressed enzymes, the product yield of D-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g-cell/h in 15 h, respectively . The accumulation of N-carbamoyl-D-hydroxyphenylglycine was significant over the reaction time . On the other hand, use of coexpressed enzymes resulted in 98% product yield of D-hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture . The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g-cell/h in 15 h . The coexpressed system was tested for an elevated concentration of D,L-p-hydroxyphenylhydantoin, and efficient production can be achieved.

Can J Microbiol, 2000 Jul, 46(7), 600 - 6
Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms; Oehrle NW et al.; Internally seedborne microorganisms are those surviving common surface sterilization procedures . Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum . Bacterial isolates from surface-sterilized soybean seed, cv . Williams 82 and cv . Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis . Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G . The effects of this antibiotic on seedling development and on B . japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here . Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development . No differences in nodulation kinetics, nitrogen fixation onset or rates were observed . However, the number of B . japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment . Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B . japonicum to the soybean root, without affecting plant development.

Syst Appl Microbiol, 2000 Jun, 23(2), 238 - 44
Phylogenetic analysis of 23S rRNA gene sequences of . Agrobacterium, Rhizobium and Sinorhizobium strains; Pulawska J et al.; The phylogenetic relationship among twelve Agrobacterium, four Rhizobium, and two Sinorhizobium strains originating from various host plants and geographical regions was studied by analysis of the 23S rDNA sequences . The study included Agrobacterium strains belonging to biovars 1, 2 (with tumor- or hairy-root inducing and non-pathogenic strains), A . vitis, A . rubi; representative species of the Rhizobium genus: R . galegae, R . leguminosarum and R . tropici and Sinorhizobium meliloti strains . The phylogenetic analysis showed that within Agrobacterium, the biovar designation was reflected in the 23S rDNA similarity and that strains of Agrobacterium and Rhizobium are closely related to each other . The results suggest that the taxonomic definition of Agrobacterium and Rhizobium should be considered for revision and that the Agrobacterium-biovar identity is probably a reliable taxonomic trait.

Plant J, 2000 Jul, 23(2), 279 - 84
Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes; Eklof S et al.; Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins . All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross . Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type . Unexpectedly, hormone levels in the cross were very similar to wild-type levels . Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced . The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs . As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.

Genetics, 2000 Aug, 155(4), 1875 - 87
The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana; Bechtold N et al.; In planta transformation methods are now commonly used to transform Arabidopsis thaliana by Agrobacterium tumefaciens . The origin of transformants obtained by these methods has been studied by inoculating different floral stages and examining gametophytic expression of an introduced beta-glucuronidase marker gene encoding GUS . We observed that transformation can still occur after treating flowers where embryo sacs have reached the stage of the third division . No GUS expression was observed in embryo sacs or pollen of plants infiltrated with an Agrobacterium strain bearing a GUS gene under the control of a gametophyte-specific promoter . To identify the genetic target we used an insertion mutant in which a gene essential for male gametophytic development has been disrupted by a T-DNA bearing a Basta resistance gene (B(R)) . In this mutant the B(R) marker is transferred to the progeny only by the female gametes . This mutant was retransformed with a hygromycin resistance marker and doubly resistant plants were selected . The study of 193 progeny of these transformants revealed 25 plants in which the two resistance markers were linked in coupling and only one plant where they were linked in repulsion . These results point to the chromosome set of the female gametophyte as the main target for the T-DNA.

Appl Environ Microbiol, 2000 Aug, 66(8), 3499 - 505
Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants; Yuan Q et al.; The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model . An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S(2) . After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration . Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring . The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb) . By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell . Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb . The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb . Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.

Bone Marrow Transplant, 2000 Jul, 26(1), 101 - 4
Agrobacterium yellow group: bacteremia and possible septic arthritis following peripheral blood stem cell transplantation; Chalandon Y et al.; A 47-year-old male patient developed sepsis and monoarticular arthritis following autologous stem cell transplantation for recurrent Hodgkin's disease . Blood cultures were positive for Agrobacterium yellow group . The knee pain and swelling responded promptly to the institution of empirical broad-spectrum antibiotics . Recurrent bacteremia developed necessitating Hickman line removal for eventual resolution of the infection . Transplant physicians should be aware of this unusual pathogen and the potential for both persistent line-related sepsis and possible septic arthritis.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1255 - 62
Purification and characterization of a novel lactonohydrolase from Agrobacterium tumefaciens; Kataoka M et al.; A novel lactonohydrolase, catalyzing the stereospecific hydrolysis of L-pantoyl lactone to L-pantoic acid, was purified 2,400-fold to apparent homogeneity with a 1.96% overall recovery from Agrobacterium tumefaciens AKU 316 through a purification procedure including ammonium sulfate fractionation, and column chromatographies on DEAE-Sephacel, phenyl-Sepharose CL-4B, Sephacryl S-200, Mono-Q and alkyl-Superose . The relative molecular mass of the native enzyme estimated on high-pressure gel permeation chromatography was 62,000 Da, and the subunit molecular mass was estimated to 26,500 Da on SDS-polyacrylamide gel electrophoresis . The enzyme hydrolyzes several aromatic lactones, such as 3,4-dihydrocoumarin and homogentisic acid lactone, other than L-pantoyl lactone . The Km and Vmax for L-pantoyl lactone were 3.59 mM and 13.7micromol/min/mg, respectively . The enzymatic activity was inhibited by several chelating reagents, Fe2+, Sn2+, Pb2+, and Fe3+.

Genetika, 2000 Jun, 36(6), 792 - 8
{Effect of a selective agent and a plant intron on the effectiveness of transformation and expression of heterologous genes in the pear (Pyrus communis L.)}; Lebedev VG et al.; The effect of selective agents on the efficiency of Agrobacterium-mediated transformation of pear was shown . The transformation frequency of the pear stock PS no . 217 by a binary vector carrying the nptII gene conferring kanamycin resistance was 0.4 or 3.1%, while the hpt gene for hygromycin resistance used as a selective marker increased transformation frequency to 6.2 (11.5%) . In addition, upon selection on hygromycin B, the proportion of pseudotransgenic regenerants considerably decreased . In four transformation experiments, twenty independent clones were recovered, and their transgenic nature was confirmed by PCR, histochemical, and fluorometric analyses of GUS activity . The presence of introns in the coding region of a heterologous gene was shown to influence the efficiency and stability of transgene expression in plant tissues . Fluorometric determination of GUS activity conducted for a period of two years demonstrated a threefold increase in transgene expression in the case that an intron-containing construct was used for transformation . The expression level was rather stable across several years . The transformation procedure developed may be used for successful expression of heterologous genes controlling agronomic characters in pear plants.

Trends Microbiol, 2000 Aug, 8(8), 361 - 9
The T-pilus of Agrobacterium tumefaciens; Lai EM et al.; T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface . T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.

Trends Microbiol, 2000 Aug, 8(8), 354 - 60
Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells; Christie PJ et al.; Several bacterial pathogens utilize conjugation machines to export effector molecules during infection . Such systems are members of the type IV or 'adapted conjugation' secretion family . The prototypical type IV system is the Agrobacterium tumefaciens T-DNA transfer machine, which delivers oncogenic nucleoprotein particles to plant cells . Other pathogens, including Bordetella pertussis, Legionella pneumophila, Brucellaspp . and Helicobacter pylori, use type IV machines to export effector proteins to the extracellular milieu or the mammalian cell cytosol.

J Bacteriol, 2000 Aug, 182(16), 4505 - 11
VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens; Hapfelmeier S et al.; VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants . Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors . Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively . Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels . Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly . In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished . This effect could not be restored by expression of VirB6 in trans . Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain . VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.

Planta Med, 2000 Jun, 66(5), 452 - 7
Production of amarogentin in root cultures of Swertia chirata; Keil M et al.; Conventional and Agrobacterium rhizogenes-transformed root cultures were studied with respect to growth and amarogentin content following cultivation in various growth media . The fastest growth rate was observed using Nitsch medium . The best amarogentin content was obtained after cultivation in root culture (RC) medium for which the slowest growth rate was noticed . Addition of sucrose at 6% and 9% (w/v), respectively, also resulted in better growth rates and increased total but unaltered relative amarogentin content compared to 3% (w/v) sucrose . No change in amarogentin content was observed upon addition of elicitors, putative precursors of amarogentin biosynthesis, and plant growth hormones with the exception of salicylic acid and chitosan: at 100 mM salicylic acid a reduction and at 25 mg/L chitosan an increase of amarogentin were observed at significant levels . The cultivation of S . chirata roots in a 2-L stirred-tank bioreactor was successful only with a stainless-steel mesh fitted inside the culture vessel for immobilization of the roots . A 15-fold enhancement of amarogentin content in the medium was achieved by a root permeabilisation treatment using Tween 20 at 1.3% (v/v) final concentration in the bioreactor.

Planta Med, 2000 Jun, 66(5), 448 - 51
The regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare; Argolo AC et al.; Transgenic roots of Solanum spp . containing extra copies of an hmgr gene derived from Artemisia annua have been obtained via transformation with Agrobacterium rhizogenes . Hairy root clones of Solanum aviculare which were transgenic for hmgr typically grew faster than those which did not contain extra copies of this gene and also accumulated up to 4.2 times more solasodine when grown under dark, but not light, conditions . The implications of these findings with respect to the regulation of solasodine production in Solanum spp . are considered.

DNA Res, 2000 Jun 30, 7(3), 157 - 63
Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library; Moriguchi K et al.; Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants . Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants . To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A . rhizogenes strain 1724 . By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids . As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes . In addition, a virulence-related gene, tzs was located independently of the vir region.

Structure Fold Des, 2000 Jul 15, 8(7), 729 - 37
Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases; Nakai T et al.; BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics . To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp . strain KNK712 . RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution . The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold . The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases . CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171 . Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46 . DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Mol Cells, 2000 Jun 30, 10(3), 263 - 8
Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae; Yeo ET et al.; In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1 . Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation . The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation . The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development . However, the plants recovered from these negative growth effects when grown in a soil mixture . The TPS1 transgenic potato plants showed significantly increased drought resistance . These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.

J Bacteriol, 2000 Aug, 182(15), 4137 - 45
Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase; Rashkova S et al.; The Agrobacterium tumefaciens VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells . In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo . A chimeric protein composed of VirB11 fused to the DNA binding domain of lambda cI repressor protein formed dimers, as shown by immunity of Escherichia coli to lambda superinfection . An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type A . tumefaciens cells . Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP . In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized A . tumefaciens cell extracts . VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5' and 3' halves of virB11 . Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (Delta GKT174-176) . By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein . Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein . Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.

Parasitol Res, 2000 Jun, 86(6), 444 - 52
Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70; Schnittger L et al.; A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T . annulata-infected bovine lymphoblastoid cell line with an antibody raised against T . annulata schizonts . The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens) . The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle . The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins . The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 degrees C in comparison with cells cultured at 37 degrees C . By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells . Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites . However, it was also found in small amounts in the cytoplasm of the parasite and host cell . This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm.

Eur J Drug Metab Pharmacokinet, 1999 Oct-Dec, 24(4), 303 - 8
A study of the production of essential oils in chamomile hairy root cultures; Maday E et al.; The active substances in chamomile (Matricaria recutita L.) belong to chemically different structural types . The largest group of medically important compounds forming the essential oils are primarily chamazulene, (-)-alpha-bisabolol, bisabololoxides, bisabolonoxide A, trans-beta-farnesene, alpha-farnesene, spathulenol and the cis/trans-en-in-dicycloethers . Flavonoids, coumarins, mucilages, mono- and oligosaccharides also have pharmacological effects . We studied the production of essential oils in genetically transformed cultures . Sterile juvenile chamomile plants were infected with A4-Y strains of Agrobacterium rhizogenes . They are known plant pathogens and are capable of inducing so-called hairy roots . The transfer DNA segment of the Ri-virulence plasmid of A . rhizogenes becomes integrated in the genome of the plant cells . The isolated hairy roots grow rapidly on hormone-free media . In order to obtain bacteria-free media, we cultured the transformed roots on Murashige-Skoog (MS) medium supplemented with carbenicillin (800 mg/l) . To study the production of essential oils, the clones were propagated on liquid and solid MS and Gamborg (B5) media, respectively . According to gas chromatography, the composition of the essential oil of hairy root cultures on different media was found to be similar, but differing in proportion . The main component of the essential oil which was identified by gas chromatography and mass spectrometry was trans-beta-farnesene, as in the intact roots.

Plant Mol Biol, 2000 Apr, 42(6), 883 - 97
Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.); Takakura Y et al.; A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils . Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs . OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants . The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice . The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding . The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate . Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family . The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated . The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis . The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation . The flower-predominant gene expression was identified also in the transgenic rice plants . The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants . The possible functions of OsChia 1;175 are discussed.

Plant Mol Biol, 2000 Apr, 42(6), 819 - 32
pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation; Hellens RP et al.; Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols . The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species . This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids . Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures . pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain . pSoup provides replication functions in trans for pGreen . The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum . Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors . Information on the pGreen plasmid system is supplemented by an Internet site through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

Plant Physiol, 2000 Jul, 123(3), 1069 - 76
Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene; Cho HJ et al.; A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agrobacterium rhizogenes with kanamycin selection . The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS . Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 microM 5MT, whereas the controls were completely inhibited by 15 microM 5MT . Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp . Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 alpha-subunit can interact with the native A . sinicus beta-subunit to form an active enzyme . The ASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots . Thus, we show for the first time that ASA2 can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.

Plant Physiol, 2000 Jul, 123(3), 895 - 904
Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method; Desfeux C et al.; The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture . To facilitate use with other plant species, we investigated the mechanisms that underlie this method . In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48% . Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase {GUS}) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed . Transformants derived from the same seed pod contained independent T-DNA integration events . In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis . In correlation with this fact, we found that the timing of Agrobacterium infection was critical . Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis . A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium . Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.

Yi Chuan Xue Bao, 2000, 27(2), 158 - 64
{Cloning and expression of trehalose synthase genes in Escherchia coli}; Dai XY et al.; Escherichia coli trehalose synthase otsBA genes have been cloned by using transposon Mu in vivo . The cloning frequency was 1.45 x 10(-3)/Kanr transductant . Genetic complements, endonuclase digestion and partial nucleotide sequencing analysis of these clones indicated that otsBA gene was on plasmid Mud5005 . Subcloning 2.87 kb DNA fragment onto expression plasmids with higher or lower copy number and transforming into E . coli recipient strain FF4050, which have otsBA genes deleted respectively, the transformed strains obtained ability of growth on medium containing 0.5 mol/L NaCl and increased trehalose synthesis under high osmotic pressure . Production and accumulation of trehalose may play an important role in crop breeding . These studies will lay an important foundation for constructing an expression vector with the otsBA genes and transformation to tobacco mediated by Agrobacterium, and obtaining ability for drought resistance and high osmotic pressure tolerance.

Plant J, 2000 Jun, 22(6), 553 - 60
Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells; Ferrando A et al.; Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo . Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells . We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium . Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection . This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.

Plant J, 2000 Jun, 22(6), 543 - 51
In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves; Yang Y et al.; A convenient, Agrobacterium-mediated transient expression assay has been evaluated for rapid analysis of plant promoters and transcription factors in vivo . By simple infiltration of Agrobacterium cells carrying appropriate plasmid constructs into tobacco leaves in planta, reproducible expression assays could be conducted in as little as 2-3 days without using expensive equipment (e.g . biolistic gun or electroporation apparatus) or complicated procedures (e.g . preparation of protoplasts) . Biotic and abiotic treatments could be applied to the intact plant to examine their influence on promoter activity and gene expression . Using this method, we have tested the stress-responsive as-1 and heat shock elements, yeast GAL4 transactivation system, two promoters of pathogenesis-related (PR) genes as well as a heat shock promoter . Through deletion analyses of tobacco PR1a promoter and a novel myb1 promoter, we have also successfully identified the cis-regulatory regions in these promoters that are responsive to salicylic acid treatment or tobacco mosaic virus infection . Together, our results demonstrate that Agrobacterium-mediated transient expression is a simple and efficient method for in vivo assays of plant promoters and transcription factors.

Plant J, 2000 Jun, 22(6), 531 - 41
Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium; Trieu AT et al.; Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula . The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium . The second method involves infiltration of young seedlings with Agrobacterium . In both cases a proportion of the progeny of the infiltrated plants is transformed . The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method . Both procedures resulted in a mixture of independent transformants and sibling transformants . The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion . These transformation systems will increase the utility of M . truncatula as a model system and enable large-scale insertional mutagenesis . T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.

Br Med Bull, 2000, 56(1), 62 - 73
Genetically modified crops: methodology, benefits, regulation and public concerns; Halford NG et al.; The genetic modification of crop plants from the methodology involved in their production through to the current debate on their use in agriculture are reviewed . Techniques for plant transformation by Agrobacterium tumefaciens and particle bombardment, and for the selection of transgenic plants using marker genes are described . The benefits of currently available genetically modified (GM) crops in reducing waste and agrochemical use in agriculture, and the potential of the technology for further crop improvement in the future are discussed . The legal requirements for containment of novel GM crops and the roles of relevant regulatory bodies in ensuring that GM crops and food are safe are summarized . Some of the major concerns of the general public regarding GM crops and food: segregation of GM and non-GM crops and cross-pollination between GM crops and wild species, the use of antibiotic resistance marker genes, the prevention of new allergens being introduced in to the food chain and the relative safety of GM and non-GM foods are considered . Finally, the current debate on the use of GM crops in agriculture and the need for the government, scientists and industry to persevere with the technology in the face of widespread hostility is studied.

Microbiology, 2000 Jul, 146 ( Pt 7), 1735 - 42
Osmotic regulation of cyclic 1,2-beta-glucan synthesis; de Iannino NI et al.; In contrast to what happens in AGROBACTERIUM: tumefaciens and RHIZOBIUM: meliloti, synthesis of periplasmic cyclic 1,2-beta-glucan in BRUCELLA: spp . was not inhibited when bacteria were grown in media of high osmolarity . Studies performed with crude membrane preparations showed that cyclic 1,2-beta-glucan synthetase of BRUCELLA: spp . was not inhibited by 0.5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A . tumefaciens A348 or R . meliloti 102F34, respectively encoded by the chvB or ndvB genes . The BRUCELLA: abortus cyclic 1, 2-beta-glucan synthetase gene (cgs) was introduced into A . tumefaciens A1011 chvB and R . meliloti GRT21s ndvB mutants . Synthesis of cyclic 1,2-beta-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0.25 M NaCl or 0.5 M mannitol) . On the other hand, when the A . tumefaciens cyclic 1, 2-beta-glucan synthetase gene was introduced into the R . meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-beta-glucan synthesis when grown in high-osmolarity media . However, the same gene introduced into a B . abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity . In vitro studies with crude membranes isolated from recombinant strains revealed that BRUCELLA: cyclic 1, 2-beta-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in AGROBACTERIUM: or RHIZOBIUM: backgrounds . It was concluded that the lack of effect of high osmolarity on 1,2-beta-glucan synthesis in BRUCELLA: is due to two convergent mechanisms: a) the presence of a cyclic 1, 2-beta-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1, 2-beta-glucan synthetase.

Appl Environ Microbiol, 2000 Jul, 66(7), 2882 - 7
Isolation and characterization of 2,3-dichloro-1-propanol-degrading rhizobia; Effendi AJ et al.; 2,3-Dichloro-1-propanol is more chemically stable than its isomer, 1, 3-dichloro-2-propanol, and is therefore more difficult to degrade . The isolation of bacteria capable of complete mineralization of 2, 3-dichloro-1-propanol was successful only from enrichments at high pH . The bacteria thus isolated were found to be members of the alpha division of the Proteobacteria in the Rhizobium subdivision, most likely Agrobacterium sp . They could utilize both dihaloalcohol substrates and 2-chloropropionic acid . The growth of these strains in the presence of 2,3-dichloro-1-propanol was strongly affected by the pH and buffer strength of the medium . Under certain conditions, a ladder of four active dehalogenase bands could be visualized from this strain in activity gels . The enzyme involved in the complete mineralization of 2,3-dichloro-1-propanol was shown to have a native molecular weight of 114,000 and consisted of four subunits of similar molecular weights.

Yi Chuan Xue Bao, 1999, 26(6), 711 - 4
{Introduction of rabbit defensin NP-1 gene into poplar (P . tomentosa) by Agrobacterium-mediated transformation}; Zhao SM et al.; Rabbit defensin NP-1 possesses a broad resistant spectrum to pathogens . In this work, we have transferred the NP-1 gene into poplar plants by Agrobacterium-mediated transformation . PCR amplification and Southern analysis showed that rabbit defensin NP-1 gene was integrated into the poplar genome . The transformation efficiency is about 15.6% . Antimicrobial activity test showed that the extract of transgenic plants inhibited the growth of the tested microbes.

DNA Cell Biol, 2000 Jun, 19(6), 377 - 82
The gene encoding the 17-kDa antigen of Bartonella henselae is located within a cluster of genes homologous to the virB virulence operon; Padmalayam I et al.; A Bartonella henselae genomic A library was screened with antiserum generated in mice against live B . henselae . One of the immunoreactive clones expressed a 17-kDa antigen that was characterized previously as an immunodominant protein of B . henselae . Sequence analysis of the recombinant clone, pBHIM-2, revealed that the open reading frame (ORF) encoding the 17-kDa antigen was situated between homologs of virB4 and virB6, two genes that belong to the virB operon . The virB operon has been associated with the transfer of oncogenic T-DNA in Agrobacterium tumefaciens and with secretion of the pertussis toxin in Bordetella pertussis . Downstream of the virB6 gene within pBHIM-2 was a partial open reading frame that was homologous to the virB8 gene . Rescreening of the library by plaque hybridization using probes specific to the 5' and 3' ends of the pBHIM-2 insert resulted in the isolation of recombinant clones containing additional virB genes . Assembly of the sequences obtained from the recombinant clones revealed that eight of the open reading frames encode homologs of the VirB proteins . The homology and colinearity with the virB genes suggest that the gene encoding the 17-kDa antigen is expressed within the virB locus of B . henselae.

Mol Plant Microbe Interact, 2000 Jul, 13(7), 787 - 90
The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity; Lemcke K et al.; The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4 . The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein . The N-terminal domain shows homology to the A . rhizogenes rolB protein and may modulate the auxin responsiveness of host cells . The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m) . We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants . Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls . We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.

Mol Plant Microbe Interact, 2000 Jul, 13(7), 715 - 23
Nodule-expressed Cyp15a cysteine protease genes map to syntenic genome regions in Pisum and Medicago spp; Vincent JL et al.; PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules . To further the understanding of nodular PsCyp15a expression, a region 5' to the coding sequence of the gene was cloned . Varying lengths of 5' untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into "hairy roots" of Vicia hirsuta . In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization . An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M . truncatula was also shown to express strongly in nodules . With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.

Plasmid, 2000 Jul, 44(1), 1 - 11
pT3.2I, the smallest plasmid of Thiobacillus T3.2; Aparicio T et al.; The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined . pT3.2I is 15,390 bp long with a 53.7% GC content . Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp) . Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids . Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB . Based on sequence comparisons, pT3 . 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group .

Planta, 2000 May, 210(6), 1018 - 22
Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts; Kato N et al.; When pumpkin (Cucurbita spp., cv . Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L . cv . Bright Yellow No . 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium . These transgenic cells showed no morphological difference from wild-type cells . However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells . We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall.

Biotechniques . 2000 Jun;28(6):1128 1130, 1132, 1134 passim.
Determination of transgene repeat formation and promoter methylation in transgenic plants; Kumar S et al.; The integration of transgenes into a plant host genome following Agrobacterium tumefaciens-mediated or direct transformation may occur as a single copy or in the form of tandem repeats . The latter has been associated with promoter methylation and silencing of transgenes . Thus, the early screening of such transgenic plants is desirable for ruling out future repeat-dependent transgene instability . We developed a simple PCR-based method in which primer pairs were specifically designed so that amplifications could only be obtained if the transgene was present in the form of multiple inserts in a transgenic line . The method was established using 35S-rolC transgenic aspen lines showing morphologically visible transgenic silencing . Later, it was possible to screen independent transgenic lines showing no visible marker gene expression . Furthermore, a method was developed in which positive PCR amplification was indicative of promoter methylation . The results were consistent and reproducible across different independent transgenic lines . The methods were quick, reliable, consistent and reproducible, and can be useful for routine screening of transgene silencing in lines derived from many different systems.

Plant Physiol, 2000 Jun, 123(2), 531 - 42
Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene; Tuominen H et al.; The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L . x Populus tremuloides Michx.) . We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes . The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA . In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression . Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA . Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants . This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.

FEMS Microbiol Lett, 2000 Jun 15, 187(2), 175 - 8
A single amino acid substitution beyond the C2H2-zinc finger in Ros derepresses virulence and T-DNA genes in Agrobacterium tumefaciens; Archdeacon J et al.; Ros is a chromosomally-encoded repressor containing a novel C2H2 zinc finger in Agrobacterium tumefaciens . Ros regulates the expression of six virulence genes and an oncogene on the Ti plasmid . Constitutive expression of these genes occurs in the spontaneous mutant 4011R derived from the octopine strain Ach-5, resulting in T-DNA processing in the absence of induction, and in the biosynthesis of cytokinin . Interestingly, the mutation in 4011R is an Arg to Cys conversion at amino acid residue 125 near the C-terminus well outside the zinc finger of Ros . Yet, Ros bearing this mutation is unable to bind to the Ros-box and is unable to complement other ros mutants.

Transgenic Res, 2000 Feb, 9(1), 71 - 8
Expression of full-length bioactive antimicrobial human lactoferrin in potato plants; Chong DK et al.; A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEK-DEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods . The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter . The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods . Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis . The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF . Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein . Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues . This is the first report of synthesis of full length, biologically active hLF in edible plants.

Transgenic Res, 2000 Feb, 9(1), 11 - 9
Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns; Fu X et al.; Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s) . This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements . We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator) . We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed . Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements . Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements . We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs . We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes . We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers . Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7545 - 50
Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB; Chen L et al.; The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid . We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins . Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2green fluorescent protein construct were not detected in the above fraction . Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2 . The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C . When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced . However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm . Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB . Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system.

J Bacteriol, 2000 Jun, 182(12), 3437 - 45
The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis; Llosa M et al.; Genetic transformation of plants by Agrobacterium tumefaciens is mediated by a virulence (vir)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4 . VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids . The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly . Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence . In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1* . Complementation of a virB1 deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain, suggesting that each domain has a unique role in tumorigenesis . The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled . When expressed independently of all other vir genes, VirB1 was processed and VirB1* was secreted . When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the virB1 deletion strain . Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to vir regulation.

J Bacteriol, 2000 Jul, 182(13), 3705 - 16
Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens; Lai EM et al.; The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells . Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined . In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation . We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation . Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A . tumefaciens strains used . With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A . tumefaciens rod-shaped cell in a polar manner . We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation . These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes . A search of the vir genes involved in controlling this biphasic reaction in induced A . tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction . The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A . tumefaciens.

Mol Microbiol, 2000 May, 36(3), 608 - 17
Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore; Kumar RB et al.; Agrobacterium tumefaciens transforms plants by transferring DNA to the plant cell nucleus . The VirB membrane proteins are postulated to form a pore for the transport of the DNA across the bacterial membranes . Immunofluorescence and immunoelectron microscopy were used to study the transport pore complex . Three likely components of the transport pore, VirB8, VirB9 and VirB10, localized primarily to the inner membrane, outer membrane and periplasm respectively . A significant amount of VirB10 was also found associated with the outer membrane . When expressed alone VirB9 and VirB10 were randomly distributed along the cell membrane . Subcellular location of both proteins changed dramatically in the presence of the other VirB proteins . Both proteins localized to fewer sites and most of the gold particles representing protein molecules were found in clusters suggesting that the two proteins are in a protein complex . VirB8, on the other hand, localized to clusters even in the absence of the other VirB proteins . To investigate the role of VirB8 in the formation of VirB9 and VirB10 protein complexes, we studied the effect of deletion of virB8 on the subcellular location of VirB9 and VirB10 . In a virB8 deletion mutant both proteins were distributed randomly on the cell membrane indicating that VirB8 is essential for complex assembly.

J Infect Dis, 2000 Jun, 181(6), 2106 - 10 Epub 2000 Jun 05.
Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens; Abuodeh RO et al.; Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis . A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A . tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia . This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia . Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy . A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio . This approach requires no special equipment that might complicate biocontainment . Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure . A . tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C . immitis and perhaps other fungi of medical importance.

Appl Environ Microbiol, 2000 Jun, 66(6), 2365 - 71
Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp . isolated on phytopathogenic bacteria; Jurkevitch E et al.; Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil . Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios . Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp . carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A . tumefaciens was used as prey . In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) PFU g(-1) . A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells . An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination . Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively . The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced . One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.

Biosci Biotechnol Biochem, 2000 Apr, 64(4), 845 - 7
Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens; Kojima M et al.; Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var . Shinano No . 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121) . The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants) . The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 microg/ml) and by detection of beta-glucuronidase (GUS) gene with PCR, indicating that 36% and 70% of the T1 plants were transformed, respectively . Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A . tumefaciens harboring a modified pBI121 for plasmid rescue . Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants.

Mol Plant Microbe Interact, 2000 Jun, 13(6), 658 - 65
Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants; De Buck S et al.; Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells . Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5% . T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells . A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining . When selection for transformation competence is applied, the picture is completely different . Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome . Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors.

Mol Plant Microbe Interact, 2000 Jun, 13(6), 649 - 57
Evidence for Agrobacterium-induced apoptosis in maize cells; Hansen G; Agrobacterium spp . can genetically transform most dicotyledonous plant cells whereas many monocot species are recalcitrant to Agrobacterium-mediated transformation . One major obstacle is that co-cultivation of Agrobacterium spp . with plant tissues often results in cell death . Report here is that, in maize tissues, this process resembles apoptosis, with characteristic DNA cleavage into oligonucleosomal fragments and morphological changes . Two anti-apoptotic genes from baculovirus, p35 and iap, had the ability to prevent the onset of apoptosis triggered by Agrobacterium spp . in maize tissues . p35 is reported to act as a direct inhibitor of a certain class of proteases (caspase) whereas i.a.p . may act upstream to prevent their activation . This evidence raises the possibility that caspase-like proteases may also be involved in the apoptotic pathway in plant cells.

Biochemistry, 2000 May 9, 39(18), 5600 - 13
Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter; Rink R et al.; Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate . The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step . The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis . Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations . Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation . The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations . The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants . This indicates that the effects of the mutations were cumulative . The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase . Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate . Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.

Arch Microbiol, 2000 Apr, 173(4), 311 - 5
Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum; Ogawa Y et al.; A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized . Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin . Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3) . Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain . Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids.

Plant Sci, 2000 Jun 29, 155(2), 179 - 185
Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L . transgenic plants via Agrobacterium tumefaciens-mediated transformation; Chen D et al.; An Agrobacterium tumefaciens-mediated transformation system was developed for Artemisia annua L . Using this system a cDNA encoding farnesyl diphosphate synthase (FDS placed under a CaMV 35S promoter) was transferred into A . annua via A . tumefaciens strain LB4404 . Leaf or leaf discs were used as explants to be infected with A . tumefaciens and an optimal concentration of 20 mg/l kanamycin was applied to select kanamycin resistant shoots . Forty-five lines of resistance kanamycin shoots transformed with FDS were established . Analysis of PCR showed that at least 20 shoots transformed with the FDS gene were PCR positive . Southern blot analysis suggested the foreign FDS gene had been integrated into the A . annua genome, and Northern blot analysis revealed that the foreign FDS gene expressed at the transcriptional level in five shoot lines (F-1, F-4, F-61, F-62 and F-73 shoot lines) . Analysis of artemisinin demonstrated that about 8 approximately 10 mg/g DW of artemisinin were then detected in transgenic plants regenerated from five shoot lines, this is about 2-3 times higher than that in the control.

Mikrobiologiia, 2000 Jan-Feb, 69(1), 81 - 8
{Conjugative transfer of pTd33-plasmid between strains of Agrobacterium}; Chumakov MI et al.; Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy . The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein . Pretreatment of agrobacterial cells with acetosyringone resulted in a six- to tenfold increase in the transfer frequency of the plasmid pTd33 at 19-25 degrees C and had almost no effect at 30 degrees C . The transfer of the plasmid pTd33 from A . tumefaciens strain GV3101 to plasmid-free A . tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells . The transfer efficiency of the plasmid pTd33 from A . tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-free A . tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-type A . tumefaciens strain GV3101 . Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26 . The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed.

Plant Physiol, 2000 May, 123(1), 393 - 402
The yeast HAL1 gene improves salt tolerance of transgenic tomato; Gisbert C et al.; Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K(+) concentration and decreasing intracellular Na(+) during salt stress . In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation . A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo . Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene . In addition, measurement of the intracellular K(+) to Na(+) ratios showed that transgenic lines were able to retain more K(+) than the control under salt stress . Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast.

Lett Appl Microbiol, 2000 May, 30(5), 406 - 10
Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation-treated soybean cotyledons; Finer KR et al.; Colonization and infection of soybean cotyledons by Agrobacterium tumefaciens and subsequent elimination of bacteria from cotyledons were monitored using bacteria expressing green fluorescent protein (GFP) . GFP provided a quick, non-destructive method to evaluate, in real time, Agrobacterium colonization of cotyledon surfaces as well as infection of internal cells . GFP was first detected 7 h following inoculation of the cotyledon . By 36 h, GFP expression was very intense, and was limited to the adaxial surface of the cotyledon . Expression of GFP also served as a useful indicator of successful elimination of the bacterium from plant tissue following antibiotic treatment.

Appl Environ Microbiol, 2000 May, 66(5), 2133 - 8
Construction and evaluation of a novel bifunctional N-carbamylase-D-hydantoinase fusion enzyme; Kim GJ et al.; A fully enzymatic process employing two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity for the production of D-amino acids . To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the D-hydantoinase . Firstly, maltose-binding protein (MBP) gene of E . coli was fused with D-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native D-hydantoinase . Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of D-hydantoinase are maintained even in a fusion state . Based on this result, we constructed an artificial fusion enzyme composed of the whole length of N-carbamylase (304 amino acids {aa}) from Agrobacterim radiobacter NRRL B11291 and D-hydantoinase (471 aa) . The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the D-amino acids . A related D-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus . The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme . The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes . Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.

Appl Environ Microbiol, 2000 May, 66(5), 1818 - 25
Characterization of plasmid-borne and chromosome-encoded traits of Agrobacterium biovar 1, 2, and 3 strains from France; Ride M et al.; We collected 111 Agrobacterium isolates from galls of various origins (most of them from France) and analyzed both their plasmid-borne and chromosome-encoded traits . Phenotypic analysis of these strains allowed their classification in three phena which exactly matched the delineation of biovars 1, 2, and 3 . A fourth phenon was identified which comprises three atypical strains . The phenotypic analysis has also allowed us to identify 12 additional characteristics which could be used to identify the three biovars of Agrobacterium . Our results also suggest that biovar 1 and 2 represent distinct species . Analysis of plasmid-borne traits confirmed that tartrate utilization is a common feature of biovar 3 strains (now named Agrobacterium vitis) and of Agrobacterium grapevine strains in general . Among pathogenic strains of Agrobacterium, several exhibited unusual opine synthesis and degradation patterns, and one strain of biovar 3 induced tumors containing vitopine and a novel opine-like molecule derived from putrescine . We have named this compound rideopine.

Biochim Biophys Acta, 2000 Jan 31, 1490(1-2), 208 - 12
A region of the Agrobacterium tumefaciens chromosome containing genes required for virulence and attachment to host cells; Matthysse AG et al.; A 29 kb region of the circular chromosome of Agrobacterium tumefaciens containing genes required for bacterial attachment to host cells and virulence has been sequenced . Transposon mutants in many of the genes have been obtained . The mutants can be divided into two groups: those which can be complemented by conditioned medium and those whose phenotype is unaffected by conditioned medium . The first group includes mutants in genes with homology to ABC transporters, one possible transcriptional regulator, and some closely linked genes immediately downstream . The second group includes mutants in two possible transcriptional regulators, one ATPase, and a number of biosynthetic genes including a transacetylase required for the formation of an acetylated capsular polysaccharide . There are also several genes with no homology to genes of identified function . The presence of such a large number of genes required for attachment to host cells suggests that the ability of A . tumefaciens to bind to plant cells may play an important role in the life of these bacteria.

J Biotechnol, 2000 Jan 7, 76(1), 73 - 81
Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors; Mukherjee S et al.; Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii . Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin . A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days . The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield . Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition . Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml . Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth . These suspension cultures showed continuous and stable production of forskolin.

Proc Natl Acad Sci U S A, 2000 Apr 25, 97(9), 4985 - 90
Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana; Chuang CF et al.; We investigated the potential of double-stranded RNA interference (RNAi) with gene activity in Arabidopsis thaliana . To construct transformation vectors that produce RNAs capable of duplex formation, gene-specific sequences in the sense and antisense orientations were linked and placed under the control of a strong viral promoter . When introduced into the genome of A . thaliana by Agrobacterium-mediated transformation, double-stranded RNA-expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference . The severity of phenotypes varied between transgenic lines . In situ hybridization revealed a correlation between a declining AG mRNA accumulation and increasingly severe phenotypes in AG (RNAi) mutants, suggesting that endogenous mRNA is the target of double-stranded RNA-mediated genetic interference . The ability to generate stably heritable RNAi and the resultant specific phenotypes allows us to selectively reduce gene function in A . thaliana.

Nucleic Acids Symp Ser, 1999, (42), 67 - 8
Genome structure of Ri plasmid (2) . Sequencing analysis of T-DNA and its flanking regions of pRi1724 in Japanese Agrobacterium rhizogenes; Maeda Y et al.; We sequenced 42.6 kb including T-DNA and its flanking regions which corresponds to about 1/5 of entire length of a mikimopine-type Ri plasmid, pRi1724 in A . rhizogenes . We identified 37 ORFs (Open Reading Frames) including genes in total . Among them, 20 ORFs are probably new genes . Those ORFs have similarity with those in Agrobacterium and 9 ORFs of them was newly found on Ri plasmids.

Mol Gen Genet, 2000 Mar, 263(2), 309 - 19
Overexpression of Arabidopsis thaliana SKP1 homologues in yeast inactivates the Mig1 repressor by destabilising the F-box protein Grr1; Schouten J et al.; The timed destruction of cell cycle regulatory proteins is of key importance in controlling cell cycle progression in eukaryotes . Recently, Skp1 from yeast (Saccharomyces cerevisiae) was shown to play an important role in the ubiquitin-mediated proteolysis of these proteins via the Skp1-Cdc53-F-box (SCF) pathway . Here we describe the fortuitous cloning of cDNAs for two Skp1 homologues from the plant Arabidopsis thaliana on account of their ability to activate reporter gene expression in yeast directed by the cyt-1 element from the promoter of the Agrobacterium tumefaciens T-cyt gene, which is essential for expression of the gene in plants . This element is strikingly similar in sequence to the binding site for the yeast Migl protein, a transcriptional repressor of genes involved in the utilisation of carbohydrates other than glucose . We report that Mig1 protein binds to the cyt-1 element with similar specificity as a previously described plant nuclear protein factor, and that the cyt-1 element is a target for an unknown yeast transcriptional activator when Mig1 itself is inactivated . Interestingly, our data further indicate that A . thaliana Skp1 inactivates Mig1 by destabilising the yeast F-box protein Grr1, which is required for cyclin degradation and is thus involved in control of the cell cycle, and for glucose-regulated gene repression . Our results suggest that the plant counterpart of yeast Skp1 is probably also instrumental in ubiquitin-mediated proteolysis of specific proteins via an SCF-like pathway.

Arch Microbiol, 2000 Mar, 173(3), 178 - 86
Involvement of the respiratory chain of gram-negative bacteria in the reduction of tellurite; Trutko SM et al.; The terminal oxidases of the respiratory chain of seven strains of gram-negative bacteria were shown to be involved in the reduction of tellurite . The rate of tellurite reduction correlated with the intensity of respiration . The inhibitors of terminal oxidases, carbon monoxide and cyanide, inhibited the reduction of tellurite . In Pseudomonas aeruginosa PAO ML4262 and P . aeruginosa PAO ML4262 (pBS 10), the respiratory chain was found to contain three types of cytochrome c, one of which (the carbon monoxide-binding cytochrome c) was involved in the reduction of tellurite . Agrobacterium tumefaciens VKM B-1219, P . aeruginosa IBPM B-13, and Escherichia coli G0-102bd++ cells contained oxidases aa3, bb3, and bd, respectively . The respiratory chain of other strains contained two oxidases: E . coli DH5alpha of bb3- and bd-type, and Erwinia carotovora VKM B-567 of bo3- and bd-type . All the strains under study reduced tellurite with the formation of tellurium crystallites . Depending on the position of the active center of terminal oxidases in the plasma membrane, the crystallites appeared either in the periplasmic space {P . aeruginosa PAO ML4262 and P . aeruginosa PAO ML4262 (pBS10)}, or on the outer surface of the membrane (A . tumefaciens VKM B-1219 and P . aeruginosa IBPM B-13), its inner surface (E . coli G0-102bd++), or on both surfaces (E . coli DHaalpha and E . carotovora VKM B-567).

Arch Microbiol, 2000 Mar, 173(3), 170 - 7
Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis; Blumer C et al.; A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes . In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite . HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2 . The hcnABC structural genes of Pseudomonas fluorescens and P . aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens . Induction of the hcn genes of P . fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron . In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system . This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase . Cyanide produced by P . fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots . Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic . However, in this case, as well as in other situations, the physiological function of cyanide is unknown.

Mol Microbiol, 2000 Mar, 35(6), 1326 - 34
The spliceosomal intron of the rolA gene of agrobacterium rhizogenes is a prokaryotic promoter; Pandolfini T et al.; Agrobacterium rhizogenes transfers DNA (T-DNA) from its Ri plasmid to plant cells . All T-DNA genes are expressed in plant cells . The rolA gene is the only T-DNA gene that contains an intron in the untranslated leader region of its mRNA . This paper shows that (i) the rolA gene is also transcribed in bacteria; (ii) the 85 bp corresponding to the spliceosomal intron drives prokaryotic gene expression in agrobacteria, in free-living rhizobia and in bacteroids within root nodules; and (iii) promoter activity is abolished by the deletion of 63 bp from its 5' end and is reduced by mutations changing its sequence near the putative -10 region . The expression pattern of a chimeric reporter gene shows that, in free-living bacteria, gene expression takes place during the exponential phase of growth and increases at the onset of the stationary phase . Within root nodules, reporter gene expression occurs in the invasion, nitrogen fixing and senescent zones.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 787 - 801
Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System; Tighe SW et al.; Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species . In this study, the fatty acid profiles of over 600 strains belonging to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were evaluated using the gaschromatography-based Sherlock Microbial Identification System (MIS) . Data collected with the MIS showed that the three phylogenetically defined biovars of the genus Agrobacterium formed discrete clusters, whilst species belonging to the genus Mesorhizobium formed three subclusters which were easily distinguished . These three subclusters contained Mesorhizobium ciceri and Mesorhizobium mediterraneum, Mesorhizobium tianshanense fatty acid group I and Mesorhizobium plurifarium, and Mesorhizobium huakuii and Mesorhizobium loti . The genus Sinorhizobium was composed of an individual position for Sinorhizobium meliloti and a large cluster comprising Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium kostiense and Sinorhizobium arboris . S . meliloti contained significantly higher levels of the fatty acid 19:0 cyclo omega 8 cis and clustered with Rhizobium sp . (Hedysarum coronarium) . However, discrimination between the species of genera Sinorhizobium and Rhizobium was a function of the concentration of 16:0 3-OH . The genus Rhizobium contained a single cluster containing Rhizobium sp . (Hedysarum coronarium), Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium etli, along with individual positions for Rhizobium giardinii, Rhizobium tropici, Rhizobium galegae and Rhizobium hainanense . R . tropici and R . hainanense exhibited similarity to Agrobacterium biovar 2, whilst R . galegae was similar to Agrobacterium biovar 1 . R . giardinii appeared unique, with comparatively little similarity to the other species . Analysis of the genus Bradyrhizobium revealed large differences from the other genera studied . Two subgroups of Bradyrhizobium elkanii were detected and easily distinguished from Bradyrhizobium japonicum . Bradyrhizobium liaoningense and Bradyrhizobium sp . (Arachis hypogaea), a group isolated from Chinese peanut plants, showed similarities to B . japonicum, whilst a subgroup of M . tianshanense appeared identical to Bradyrhizobium sp . (Arachis hypogaea).

Plant J, 2000 Mar, 21(6), 519 - 28
Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1; Liu Z et al.; To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated . UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light . Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism . The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location . Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant . The cosmid encodes a protein similar to the S . cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER) . The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon . These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease . The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants . The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.

Plant J, 2000 Feb, 21(3), 259 - 67
Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content; Geelen D et al.; In animals and yeast, voltage-dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plasma membrane excitability, and control of pH and membrane potential in intracellular compartments . To assess the function of CLCs in plants, we searched for CLC insertion mutants in a library of Arabidopsis lines transformed by Agrobacterium tumefaciens transferred DNA (T-DNA) . Using a polymerase chain reaction-based screening procedure, an Arabidopsis line that carries a T-DNA insertion within the C-terminus of the AtCLC-a coding sequence was identified . Progeny from this plant line, clca-1, showed dramatically altered transcription of the AtCLC-a gene . Plants homozygous for the clca-1 mutation exhibited normal development and a morphology indistinguishable from the wild-type . However, their capacity to accumulate nitrate under conditions of nitrate excess was reduced in roots and shoots, by approximately 50%, while chloride, sulphate and phosphate levels were similar to the wild-type . In addition, the herbicide chlorate, an analogue of nitrate, induced a faster and more pronounced chlorosis in mutant plants . Hypersensitivity to chlorate as well as decreased nitrate levels co-segregated with the T-DNA insertion . They were found at various time points of the clca-1 life cycle, supporting the idea that AtCLC-a has a general role in the control of the nitrate status in Arabidopsis . Concordant with such a function, AtCLC-a mRNA was found in roots and shoots, and its levels rapidly increased in both tissues upon addition of nitrate but not ammonium to the culture medium . The specificity of AtCLC-a function with respect to nitrate is further supported by a similar free amino acid content in wild-type and clca-1 plants . Although the cellular localization of AtCLC-a remains unclear, our results suggest that AtCLC-a plays a role in controlling the intracellular nitrate status.

Genetics, 2000 Mar, 154(3), 1323 - 33
Segregation distortion of T-DNA markers linked to the self-incompatibility (S) locus in Petunia hybrida; Harbord RM et al.; In plants with a gametophytic self-incompatibility system the specificity of the pollen is determined by the haploid genotype at the self-incompatibility (S) locus . In certain crosses this can lead to the exclusion of half the gametes from the male parent carrying a particular S-allele . This leads to pronounced segregation distortion for any genetic markers that are linked to the S-locus . We have used this approach to identify T-DNA insertions carrying a maize transposable element that are linked to the S-locus of Petunia hybrida . A total of 83 T-DNA insertions were tested for segregation distortion of the selectable marker used during transformation with Agrobacterium . Segregation distortion was observed for 12 T-DNA insertions and at least 8 of these were shown to be in the same linkage group by intercrossing . This indicates that differential transmission of a single locus (S) is probably responsible for all of these examples of T-DNA segregation distortion . The identification of selectable markers in coupling with a functional S-allele will allow the preselection of recombination events around the S-locus in petunia . Our approach provides a general method for identifying transgenes that are linked to gametophytic self-incompatibility loci and provides an opportunity for transposon tagging of the petunia S-locus.

Mol Plant Microbe Interact, 2000 Apr, 13(4), 465 - 9
Comparison of the hypersensitive response induced by the tomato Cf-4 and Cf-9 genes in Nicotiana spp; Thomas CM et al.; We have previously shown that tomato Cf-9 induces an Avr9-dependent hypersensitive response (HR) in Nicotiana tabacum and potato . We show here that Cf-4 also induces an Avr4-dependent HR in two tobacco species (N . tabacum and N . benthamiana) . The HR induced by Cf-4 and Cf-9 was compared in stable tobacco transgenics by a seedling lethal assay and resistance to recombinant Potato virus X expressing Avr4 or Avr9 . We also compared HR induction with Agrobacterium-mediated transient expression . The Cf-4/Avr4 combination induced a more rapid HR than Cf-9/Avr9 . Sensitive assays for Cf-9 and Cf-4 function should prove useful for structure/function analyses of these resistance proteins in tobacco.

Mol Plant Microbe Interact, 2000 Apr, 13(4), 439 - 46
Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis; Van der Hoorn RA et al.; The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively . We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration) . In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration . By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition . Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses . Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4 . Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities . Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species . Most non-solanaceous species did not develop specific Avr/Cf-induced responses . However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.

Genetika, 2000 Feb, 36(2), 209 - 16
{Chromosomal variability of ginseng cells transformed with plant oncogene rolC}; Bulgakov VP et al.; Chromosome numbers were was studied in ginseng cell line 1c transformed with Agrobacterium rhizogenes strain A4, which carried plasmid pRiA4, and with A . tumefaciens strain GV3101, which carried vector pPCV002-35S rolC . As compared with the nontransformed cell line 1c, tumor cell cultures 1c-A4 and 1c-rolC and the tissues of rolC teratoma (excluding leaves) displayed higher polyploidy and aneuploidy . The 1c-A4 and 1c-rolC hairy-root cultures also had aneuploid and polyploid cells, but the chromosome variation was lower than in tumor cells or the initial culture 1c . Generally, an increase of chromosome variation in cultivated cells was the main effect of the integration of several oncogenes, which were in the A . rhizogenes A4 T-DNA, or of the individual rolC gene in the ginseng genome . Another effect consisted in stabilization of the chromosome number in some differentiated transgenic tissues . Possible reasons for this effect are discussed.

Genetika, 2000 Feb, 36(2), 203 - 8
{The role of phytohormones in tumor formation in radish}; Matveeva TV et al.; Tumor formation was studied in inbred radish lines that produce tumors on plant roots during flowering . In all radish lines under consideration, the sequences homologous to oncogenes tmr/tml of Agrobacterium tumefaciens were revealed by Southern hybridization . No sequences homologous to the tms locus of A . tumefaciens and the oncogenes of A . rhizogenes were determined . It was found that auxin sensitivity and the tumor-producing capacity were coinherited . We suggest that tumor phenotype arise as a result of a combination between agrobacterial "cytokinin" oncogenes and certain alleles of "auxin" radish genes.

Plant Cell Physiol, 2000 Jan, 41(1), 42 - 8
The promoter for the maize C4 pyruvate, orthophosphate dikinase gene directs cell- and tissue-specific transcription in transgenic maize plants; Taniguchi M et al.; The pyruvate,orthophosphate dikinase (PPDK) gene coding the chloroplast enzyme involved in C4 photosynthesis has a dual promoter system . The first promoter is responsible for the transcription of a larger transcript and its product is targeted to the chloroplast (hence, it is designated as C4Pdk promoter) while the second promoter is responsible for the transcription of a smaller transcript and its product remains in the cytosol . In this study, chimeric maize C4Pdk promoter (0.9 or 1.5 kb)-beta-glucuronidase or luciferase fusion genes were introduced into maize plants by Agrobacterium-mediated transformation . The cell- and tissue-specificities of the maize C4Pdk promoter in the transgenic maize plants were examined by histochemical and enzymic activity analyses of the reporters in different photosynthetic cells and tissues . The results showed that the reporter proteins are almost exclusively localized in leaf mesophyll cells . Among the tissues tested, leaf blade had the highest reporter activities with sheath exhibiting about 10% of the activities in blade . Husk, stem, tassel and root had no or very little reporter activities . Taken together, these results suggest that the maize C4Pdk promoter is specifically transcribed in the mesophyll cells of leaf blade and to a much less extent in the mesophyll cells of sheath, but not in leaf bundle sheath cells or other tissues . Furthermore, the 0.9 kb maize C4Pdk promoter sequences appear to contain the necessary cis-acting elements for its cell- and organ-specific expression.

DNA Cell Biol, 2000 Mar, 19(3), 141 - 7
Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region; Schmiederer M et al.; A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized . This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene . Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids) . An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene . Sequence analysis revealed three ORFs upstream of the gene . The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A . tumefaciens . In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions . These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B . henselae and other gram-negative bacteria such as A . tumefaciens.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3718 - 23
Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency; Cutler SR et al.; We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana . A library of Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3' end of the green fluorescent protein (GFP) coding sequence . The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transformation . Fluorescence imaging of 5,700 transgenic plants indicated that approximately 2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP . About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs) . This observation suggests that some targeting signals are of sufficiently low information content that they can be generated frequently by chance . The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution . Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells.

Plant Mol Biol, 1999 Dec, 41(6), 825 - 35
Large alkyl side-chains of isoleucine and leucine in the NPIRL region constitute the core of the vacuolar sorting determinant of sporamin precursor; Matsuoka K et al.; The N-terminal propeptide of the sporamin precursor contains vacuolar targeting information within the Asn-26/Pro-27/Ile-28/Arg-29/Leu-30 (NPIRL) sequence . An Agrobacterium-mediated transient expression assay with tobacco BY-2 cells was employed to investigate the role of each amino acid of the NPIRL region in vacuolar targeting . Replacement of Asn-26, Pro-27, Ile-28 and Leu-30 with several amino acids caused secretion of the mutant prosporamin . Leu was the only amino acid that could be substituted for Ile-28 without affecting transport . Exchange of Leu-30 for amino acids with small side-chains abolished vacuolar delivery . These results indicate that the consensus composition of the NPIRL sequence is {preferably Asn}-{not acidic}-{Ile or Leu}-{any amino acid}-{large and hydrophobic} and suggest that the large alkyl side-chains of Ile-28 and Leu-30 constitute the core of the vacuolar sorting determinant.

Plant Mol Biol, 1999 Dec, 41(6), 765 - 76
Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid, pTiC58; Otten L et al.; The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection . Part of this DNA (the 'common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific . Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced . We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM . Gene c codes for a new unknown T-DNA protein . Gene a is homologous to the agrocinopine synthase gene . Genes b, c', d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3' . Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes . Our studies show that gene 3' (located on the TR-DNA of octopine strains) is also oncogenic . Although the b-e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora . Shoot formation could be restored by genes c and c' in co-infection experiments . Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.

Plant Sci, 2000 May 29, 154(2), 171 - 181
Transgenic expression of a gene encoding a synthetic antimicrobial peptide results in inhibition of fungal growth in vitro and in planta; Cary JW et al.; Transgenic tobacco plants producing the synthetic antimicrobial peptide D4E1, encoded by a gene under the control of an enhanced cauliflower mosaic virus 35S RNA promoter, were obtained by Agrobacterium-mediated transformation . Successful transformation was demonstrated by PCR and Southern hybridization analysis of tobacco DNAs . Expression of the synthetic D4E1 gene was shown by RT-PCR of tobacco mRNA . Crude protein extracts from leaf tissue of transformed plants significantly reduced the number of fungal colonies arising from germinating conidia of Aspergillus flavus and Verticillium dahliae by up to 75 and 99%, respectively, compared to extracts from plants transformed with pBI121 . Compared to negative controls, tobacco plants expressing the D4E1 gene showed greater levels of disease resistance in planta to the fungal pathogen, Colletotrichum destructivum, which causes anthracnose.

DNA Seq, 1999, 10(4-5), 349 - 54
Molecular analysis of a tryptophan-2-monooxygenase gene (IaaM) of Agrobacterium vitis; Oetiker JH et al.; Tryptophan-2-monooxygenase genes occur in a number of bacteria and encode the conversion of tryptophan to the plant hormone precursor indole-3-acetamide . The role of these genes in the plant-bacteria interaction is often unclear . However, their function as a virulence determinant is established for Pseudomonas savastanoi and Agrobacterium tumefaciens . Some members of the Agrobacteria, such as Agrobacterium vitis have a limited host range . We have characterized the tryptophan-2-monooxygenase (iaaM) gene of A . vitis strain AG162 and show it is different from other A . vitis strains and related to iaaM of A . rhizogenes . The sequence of AG162 iaaM was deposited in the Genbank database under the accession number AF142716.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3358 - 63
RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium; Reiss B et al.; Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco . Here we show that RecA plays a direct role in DNA strand exchange in vivo . The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants . Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation . However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold . Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis . Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Gene, 2000 Jan 25, 242(1-2), 331 - 6
Complete nucleotide sequence of a plant tumor-inducing Ti plasmid; Suzuki K et al.; Crown gall tumor disease in dicot plants is caused by Agrobacterium tumefaciens harboring a giant tumor-inducing (Ti) plasmid . Here, for the first time among agrobacterial plasmids, the nucleotide sequence of a typical nopaline-type Ti plasmid (pTi-SAKURA) was determined completely . In total, 195 open reading frames (ORFs) were estimated in the 206479 bp long sequence . 20 genes for conjugation, three for replication, 22 for pathogenesis and 37 for genetic colonization of host plants were found within two-thirds of the plasmid . These genes formed seven functional gene clusters with narrow inter-cluster spaces . In the remaining one-third of the plasmid, novel genes including homologs of mutT, Rhizobium nodQ and Sphingomonas ligE genes were found, which are likely to be responsible for the broad host range . Restriction fragment length variation indicates extreme plasticity of the part required for conjugational gene transfer and the above-mentioned one-third of the plasmid, even among closely related Ti plasmids.

Gene, 2000 Jan 25, 242(1-2), 105 - 14
Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter; Wang Y et al.; A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985 . J . Bacteriol . 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupre et al., 1997 . J . Bacteriol . 179, 78-89) has been developed . This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A . tumefaciens . The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters . This expression system will enable a more detailed analysis of the activation events in signal transduction in A . tumefaciens, and we expect it to be useful in other prokaryotes.

Gene, 2000 Jan 25, 242(1-2), 87 - 95
Cryptic polyadenylation sites within the coding sequence of three yeast genes expressed in tobacco; Grec S et al.; Three yeast genes, MIP (mitochondrial DNA polymerase) and two genes, YCF1 (yeast cadmium factor 1) and PDR5 (pleiotropic drug resistance 5), conferring multidrug resistance, were provided with the cauliflower mosaic virus 35S transcription promoter and introduced into tobacco using an Agrobacterium tumefaciens T-DNA-derived vector . Transcripts of each gene much shorter than those expected were found in the transgenic plants . RT-PCR and S1 nuclease mapping of the PDR5 and MIP transcripts demonstrated the presence of one (PDR5), or several close (MIP), cryptic polyadenylation site(s) within the coding sequence of these yeast genes . Possible sequences involved in polyadenylation are discussed.

Chin J Biotechnol, 1999, 15(2), 71 - 5
Transgenic potato plants expressing osmotin gene inhibits fungal development in inoculated leaves; Li R et al.; Osmotin, a 24-KD protein isolated from Nicotiana tabacum 'Xanthi nc' was very toxic to Phytophthora infestans in vitro assay . Transgenic potato plants expressing wild osmotin protein delayed the emergence of symptoms of disease . Two osmotin mutants have been produced by PCR mutagenesis . One mutant, delta C18 osmotin, had a nonsense mutation near the C-terminus that deleted the carboxyl terminal prepropeptide (CTPP) of 18 amino acids . Another mutant, osmotin-P, had amber mutation at 96th amino acid . Both mutant genes driven by CaMV d35S promoter were introduced in potato by Agrobacterium mediated genetic transformation . The expression and osmotin protein accumulation in transgenic potato were detected by Southern, Northern, and Dot-blot ELISA assay . Disease resistance test using P.infestens complex race showed that expression of both osmotin mutants in transgenic potato reduced the lesion growth rate in inoculated leaves . The results showed that accumulation of osmotin protein in intercellular spaces (delta C18osmotin) inhibited the development of fungal disease in inoculated leaves . Based on the results we suggest that antifungal activity was possibly located at N end of osmotin protein (osmotin-P).

Plant Sci, 2000 Apr 25, 153(2), 161 - 170
The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions; Polowick PL et al.; Transgenic pea plants can be produced by Agrobacterium-mediated transformation of thin slices from developing embryo axes . To determine if the method is effective for different pea genotypes, seven pea breeding lines adapted to western Canadian growing conditions were tested, using three different Agrobacterium tumefaciens transformation vectors . All vectors contained the gus (uidA) gene coding for the beta-glucuronidase (GUS) protein, but with different chemical selection genes . In total, 323 transgenic plants were recovered from 39 independent transformation events . Transgenic plants were recovered from each genotype and each selection system, but not from all combinations . GUS-positive explants were obtained from seeds harvested between 24 and 31 days after flowering . The mean time from Agrobacterium treatment to planting into soil averaged 186 days . Based on the initial number of seeds used, the transformation frequency was 0.6% (i.e . six independent transgenic events per 1000 axes sliced) . The inserted genes were functional and inherited in a Mendelian fashion . Although more plants were recovered by selection on chlorsulfuron, GUS activity was generally greater in plants selected on kanamycin . GUS activity in the leaves of the original plants varied, but GUS activity in the second generation was correlated with that of the original transformants.

Plant Sci, 2000 Apr 25, 153(2), 135 - 144
Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci; Takaichi M et al.; In vitro assay indicated that the human lysozyme has lytic activity against phytopathogenic fungi and bacteria . A human lysozyme gene was placed under control of the constitutive CaMV 35S promoter and the resulting expression plasmid was introduced into two cultivars (cv.) of carrot, Kurodagosun (K5) and Nantes Scarlet (NS), by Agrobacterium tumefaciens-mediated method . Seven and fourteen transgenic plants of cv . K5 and cv . NS were regenerated, respectively, and the obtained transgenic carrots of T0 generation was tested for disease resistance against Erysiphe heraclei, a pathogenic fungi causing powdery mildew . Among the tested lines, the transgenic plant No . 12-1 and 8-1 of cv . NS showed a fairly strong resistance against E . heraclei . The strong disease resistance was also confirmed in T1 generation . Disease resistance against another pathogen of leaf blight, Alternaria dauci, were also tested using T1 transgenic lines . Significant enhanced resistance was observed in the No . 12-1 of cv . NS . Accumulation of synthesized human lysozyme protein was observed in this line, a finding consistent with observed disease resistance.

J Bacteriol, 2000 Apr, 182(7), 1935 - 41
Sinorhizobium meliloti putA gene regulation: a new model within the family Rhizobiaceae; Soto MJ et al.; Proline dehydrogenase (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate . In Sinorhizobium meliloti, as in other microorganisms, the putA gene is transcriptionally activated in response to proline . In Rhodobacter capsulatus, Agrobacterium, and most probably in Bradyrhizobium, this activation is dependent on an Lrp-like protein encoded by the putR gene, located immediately upstream of putA . Interestingly, sequence and genetic analysis of the region upstream of the S . meliloti putA gene did not reveal such a putR locus or any other encoded transcriptional activator of putA . Furthermore, results obtained with an S . meliloti putA null mutation indicate the absence of any proline-responsive transcriptional activator and that PutA serves as an autogenous repressor . Therefore, the model of S . meliloti putA regulation completely diverges from that of its Rhizobiaceae relatives and resembles more that of enteric bacteria . However, some differences have been found with the latter model: (i) S . meliloti putA gene is not catabolite repressed, and (ii) the gene encoding for the major proline permease (putP) does not form part of an operon with the putA gene.

J Bacteriol, 2000 Apr, 182(7), 1889 - 94
pTAR-encoded proteins in plasmid partitioning; Kalnin K et al.; Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind . The diminutive partition region of pTAR of Agrobacterium spp . was reported to be exceptional, encoding only a single protein, ParA (D . R . Gallie and C . I . Kado, J . Mol . Biol . 193:465-478, 1987) . However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A . tumefaciens . Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA . ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension . In certain respects the Par proteins behave normally in the foreign host Escherichia coli . In E . coli, as in A . tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression . Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

J Biol Chem, 2000 Mar 17, 275(11), 7713 - 22
The antiactivator TraM interferes with the autoinducer-dependent binding of TraR to DNA by interacting with the C-terminal region of the quorum-sensing activator; Luo ZQ et al.; Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via the transcriptional activator TraR and the acyl-homoserine lactone Agrobacterium autoinducer (AAI) . Unique to this system, the activity of TraR is negatively modulated by an antiactivator called TraM . Analyses from yeast two-hybrid studies suggest that TraM directly interacts with the activator, but the conditions under which these components interact and the region of TraR responsible for this interaction are not known . Induction of traM in a strain in which TraR was activating transcription of a reporter system led to rapid cessation of gene expression . As assessed by a genetic assay that measures AAI-dependent DNA binding, TraM inhibited TraR function before and after the transcription factor had bound to its DNA recognition site . Consistent with this observation, in gel retardation assays, purified TraM abolished the DNA binding activity of TraR in a concentration-dependent manner . Such inhibition occurred independent of the order of addition of the reactants . As assessed by far Western analyses TraM interacts with TraR by directly binding the activator . TraM in its native form interacted with native TraR and also with heat-treated TraR but only when SDS was included with the denatured protein . TraM interacted with TraR on blots prepared with total lysates of cells grown in the presence and absence of AAI . Far Western analysis of N- and C-terminal deletion mutants localized a domain of TraR contributing to TraM binding to the C-terminal portion of the activator protein . Random mutagenesis by hydroxylamine treatment and error-prone polymerase chain reaction identified several residues in this region of TraR important for interacting with TraM as well as for transcriptional activation or/and DNA binding . We conclude that TraM inhibits TraR by binding to the activator at a domain within or close to the helix-turn-helix motif located at the C terminus of the protein.

Biochem Biophys Res Commun, 2000 Mar 16, 269(2), 508 - 12
Enhancing activity of epsilon in Escherichia coli and Agrobacterium tumefaciens cells; Golshani A et al.; Epsilon (epsilon) sequence is a bacterial enhancer of translation found in the bacteriophage T7 gene 10 . It is believed that its enhancing effect of epsilon is due to a base-pairing with the nucleotides 458-467 from the helical domain 17 of Escherichia coli 16S rRNA . To prove this we have taken advantage of the difference of this domain in Agrobacterium tumefaciens and E . coli . To evaluate the significance of nucleotide complementarity for the enhancing activity of epsilon, a series of nucleotide sequences matching either E . coli or A . tumefaciens domain 17 are cloned in a binary expression vector in front of the chloramphenicol acetyltransferase (CAT) gene . The CAT assay shows that: (i) the epsilon in combination with an SD consensus sequence increases the yield of CAT in both microorganisms over that obtained with the SD alone; (ii) the epsilon sequence complementary to the A . tumefaciens domain 17 leads to a 2.71-fold increase in the yield of CAT in homologous cells but not in E . coli cells; (iii) the yield of CAT correlates with the free energy of base-pairing with the helical domain 17 in both microorganisms .

Nucleic Acids Res . 2000 Apr 1;28(7):E19.
A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination; Choi S et al.; With current plant transformation methods ( Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites . Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable . An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/ lox . Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells . By incorporating the Cre/ lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible . Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted 'position effects' caused by the random integration of exogenously introduced DNA . In an effort to make the Cre/ lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes . In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.

Amino Acids, 1999, 17(4), 357 - 68
Antimicrobial activity of o-carboranylalanine; Oros G et al.; Functionalized polyhedral carboranes, including amino acid analogs, have unique physicochemical properties and are used as experimental anticancer agents . However, our current knowledge on their effect in nonmammalian biological systems is limited . We investigated the activity spectrum in vitro of o-carboranylalanine (o-Cba), considered to be a highly lipophilic analog of phenylalanine, against representative plant pathogenic bacteria and fungi of various taxonomic position . The antibacterial effect of o-Cba against some species was comparable to that of the widely used agricultural antibiotic, streptomycin . The sensitivity of individual bacterial species to o-Cba within the same genus varied to a greater extent than the average sensitivity of various genera . In general, this carborane-containing amino acid was more toxic to Gram positive bacteria (Bacillus, Corynebacterium, Curtobacterium, Micrococcus, Rhodococcus, and Staphylococcus) than to Gram negative ones (Agrobacterium, Erwinia, Escherichia, Pseudomonas, Rhizobium, and Xanthomonas) . Compared to the commercial fungicide, prochloraz, o-Cba was weakly toxic against various fungi (Zygo- and Ascomycota) . It was also inferior to the commercial fungicide metalaxyl in inhibiting the vegetative growth of oomyceteous plant pathogens (Pythium irregulare, Phytophthora cryptogea and Plasmopara halstedii) . Against the asexual spores of P . halstedii, o-Cba, however, was over a thousandfold more active than tridemorph, a selective zoospore inhibitor fungicide . For all taxonomic groups, the observed antimicrobial effect of o-Cba could be diminished with histidine, but not with phenylalanine . In studies on healthy and mildew-infected sunflower and tobacco plants o-Cba showed neither fungicidal nor phytotoxic effects at 500 ppm . This is the first report on the biological activity spectrum of a carborane-containing amino acid.

Planta Med, 2000 Feb, 66(1), 96 - 8
Quantitative determination of secoiridoid and gamma-pyrone compounds in Gentiana lutea cultured in vitro; Menkovic N et al.; The production of secondary metabolites was studied in shoots, roots, and hairy roots of Gentiana lutea obtained in vitro . In shoots, both secoiridoid and gamma-pyrone compounds were detected in amounts similar to those found in aerial parts of plants collected from nature . The most abundant secoiridoid was gentiopicrin while mangiferin was the main compound among the gamma-pyrones . The adventitious roots obtained in vitro showed a poor biosynthetic capacity . Upon infection with Agrobacterium rhizogenes, nine hairy root clones were established which differed in the amount of secondary metabolites.

Genome, 2000 Feb, 43(1), 102 - 9
A binary vector-based large insert library for Brassica napus and identification of clones linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS); Wu Y et al.; We constructed and characterized a large DNA insert library for Brassica napus that would facilitate genome-related research and map-based cloning efforts in Brassica species . This library, consisting of 92,160 clones arrayed in 384-well microtiter dishes, was based on a conventional plant transformation vector (binary vector), and was constructed using a single ligation with transformation efficiency of over 5000 recombinants per microliter of ligation mixture . Every clone in this library contains an insert in the size range of 30-190 kb, facilitating both chromosome walking and plant transformation . Screening this library with three DNA markers (C2, F10, and CabR) that are linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS) identified at least 17 positive clones for each probe . Among the 17 positive clones identified by C2, nine are linked to the restorer locus . Marker F10 identified 21 clones, of which only two are linked to the restorer locus . None of 68 clones identified by CabR is linked to the restorer locus . A stability test using two clones identified by the C2 marker indicated that large DNA inserts are stable in this conventional vector in both Escherichia coli and Agrobacterium.

Plant Physiol, 2000 Feb, 122(2), 447 - 52
Nucleoside diphosphate kinase required for coleoptile elongation in rice; Pan L et al.; Although several nucleoside diphosphate (NDP) kinase genes have been cloned in plants, little is known about the functional significance of this enzyme during plant growth and development . We introduced a chimeric gene encoding an antisense RNA of NDP kinase under the control of the Arabidopsis heat shock protein HSP81-1 promoter into rice (Oryza sativa L.) plants using the Agrobacterium tumefaciens transformation system . The expression of antisense RNA down-regulated the accumulation of mRNA, resulting in reduced enzyme activity even under the standard growth temperature (25 degrees C) in transgenic plants . Following heat shock treatment (37 degrees C), NDP kinase activities in some transgenic rice plants were more reduced than those grown under 25 degrees C . The comparison of the coleoptile growth under submersion showed that cell elongation process was inhibited in antisense NDP kinase transgenic plants, suggesting that an altered guanine nucleotide level may be responsible for the processes.

J Bacteriol, 2000 Mar, 182(6), 1706 - 13
Identification of genes in the RosR regulon of Rhizobium etli; Bittinger MA et al.; RosR is a determinant of nodulation competitiveness and cell surface characteristics of Rhizobium etli and has sequence similarity to a family of transcriptional repressors . To understand how RosR affects these phenotypes, we mutagenized a rosR mutant derivative of R . etli strain CE3 with a mini-Tn5 that contains a promoterless gusA gene at one end, which acts as a transcriptional reporter . Using a mass-mating technique, we introduced rosR into each mutant in trans and screened for mutants that expressed different levels of beta-glucuronidase activity in the presence and absence of rosR . A screen of 18,000 mutants identified 52 insertions in genes negatively regulated by RosR and 1 insertion in a gene positively regulated by RosR . Nucleotide sequence analysis of the regions flanking the insertions suggests that RosR regulates genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism and those in a region containing sequence similarity to virC1 and virD3 from Agrobacterium tumefaciens . Two of the mutants produced colonies with altered morphology and were more competitive in nodulation than was CE3DeltarosR, the rosR parent . One mutant that contained an insertion in a gene with similarity to exsH of Sinorhizobium meliloti did not nodulate the plant host Phaseolus vulgaris without rosR . These results indicate that RosR directly or indirectly influences expression of diverse genes in R . etli, some of which affect the cell surface and nodulation competitiveness.

J Bacteriol, 2000 Mar, 182(6), 1541 - 8
TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58; Hamilton CM et al.; Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf . During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge . These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components . The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4) . A . tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome . However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome . The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors . Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector . When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome . However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid . VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome . TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge . We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge . However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge . These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

Structure Fold Des, 2000 Feb 15, 8(2), 143 - 51
The atomic-resolution structure of a novel bacterial esterase; Bourne PC et al.; Background: A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species . This esterase 713 is encoded by a 1062 base pair gene . The presence of a leader sequence of 27 amino acids suggests that this enzyme is exported from the cytosol . Esterase 713 has been over-expressed in Agrobacterium without this leader sequence . Its amino acid sequence shows no significant homology to any known protein sequence . Results: The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1 . 1 A resolution . The subunits of this dimeric enzyme comprise a single domain with an alpha/beta hydrolase fold . The catalytic triad has been identified as Ser206-His298-Glu230 . The acidic residue of the catalytic triad (Glu230) is located on the beta6 strand of the alpha/beta hydrolase fold, whereas most other alpha/beta hydrolase enzymes have the acidic residue located on the beta7 strand . The oxyanion hole is formed by the mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1, 4-benzodiazepine-2-acetic acid . Cys71 forms a disulphide bond with the neighbouring Cys72 . Conclusions: Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases . Residues of the oxyanion hole were confirmed by structural comparison with Rhizomucor miehei lipase . It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of the periplasmic space.

Mol Cells, 1999 Dec 31, 9(6), 603 - 8
Cymbidium mosaic virus coat protein gene in antisense confers resistance to transgenic Nicotiana occidentalis; Lim SH et al.; The nucleotide sequence of the 3'-terminal region of the Korean isolate of cymbidium mosaic virus (CyMV-Ca) from a naturally infected cattleya was determined . The sequence contains an open reading frame (ORF) coding for the viral coat protein (CP) at the 3'-end and three other ORFs (triple gene block or movement protein) of CyMV . The CP gene encodes a polypeptide chain of 220 amino acids with a molecular mass of 23,760 Da . The deduced CP sequence showed a strong homology with those of two CyMVs reported . A construct of the CyMV-Ca CP gene in the antisense orientation in the plant expression vector pMBP1 was transferred via Agrobacterium tumefaciens-mediated transformation into Nicotiana occidentalis which is a propagation host of CyMV . The T1 progeny of the transgenic plants were inoculated with CyMV and found to be highly resistant to CyMV infection.

J Bacteriol, 2000 Mar, 182(5), 1304 - 12
Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component; Zorreguieta A et al.; Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers . When grown on agar medium, CMC degradation occurred only directly below colonies of R . leguminosarum, suggesting that the enzymes remain attached to the bacteria . Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type . There was no CMC degradation in the region between the colonies . By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony . Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface . PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed . EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB . However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis . Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R . leguminosarum . This indicates that the surface of A . tumefaciens is inappropriate to activate CMC degradation by PlyB . Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

Planta, 2000 Jan, 210(2), 252 - 60
Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings; Migge A et al.; The impact of increased plastidic glutamine synthetase (GS-2; EC 6.1 . 3.2) activity on foliar amino-acid levels and on biomass production was examined in transgenic tobacco . For that, tobacco was transformed via Agrobacterium tumefaciens with a binary vector containing a tobacco GS-2 cDNA downstream of the leaf-specific soybean ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promotor . Two transgenic tobacco lines with 15- to 18-fold higher foliar GS-2 transcript levels than the wild type were obtained . The GS-2 protein pools and the specific GS-2 activities were, however, only 2- to 2.3-fold higher in the leaves of the transgenic plants than in the leaves of the wild type . This discrepancy may reflect a post-transcriptional control of GS-2 protein accumulation . The increased GS-2 activity was correlated with a decrease in the leaf ammonium pool (3.7-fold) and an increase in the levels of some free amino acids, including glutamate (2 . 5-fold) and glutamine (2.3-fold) . The accumulation of soluble protein per unit fresh weight, however, remained unchanged . This result indicates that a process downstream of the synthesis of the primary organic products of N-assimilation is limiting leaf protein accumulation . Nevertheless, the overexpression of GS-2 stimulated the growth rate of the transgenic tobacco seedlings which, consequently, were larger (20-30% on a fresh-weight basis) than wild-type seedlings grown under identical conditions . This result suggests that GS-2 is the rate-limiting enzyme during biomass production in tobacco seedlings . The requirement for glutamate as the ammonium acceptor in the reaction catalysed by GS-2 may imply that there is co-regulation of GS-2 and ferredoxin dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) gene expression . Increased leaf GS-2 activity had, however, no influence on the foliar Fd-GOGAT protein abundance . This result suggests that in tobacco leaves, more Fd-GOGAT is present than required to meet the demands of primary ammonium assimilation and that there is no strong interdependence between GS-2 and Fd-GOGAT protein expression.

Planta, 2000 Jan, 210(2), 232 - 40
Transgene expression driven by heterologous ribulose-1, 5-bisphosphate carboxylase/oxygenase small-subunit gene promoters in the vegetative tissues of apple (Malus pumila mill.); Gittins JR et al.; It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity . To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max {h.} Mers.), to drive expression of the beta-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.) . Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter . These quantitative GUS data were assessed for their relationship to the copy number of transgene loci . The precise location of GUS activity in leaves was identified histochemically . The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter . The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter . Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf . The influence of light on expression was also determined . The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be . Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple.

Planta, 2000 Jan, 210(2), 195 - 204
High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode; Cho HJ et al.; Cotyledon explants of 10 soybean {Glycine max (L.) Merr.} cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively . Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin . Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction . All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors . Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes . Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP . Attempts to induce shoot formation from the hairy roots failed . Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H . glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation . Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP . This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

Chem Biol, 2000 Jan, 7(1), 65 - 76
Xenognosin sensing in virulence: is there a phenol receptor in Agrobacterium tumefaciens?
Campbell AM, Tok JB, Zhang J, Wang Y, Stein M, Lynn DG, Binns AN.
BACKGROUND: The mechanisms of signal perception and transmission in the 'two-component' autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known . Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols . RESULTS: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid . CONCLUSIONS: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception . This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.

J Antibiot (Tokyo), 1999 Nov, 52(11), 983 - 7
Agrochelin, a new cytotoxic antibiotic from a marine Agrobacterium . Taxonomy, fermentation, isolation, physico-chemical properties and biological activity; Acebal C et al.; Agrochelin, a new alkaloid cytotoxic substance, was produced by the fermentation of Agrobacterium sp . The compound was obtained from the bacterial cells by solvent extraction and purified by silica gel chromatography . Agrochelin (1) and its acetyl derivative (2) exhibited cytotoxic activity.

J Clin Pathol, 1999 Sep, 52(9), 653 - 7
Modulation of Helicobacter pylori induced interleukin-8 synthesis in gastric epithelial cells mediated by cag PAI encoded VirD4 homologue; Crabtree JE et al.; BACKGROUND: Strains of Helicobacter pylori carrying the virulence associated cag pathogenicity island (PAI) induce gastric epithelial synthesis of the chemokine interleukin-8 (IL-8), a neutrophil chemoattractant, and thereby a strong inflammatory response during chronic infection of the human gastric mucosa . Previous mutational analyses have shown that many genes in the cag PAI are needed to elicit IL-8 synthesis in gastric epithelial cells, and also that some genes are not involved . AIM: To test the possibility that certain genes in the cag PAI also downregulate (modulate) the inflammatory response elicited by cag+ H pylori infection . METHODS: Cells of L5F11, a derivative of the Kato-3 gastric epithelial cell line that carries an engineered IL-8 promoter-luciferase reporter gene fusion, were cocultured with H pylori strain 26695 or with an isogenic mutant in which most of the cag PAI ORF 10 gene, an Agrobacterium virD4 homologue, was deleted . Luciferase activity was measured to assess IL-8 gene transcription and secreted IL-8 was measured by enzyme linked immunosorbent assay to assess synthesis and release of IL-8 protein from gastric epithelial cells . RESULTS: Inactivation of ORF10 led to a 2.8-fold increase in IL-8 gene transcription and a 3.6-fold increase in IL-8 synthesis and secretion . CONCLUSIONS: The results suggest that this VirD4 homologue participates in the control of inflammation that H pylori infection elicits by downregulating (modulating) the strong induction of IL-8 synthesis mediated by other cag encoded proteins.

Plant J, 2000 Jan, 21(1), 73 - 81
Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato; Bendahmane A et al.; Rx2 confers resistance against potato virus X (PVX) . To clone Rx2, we developed a system based on Agrobacterium-mediated transient expression of candidate R genes in transgenic tobacco leaves expressing the PVX coat protein elicitor of Rx2-mediated resistance . Using this system, a potato gene eliciting HR specifically in the presence of the elicitor was identified . Based on genetical and functional analysis, it is concluded that the cloned gene is Rx2 . The transient expression system is potentially adaptable to cloning of any other resistance gene . The Rx2 locus is on chromosome V of potato and the encoded protein is highly similar to the products of Rx1 and Rxh1 encoded on potato chromosome XII . Rxh1 has been shown elsewhere to encode a potato cyst nematode resistance gene Gpa2 . All three proteins are in the leucine zipper-nucleotide binding site-leucine rich repeat class of resistance gene products . Rx1 and Rx2 are functionally identical and are almost identical in the C terminal region consistent with a role of the leucine rich repeats in recognition of the PVX coat protein . In the N terminal, half there are some regions where the Rx1 and Rx2 proteins are more similar to each other than to the Rxh1 protein . However, in other regions these proteins are more similar to Rxh1 than to each other . Based on this mosaic pattern of sequence similarity, we conclude that sequence exchange occurs repeatedly between genetically unlinked disease resistance genes through a process of gene conversion.

Plant J, 2000 Jan, 21(1), 9 - 16
Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation; Mysore KS et al.; Germ-line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens . We have recently identified several Arabidopsis ecotypes and T-DNA-tagged mutants that are recalcitrant to Agrobacterium-mediated transformation of cut root segments . Some of these ecotypes and mutants are deficient in their ability to bind bacteria . Some are deficient in T-DNA integration . We report here that using a germ-line transformation protocol we transformed these ecotypes and mutants, including attachment- and integration-defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild-type plants . However, we could not transform otherwise highly susceptible Arabidopsis plants by germ-line or root transformation using several vir and attachment-deficient Agrobacterium mutants . These results indicate that certain plant factors important for transformation may exist in germ-line tissue but may be lacking in some somatic cells.

Mol Microbiol, 2000 Jan, 35(2), 407 - 14
An Agrobacterium catalase is a virulence factor involved in tumorigenesis; Xu XQ et al.; Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component . However, the soil-borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T-DNA that causes crown gall tumours on many plant species . A . tumefaciens does not elicit a typical HR on those plants . Here, we report that inactivation of one of A . tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2 . This provides the first genetic evidence that the Agrobacterium-plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen.

FEBS Lett, 2000 Jan 21, 466(1), 40 - 4
The E358S mutant of Agrobacterium sp . beta-glucosidase is a greatly improved glycosynthase; Mayer C et al.; Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides . The 'original' glycosynthase, derived from Agrobacterium sp . beta-glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% {Mackenzie, L.F., Wang, Q., Warren, R.A.J . and Withers, S.G . (1998) J . Am . Chem . Soc . 120, 5583-5584} . This mutant has now been re-cloned with a His(6)-tag into a pET-29b(+) vector, allowing gram scale production and single step chromatographic purification . A dramatic, 24-fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire . Thus poor acceptors for Abg E358A, such as PNP-GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP-beta-LacNAc . The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the alpha-glycosyl fluoride.

J Bacteriol, 2000 Feb, 182(4), 1080 - 8
Quorum sensing but not autoinduction of Ti plasmid conjugal transfer requires control by the opine regulon and the antiactivator TraM; Piper KR et al.; Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is controlled by autoinduction via the transcriptional activator TraR and the acyl-homoserine lactone ligand, Agrobacterium autoinducer (AAI) . This control process is itself regulated by opines, which are small carbon compounds produced by the crown gall tumors that are induced by the bacteria . Opines control autoinduction by regulating the expression of traR . Transfer of pTiC58 from donors grown with agrocinopines A and B, the conjugal opines for this Ti plasmid, was detected only after the donors had reached a population level of 10(7) cells per cm(2) . Donors incubated with the opines and AAI transferred their Ti plasmids at population levels about 10-fold lower than those incubated with opines only . Transcription of the tra regulon, as assessed by monitoring a traA::lacZ reporter, showed a similar dependence on the density of the donor population . However, even in cultures at low population densities that were induced with opines and AAI, there was a temporal lag of between 15 and 20 h in the development of conjugal competence . Moreover, even after this latent period, maximal transfer frequencies required several hours to develop . This lag period was independent of the population density of the donors but could be reduced somewhat by addition of exogenous AAI . Quorum-dependent development of conjugal competence required control by the opine regulon; donors harboring a mutant of pTiC58 deleted for the master opine responsive repressor accR transferred the Ti plasmid at maximum frequencies at very low population densities . Similarly, an otherwise wild-type derivative of pTiC58 lacking traM, which codes for an antiactivator that inhibits TraR activity, transferred at high frequency in a population-independent manner in the absence of the conjugal opines . Thus, while quorum sensing is dependent upon autoinduction, the two phenomena are not synonymous . We conclude that conjugal transfer of pTiC58 is regulated in a quorum-dependent fashion but that supercontrol of the TraR-AAI system by opines and by TraM results in a complex control process that requires not only the accumulation of AAI but also the expression of TraR and the synthesis of this protein at levels that overcome the inhibitory activity of TraM.

Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 948 - 53
An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration; Mysore KS et al.; Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, the T-DNA, to the plant and integrating the T-DNA into the plant genome . Little is known about the T-DNA integration process, and no plant genes involved in integration have yet been identified . We characterized an Arabidopsis mutant generated by T-DNA insertional mutagenesis, rat5, that is resistant to Agrobacterium root transformation . rat5 contains two copies of T-DNA integrated as a tandem direct repeat into the 3' untranslated region of a histone H2A gene, upstream of the polyadenylation signal sequence . Transient and stable beta-glucuronidase expression data and assessment of the amount of T-DNA integrated into the genomes of wild-type and rat5 Arabidopsis plants indicated that the rat5 mutant is deficient in T-DNA integration . We complemented the rat5 mutation by expressing the RAT5 histone H2A gene in the mutant plant . Overexpression of RAT5 in wild-type plants increased Agrobacterium transformation efficiency . Furthermore, transient expression of a RAT5 gene from the incoming T-DNA was sufficient to complement the rat5 mutant and to increase the transformation efficiency of wild-type Arabidopsis plants.

J Bacteriol, 2000 Feb, 182(3), 758 - 63
The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another; Das A et al.; The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants . The assembly of the transport pore will require interaction among the constituent proteins . The identification of proteins that interact with one another can provide clues to the assembly of the transport pore . We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10 . Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another . In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself . These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore . Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9 . All interaction domains of both proteins mapped to the N-terminal half of the proteins . Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer . We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function . Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9 . The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.

J Dairy Sci, 1999 Dec, 82(12), 2620 - 4
A study on the prevalence of gram-negative bacteria in bulk tank milk; Jayarao BM et al.; Bulk tank milk from 131 dairy herds in eastern South Dakota and western Minnesota were examined for coliforms and noncoliform bacteria . Coliforms were detected in 62.3% of bulk tank milk samples . Counts ranged from 0 to 4.7 log10 cfu/ml . The mean count was 3.4 log10 cfu/ml . Gram-negative noncoliform bacteria were observed in 76.3% of bulk tank milk . Counts ranged from 0 to 6.2 log10 cfu/ml . The mean count was 4.8 log10 cfu/ml . A total of 234 isolates from bulk tank milk were examined to species level; 205 isolates belonged to 28 species . Coliforms and gram-negative noncoliform bacteria accounted for 32.9 and 67.1% of the total isolates, respectively . Organisms such as Agrobacterium radiobacter, Bordetella spp., Comamonas testosteroni, Listonella damsela, Ochrobactrum anthropi, and Oligella urethralis were isolated from bulk tank milk in this study . These organisms have not been reported previously in bulk tank milk . A total of 116 isolates of Pseudomonas spp . were isolated from raw milk; 98 isolates belonged to nine Pseudomonas spp., and the remaining 18 isolates could not be identified to their species level . Pseudomonas was the most predominant genus . Pseudomonas fluorescens was the most predominant species isolated from bulk tank milk and accounted for 29.9% of all isolates examined . The results of the study suggest that counts of coliforms and noncoliform bacteria in bulk tank milk vary considerably . The isolates represent a wide variety of Gram-negative bacterial species . Examination of bulk tank milk for coliforms and noncoliform bacteria could provide an indication of current and potential problems associated with bacterial counts and milk quality.

Mol Gen Genet, 1999 Dec, 262(4-5), 608 - 17
The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers; Guevara-Garcia A et al.; A 479-bp bi-directional promoter controls the expression of two genes (mas1' and mas2') that encode enzymes for the synthesis of the opine mannopine in plant tissues infected with Agrobacterium tumefaciens . This 5' regulatory region (mas promoter) contains all the cis-acting elements involved in mediating the complex regulatory properties of these genes in plants . Using different mas promoter regions fused to a minimal 35S promoter (35Sdelta108), we found that the regulatory properties of these divergent promoters result from the presence of orientation-dependent negative and positive regulatory regions . Some of these elements have the unusual property of acting as enhancers in one orientation and as silencers in the other . Using electrophoretic mobility shift analysis (EMSA), we showed that the functional mas promoter regions identified by fluorometric and histochemical assays for reporter gene activity in transgenic plants have the ability specifically to bind nuclear protein factors from Nicotiana tabacum, Phaseolus vulgaris, Solanum tuberosum, and Arabidopsis thaliana.

J Ethnopharmacol, 1999 Dec 15, 68(1-3), 47 - 53
Investigation of bioactivity of extracts from Moroccan solitary tunicate Cynthia savignyi; Abourriche A et al.; Extracts of the tunicate Cynthia savignyi from the Moroccan Atlantic sea have been investigated in five bioassays . The first is an antibacterial test against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Agrobacterium tumefaciens; the second is an antifungal test against three tomato pathogenic fungi, Botrytis cinerea, Fusarium oxysporum and Verticillium albo-atrum; the third is a test based to the ability to reduce DNA peak size in procedures using an HPLC system for detection of antitumour agents; the fourth is a toxicity test using larva of Artemia salina; and the last is an enzymatic inhibitory test against papain and trypsin . For all the bioassays, extracts of C . savignyi were found to be bioactive . This result suggests that this tunicate is able to produce biologically active agents required for an overall defence against their predators.

Appl Environ Microbiol, 2000 Jan, 66(1), 92 - 7
A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies; Santini JM et al.; A previously unknown chemolithoautotrophic arsenite-oxidizing bacterium has been isolated from a gold mine in the Northern Territory of Australia . The organism, designated NT-26, was found to be a gram-negative motile rod with two subterminal flagella . In a minimal medium containing only arsenite as the electron donor (5 mM), oxygen as the electron acceptor, and carbon dioxide-bicarbonate as the carbon source, the doubling time for chemolithoautotrophic growth was 7.6 h . Arsenite oxidation was found to be catalyzed by a periplasmic arsenite oxidase (optimum pH, 5.5) . Based upon 16S rDNA phylogenetic sequence analysis, NT-26 belongs to the Agrobacterium/Rhizobium branch of the alpha-Proteobacteria and may represent a new species . This recently discovered organism is the most rapidly growing chemolithoautotrophic arsenite oxidizer known.

J Agric Food Chem, 1999 Dec, 47(12), 5038 - 43
Genetically modified organisms in food-screening and specific detection by polymerase chain reaction; Vollenhofer S et al.; PCR methods for the detection of genetically modified organisms (GMOs) were developed that can be used for screening purposes and for specific detection of glyphosate-tolerant soybean and insect-resistant maize in food . Primers were designed to amplify parts of the 35S promoter derived from Cauliflower Mosaic Virus, the NOS terminator derived from Agrobacterium tumefaciens and the antibiotic marker gene NPTII (neomycin-phosphotransferase II), to allow for general screening of foods . PCR/hybridization protocols were established for the detection of glyphosate-tolerant RoundUp Ready soybean and insect-resistant Bt-maize . Besides hybridization, confirmation of the results using restriction analysis was also possible . The described methods enabled a highly sensitive and specific detection of GMOs and thus provide a useful tool for routine analysis of raw and processed food products.

J Bacteriol, 1999 Dec, 181(24), 7509 - 15
Differential and independent roles of a sigma(32) homolog (RpoH) and an HrcA repressor in the heat shock response of Agrobacterium tumefaciens; Nakahigashi K et al.; The heat shock response in alpha proteobacteria is unique in that a combination of two regulators is involved: a positive regulator, RpoH (sigma(32) homolog), found in the alpha, beta, and gamma proteobacteria, and a negative regulator, HrcA, widely distributed in eubacteria but not in the gamma proteobacteria . To assess the differential roles of the two regulators in these bacteria, we cloned the hrcA-grpE operon of Agrobacterium tumefaciens, analyzed its transcription, and constructed deletion mutants lacking RpoH and/or HrcA . The DeltarpoH mutant and DeltarpoH DeltahrcA double mutant were unable to grow above 30 degrees C . Whereas the synthesis of heat shock proteins (e.g., DnaK, GroEL, and ClpB) was transiently induced upon temperature upshift from 25 to 37 degrees C in the wild type, such induction was not observed in the DeltarpoH mutant, except that GroEL synthesis was still partially induced . By contrast, the DeltahrcA mutant grew normally and exhibited essentially normal heat induction except for a higher level of GroEL expression, especially before heat shock . The DeltarpoH DeltahrcA double mutant showed the combined phenotypes of each of the single mutants . The amounts of dnaK and groE transcripts before and after heat shock, as determined by primer extension, were consistent with those of the proteins synthesized . The cellular level of RpoH but not HrcA increased significantly upon heat shock . We conclude that RpoH plays a major and global role in the induction of most heat shock proteins, whereas HrcA plays a restricted role in repressing groE expression under nonstress conditions.

J Bacteriol, 1999 Dec, 181(24), 7485 - 92
Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens; Schmidt-Eisenlohr H et al.; Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material . Recently, VirB2 was indeed shown to be its major component (E.-M . Lai and C . I . Kado, J . Bacteriol . 180:2711-2717, 1998) . Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed . Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction . In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions . The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium . In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation . VirB5 may therefore be directly involved in pilus assembly, possibly as minor component . In contrast, secreted VirB1* showed no association with the T pilus . In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures . Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.

Planta, 1999 Nov, 210(1), 19 - 26
Expression of a conifer glutamine synthetase gene in transgenic poplar
Gallardo F, Fu J, Canton FR, Garcia-Gutierrez A, Canovas FM, Kirby EG.
The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M . Lam et al . 1995, Plant Cell 7: 887-898) . We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene . A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula x P . alba) via Agrobacterium tumefaciens . Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin . The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis . Transgene expression was detected in all selected poplar lines at the mRNA level . The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes . Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees . Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months . Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.

Yi Chuan Xue Bao, 1999, 26(3), 262 - 8
{Insect-resistant transgenic plants of Brassica napus and analysis of resistance in the plants}; Li XB et al.; Cotyledons, each with a 1-2 mm petiole at its base, were cut from axenic seedlings and infected with Agrobacterium tumefaciens . After 2-3 days of cocultivation, the cotyledon explants were transferred to MS selection medium containing 15 mg/L kanamycin and 4.5 mg/L 6-BA to induce shoot differentiation . Kanamycin-resistant shoots were subcultured on selection medium with 20-50 mg/L kanamycin for 3-6 months for eliminating escaped non-transformants, and then rooted on MS medium containing 25 mg/L kanamycin and 0.1 mg/L NAA . Whole plants were transplanted into soil and grew in the field . DNA Southern blot hybridization and polymerase chain reaction showed that some of the plants were positive when probed with the insecticidal crystal protein gene . The transgenic plants exhibited tolerant to pest insects such as Laphygma exigua and Pieris rapae in leaf feeding experiments Kanamycin-resistance and insect-resistance were maintained in the progeny . The foreign genes were delivered to the progeny according to Mendelian Law of single gene segregation.

Biotechnol Prog, 1999 Nov-Dec, 15(6), 1039 - 45
One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains; Chao YP et al.; The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction . A positive clone was scored, and its nucleotide sequence was further analyzed . The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site . By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A . radiobacter NRRL B11291 . Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A . radiobacter NRRL B11291 attained 20% within the same period of reaction time . These results illustrate the feasibility in employing recombinant E . coli to accomplish one-step conversion of DL-HPH to D-HPG . In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate . It suggests that carbamoylase is the step setting the pace of the reaction . Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system . This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice . In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.

Microbes Infect, 1999 Dec, 1(14), 1211 - 9
Intracellular lifestyle of Brucella spp . Common genes with other animal pathogens, plant pathogens, and endosymbionts; Ugalde RA; Brucella spp . are intracellular pathogens that belong, like Agrobacterium, Rhizobium and Rickettsia, to the alpha-2-subgroup of proteobacteria . The genome organization of most Brucella spp . is characterized by the presence of two chromosomes . The intracellular lifestyle of Brucella, as well as the possible genes involved in pathogenesis and host cell signaling, are discussed, including the presence of genes with high similarity to those from other animal pathogens, plant pathogens and endosymbionts.

J Gen Virol, 1999 Nov, 80 ( Pt 11), 2813 - 22
Transformation of tobacco and potato with cDNA encoding the full-length genome of potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication; Franco-Lara LF et al.; A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation . Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants . PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants . Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants . Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted . In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants . In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants . In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants . In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus . From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.

Plant Mol Biol, 1999 Sep, 41(2), 217 - 31
A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs; Stromvik MV et al.; We report the isolation of a novel soybean gene, Msg, which is highly expressed in developing soybean pods . The gene shows significant homology to a family of fruit- and flower-specific genes, designated the major latex protein (MLP) homologues, so far reported in only a few species and whose functions are unknown . The MLPs are more distantly related to a group of pathogenesis-related proteins (IPR or PR-10) whose functions are likewise unknown . This is the first report of a MLP homologue in a plant for which there is already an IPR-protein reported . We performed an analysis of the Msg promoter with 14 different promoter fragments ranging from 0.65 kb to 2.26 kb, fused to the uidA (GUS) gene . High transient expression was obtained with all the constructs upon particle bombardment in soybean and green bean pods . Stable Arabidopsis transformants were obtained with the Agrobacterium vacuum infiltration method . The promoter is fully active in Arabidopsis only in plants transformed with the 2.26 kb fragment promoter, expressing GUS in nectaries, nodes, short style and in guard cells of the silique, pedicel and stem but not in mature leaves . Surprisingly, the proximal 650 bp TATA-containing region cannot function on its own in Arabidopsis and can be deleted without a change in expression pattern in both Arabidopsis and soybean . Thus, tissue-specific regions of the complex Msg promoter reside in the distal 5' regions upstream of a dispensable TATA box in contrast to many examples of tissue-specific elements that reside much closer to the TATA box.

Genetika, 1999 Sep, 35(9), 1214 - 22
{Insertional mutagenesis of Arabidopsis thaliana: increase of germinating seeds transformation efficiency after sonication}; Tomilov AA et al.; An efficient procedure of transforming Arabidopsis thaliana germinating seeds was elaborated on the basis of the method of Feldmann and Marks . The procedure involves microdamaging T1 seeds by sonication before culturing with a vector Agrobacterium strain and yields more than 1% T2 transformants . Germinating seeds were transformed with Agrobacterium timefaciens hypervirulent strain A281 carrying plasmid pLD3 derived from pBI121 . A collection of 54 A . thaliana T2 transformants was obtained; some of them showed marked morphological alterations . The transgenic nature of plants that acquired resistance to a marker antibiotic was confirmed histochemically and by PCR amplification of a T-DNA region.

Plant J, 1999 Sep, 19(6), 727 - 34
A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences; Hanson B et al.; A simple modification to standard binary vector design has been utilized to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences . A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker . The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors) . The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int) . To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted . Populations of 50 transgenic plants were scored for LUC expression . The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector . Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector . Importantly, the efficiency with which transformed tobacco plants was obtained was reduced by no more than 30% . The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences . Similar transformation results were obtained with tomato and grape . The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.

Plant J, 1999 Nov, 20(3), 295 - 304
The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration; De Buck S et al.; After Agrobacterium-mediated plant transformation, multiple T-DNAs frequently integrate at the same position in the plant genome, resulting in the formation of inverted and direct repeats . Because these inverted repeats cannot be amplified and analyzed by PCR, Arabidopsis root cells were co-transformed with two different T-DNAs with distinct sequences adjacent to the T-DNA borders . Nine direct or inverted T-DNA border junctions were analyzed at the sequence level . Precise end-to-end fusions were found between two right border ends, whereas imprecise fusions and filler DNA were present in T-DNA linkages containing a left border end . The results suggest that end-to-end ligation of double-stranded T-DNAs occurs especially between right T-DNA ends and that illegitimate recombination on the basis of microhomology, deletions, repair activities and insertions of filler DNA is involved in the formation of left border T-DNA junctions . Therefore, a similar illegitimate recombination mechanism is proposed that is involved in the formation of complex T-DNA inserts as well as in the integration of the T-DNA in the plant genome.

Plant J, 1999 Oct, 20(2), 237 - 43
Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serine acetyltransferase; Blaszczyk A et al.; Plant expression cassettes containing the Escherichia coli cysE gene alleles (encoding SAT) were constructed . After the Agrobacterium-mediated transformation of tobacco, we identified stable transformed plants containing several-fold higher SAT activity in comparison to the control plant . Determination of non-protein thiol contents indicated two- to threefold higher cysteine and glutathione levels in some of these transgenic plants . The maximal elevation of the cysteine level was about fourfold while that of GSH was about twofold higher than in the controls . The most striking physiological consequence of the modification of sulfur metabolite levels in the transgenic plants, however, was their several-fold increased resistance to oxidative stress generated by exogenous hydrogen peroxide.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 14153 - 8
Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato; Tai TH et al.; The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv . vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2 . The involvement of avrBs2 in pathogen fitness and its prevalence in many X . campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species . Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay . A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified . This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2 . Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

J Agric Food Chem, 1999 Apr, 47(4), 1776 - 80
Phenolic content in differentiated tissue cultures of untransformed and Agrobacterium-transformed roots of anise (Pimpinella anisum L.); Andarwulan N et al.; To investigate the role of differentiation of anise tissue cultures on total phenolic and anethole contents, benzylaminopurine- and thidiazuron-induced shoot cultures were generated from roots of the A-8 clonal line and its Agrobacterium rhizogenes-induced genetically transformed derivative JB-10 . Embryogenic cultures were induced following 2,4-D treatment . Root cultures were multiplied on hormone-free medium . The effect of proline on differentiation and phenolic synthesis was also investigated . GC/MS studies indicate that anethole was not produced in root or other differentiated cultures . The predominant phenolic metabolite, however, was an anethole precursor, epoxypseudoisoeugenol-2-methylbutyrate (EPB) . Total phenolics and EPB contents were highest in root cultures, which also correlated with higher proline content . Embryo and shoot cultures had reduced phenolic level and EPB and proline contents . Antioxidant activity in all differentiating cultures was high on day 60 compared to that on day 30, and there was no significant difference between differentiating tissues . This indicated that antioxidant protection might be linked not only to phenolics but to other nonphenolic metabolites as well.

Mol Microbiol, 1999 Nov, 34(3), 512 - 22
The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid; Kalogeraki VS et al.; Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450-type monooxygenases . Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon . We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds . We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2 . Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH-proficient host . For all tested compounds, VirH2 appeared to be solely responsible for this effect . One such compound, ferulic acid, was chosen for biochemical analysis . Ferulic acid was degraded by a VirH-proficient host but not by a VirH mutant . The wild-type strain released large amounts of a more hydrophilic compound into the cell supernatant . This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid . This indicates that wild-type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O-demethylation reaction . Ferulic acid was far more toxic than caffeic acid to the wild-type strain, although the wild-type strain was more resistant to ferulic acid than was the virH mutant . Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.

Mol Microbiol, 1999 Oct, 34(2), 282 - 94
Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes; Hwang I et al.; Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum-sensing system involving the transcriptional activator TraR and the acyl homoserine lactone autoinducer N-(3-oxo-octanoyl)-L-homoserine lactone (AAI) . Activation of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid . Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activator protein . Using the yeast two-hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmid pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 produced beta-galactosidase and grew on medium lacking leucine, both phenotypes indicative of an interaction between the two proteins . Early termination mutants and substitution mutants mapping to the C-terminus of TraM were isolated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens . These mutants all failed to interact with the TraR fusion in the two-hybrid system . An N-terminal deletion mutant of TraM lacking the first 27 residues weakly interacted with TraR in the two-hybrid system whereas deletions of 48 amino acids or more abolished the interaction . As assessed by Western blot analysis, the mutant fusion proteins were produced at levels indistinguishable from that of the wild-type TraM in the yeast tester strain . Mutants of TraR that were not inhibited by TraM in A . tumefaciens were isolated and fell into two classes . In the first, the mutation resulted in increased expression of wild-type TraR . In the second, a proline residue at position 176 was changed to serine (P176 --> S) or to leucine (P176 --> L) . The P176 --> S mutant interacted with wild-type TraM, but at a detectably lower level, in the two-hybrid assay . Mutants of TraR with N-terminal deletions as large as 105 amino acids interfered with the ability of TraM to inhibit wild-type TraR in A . tumefaciens . Two-hybrid assays indicated that these mutants, as well as a C-terminal 49 residue fragment of TraR, can interact with TraM . We conclude that TraM and TraR interact in vivo and that this interaction is responsible for inhibition of TraR-mediated activation . We also conclude that the two proteins interact with each other through domains located at their respective C-termini.

Mol Vis . 1999 Oct 29;5:23.
A heterologous expression system for bovine lens transmembrane main intrinsic protein (MIP) in Nicotiana tabacum plants; de Peyer OS et al.; We have developed a heterologous expression system for transmembrane lens main intrinsic protein (MIP) in Nicotiana tabacum plant tissue . A native bovine MIP26 amplicon was subcloned into an expression cassette under the control of a constitutive Cauliflower Mosaic Virus promoter, also containing a neomycin phosphotransferase operon . This cassette was transformed into Agrobacterium tumefaciens by triparental mating and used to infect plant tissue grown in culture . Recombinant plants were selected by their ability to grow and root on kanamycin-containing media . The presence of MIP in the plant tissues was confirmed by PCR, RT-PCR and immunohistochemistry . A number of benefits of this system for the study of MIP will be discussed, and also its application as a tool for the study of heterologously expressed proteins in general.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13229 - 34
Horizontal gene transfer and mutation: ngrol genes in the genome of Nicotiana glauca; Aoki S et al.; Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca . It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana . Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N . glauca and plants transformed with Ngrol genes did not exhibit this phenotype . Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations . Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene . When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35S-RirolB plants . In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria . Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N . glauca.

Plant Physiol, 1999 Nov, 121(3), 957 - 964
The Effect of Elevated Concentrations of Fructose 2,6-Bisphosphate on Carbon Metabolism during Deacidification in the Crassulacean Acid Metabolism Plant Kalanchöe daigremontiana; Truesdale MR et al.; In C(3) plants, the metabolite fructose 2,6-bisphosphate (Fru 2,6-P(2)) has an important role in the regulation of carbon partitioning during photosynthesis . To investigate the impact of Fru 2,6-P(2) on carbon metabolism during Crassulacean acid metabolism (CAM), we have developed an Agrobacterium tumefaciens-mediated transformation system in order to alter genetically the obligate CAM plant Kalanchoe daigremontiana . To our knowledge, this is the first report to use genetic manipulation of a CAM species to increase our understanding of this important form of plant metabolism . Transgenic plants were generated containing a modified rat liver 6-phosphofructo-2-kinase gene . In the plants analyzed the activity of 6-phosphofructo-2-kinase ranged from 175% to 198% of that observed in wild-type plants, resulting in Fru 2,6-P(2) concentrations that were 228% to 350% of wild-type plants after 2 h of illumination . A range of metabolic measurements were made on these transgenic plants to investigate the possible roles of Fru 2,6-P(2) during Suc, starch, and malic acid metabolism across the deacidification period of CAM . The results suggest that Fru 2,6-P(2) plays a major role in regulating partitioning between Suc and starch synthesis during photosynthesis . However, alterations in Fru 2,6-P(2) levels had little effect on malate mobilization during CAM fluxes.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1457 - 69
Characterization of bacteria isolated from wild legumes in the north-western regions of China; Tan ZY et al.; Nodule isolates from 11 species of wild legumes in north-western China were characterized by numerical taxonomy, PCR-based 16S rRNA gene RFLP and sequence analyses, DNA-DNA hybridization, restriction patterns of nodDAB and nifH genes, and symbiotic properties . Based on the results of numerical taxonomy, most of the 35 new isolates were grouped into five clusters (clusters 7, 9, 12, 14 and 15) . Clusters 7 and 12 were identified as Mesorhizobium amorphae and Agrobacterium tumefaciens, respectively, based on their high DNA homologies with the reference strains for these species, their 16S rRNA gene analysis and their phenotypic features . Results of 16S rDNA PCR-RFLP analysis showed that cluster 9 belonged to Rhizobium . Clusters 14 and 15 were identified as Mesorhizobium based on their moderately slow-growing, acid-producing characters and the high similarity of their 16S rDNA PCR-RFLP patterns to those of Mesorhizobium species . These two clusters were genomic species distinct from all described species based on analysis of DNA relatedness within this genus . The isolates in cluster 12 (Agrobacterium tumefaciens) failed to nodulate their original host and other selected hosts and they did not hybridize to nif or nod gene probes . The possibility of opportunistic nodulation of these isolates is discussed . Identical restriction patterns were obtained in the nif or nod gene hybridization studies from the three isolates within cluster 15, which were isolated from the same host species . The isolates from different host plants in each of clusters 9 and 14 produced different nodDAB RFLP patterns, but similar nifH RFLP patterns appeared (one band for each isolate) . Different patterns were observed among different clusters from both the nod and nif gene hybridization studies . Crossnodulation was recorded among the isolates and the host plants in the same cluster and promiscuous properties were found among some of the hosts tested.

New Microbiol, 1999 Oct, 22(4), 375 - 82
Emerging gram-negative pathogens in the immunocompromised host: Agrobacterium radiobacter septicemia during HIV disease; Manfredi R et al.; Three out of 2,412 consecutive HIV-infected patients hospitalized since 1990, developed Agrobacterium radiobacter septicemia . All patients were severely immunocompromised, showing a prior diagnosis of AIDS, concurrent opportunistic infections, a mean CD4+ lymphocyte count below 100 cells/microL, and neutropenia . Nosocomial A . radiobacter sepsis occurred in two cases of three, and was related to a lower neutrophil and CD4+ cell count . Antibiotic and cotrimoxazole treatment were carried out during the month preceding disease onset by two and three patients, respectively . Antimicrobial susceptibility assays showed resistance to ureidopenicillins and aztreonam, and complete sensitivity to carbapenems, amikacin, and ciprofloxacin . A therapeutic regimen including amikacin plus ceftriaxone or ceftazidime obtained clinical and microbiological cure in all cases, in the absence of related mortality or relapses . Only two episodes of HIV-associated A . radiobacter complications have been described to date: one case of sepsis and one patient with pneumonia . Despite their low frequency, gram-negative non-fermenting bacilli should be considered in HIV-infected patients with a suspected bacterial complication, because of their cumbersome identification procedures, and their unpredictable antibiotic susceptibility, with elevated resistance to many compounds expected to be effective against gram-negative organisms . A . radiobacter may play a pathogenic role in patients with advanced HIV disease, even when some commonly recognized risk factors are lacking (in-dwelling catheters and instrumentation), while a very low CD4+ lymphocyte count, leukopenia-neutropenia, hospitalization, and concurrent AIDS-related infectious complications, may act as predisposing factors.

Planta, 1999 Oct, 209(4), 453 - 61
The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings
Galis I I, Simek P, Macas J, Zahradnickova H, Vlasak J, Wabiko H, Van Dongen W, Van Onckelen HA, Ondrej M.
The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin . To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L . cv . Samsun) plants were transformed with the A . tumefaciens C58-6b gene . Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA) . The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited . Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment . Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth . Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings . Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Planta, 1999 Oct, 209(4), 399 - 405
Identification and localization of transformed cells in agrobacterium tumefaciens-induced plant tumors
Rezmer C, Schlichting R, Wachter R, Ullrich CI.
Agrobacterium tumefaciens-induced tumors of dicotyledonous plants consist of well-defined vascular bundle-like structures originating from transformed cells . The current view that 25% of the tumor cells are transformed has been re-investigated by using beta-glucuronidase (gus)-gene-containing wild-type bacteria (A281 p35S gus-int) . Regularly growing stem and leaf tumors showed irregular GUS-staining patterns in the different plant species, Ricinus communis L., Cucurbita maxima L., Vicia faba L . and Kalanchoe daigremontiana Hamet et Perrier . Variable staining and inconsistency between staining and tumor growth suggested an inhibition of gus expression . By polymerase chain reaction (PCR) and reverse transcriptase-PCR analyses it became evident that gus is also integrated into the DNA of unstainable tumor parts but not expressed . These results and area calculations of tissues unable to contain the bacterial transferred-DNA with gus provide strong evidence that in A . tumefaciens-induced tumors most cells, or even all, are transformed, i.e . ca . 100%.

J Bacteriol, 1999 Nov, 181(21), 6850 - 5
The Agrobacterium tumefaciens chaperone-like protein, VirE1, interacts with VirE2 at domains required for single-stranded DNA binding and cooperative interaction; Sundberg CD et al.; Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants . Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2 . VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 332 - 7
Succinoglycan production by solid-state fermentation with Agrobacterium tumefaciens; Stredansky M et al.; Succinoglycan was produced by cultivating Agrobacterium tumefaciens on various solid substrates, including agar medium, spent malt grains, ivory nut shavings, and grated carrots, impregnated with a nutrient+ solution . Fermentations were performed on a laboratory scale, both under static conditions and with agitation, using bottles and a prototype horizontal bioreactor . Several fermentation parameters were examined and optimized, including carbon and nitrogen composition, water content and layer thickness of the substrate . The yields and rheological properties of the polymers obtained under different fermentation conditions were compared . The highest succinoglycan yield was achieved in static cultivation, reaching 42 g/l of impregnating solution, corresponding to 30 g/kg of wet substrate . The polymer production in the horizontal bioreactor was faster, but the final yield was lower (29 g/l of impregnating solution).

Plant Cell, 1999 Oct, 11(10), 1995 - 2012
Glutamine synthetase in the phloem plays a major role in controlling proline production
Brugiere N, Dubois F, Limami AM, Lelandais M, Roux Y, Sangwan RS, Hirel B.
To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia . After Agrobacterium-mediated transformation, two transgenic N . tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained . Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants . Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium . In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent . A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability . After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves . We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.

DNA Seq, 1998, 9(2), 125 - 8
Diversity of the limited-host range iaaH gene of Agrobacterium vitis strain Ag162; Oetiker JH et al.; The indole-3-acetamide hydrolase gene (iaaH) of the limited-host range strain AG162, a biotype III strain of Agrobacterium tumefaciens has been the subject of several studies and reviews, but its primary structure has not been previously reported . In the course of our own work we found that this gene hybridizes only weakly to a nucleic acid probe corresponding to the iaaH gene from a biotype I strain of A . tumefaciens . Analysis of the primary structure of the Ag162 iaaH gene revealed that it is diverse from biotype I iaaH genes and, surprisingly, also from the iaaH genes of previously characterized biotype III Agrobacterium strains.

Mol Cells, 1999 Aug 31, 9(4), 376 - 83
Gene silencing-mediated resistance in transgenic tobacco plants carrying potato virus Y coat protein gene; Han SJ et al.; Unlike other pathogens, plant viruses are hardly controlled by chemical agents . Potato virus Y (PVY) is distributed around the world, and causes a great loss economically . In an attempt to minimize the damage by viruses, the PVY coat protein (CP) gene was introduced into tobacco by Agrobacterium-mediated transformation . A significant proportion of the transgenic plants displayed resistance to PVY and showed substantially decreased CP transgene expression at both protein and steady-state mRNA levels compared to susceptible transgenic or nontransgenic plants . A resistant plant was selected and self-fertilized for several generations until T4 progenitor lines were obtained . Most of these T4 plants accumulated extremely low levels of CP protein and steady-state mRNA, and exhibited almost complete resistance to PVY . DNA gel blot analysis revealed that the transgenic plants typically had two or three copies of the transgene . These results are characteristic of pathogen-derived resistance, in which the resistance against virus is the consequence of post-transcriptional gene silencing directed by homologous transgenes . To uncover factors that may play roles in gene silencing, sequences in the 3' part of the transcribed region of the CP gene were transcribed in vitro and the RNA fragments were incubated with cell extracts from transgenic plants . A ribonuclease activity was detected that appeared to be specific for this transcript in the PVY-resistant transgenic plants.

Membr Cell Biol, 1999, 12(6), 907 - 9
Localization of VirB1 protein on the agrobacterial cell surface; Chumakov MI et al.; Using colloidal gold-labelled VirB1-specific antibodies, it was found that VirB1 proteins are included into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells.

J Ind Microbiol Biotechnol, 1999 Aug, 23(2), 143 - 148
Optimal pH control of batch processes for production of curdlan by Agrobacterium species; Lee JH et al.; We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species . The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin . The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters . The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion . By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L-1) compared to that of constant pH operation (36 g L-1).

Mol Microbiol, 1999 Sep, 33(6), 1210 - 20
A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis; O'Callaghan D et al.; Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B . suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis . DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots . Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently . Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages . The virulence was restored by complementation with a plasmid containing the full virB region . The virB region appears to be essential for the intracellular survival and multiplication of B . suis.

FASEB J, 1999 Oct, 13(13), 1796 - 9
A plant-derived edible vaccine against hepatitis B virus; Kapusta J et al.; The infectious hepatitis B virus represents 42 nm spherical double-shelled particles . However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein . These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast . Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization . Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv . Burpee Bibb expressing envelope surface protein . Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies . Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.

Appl Environ Microbiol, 1999 Oct, 65(10), 4575 - 81
Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp . strain M15-3; Bosma T et al.; Trihalogenated propanes are toxic and recalcitrant organic compounds . Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful . Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp . strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better . Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp . strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc) . The resulting plasmids yielded functional expression in several gram-negative bacteria . A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth . The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources . Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.

Plant J, 1999 Sep, 19(5), 615 - 23
Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system; Chin HG et al.; In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds . We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation . In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency . The 3' end of Ds was examined for putative splicing donor sites . As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice . Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter . Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings . About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures . Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles . With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization . Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds . Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice . We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.

Chin J Biotechnol, 1998, 14(4), 249 - 54
Studies on dynamics of growth and biosynthesis of artemisinin in hairy roots of Artemisia annua L; Liu B et al.; Seven hairy root lines with the properties of fast growth and high artemisinin contents were selected from 747 hairy roots induced by transformation of Artemisia annua L . strain 025 with Agrobacterium rhizogenes ATCC15834 . The differences of growth rates and artemisinin contents among the 7 selected hairy root lines were extremely significant, of which HR-9 gave the highest yield of artemisinin, reaching 33.25 mg/y.L . The differences of growth rates and artemisinin contents among hairy roots, untransformated roots and callus were also significant . There were no obvious lag phase in batch culture of hairy roots of Artemisia annua L . The exponential growth phase was 7 approximately 15 days after inculation . The growth rate reached the highest on the 11th day, and the cultures reached a stationary phase on the 20th day . The properties of artemisinin content of hairy roots were obviously "related to growth" . The artemisinin content decreased slowly during the exponential phase, increased while the growth rate slowed down and remained consistent after the growth stopped . The optimum culture time for hairy roots of Artemisia annua L . was 21 days in our system.

Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11128 - 33
Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves; Vaquero C et al.; To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system . A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84 . 66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector . Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors . In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium . Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week . His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography . Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.

J Bacteriol, 1999 Sep, 181(18), 5758 - 65
Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells; Bravo-Angel AM et al.; Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2 . Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria . A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed . We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2 . MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus . However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies . In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end . Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another . These complex integration patterns indicate a specific deficiency in MobA . The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins.

J Bacteriol, 1999 Sep, 181(18), 5563 - 71
TraC of IncN plasmid pKM101 associates with membranes and extracellular high-molecular-weight structures in Escherichia coli; Schmidt-Eisenlohr H et al.; Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components . Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer . Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S . C . Winans, and G . C . Walker, J . Bacteriol . 161:402-410, 1985) . Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coli allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography . Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery . Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery, traM, traB, traD, and traE but not eex, coding for an entry exclusion protein not required for DNA transfer . Cross-linking identified protein-protein interactions of TraC in E . coli carrying pKM101 but not derivatives with transposon insertions in essential tra genes . Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex . Heterologous expression of TraC in A . tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili . In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu . Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

Transgenic Res, 1999 Apr, 8(2), 105 - 17
Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance; Kenward KD et al.; Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization . This extensively disulfide-bonded 14 kDa protein was targeted for accumulation in its pro- and mature forms in the cytosol and apoplast of transgenic tobacco plants . Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciens-mediated transformation . AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% the total protein present in the apoplast . Plant-produced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wild-type AFP in thermal hysteresis activity and its effect on ice crystal morphology . Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for large-scale production: but no difference in frost resistance was observed between transgenic and wild-type plants during the onset of early fall frosts.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 169 - 74
Lack of expression of bundle-forming pili in some clinical isolates of enteropathogenic Escherichia coli (EPEC) is due to a conserved large deletion in the bfp operon; Bortolini MR et al.; Enteropathogenic Escherichia coli (EPEC) produces a plasmid-encoded type IV pilus, called the bundle-forming pilus (BFP), involved in the formation of the localized adhesion onto epithelial cells . In this study, we demonstrate that clinical isolates of serotypes O128ab:H2 and O119:H2 contain a ca . 13-kb deletion in the bfp operon, resulting in a lack of expression of these pili . An IS sequence with homology to the IS66 of Agrobacterium tumefaciens replaced the deleted bfp genes . These results suggest that the bfp operon was deleted through a transpositional event and that other adherence factors may mediate attachment of these bacteria to the host cells.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 141 - 6
GFP-aided confocal laser scanning microscopy can monitor Agrobacterium tumefaciens cell morphology and gene expression associated with infection; Li L et al.; We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon containing a promoterless gene encoding a green fluorescent protein (GFP) . Some of the GFP-tagged individual bacterial cells exhibited strong green fluorescence, which reflected the expression levels of the GFP-tagged genes . Those cells could be readily detected with a confocal laser scanning microscope (CLSM) . We observed that the fluorescence and morphology of A . tumefaciens cells grown in plant tissues resembled those grown in a minimal medium of low pH, which is required for expression of the virulence genes responsible for tumorigenesis . This suggests that GFP-aided CLSM can be used to determine which growth medium is more representative of the nutritional conditions that a pathogen encounters in plant tissues . We also observed that the fluorescence and morphology of A . tumefaciens cells changed dramatically during the course of infection . Our data suggested that A . tumefaciens cells were probably better fed upon successful colonization . We believe that GFP-aided CLSM can help study the fate of A . tumefaciens cells inside plant tissues by monitoring cell morphology and gene expression associated with the infection process in situ.

FEMS Microbiol Lett, 1999 Oct 1, 179(1), 37 - 42
A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens; Tang X et al.; A mini-Tn5 transposon derivative, mini-Tn5gfp-km, has been constructed which contained a promoter-less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII) . When this transposon was used to mutagenize Agrobacterium tumefaciens, all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer . The transposon appeared to be bifunctional and could provide both selection and reporter functions . Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin . This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non-lethal . This system was used to identify A . tumefaciens genes that were upregulated in response to acidic pH . Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.

Plant Mol Biol, 1999 Jul, 40(4), 711 - 7
A mini binary vector series for plant transformation; Xiang C et al.; A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb) . This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector . The vector still retains all the essential elements required for a binary vector . These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning . Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning . Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating . This problem can be easily resolved by direct transformation . The mini binary vector has been demonstrated to successfully transform Arabidopsis plants . Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.

Transgenic Res, 1999 Jun, 8(3), 203 - 13
Turnip mosaic potyvirus resistance in Nicotiana benthamiana derived by post-transcriptional gene silencing; Jan FJ et al.; The coat protein (CP) gene of turnip mosaic potyvirus isolate ESC8 (TuMV-ESC8) was cloned and sequenced . Comparisons of the 867-nucleotide (nt) CP region with those of 11 TuMV isolates showed 86.7-89.3% nucleotide identity and 92.4-95.5% amino acid identity . The CP gene was cloned into a plant expression vector and transformed into Nicotiana benthamiana plants via Agrobacterium tumefaciens-mediated leaf disk transformation . Progeny from R0 lines was screened for resistance to TuMV-ESC8 . Five of 29 tested lines showed TuMV protection in more than 50% of their progeny . Interestingly, some of the resistant plants transformed with the CP gene of TuMV displayed mild mosaicism in the new growing leaves at the later stages of evaluation; but these mosaic symptoms disappeared when the leaves were fully expanded . Collective data from steady-state RNA analysis and nuclear run-on assay of a line showed that the resistance was RNA-mediated through the post-transcriptional gene silencing mechanism.

Plant J, 1999 Aug, 19(3), 249 - 57
Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation; Ye GN et al.; The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens . High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved . The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants . GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration . In addition, all ovules from a single infiltrated plant were examined every other day . GUS expression frequencies of up to 1% of pollen were observed 3-5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5-11 days post-infiltration . Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.

Nat Biotechnol, 1999 Sep, 17(9), 916 - 9
Inducible isopentenyl transferase as a high-efficiency marker for plant transformation; Kunkel T et al.; Overexpression of the isopentenyltransferase gene (ipt) from the Ti-plasmid of Agrobacterium tumefaciens increases cytokinin levels, leading to generation of shoots from transformed plant cells . When combined with a dexamethasone-inducible system for controlling expression, ipt expression can be used to select for transgenic regenerants without using an antibiotic-resistance marker . The combined system allows efficient cointroduction of multiple genes (in addition to ipt) and produces transgenic plants without morphological or developmental defects.

Appl Environ Microbiol, 1999 Sep, 65(9), 4197 - 206
Ti plasmids from Agrobacterium characterize rootstock clones that initiated a spread of crown gall disease in Mediterranean countries; Pionnat S et al.; Crown gall caused by Agrobacterium is one of the predominant diseases encountered in rose cultures . However, our current knowledge of the bacterial strains that invade rose plants and the way in which they spread is limited . Here, we describe the integrated physiological and molecular analyses of 30 Agrobacterium isolates obtained from crown gall tumors and of several reference strains . Characterization was based on the determination of the biovar, analysis of 16S ribosomal DNA restriction fragment length polymorphisms by PCR (PCR-RFLP), elucidation of the opine type, and PCR-RFLP analysis of genes involved in virulence and oncogenesis . This study led to the classification of rose isolates into seven groups with common chromosome characteristics and seven groups with common Ti plasmid characteristics . Altogether, the rose isolates formed 14 independent groups, with no specific association of plasmid- and chromosome-encoded traits . The predominant Ti plasmid characteristic was that 16 of the isolates induced the production of the uncommon opine succinamopine, while the other 14 were nopaline-producing isolates . With the exception of one, all succinamopine Ti plasmids belonged to the same plasmid group . Conversely, the nopaline Ti plasmids belonged to five groups, one of these containing seven isolates . We showed that outbreaks of disease provoked by the succinamopine-producing isolates in different countries and nurseries concurred with a common origin of specific rootstock clones . Similarly, groups of nopaline-producing isolates were associated with particular rootstock clones . These results strongly suggest that the causal agent of crown gall disease in rose plants is transmitted via rootstock material.

Arch Virol, 1999, 144(2), 259 - 71
In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao; Jacquot E et al.; Cacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome . A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease . As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao . A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin 19 and was transferred into young plants via Agrobacterium tumefaciens . Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively . Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms . Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta . In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells . Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

Phytochemistry, 1999 Sep, 52(1), 37 - 40
Methoxybifurcarenone: an antifungal and antibacterial meroditerpenoid from the brown alga Cystoseira tamariscifolia; Bennamara A et al.; A meroditerpenoid metabolite has been isolated from the brown alga Cystoseira tamariscifolia and characterized as methoxybifurcarenone, by spectral analysis . Methoxybifurcarenone possesses antifungal activity against three tomato pathogenic fungi: Botrytis cinerea, Fusarium oxysporum sp . mycopersici and Verticillium alboatrum and antibacterial activity against Agrobacterium tumefaciens and Escherichia coli.

Yi Chuan Xue Bao, 1998 Dec, 25(6), 517 - 24
{Obtaining transgenic rice plants and their progenies using Agrobacterium tumefaciens}; Yin ZC et al.; Rice (Oriza sativa L.) suspension cells of Taipei 309 were co-cultivated with A . tumefaciens stran EHA101 harbouring binary vector pBYT2 for 3 days in the presence of vir inducer, 100 mumol/L acetosyringone (AS) . After 2 months of continuous selection, 17 stable hygromycin-resistant, GUS-positive calli were recovered from 364 suspension cell clusters co-cultivated with A . tumefaciens . 10 putative transgenic R0 plants obtained from 8 tansformed calli and their progenies were analyzed for the integration and expression of foreign genes . Southern blot analysis of R0 and R1 generations indicated that foreign genes had been stably integrated in the genome of transgenic rice and sexually transmitted . One of the transgenic lines showed 5 copies of T-DNA integration, while the others had only one copy . Histochemical staining observation and fluorometric assay of GUS activity in transgenic rice cells and plants showed ubiquitin promoter from maize was highly effective in driving the expression of gus reporter gene in transgenic rice cells . GUS protein and its activity were also investigated through ndPAGE-X-Gluc staining assay, and it was found that the GUS protein in transgenic rice cells was smaller in size than the standard GUS protein (Sigma Co . G0786) but as large as that from E.coli HB101 (pBI121) . This study suggested that Agrobacterium-mediated transformation of plant is an efficient and reliable method to introduce foreign genes into rice.

J Bacteriol, 1999 Sep, 181(17), 5160 - 6
Combined genetic and physical map of the complex genome of Agrobacterium tumefaciens; Goodner BW et al.; A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes . The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome . The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.

J Bacteriol, 1999 Sep, 181(17), 5280 - 7
Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae; Abola AP et al.; We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis . Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4) . The third gene has homology to the E . coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase . In order to determine the specificity of the R . meliloti reductase, the R . meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro . Like the A . thaliana reductases, the histidine-tagged R . meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM . In order to determine whether this preference for APS is unique to R . meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R . leguminosarum, Rhizobium sp . strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity . Cell extracts from all four species also preferentially reduce APS over PAPS.

Microbiology, 1999 Aug, 145 ( Pt 8), 1967 - 75
A simplified subtractive hybridization protocol used to isolate DNA sequences specific to Xylella fastidiosa; Ferreira H et al.; A simplified protocol of subtractive hybridization based on the technique of L . M . Kunkel, A . P . Monaco, W . Middlesworth, H . D . Ochs & S . A . Latt (1985, Proc Natl Acad Sci USA, 82, 4778-4782) was used to obtain DNA sequences specific to Xylella fastidiosa isolated from diseased citrus plants . As a driver, DNA extracted from bacteria showing different degrees of relatedness was used: Xy . fastidiosa 788 isolated from another host (plum), Xanthomonas campestris pv . campestris and Burkholderia gladioli strains . A DNA fragment, f14, showing no hybridization to the driver DNA, was used as a probe specific to Xy . fastidiosa from citrus and oleander . This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of Escherichia coli serotypes H1 and H12 . A pair of internal primers (f14-1 and f14-2) was designed for amplification of Xy . fastidiosa DNA . These primers detected Xy . fastidiosa strains isolated from citrus and oleander and yielded an amplification product of about 600 bp . They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease . No amplification reaction was obtained using DNA extracted from other species and pathovars of Xanthomonas, Pseudomonas cichorii, Erwinia carotovora, Agrobacterium tumefaciens and phytopathogens of citrus (Xanthomonas axonopodis pv . citri) and coffee (Burkholderia andropogonis, P . cichorii, Pseudomonas syringae pv . garcae) . The isolation of a DNA fragment specific to Xy . fastidiosa from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other micro-organisms.

J Ind Microbiol Biotechnol, 1999 Jun, 22(6), 590 - 599
Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon; Shaw N et al.; The E . coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression . The effects of the modifications on the bio operon were studied in E . coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis . The modified E . coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material . Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Mol Ecol, 1999 Aug, 8(8), 1273 - 84
Direct conjugal transfers of Ti plasmid to soil microflora; Teyssier-Cuvelle S et al.; The bacterial species in soil that can receive a Ti plasmid by conjugation from Agrobacterium spp . were investigated . In order to have direct access to the potential reservoir of Ti plasmid amongst soil microflora, the conjugal system consisting of a multiply auxotrophic derivative of C58 (ST-96-4) and a derivative of pTiC58Delta(acc)R (pSTiEGK) containing a triple antibiotic-resistance cassette in traM was used to transfer the Ti plasmid in a complex soil microflora used as the recipient . Numerous transconjugants were obtained by this method but none was identified as Agrobacterium . This could be explained by the low density of Agrobacterium in the tested soil . As indicated by analysis of the ribosomal gene rrs, transconjugants recovered directly from soil were found to be new bacterial species which appeared to be closely related to Sinorhizobium spp.

Biotechnol Prog, 1999 Jul-Aug, 15(4), 603 - 7
Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains; Chao YP et al.; The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli . Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E . coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid . In addition, the recombinant plasmids were very stable in the E . coli strain ATCC11303 but not in JCL1258 tested here . Employing the recombinant E . coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h) . In comparison with A . radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production . This result suggests the potential application of recombinant E . coli strains for the transformation reaction.

J Bacteriol, 1999 Aug, 181(16), 5033 - 41
Essential components of the Ti plasmid trb system, a type IV macromolecular transporter; Li PL et al.; The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid . The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family . The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI) . Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon . This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI . Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids . When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58 . However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid . The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors . Only the trbK mutant was unaffected in conjugal transfer from either donor . Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene . An insertion mutation in traI abolished the production of AAI and also conjugal transfer . This defect was restored by culturing the mutant donor in the presence of AAI . We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58 . We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9009 - 14
Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity; Luo ZQ et al.; TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmid conjugal transfer . The protein requires as coinducer an acyl-homoserine lactone signal molecule called AAI (Agrobacterium autoinducer) that is produced by the bacteria themselves . TraR only activates its target genes, making it difficult to determine whether interaction with AAI is required for binding DNA or for initiating transcription . To assess this, we converted TraR into a repressor by placing a copy of the tra box, an 18-bp inverted repeat believed to be the recognition site for this protein, over the -10 region of a promoter driving expression of lacZ . Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of Vibrio fischeri . C-terminal deletions as short as 2 aa and N-terminal deletions as short as 4 aa in TraR abolished both repressor and activator functions . The C-terminal mutants were strongly dominant over TraR, suggesting that they can form heteromultimers with the wild-type activator . Mutants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric promoter in an AAI-dependent manner . We conclude that TraR recognizes the tra box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation but not for multimerization, for interaction with the acyl-homoserine lactone, or for DNA binding.

J Biol Chem, 1999 Aug 6, 274(32), 22548 - 55
Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits; Eisenbrandt R et al.; TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli . Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens . TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively . TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease . The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond . The T pilin does not undergo additional truncation but is likewise cyclized . The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.

Plant Cell Physiol, 1999 May, 40(5), 482 - 8
Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium; Koyama H et al.; A mitochondrial citrate synthase (CS) of Arabidopsis thaliana was introduced into carrot (Daucus carota L . cv . MS Yonsun) cells by Agrobacterium tumefaciens-mediated transformation . Transgenic cell lines had high CS activity, the highest value observed was 0.24 mumol (mg protein)-1 min-1 which was 1.9-fold of that in wild-type cells . Transcript levels of DcCS were similar between transgenic lines, but those of AtCS were increased as the CS activity of cells was increased . Isoelectric focussing revealed that the CS polypeptide of the transgenic lines had a pI value different from that of the wild-type cells, although the molecular mass was the same . These results indicate that the CS polypeptides of A . thaliana were expressed and processed to the mature form in carrot cells . The growth rate and excretion was 2.2-2.8 and 2.8-4.0 fold greater in the transgenic cells than in the wild type cells, respectively . Phosphate uptake from Al-phosphate also increased in transgenic cells . It appears, the overexpression of mitochondrial citrate synthase in carrot cells improves the growth rate in Al-phosphate medium possibly as a result of increased citrate excretion.

Appl Environ Microbiol, 1999 Aug, 65(8), 3754 - 6
The viable-but-nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum; Alexander E et al.; Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state . We have determined that copper can induce nutrient-starved Agrobacterium tumefaciens and Rhizobium leguminosarum cells to become viable but nonculturable . This is the first report of a chemical inducer of this condition.

Appl Environ Microbiol, 1999 Aug, 65(8), 3622 - 6
Soil bacterial community shift correlated with change from forest to pasture vegetation in a tropical soil; Nusslein K et al.; The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community . DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction . All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution . The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67% . None of the dominant phylotypes found in the forest soil were detected in the pasture soil . For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium-Agrobacterium assemblages in the pasture soil.

J Bacteriol, 1999 Aug, 181(15), 4533 - 9
Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A . tumefaciens RpoA gene, encoding the alpha subunit of RNA polymerase; Lohrke SM et al.; The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens . However, virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A . tumefaciens genes . In a search for these additional genes, we have identified the rpoA gene, encoding the alpha subunit of RNA polymerase (RNAP), which confers significant expression of a virB promoter (virBp)::lacZ fusion in E . coli in the presence of an active transcriptional regulator virG gene . We conducted in vitro transcription assays using either reconstituted E . coli RNAP or hybrid RNAP in which the alpha subunit was derived from A . tumefaciens . The two forms of RNAP were equally efficient in transcription from a sigma(70)-dependent E . coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBp in a virG-dependent manner . In addition, we provide evidence that the alpha subunit from A . tumefaciens, but not from E . coli, is able to interact with the VirG protein . These data suggest that transcription of virulence genes requires specific interaction between VirG and the alpha subunit of A . tumefaciens and that the alpha subunit from E . coli is unable to effectively interact with the VirG protein . This work provides the basis for future studies designed to examine vir gene expression as well as the T-DNA transfer process in E . coli.

Infect Immun, 1999 Aug, 67(8), 3893 - 9
Multiple genes in the left half of the cag pathogenicity island of Helicobacter pylori are required for tyrosine kinase-dependent transcription of interleukin-8 in gastric epithelial cells; Li SD et al.; Helicobacter pylori strains that contain the cag pathogenicity island (PAI) elicit increased synthesis of gastric C-X-C chemokines, promote neutrophilic infiltration into the gastric epithelium, and stimulate the synthesis of interleukin-8 (IL-8) in cultured gastric epithelial cells . To investigate the effects of cag PAI genes on the transcription of the IL-8 gene, the Kato-3 gastric epithelial cell line was stably transfected with plasmid DNA containing the IL-8 gene promoter fused to a luciferase reporter gene . The resulting reporter cell line, L5F11, was used to monitor the effects of infection in cell culture by H . pylori 26695 and isogenic derivatives with null mutations in genes in the cag PAI on transcription of the IL-8 gene . We found that null mutations in eight open reading frames, including homologs of the Agrobacterium virB9, virB10, and virB11 genes, in the left half of the cag PAI abrogated the induction of IL-8 gene transcription . Further studies with the L5F11 cell line showed that IL-8 gene transcription induced by H . pylori was blocked by the protein tyrosine kinase inhibitor herbimycin A but not by the protein kinase C inhibitor calphostin C or by the protein kinase G inhibitor KT5823 . IL-8 gene transcription in L5F11 cells could also be induced by the cytokine tumor necrosis factor alpha (TNF-alpha) without exposure to H . pylori . This TNF-alpha-induced IL-8 transcription was inhibited by the protein kinase A inhibitor H7, which had no significant effect on H . pylori-induced IL-8 transcription . These studies show that multiple genes in the left half of the cag PAI are essential for the transcription of the IL-8 gene in gastric epithelial cells and that this depends on protein tyrosine kinase activation.

Plant Mol Biol, 1999 May, 40(2), 223 - 35
Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene; Gleave AP et al.; Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes . Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome . Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene . To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase . Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection . Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase . 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA . In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome . The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny . Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing . This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.

Microbiology, 1999 Jun, 145 ( Pt 6), 1397 - 407
Agrobacterium tumefaciens possesses a fourth flagelin gene located in a large gene cluster concerned with flagellar structure, assembly and motility; Deakin WJ et al.; The authors have identified a fourth flagellin gene in a 21850 bp region of the Agrobacterium tumefaciens C58C1 chromosome containing at least 20 genes concerned with flagellar structure, assembly and function . Three flagellin genes, flaA, flaB and flaC, orientated rightward, are positioned in a tandem array at the right end, with the fourth, substantially larger gene, flaD, in the opposite orientation, at the left end . Between these lie four apparent operons, two transcribed in each direction (motA, flhB leftward; flgF, flgB rightward) from a divergent position approx 7.5 kb from the left end . This unifies the previously published motA, flgB and flaABC sequences into a single region, also containing the homologues of flhB, flgF and fliI . Site-specific mutagenesis of fliI results in a non-flagellate phenotype, while a Tn5-induced flhB mutant possesses abnormal flagella . Mutagenesis and protein profiling demonstrate that all four flagellins contribute to flagellar structure: FlaA is the major protein, FlaB and FlaC are present in lesser amounts, and FlaD is a minor component . FlaA has anomolous electrophoretic mobility, possibly due to glycosylation.

J Bacteriol, 1999 Jul, 181(14), 4342 - 52
Mutagenesis of the Agrobacterium VirE2 single-stranded DNA-binding protein identifies regions required for self-association and interaction with VirE1 and a permissive site for hybrid protein construction; Zhou XR et al.; The VirE2 single-stranded DNA-binding protein (SSB) of Agrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells . By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1 . VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers . By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1 . These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1 . The capacity of VirE2 mutants to interact with full-length VirE2 in the yeast Saccharomyces cerevisiae correlated with the abundance of the mutant proteins in A . tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium . We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2 . A PvirB::virE2 construct yielded functional VirE2 protein as defined by complementation of a virE2 null mutation . By contrast, PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2 . Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function . However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A . tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant . We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.

Mol Gen Genet, 1999 Jun, 261(4-5), 623 - 6
Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation; Preuss SB et al.; Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome . Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist . It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis . Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.

Clin Diagn Lab Immunol, 1999 Jul, 6(4), 627 - 9
Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae; Cloeckaert A et al.; Brucellae have been reported to be phylogenetically related to bacteria of the family Rhizobiaceae . In the present study, we used a panel of monoclonal antibodies (MAbs) to Brucella outer membrane proteins (OMPs) to determine the presence of common OMP epitopes in some representative bacteria of this family, i.e., Ochrobactrum anthropi, Phyllobacterium rubiacearum, Rhizobium leguminosarum, and Agrobacterium tumefaciens, and also in bacteria reported to serologically cross-react with brucella, i.e., Yersinia enterocolitica O:9, Escherichia coli O:157, and Salmonella urbana . In particular, most MAbs to the Brucella outer membrane lipoproteins Omp10, Omp16, and Omp19 cross-reacted with O . anthropi and P . rubiacearum, which are actually the closest relatives of brucellae . Some of them also cross-reacted, but to a lower extent, with R . leguminosarum and A . tumefaciens . The putative Omp16 and Omp19 homologs in these bacteria showed the same apparent molecular masses as their Brucella counterparts . None of the antilipoprotein MAbs cross-reacted with Y . enterocolitica O:9, E . coli O:157, or S . urbana.

Genetics, 1999 Jul, 152(3), 1173 - 81
Double-strand break-induced recombination between ectopic homologous sequences in somatic plant cells; Puchta H; Homologous recombination between ectopic sites is rare in higher eukaryotes . To test whether double-strand breaks (DSBs) can induce ectopic recombination, transgenic tobacco plants harboring two unlinked, nonfunctional homologous parts of a kanamycin resistance gene were produced . To induce homologous recombination between the recipient locus (containing an I-SceI site within homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants . Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI expression, corresponding to approximately one event in 10(5) transformations . After regeneration, the F1 generation of all recombinants showed Mendelian segregation of kanamycin resistance . Molecular analysis of the recombinants revealed that the resistance gene was indeed restored via homologous recombination . Three different kinds of reaction products could be identified . In one recombinant a classical gene conversion without exchange of flanking markers occurred . In the three other cases homologous sequences were transferred only to one end of the break . Whereas in three cases the ectopic donor sequence remained unchanged, in one case rearrangements were found in recipient and donor loci . Thus, ectopic homologous recombination, which seems to be a minor repair pathway for DSBs in plants, is described best by recombination models that postulate independent roles for the break ends during the repair process.

Nat Biotechnol, 1999 Jun, 17(6), 598 - 601
Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination; Gouka RJ et al.; Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome . Here, we show that when this Ti DNA shares homology with the A . awamori genome, integration can also occur by homologous recombination . On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A . tumefaciens-mediated transformation . Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA . Recombinant A . awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus . This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects . Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level . This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.

J Bacteriol, 1999 Jul, 181(13), 4133 - 6
Swarming by Pseudomonas syringae B728a requires gacS (lemA) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI; Kinscherf TG et al.; Pseudomonas syringae pv . syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates . Filamentation of swarming cells of B728a was not observed . Mutations in either the gacS (formerly lemA) or gacA gene of B728a eliminate the ability of this P . syringae isolate to swarm without obvious effects on bacterial motility . Three field isolates showed a similar dependence on gacS for swarming . Since gacS and gacA mutants are known to be deficient in N-acyl-L-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the ahlI gene of B728a . This mutant did not make any acyl-HSL detectable by the Agrobacterium traG::lacZ reporter system, yet was unaffected in its ability to swarm . Other phenotypes of gacS and gacA mutations were similarly unaffected in the ahlI mutant.

Mol Microbiol, 1999 Jun, 32(6), 1239 - 53
Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter; Dang TA et al.; The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells . Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells . This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity . VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to lambda phage infection . VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography . Walker A sequence mutants directed repressor dimerization in E . coli and interacted with His-VirB4 in A . tumefaciens, indicating that ATP binding is not required for self-association . A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues . A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors . VirB4'312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells . In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4 . These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system . The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.

Biosci Biotechnol Biochem, 1999 May, 63(5), 785 - 91
Purification and characterization of D-glucosaminitol dehydrogenase from Agrobacterium radiobacter; Iwamoto R et al.; D-Glucosaminitol dehydrogenase, which catalyzes the conversion of D-glucosaminitol to 3-keto-D-glucosaminitol, was purified to apparent homogeneity from extracts of Agrobacterium radiobacter . This organism has constitutively depressed levels of the enzyme but expression of the enzyme is induced by addition of D-glucosamine to the medium . Purification included ammonium sulfate fractionation and chromatography on columns of DEAE-Sephacel, Octyl-Sepharose CL-4B, and Cellulofine . The purified enzyme migrated as a single band, coinciding with dehydrogenase activities specific for D-glucosaminitol and ethanol, when electrophoresed on a 7.5% polyacrylamide gel at pH 8.0 . Electrophoresis on a 12.5% PAGE in the presence of 1% SDS also yielded a single band . The enzyme had an apparent molecular mass of 79 kDa, as measured by the pattern of elution from a column of Cellulofine . The results indicated that the enzyme was a dimer of identical (or nearly identical) subunits of 39.5 kDa . D-Glucosaminitol dehydrogenase required NAD+ as a cofactor and used ethanol as the preferred substrate, as well as aliphatic alcohols with 2 to 4 carbon atoms, D-glucosaminitol, D-glucosaminate, DL-allothreonine, glycerol, and erythritol as additional substrates . In 50 mM Tris-HCl buffer (pH 9.0) at 25 degrees C, the K(m) for D-glucosaminitol, ethanol, and NAD+ were 2.2, 2.0, and 0.08 mM, respectively . The enzyme had a pH optimum of 10 for D-glucosaminitol and 8.5 for ethanol . The enzyme lost substantial activity when treated with pyrazole, with certain reagents that react with sulfhydryl groups and with Zn2+ ion . The various results together suggest that the enzyme exploits different amino acid residues for the dehydrogenation of ethanol and of D-glucosaminitol.

Microbiology, 1999 May, 145 ( Pt 5), 1253 - 62
Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv . trifolii; Ausmees N et al.; Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv . trifolii were identified using Tn5 mutagenesis . Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE . These genes are organized similarly in R . leguminosarum bv . trifolii . In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis . The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators . This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis . Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R . leguminosarum bv . trifolii is induced upon close contact with plant roots during the attachment process.

J Bacteriol, 1999 Jun, 181(12), 3816 - 23
Analysis of quorum-sensing-dependent control of rhizosphere-expressed (rhi) genes in Rhizobium leguminosarum bv . viciae; Rodelas B et al.; The rhi genes of Rhizobium leguminosarum biovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea . Previously (K . M . Gray, J . P . Pearson, J . A . Downie, B . E . A . Boboye, and E . P . Greenberg, J . Bacteriol . 178:372-376, 1996) the rhiABC operon had been shown to be regulated by RhiR and to be induced by added N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3OH, C14:1-HSL) . Mutagenesis of a cosmid carrying the rhiABC and rhiR gene region identified a gene (rhiI) that affects the level of rhiA expression . Mutation of rhiI slightly increased the number of nodules formed on the pea . The rhiI gene is (like rhiA) regulated by rhiR in a cell density-dependent manner . RhiI is similar to LuxI and other proteins involved in the synthesis of N-acyl-homoserine lactones (AHLs) . Chemical analyses of spent culture supernatants demonstrated that RhiI produces N-(hexanoyl)-L-homoserine lactone (C6-HSL) and N-(octanoyl)-L-homoserine lactone (C8-HSL) . Both of these AHLs induced rhiA-lacZ and rhiI-lacZ expression on plasmids introduced into an Agrobacterium strain that produces no AHLs, showing that rhiI is positively regulated by autoinduction . However, in this system no induction of rhiA or rhiI with 3OH,C14:1-HSL was observed . Analysis of the spent culture supernatant of the wild-type R . leguminosarum bv . viciae revealed that at least seven different AHLs are made . Mutation of rhiI decreased the amounts of C6-HSL and C8-HSL but did not block their formation, and in this background the rhiI mutation did not significantly affect the expression levels of the rhiI gene or rhiABC genes or the accumulation of RhiA protein . These observations suggest that there are additional loci involved in AHL production in R . leguminosarum bv . viciae and that they affect rhiI and rhiABC expression . We postulate that the previously observed induction of rhiA by 3OH,C14:1-HSL may be due to an indirect effect caused by induction of other AHL production loci.

Plasmid, 1999 May, 41(3), 226 - 39
Characterization of the Tra2 region of the IncHI1 plasmid R27; Rooker MM et al.; In this study, the DNA sequence of one of the transfer regions of the IncHI1 plasmid R27 was determined . This region, which corresponds to coordinates 0-40 on the R27 map has been called the Tra2 region, and is believed to be involved in mating pair formation . DNA sequence analysis of the transfer region identified 11 open reading frames which showed similarities to the transfer genes from other conjugative systems . The R27 transfer genes appear to most closely resemble the genes from the F plasmid and Sphingomonas aromaticivorans plasmid pNL1, both within the individual genes and in the overall gene order . The Tra2 region is also distinct in that replication, partitioning, and stability genes are found in the middle of the transfer region . The R27 Tra2 region also contains a gene, trhF, which appears to be related to the TraF genes of Agrobacterium and Rhizobium species . This, along with the temperature-sensitive transfer system found in both H plasmids and Agrobacterium, leads to the speculation that the R27 transfer region evolved from both ancestral F-like and P-like plasmids .

Mol Microbiol, 1999 Jun, 32(5), 1077 - 89
Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium; Piper KR et al.; Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum-sensing system . Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoinducer (AAI) . These last two elements combine to activate expression of the tra system at high population densities . Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR . Complementation analysis of mutations in the genes 5' to traR showed that the other members of the arc operon are not required for conjugation . Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR . Deletion analysis showed that AccR-regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon . Reverse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR . These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine-responsive transcriptional regulator.

Mol Microbiol, 1999 May, 32(4), 703 - 14
Characterization of a sugar-binding protein from Azospirillum brasilense mediating chemotaxis to and uptake of sugars; Van Bastelaere E et al.; Our approach to the isolation of plant-inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein . Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation . The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence . The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators . The function of the ChvE-like protein in A . brasilense was investigated further . The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D-galactose and functions in the chemotaxis of A . brasilense towards several sugars, including D-galactose, L-arabinose and D-fucose . Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A . brasilense cells.

FEBS Lett, 1999 May 14, 451(1), 45 - 50
Gene for a protein capable of enhancing lateral root formation; Mikami Y et al.; Analysis of genes preferentially expressed in hairy roots caused by infection with Agrobacterium rhizogenes has provided insights into the regulation of lateral root formation . A hairy root preferential cDNA, HR7, has been cloned from hairy roots of Hyoscyamus niger . HR7 encodes a novel protein partially homologous to a metallocarboxypeptidase inhibitor and is expressed exclusively in the primordium and base of lateral roots in hairy roots . Overexpression of HR7 in transgenic roots of H . niger dramatically enhances the frequency of lateral root formation . The results of this study indicate that expression of HR7 plays a critical role in initiating lateral root formation.

Plant Mol Biol, 1999 Mar, 39(4), 683 - 93
Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene; Sommer S et al.; The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC . This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants . The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter . This construct was introduced into L . erythrorhizon by Agrobacterium rhizogenes-mediated transformation . The resulting hairy root cultures showed high CPL activity . 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase . A feeding experiment with {1,7-13C2}shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca . 20% of the overall 4HB biosynthesis in the transgenic cultures . ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.

Infect Control Hosp Epidemiol, 1999 May, 20(5), 345 - 7
Agrobacterium radiobacter as a cause of pseudobacteremia; Rogues AM et al.; Agrobacterium radiobacter was isolated from 15 blood cultures collected from 15 newborns . Contamination of blood cultures was suspected because, in most cases, the babies' illnesses seemed incompatible with infection . A radiobacter was isolated from citrated tubes used for clotting-factor studies . Review of venipuncture technique revealed that occasionally the coagulation study tubes were being inoculated before the blood-culture bottles . This investigation demonstrated how an environmental source coupled with faulty technique caused a cluster of false-positive blood cultures.

Appl Environ Microbiol, 1999 Jun, 65(6), 2798 - 801
Conjugal transfer but not quorum-dependent tra gene induction of pTiC58 requires a solid surface; Piper KR et al.; Donors of Agrobacterium tumefaciens harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium . However, as measured with a lacZ reporter fusion, the tra genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium . Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium . We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate tra gene expression to equivalent levels in liquid and on solid surfaces.

FEMS Microbiol Lett, 1999 May 15, 174(2), 333 - 7
Structure and function of a conserved DNA region coding for tartrate utilization in Agrobacterium vitis; Salomone JY et al.; Three tartrate utilization regions from Agrobacterium vitis strains involved in host specificity have been compared, to clearly define the borders of these regions and eventually identify specific sequences that could provide a mechanism of duplication of this region . A 10.8-kb conserved DNA fragment called the TAR element, found in different genetic contexts, was defined . A comparison of the two tartrate dehydrogenase genes (ttuC and ttuC') in each of the three TAR elements suggests that these genes co-evolve.

Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6535 - 40
Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning; Liu YG et al.; To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable . Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens . Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants . We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably . Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny . Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis . A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca . 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line . Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.

J Biol Chem, 1999 May 21, 274(21), 14579 - 86
The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1 . An enzyme to detoxify harmful epoxides; Nardini M et al.; Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides . They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants . The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-A resolution . The enzyme shows a two-domain structure with the core having the alpha/beta hydrolase-fold topology . The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains . A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275 . Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate . The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.

J Mol Biol, 1999 May 21, 288(5), 811 - 24
An Lrp-type transcriptional regulator from Agrobacterium tumefaciens condenses more than 100 nucleotides of DNA into globular nucleoprotein complexes; Jafri S et al.; The PutR protein of Agrobacterium tumefaciens positively regulates expression of the putA gene in response to exogenous proline, resulting in the utilization of proline as a source of carbon and nitrogen . PutR activity required a region of DNA extending more than 106 nt upstream of the putA transcription start site . Purified PutR bound to this region with high degree of affinity and repressed expression of the putR promoter in vitro . PutR also activated the putA promoter in vitro in the presence of proline, though less strongly than in whole cells . PutR protected a DNA interval extending from nucleotides -30 to -140, but protected only one helical face over most of this interval, suggesting that it may bind only to this face of the DNA . The addition of proline caused a slight decrease in binding affinity and altered DNase I protection patterns along the entire length of the binding site . PutR-DNA complexes were found by atomic force microscopy to be globular rather than elongated . Although the DNA fragment in these complexes was 190 nm in length, the length of the visible DNA was only 150 nm, indicating that 40 nm of DNA (115 nt) must be condensed with protein . PutR caused a net bend of this binding site, and under some conditions, proline shifted the center of this bend by one helical turn .

Mol Gen Genet, 1999 Apr, 261(3), 429 - 38
Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium; Nam J et al.; We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants) . These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli . The results of transient GUS (beta-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus . Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions . In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA . In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait.

Appl Environ Microbiol, 1999 May, 65(5), 2072 - 7
Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection
Boncompagni E, Osteras M, Poggi MC, le Rudulier D.
The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated . When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants . However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum . Except for B . japonicum, all strains exhibit transport activity for glycine betaine and choline . When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R . leguminosarum and S . meliloti or, more surprisingly, reduced in R . tropici, S . fredii, and M . loti . The transport of glycine betaine was increased by growing the cells in the presence of the substrate . With the exception of B . japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources . This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere . Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M . huakuii and B . japonicum . The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.

Appl Environ Microbiol, 1999 May, 65(5), 1936 - 40
Cocolonization of the rhizosphere by pathogenic agrobacterium strains and nonpathogenic strains K84 and K1026, used for crown gall biocontrol
Penyalver R, Lopez MM.
The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra- deletion mutant of pAgK84), K84 Agr- (lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach x almond seedlings planted in infested soil . Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions . In spite of the efficient biocontrol observed, average populations consisting of 10(2) and 10(6) pathogenic agrobacteria per g of root were found 8 months after planting . The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants . Strain K84 and the genetically engineered organism K1026 survived at a level of 10(6) agrocin 84-producing bacteria per g of root . The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation . Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system . In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.

Proc Natl Acad Sci U S A, 1999 Apr 27, 96(9), 4832 - 7
Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells; Zhu J et al.; TraR is an Agrobacterium transcriptional regulator whose activity requires the pheromone N-3-oxooctanoyl-L-homoserine lactone . TraR was purified as a complex with the pheromone and contained one pheromone molecule per protein monomer . TraR-pheromone complexes bound to a single DNA site and activated two promoters that flank this site . Promoter expression was elevated 30-fold by using a supercoiled template . Pheromone binding increased the affinity of TraR for this binding site . Pheromone also increased TraR abundance in vivo by causing a 20-fold decrease in TraR turnover rates.

Philos Trans R Soc Lond B Biol Sci, 1999 Mar 29, 354(1383), 653 - 8
Interactions between tobacco mosaic virus and the tobacco N gene; Erickson FL et al.; The interaction between tobacco mosaic virus (TMV) and tobacco harbouring the N gene is a classical system for studying gene-for-gene interactions in disease resistance . The N gene confers resistance to TMV by mediating defence responses that function to limit viral replication and movement . We isolated the N gene and determined that N belongs to the nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) class of plant disease resistance genes, and encodes both full-length and truncated proteins . Sequence homologies and mutagenesis studies indicated a signalling role for the N protein similar to that seen for proteins involved in defence responses in insects and mammals . The N gene confers resistance to TMV in transgenic tomato, demonstrating the use of the NBS-LRR class of disease resistance genes in engineering crop resistance . From the pathogen side of this interaction, the TMV 126 kDa replicase protein has been implicated as the avirulence factor that triggers N-mediated defence responses . We employed Agrobacterium-mediated expression strategies to demonstrate that expression of the putative helicase region of the replicase protein is sufficient to elicit N-mediated defences . The thermosensitivity of the N-mediated response to TMV is retained when induced by expression of this replicase fragment . Thus, both components of this gene-for-gene interaction are now available for studies that address the molecular mechanisms involved in N-mediated TMV resistance.

J Appl Microbiol, 1999 Apr, 86(4), 591 - 602
A simple and efficient PCR method for the detection of Agrobacterium tumefaciens in plant tumours; Cubero J et al.; A simple PCR protocol was developed for identifying Agrobacterium as the causal agent of the tumours produced by this bacterium in plant material . The sensitivity of this method was compared with that of bacterial isolation using common and selective media with a previous enrichment step . More than 200 samples from tumours of naturally infected and inoculated plants from several hosts including almond, peach x almond hybrids, apricot, rose, tobacco, tomato, raspberry, grapevine and chrysanthemum, were analysed by both methods . PCR was the most efficient method for detecting the bacterial aetiology of the plant tumours . Agrobacterium tumefaciens was better detected in crown and root tumours than in aerial tumours with all the methods assayed in inoculated plants . A comparison between the efficiency of the diagnosis by analysing pieces from the external and internal part of the tumour showed no differences between them.

Food Addit Contam, 1998 Oct, 15(7), 767 - 74
Development and application of a selective detection method for genetically modified soy and soy-derived products; Hoef AM et al.; A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e . the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company . Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene . The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure . Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean) . The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products . The results obtained are discussed with respect to practical application of the detection method developed.

Mol Microbiol, 1999 Mar, 31(6), 1795 - 807
VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium; Deng W et al.; Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell . The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA . A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells . The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation . In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays . Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2 . VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay . In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially . Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain . Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E . coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction . In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction . In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed . When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected . These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state . Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems . We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.

Biochem Mol Biol Int, 1999 Mar, 47(3), 361 - 7
Purification and characterization of a cell-surface lectin (Lectin II) from Agrobacterium radiobacter NCIM 2443; Syed FB et al.; A lectin was isolated from Agrobacterium radiobacter cell surface and purified . It is a monomer of 40 kDa as shown by SDS-PAGE . The lectin has a pI of 9.15 and amino acid composition of the lectin shows that 44% of the amino acids are hydrophobic . The lectin agglutinates rabbit erythrocytes but does not agglutinate human erythrocytes . It does not show specificity for monosaccharides except for D-glucosamine . Fetuin and its N-linked glycopeptide also inhibit the activity of the lectin but greater inhibition is shown by locust bean gum and Nicotiana tobaccum (tobacco) tissue extracts.

Plant Physiol, 1999 Apr, 119(4), 1507 - 16
Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco; Garcia I et al.; 4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen . In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine . We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues . We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp . Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene . We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105 . The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation . We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections . These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast . Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Plant Physiol, 1999 Apr, 119(4), 1447 - 56
Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the andean crop Mirabilis expansa; Vivanco JM et al.; Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop . The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography . The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0 . Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs . Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2 . ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP . Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity . Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others . ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed . Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Biotechnol Prog, 1999 Mar-Apr, 15(2), 278 - 82
Direct Agrobacterium tumefaciens-mediated transformation of Hyoscyamus muticus hairy roots using green fluorescent protein; Merritt CD et al.; Hyoscyamus muticus hairy root segments were infected with Agrobacterium tumefaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter . The roots were incubated on callus-inducing medium to generate transformed cells . Transformants were selected on medium containing 50 and 100 mg/L kanamycin and screened by visual inspection for GFP expression . Highly fluorescent cells were incubated on phytohormone-free medium for regeneration of the hairy root phenotype . This infection technique can be applied directly to existing hairy root cultures which have been previously characterized and selected for desirable physiological traits . These studies also indicate that GFP is not toxic to H . muticus plant tissue and that H . muticus hairy roots have minimal autofluorescence which allows for clear observation of GFP.

Planta Med, 1999 Mar, 65(2), 144 - 8
Datura metel: in vitro production of tropane alkaloids; Cusido RM et al.; Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium . The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts . In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7% to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture . Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25 . After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular) . The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.

Mol Gen Genet, 1999 Mar, 261(2), 236 - 41
Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product; Park YW et al.; A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E . coli DH5alpha . A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da . BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues . The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens . Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8 . The apparent molecular mass of the protein, when expressed in E . coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space . The enzyme is optimally active at pH 7 and a temperature of 40 degrees C.

Mol Gen Genet, 1999 Mar, 261(2), 226 - 35
pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities; McCormac AC et al.; A new plasmid series has been created for Agrobacterium-mediated plant transformation . The pBECKS2000 series of binary vectors exploits the Cre/ loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors . The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions . The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors . A 'shuttle' or intermediate plasmid approach has been employed . This permits independent ligation strategies to be used for two gene sets . The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system . This reaction is carried out within Agrobacterium cells . Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid . This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.

Biotechnol Bioeng, 1999 Apr 20, 63(2), 224 - 32
Production of glomus intraradices propagules, an arbuscular mycorrhizal fungus, in an airlift bioreactor
Jolicoeur M, Williams RD, Chavarie C, Fortin JA, Archambault J.
This work addresses the symbiotic culture of the arbuscular mycorrhizal (AM) fungus Glomus intraradices with Daucus carota hairy roots transformed by Agrobacterium rhizogenes, in two submerged culture systems: Petri dish and airlift bioreactor . AM fungi play an active role in plant nutrition and protection against plant pathogens . These fungi are obligate biotrophs as they depend on a host plant for their needs in carbohydrates . The effect of the mycorrhizal roots inoculum-to-medium volume ratio on the growth of both symbionts was studied . A critical inoculating condition was observed at approximately 0.6 g dry biomass (DW) . L-1 medium, above which root growth was significantly reduced when using a low-salt minimal (M) liquid medium previously developed for hairy root-AM fungi co-culture . Below critical inoculum conditions the maximum specific root growth and specific G . intraradices spore production rates of 0.021 and 0.035 d-1, respectively, were observed for Petri dish cultures . Maximum spore production in the airlift bioreactor was ten times lower than that of Petri dish cultures and obtained with the lowest inoculum assessed (0.13 g DW . L-1 medium) with 1.82 x 10(5) +/- 4.05 x 10(4) (SEM) spores (g DW inoculum)-1 (L medium)-1 in 107 d . This work proposes a second-generation bioprocess for AM fungi propagule production in bioreactors . Copyright 1999 John Wiley & Sons, Inc.

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